Handbook of Algal Science, Technology and Medicine [1 ed.] 0128183055, 9780128183052

Handbook of Algal Science, Microbiology, Technology and Medicine provides a concise introduction to the science, biology

854 76 35MB

English Pages 800 [684] Year 2020

Report DMCA / Copyright

DOWNLOAD FILE

Polecaj historie

Handbook of Algal Science, Technology and Medicine [1 ed.]
 0128183055, 9780128183052

Table of contents :
Cover
Handbook of Algal Science,
Technology and Medicine
Copyright
Contributors
Editor biography
Preface
References
Acknowledgments
Part I: Introduction to the algal science, technology, and medicine
1
The scientometric analysis of the research on the algal science, technology, and medicine
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Discussion
Conclusion
Appendix. The keyword sets
Acknowledgments
References
2
100 Citation classics in the algal science, technology, and medicine: A scientometric analysis
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Algal bioenergy and biofuels
Algal biomedicine
Algal photosynthesis
Algal ecology
Algal toxicology
Algal food
Algal genomics
Discussion
Conclusion
Appendix. The keyword sets
Acknowledgments
References
Part II: Algal structures
3
The scientometric analysis of the research on the algal structures
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Composition
Flagellar structures
Phylogeny
Evolution
Silica structures in diatoms
Other fronts
Discussion
Conclusion
Appendix. The keyword sets
Acknowledgments
References
4
Anatomy of Euglena gracilis
General overview
Pellicle and metaboly
Flagellar apparatus
Mitochondrion
Chloroplasts
Nucleus
Locomotion
Photoreceptor system
Photoreception
Practical importance
References
5
Biology and ecology of Northwest Atlantic seaweeds
Introduction
General seaweed biology
What are algae?
Types of seaweeds and their habitats
Anatomy and morphology
Life history patterns
Seasonality and phenotypic patterns
Seaweed communities and zonation patterns
Rocky open coastal systems
Spray zone
Intertidal zone
Subtidal zone
Estuaries and salt marshes
Drift and unattached macroalgae
Biogeographic patterns
Introduced species
Global warming and sea level rise
Pollution and algal blooms
Acknowledgments
References
Further reading
6
Autecology of Northwest Atlantic fucoid algae
Introduction
General reproductive and growth patterns
Reproductive biomass patterns
Limicolous populations
Unique brackish water populations
Effects of water motion
Ice damage
Functional roles of fucoids in estuarine habitats
Acknowledgments
References
Part III: Algal genomics
7
The scientometric analysis of the research on the algal genomics
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Phylogeny of algae
Microalgae
Diatoms
Cyanobacteria
Dinoflagellates
Genomic methodology of algae
Cyanobacteria
Microalgae
Ecology of algae
Cyanobacteria
Microalgae
Other research fronts
Algal structures
Algal biofuels
Algal photosynthesis
Discussion
Conclusion
Appendix. The keyword sets used for the search of the literature on the algal genomics
Genomics related keywords
Algae-related keywords
Algae general
Dinoflagellates and coccolithophores
Microalgae
Macroalgae
Diatoms
Cyanobacteria
Algae-related journals
Acknowledgments
References
8
Metabolic engineering of cyanobacteria for production of platform chemicals: A synthetic biology approach
Introduction
Cyanobacteria: A chassis for platform chemicals
Strategies for enhancing the titers of platform chemicals in cyanobacterial strains
Engineering model cyanobacterial strains for platform chemicals
Pyruvate derived products
Lactate
2,3-Butanediol
Isobutylaldehyde, isobutanol, 1-butanol and 2-methyl-1-butanol
Isoprene and squalene
Acetyl-CoA derived products
Acetone and isopropanol
Tricarboxylic acid cycle derived products
Succinate
Ethylene
DHAP derived products
1,2-Propanediol and 1,3-propanediol
Synthetic biology tools for enhancing the yields in model cyanobacterial strains
Promoters
Transcriptional terminators
Ribosomal binding sites
Future outlook and challenges
Acknowledgments
References
9
Genomics perspectives on cyanobacteria research
Cyanobacteria, their toxins and natural products
PCR based methods
Non-PCR based methods
Next-generation sequencing
Phylogeny and biogeography
Biodiscovery of new compounds
Water quality management
References
10
Using new techniques to study old favorites: A case study of Euglena
Introduction to Euglena
Historical research
Sequencing Euglena
Transcriptome sequencing
Genome sequencing
Plastid genome
Mitochondrial genome
Nuclear genome
Euglena proteomics
Metabolites in Euglena
Central metabolites
Specialized metabolites
Euglena glycomics
Paramylon
Protein glycosylation
Surface glycans
Biotechnology exploiting Euglena
Conclusions
References
11
Exploring ‘omics’ approaches: Towards understanding the essence of stress phenomena in diatoms and haptophytes
Introduction
Revolution of ‘omics’ approaches in diatoms and haptophytes
Genomics
Transcriptomics
Proteomics
Metabolomics and lipidomics
Role of ‘omics’ approaches in understanding stress response in diatoms
Role of ‘omics’ approaches in understanding stress response in haptophytes
Potential application of ‘omics’ tools in reconstruction of biosynthetic pathways for the production of commercia ...
Conclusions and future prospects
Acknowledgments
References
Part IV: Algal photosystems and photosynthesis
12
The scientometric analysis of the research on the algal photosystems and photosynthesis
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Photosystems
Algae/phytoplankton
Cyanobacteria
Photosynthesis
Algae/phytoplankton
Microalgae
Cyanobacteria
Carbon concentration mechanisms
Algae/phytoplankton
Cyanobacteria
Discussion
Conclusion
Appendix: The keyword sets
Photosystems and photosynthesis related keywords
Algae related keywords
Algal general
Dinoflagellates & coccolithophores
Microalgae
Macroalgae
Diatoms
Cyanobacteria
Journals
Cross-subject keywords
References
Further reading
13
Photosynthesis in diatoms
Introduction
Diatom lipids
Lipid composition of thylakoid membranes
Fatty acid composition of thylakoid membrane lipids
Changes of lipid and fatty acid composition by abiotic stress or nutrient deprivation
Function of thylakoid membrane lipids
Diatom light-harvesting complexes
FCP proteins
Native structure of FCPs
Function of FCPs
Regulation of photosynthetic electron transport
Nonphotochemical quenching
Alternative electron transport
Dark reactions in photosynthesis
References
14
The pioneering research on the cyanobacterial photosystems and photosynthesis
Introduction
Materials and methodology
Results
The research landscape
The pioneering research on the cyanobacterial photosystems
The pioneering research on the cyanobacterial photosynthesis
Discussion
The research landscape
The pioneering research on the cyanobacterial photosystems
The pioneering research on the cyanobacterial photosynthesis
Conclusion
Appendix. The keyword sets
Photosynthesis related keywords
Cyanobacteria-related keywords
Cross-subject keywords
Acknowledgment
References
15
Carbon and nitrogen metabolism in cyanobacteria: Basic traits, regulation and biotechnological application
Introduction
Carbon (C) metabolism
Nitrogen (N) metabolism
Regulation and coordination between C and N metabolic pathways
Concluding remarks
Acknowledgments
Competing interests
References
Part V: Algal ecology
16
The scientometric analysis of the research on the algal ecology
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Phytoplankton growth
Harmful algal blooms
Primary production
Climate
Other research fronts
Discussion
Conclusion
Appendix: The keyword sets
Keywords related to ecology
Keywords related to the algae
Excluding keywords
References
17
Macroalgae as a tool for coastal management in the Mediterranean Sea
Introduction
What are macroalgae?
Why can macroalgae constitute a valuable tool for coastal management?
Take care: Misidentifications can spoil a powerful tool
Biological indicators
Delineation of biogeographic provinces
Indicators of global warming, based on changes in their distribution area
Multispecies indices of water quality
Ecosystem-based quality indices (EBQIs)
Management of outstanding species
Discussion and conclusion
Acknowledgments
References
18
Linking phytoplankton community structure to aquatic ecosystem functioning: A mini-review of the current status and future ...
Phytoplankton use in biomonitoring activities
Why use phytoplankton community structure as an index of ecosystem functioning?
Stressor-response relationships within aquatic ecosystems
Traits in river phytoplankton biomonitoring
Estuarine phytoplankton community shifts: What’s in a life form?
Framework around phytoplankton biomonitoring
Current status and future direction
References
19
Microalgae culture technology for carbon dioxide biomitigation
Introduction
Mechanism of CO 2 assimilation via microalgae photosynthesis
Algal cultivation conditions
Photoautotrophic cultivation
Heterotrophic cultivation
Mixotrophic cultivation
Photoheterotrophic cultivation
Algal cultivation systems
Factors effecting microalgae cultivation process
Light intensity
Light wavelength, λ max
Macro and micro-nutrients
pH of the algal culture
Temperature of the algal culture
Carbon dioxide fixation strategies
Conclusions
References
Part V: Algal ecology
20
The scientometric analysis of the research on the algal bioenergy and biofuels
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Biodiesel and lipid production
Biofuel production
Other research fronts
Discussion
Conclusion
Appendix. The keyword sets
Energy and fuels keywords
Core energy journals
Energy and fuels keywords
Algae keywords
Algal general
Dinoflagellates and coccolithophores
Microalgae
Macroalgae
Diatoms
Cyanobacteria
Journals
Excluding keywords
Acknowledgment
References
Further reading
21
Overview of microalgal cultivation, biomass processing and application
Introduction
Environmental and nutritional requirements for microalgal growth
Light
Temperature
Nutrients
Microalgal cultivation systems for biomass production
Open cultivation systems
Closed cultivation systems
Microalgae growing techniques
Microalgal downstream processing chain
Harvesting
Drying
Cell disruption and compounds extraction
Microalgal applications
Biofuels
Wastewater treatment
Food and feed
Other applications
Conclusions
References
22
Application of microalgae for the production of biodiesel fuel
Advantages of the use of microalgae biomass for biofuel production
Triglyceride transesterification process
Use of homogeneous catalysis in biodiesel synthesis from microalgal oil
Application of acid catalysis for microalgal oil transesterification
Application of alkaline catalysis for microalgal oil transesterification
Biodiesel fuel synthesis applying heterogeneous catalysis
Heterogeneous catalysis with enzymes as catalysts
Properties of biodiesel obtained from microalgal oil
Conclusions
References
23
Microalgal hydrogen production in the framework of bioeconomy
Biological hydrogen production as renewable energy
Hydrogen production with microalgae: Physiology and metabolism
Biotechnology of microalgae for hydrogen production: Improvement of H 2 production efficiency
Microalgal utilization of wastewaters as substrate for growth and hydrogen production with microalgae
Role of biohydrogen production with microalgae in bioeconomy
Utilization of solar energy
Recovery of waste material and conversion into biofuel
Production of high added value bio-products
Future perspectives
References
24
Bioelectricity generation in algal microbial fuel cells
Introduction
Role of algae in microbial fuel cells
Algae as a biocatholyte
Algae as substrate
Algal biofilm assisted microbial fuel cells
Electron transport and transfer mechanisms in algae
Performance evaluation of algal microbial fuel cells
Algal MFCs and bioelectricity generation
Algal MFCs and wastewater treatment
Beneficial algal byproducts in MFCs
Conclusions
References
25
The pioneering research on the bioethanol production from green macroalgae
Introduction
Materials and methodology
Results
The research landscape
Pioneering research on the pretreatments of green macroalgae for bioethanol production
Pioneering research on the acid hydrolysis of green macroalgae for bioethanol production
Pioneering research on the enzymatic hydrolysis of green macroalgae for bioethanol production
Pioneering research on the fermentation of green macroalgae for bioethanol production
Discussion
The research landscape
Pioneering research on the pretreatments of green macroalgae for bioethanol production
Pioneering research on the acid hydrolysis of green macroalgae for bioethanol production
Pioneering research on the enzymatic hydrolysis of green macroalgae for bioethanol production
Pioneering research on the fermentation of green macroalgae for bioethanol production
Conclusion
Appendix. The keyword sets
Ethanol-related keywords
Green macrolgae-related keywords
References
Further reading
Part VII: Algal biomedicine
26
The scientometric analysis of the research on the algal biomedicine
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Alginates
Agaroses
Channelrhodopsins
Other research fronts
Discussion
Conclusion
Appendix. The keyword sets
Biomedicine related keywords
Keywords related to the subject categories
Keywords related to the journal titles
Keywords related to the paper titles
Keywords related to the algae
Algae in general
Dinoflagellates and coccolithophores
Microalgae
Macroalgae
Diatoms
Cyanobacteria
Journals
Cross-subject keywords
References
27
Microalgal biotechnology applied in biomedicine
Introduction
Microalgae
Bioactive compounds and their properties
Proteins
Lipids
Carbohydrates
Phenolic compounds
Vitamins
Bioengineering of microalgae for the synthesis of bioactive compounds
Microalgal biomass applied in biomedicine
Future perspectives
Conclusion
References
28
Applications of cyanobacteria in biomedicine
Introduction (scope of the review)
Improving access to natural products as biomedical compounds
New approaches to the value of biomedical natural products
Activity profiling of extracts of cyanobacteria
Natural products as novel pharmaceutical drugs and biomedical tools
Role of systems biology in cyanobacterial drug discovery and biomedical compounds
Model for systems biology based cyanobacterial drug discovery
Identification of drug targets
Virtual screening
Cyanobacteria involvement in production of biomedical metabolites
Conclusions
References
Further reading
29
Health benefits of bioactive seaweed substances
Introduction
Applications of bioactive seaweed substances in health products
Health benefits of bioactive seaweed substances
Anti-tumor effects
Immunoregulation properties
Anti-oxidant properties
Reduction of blood pressure
Reduction of blood sugar
Reduction of blood fat
Anticoagulant and antithrombotic properties
Anti-inflammatory and anti-allergic properties
Anti-bacteria and anti-virus properties
Anti-HIV properties
Anti-fatigue properties
Anti-aging properties
Absorption of heavy metal ions
Suppression of esophageal and esophageal reflux
Bulking of fecal contents and relief of constipation
Slimming properties
Anti-diabetic properties
Deodorant properties
Anti-acne properties
Anti-depression properties
Protection against radiation
Inhibition of matrix metalloproteinase
Skin whitening effect
Summary
Acknowledgments
References
30
The pioneering research on the wound care by alginates
Introduction
Materials and methodology
Results
The research landscape
The pioneering research on the wound care by alginates only
The pioneering research on the wound care by alginates and chitosan
The pioneering research on the wound care by alginates and other biomaterials
Discussion
The research landscape
The pioneering research on the wound care by alginates only
The pioneering research on the wound care by alginates and chitosan
The pioneering research on the wound care by alginates and other biomaterials
Conclusion
Appendix. The keyword sets
Alginate-related keywords
Wound-related keywords
References
Part VIII: Algal foods
31
The scientometric analysis of the research on the algal foods
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Sulfated polysaccharides
Fucoidans
Algal foods
Other research fronts
Discussion
Conclusion
Appendix. The keyword sets
Algae and foods separate keywords
Foods
Keywords related to the algae
Algal general
Dinoflagellates and coccolithophores
Microalgae
Macroalgae
Diatoms
Cyanobacteria
Journals
Algal foods- cross-subject keywords
Acknowledgments
References
32
Microalgae: A new and promising source of food
Introduction
Microalgae composition
Microalgae as source of food
Nutritional properties
Nutraceutical properties
Techno-functional properties
Microalgae as feed
Final considerations
References
Part IX: Algal toxicology
33
The scientometric analysis of the research on the algal toxicology
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Algal toxins
Dinoflagellates-okadaic acid
Cyanobacteria-microcystins
Cyanobacterial toxins in general
Harmful algal blooms
Discussion
Conclusion
Appendix. The keyword sets
Algal toxicology direct keywords
Combined keyword sets
Toxicology
Algae
Algal general
Phytoplankton, dinoflagellates, coccolithophores
Microalgae
Macroalgae
Diatoms
Cyanobacteria
Journals
References
Further reading
34
Toxic effects of harmful algal blooms on finfish and shellfish
Introduction
Impact of HAB species on finfish and shellfish industries
Fish-killing species and its mechanism of detrimental effects
Chattonella
Karenia
Cochlodinium
Heterosigma
Other species
Effects of fish species and physiological conditions on survival rate
Mass mortality and detrimental effects on molluscan shellfish
Karenia mikimotoi
Aureococcus anophagefferen
Cochlodinium polykrikoides
Heterocapsa circularisquama
Other potentially toxic species to shellfish
Adverse effects on gastropod species
Conclusions
Acknowledgements
References
35
Cyanobacterial toxins and their effects on human and animal health
Introduction
Types of toxins produced by cyanobacteria
Hepatotoxins
Cytotoxins
Neurotoxins
Anatoxin-a
Anatoxin-a(S)
Saxitoxins
BMAA
Dermatotoxins
LPS
Routes of exposure and poisoning incidents
Animals
Humans
Water
Recreational exposure
Drinking water
Aerosol
Medical procedures
Food
Air
Bioassays for cyanobacterial toxins
Small animal
Enzyme and molecular systems
Detoxication, metabolism and deposition
Conclusion
References
Further reading
36
Different intoxication mechanism between paralytic shellfish toxin (PST)- and/or tetrodotoxin-contaminated xanthid crabs an ...
Introduction
Food poisoning cases with xanthid crabs
Toxicity of xanthid crabs
Z. aeneus
A. floridus
P. granulosa
Anatomical distribution of toxicity
Origin of crab toxin
Resistibility of toxic crabs to PST and TTX
Live crabs
Crab neurofibers
PST contamination of edible shore crabs T. acutidens and C. japonica
Some other toxic crabs
D. perlata
Horseshoe crab Carcinoscorpius rotundicauda
‘Palytoxin’ (PTX)-bearing crabs
Discussion
Acknowledgments
Declaration of interest
References
37
Microcystins as environmental and human health hazards
Introduction
What are microcystins?
Structure, synthesis, and possible functions
Mode of action and toxicity
Environmental fate and risk posed
Human exposure risk
Detoxification
Countermeasures: The Green Liver System
Concluding remarks
References
Part X: Algal bioremediation
38
The scientometric analysis of the research on the algal bioremediation
Introduction
Materials and methodology
Results
Documents and indexes
Keywords
Authors
Countries
Institutions
Research funding bodies
Publication years
Source titles
Subject categories
Research fronts
Citation classics
Scientometric overview of the citation classics
Brief overview of the content of the citation classics
Heavy metal bioremediation by algae
Macroalgae
Microalgae
Algae
Wastewater bioremediation by algae
CO 2 bioremediation by algae
Discussion
Conclusion
Appendix: The keyword sets
Keywords related to bioremediation
Keywords related to the algae
Algal general
Dinoflagellates and coccolithophores
Microalgae
Macroalgae
Diatoms
Cyanobacteria
Journals
Acknowledgment
References
39
Spirulina platensis as a model object for the environment bioremediation studies
The necessity and importance of bioremediation studies
Advisability of the use of Spirulina as a model object in bioremediation studies
Sorption and accumulation of metals by Spirulina in mono and multicomponent systems
Biosorption
Biosorption isotherm models
Kinetic models
Bioaccumulation
Detoxification of persistent organic pollutants by Spirulina
Conclusions
References
40
Exploring the potential of microalgae for the bioremediation of agro-industrial wastewaters
Introduction
Microalgae-based treatment of agroindustrial wastewaters
Wastewater in the agroindustrial sector
Fundamentals of pollutant removal in algal-bacterial systems
Optimal design of photobioreactors for wastewater treatment
Microbiology of microalgae-based wastewater treatment
Harvesting of algal-bacterial biomass
Optimization of harvesting strategy
Coagulation-flocculation
Bio-flocculation
Valorization of the residual algal-bacterial biomass
Biofertilizers
Aquafeed
Biogas
Sustainability of the process
Cultivation
Harvesting
Transformation
CO 2 fixed as algal biomass
Comparison of recent research
Future trends
Acknowledgments
References
Further reading
41
Microalgal bioremediation of heavy metals and dyes
Introduction
Textile operations
Sizing and desizing
Bleaching
Mercerization
Dyeing and printing
Finishing
Regulations for wastewater discharge and harmful effects of dyes and heavy metal ions
Impact of azo dyes
Dyes
Treatment processes applied for textile wastewater and heavy metal ions
Physical processes
Adsorption
Ion exchange
Membrane filtration technologies
Chemical treatment methods
Chemical precipitation
Oxidation methods
Physicochemical treatment (coagulation-flocculation)
Biological treatment methods
Dyes treatment employing biological approaches
Heavy metal ions treatment employing biological approaches
Employing algae for textile wastewater treatment and heavy metals removal
Algae and dyes
Algae and heavy metals
Metabolism-dependent biosorption
Metabolism-independent biosorption
Detoxification mechanism of heavy metals by microalgae
Conclusion
References
42
Biosorption of chemical species by Sargassum algal biomass: Equilibrium data, part I
Introduction
Data analysis using the Langmuir equation
Free Gibbs energy of biosorption
The pH effect
The temperature effect
Analysis of the chemical composition of native Sargassum worldwide
Conclusions
Acknowledgments
References
Index
A
B
C
D
E
F
G
H
I
K
L
M
N
O
P
R
S
T
U
V
W
X
Y
Z
Back Cover

Citation preview

Handbook of Algal Science, Technology and Medicine

Handbook of Algal Science, Technology and Medicine

Edited by

Ozcan Konur

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-818305-2 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisitions Editor: Linda Versteeg-Buschman Editorial Project Manager: Sam W. Young Production Project Manager: Debasish Ghosh Cover Designer: Andre G. Wolff Typeset by SPi Global, India

Contributors Numbers in parenthesis indicate the pages on which the authors’ contributions begin.

Charles F. Boudouresque (277), Aix-Marseille University, University of Toulon, CNRS, Marseille, France

Francisco Gabriel Acién-Fernández (641), University of Almería, Almería, Spain

Cristiane Canan (507), Federal Technological University of Parana, Medianeira, Brazil

Hossein Ahmadzadeh  (659), Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, Iran

Naira Valle de Castro (245), Federal University of Viçosa, Viçosa, Brazil

Cynthia Alcántara (641), Valladolid University, Valladolid, Spain

Liliana Cepoi  (629), Institute of Microbiology and Biotechnology; State University “Dimitrie Cantemir”, Chisinau, Republic of Moldova

Allan Victor Martins Almeida  (245), Federal University of Viçosa, Viçosa, Brazil Agostinho Antunes  (147), CIIMAR, Interdisciplinary Centre of Marine and Environmental Research; Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal

Channakeshavaiah Chikkaputtaiah  (171), Academy of Scientific and Innovative Research (AcSIR), CSIRNEIST; Biological Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, India

Osamu Arakawa  (575), Nagasaki University, Nagasaki, Japan

Ann D. Christy  (377), Ohio State University, Columbus, OH, United States

Wagner L. Araújo  (245), Federal University of Viçosa, Viçosa, Brazil

Eliane Colla  (507), Federal Technological University of Parana, Medianeira, Brazil

Neha Arora (127), Indian Institute of Technology Bombay, Mumbai, India

Jorge Alberto Vieira Costa  (429), Federal University of Rio Grande, Rio Grande, Brazil

Manabu Asakawa (575), Hiroshima University, Hiroshima, Japan

Rosana Aparecida da Silva-Buzanello  (507), Federal Technological University of Parana, Medianeira, Brazil

Jose L. Barriada  (675), University of Coruna, Coruna, Spain Laura Barsanti (61), National Council of Research (CNR), Pisa, Italy Eloy Bécares  (641), Institute of the Environment, Leon, Spain Saúl Blanco  (441,641), Departamento de Biodiversidad y Gestión Ambiental, Facultad de Ciencias Biológicas y Ambientales,Universidad de León, León, Spain; Institute of the Environment, Leon, Spain Aurélie Blanfuné  (277), Aix-Marseille University, University of Toulon, CNRS, Marseille, France Silvia Bolado  (641), Valladolid University, Valladolid, Spain; Institute of Sustainable Processes (ISP), Valladolid, Spain

Kinue Daigo (575), Tokyo Healthcare University Graduate School, Tokyo; Nagasaki University, Nagasaki; National Research Institute of Fisheries Science, Yokohama; Hiroshima University, Hiroshima, Japan Tatenda Dalu  (291), University of Venda, Thohoyandou; South African Institute for Aquatic Biodiversity, Grahamstown, South Africa Clinton J. Dawes (71,91), University of New Hampshire, Durham, NH, United States Deepi Deka  (171), Biological Sciences Division, CSIRNorth East Institute of Science and Technology, Branch Laboratory; Academy of Scientific and Innovative Research (AcSIR), CSIR-NEIST, Jorhat, India Deisy Alessandra Drunkler (507), Federal Technological University of Parana, Medianeira, Brazil

xvii

xviii  Contributors

Maranda Esterhuizen-Londt  (591), University of Helsinki, Lahti, Finland; Korean Institute of Science and Technology Europe (KIST Europe), Saarbrucken, Germany Letícia Schneider Fanka (429), Federal University of Rio Grande, Rio Grande, Brazil Cecilia Faraloni (367), IBE (Institute for the Bioeconomy), National Research Council (CNR), Florence, Italy José María Fernández-Sevilla  (641), University of Almería, Almería, Spain Pedro García-Encina  (641), Valladolid University, Valladolid, Spain; Institute of Sustainable Processes (ISP), Valladolid, Spain María Cruz García-González  (641), Agricultural Technological Institute of Castilla and Leon (ITACyL), Valladolid, Spain Reimund Goss  (217), University of Leipzig, Leipzig, Germany Paolo Gualtieri (61), National Council of Research (CNR), Pisa, Italy David Hernández  (641), Agricultural Technological Institute of Castilla and Leon (ITACyL), Valladolid, Spain Roberto Herrero  (675), University of Coruna, Coruna, Spain Rubén Irusta  (641), Valladolid University, Valladolid, Spain; Institute of Sustainable Processes (ISP), Valladolid, Spain Damini Jaiswal  (127), Indian Institute of Technology Bombay, Mumbai, India Torsten Jakob  (217), University of Leipzig, Leipzig, Germany Daneysa Lahis Kalschne  (507), Federal Technological University of Parana, Medianeira, Brazil Ozcan Konur  (3,19,41,105,195,231,257,319,385,405, 467,485,521,607), Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey Roberta da Costa Kosinski  (429), Federal University of Rio Grande, Rio Grande, Brazil

Violeta Makareviciene (353), Vytautas Magnus University, Agriculture Academy. Kaunas, Lithuania Giorgos Markou  (343), Institute of Technology of Agricultural Products, Hellenic Agricultural, Organization-Demeter (ELGO-Demeter), Athens, Greece Joana Martins  (147), CIIMAR, Interdisciplinary Centre of Marine and Environmental Research; Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal Arthur C. Mathieson  (71,91), University of New Hampshire, Durham, NH, United States Yukihiko Matsuyama  (543), Fisheries Research and Education Agency, Nagasaki, Japan Anne Luize Lupatini Menegotto  (507), University Regional Integrated High Uruguay and Missions (URI), Erechim, Brazil J.S. Metcalf  (561), Brain Chemistry Labs, Jackson, WY, United States Keisuke Miyazawa  (575), Tokyo Healthcare University Graduate School, Tokyo; Nagasaki University, Nagasaki; National Research Institute of Fisheries Science, Yokohama; Hiroshima University, Hiroshima, Japan Marziyeh Molazadeh  (659), Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, Iran Michele Greque de Morais  (429), Federal University of Rio Grande, Rio Grande, Brazil Cristiana Moreira  (147), CIIMAR, Interdisciplinary Centre of Marine and Environmental Research; Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal Juliana Botelho Moreira (429), Federal University of Rio Grande, Rio Grande, Brazil Raúl Muñoz  (641), Valladolid University, Valladolid, Spain; Institute of Sustainable Processes (ISP), Valladolid, Spain Tamao Noguchi  (575), Tokyo Health University, Tokyo, Japan

Ana Larrán (641), Agricultural Technological Institute of Castilla and Leon (ITACyL), Valladolid, Spain

Bahareh Nowruzi (441), Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

Daniel A. Lemley (291), Nelson Mandela University, Port Elizabeth, South Africa

Adriano Nunes-Nesi (245), Federal University of Viçosa, Viçosa, Brazil

Pablo Lodeiro  (675), GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany

Ellis O’Neill  (161), School of Chemistry, University of Nottingham, Nottingham, United Kingdom

Stephen Lyon  (659), SRL-Environmental, LLC, Racine, WI, United States

Tatsuya Oda (543), Nagasaki University, Nagasaki, Japan

Contributors  xix

Hiroshi Oikawa  (575), National Research Institute of Fisheries Research Agency, Yokohama, Japan

Thierry Thibaut  (277), Aix-Marseille University, University of Toulon, CNRS, Marseille, France

Sheyla Ortíz (641), Valladolid University, Valladolid, Spain

Cristina Tomás (641), Agricultural Technological Institute of Castilla and Leon (ITACyL), Valladolid, Spain

Stephan Pflugmacher (591), University of Helsinki, Lahti, Finland; Korean Institute of Science and Technology Europe (KIST Europe), Saarbrucken, Germany Yimin Qin  (455), State Key Laboratory of Bioactive Seaweed Substances, Qingdao, China Berta Riaño (641), Agricultural Technological Institute of Castilla and Leon (ITACyL), Valladolid, Spain Pilar Rodriguez-Barro  (675), University of Coruna, Coruna, Spain Sandrine Ruitton  (277), Aix-Marseille University, University of Toulon, CNRS, Marseille, France Beenish Saba (377), Ohio State University, Columbus, OH, United States Gisoo Sarvari  (441), Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Chemistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Manuel E. Sastre de Vicente (675), University of Coruna, Coruna, Spain Egle Sendzikiene  (353), Vytautas Magnus University, Agriculture Academy. Kaunas, Lithuania Shinjinee Sengupta (127), Indian Institute of Technology Bombay, Mumbai, India Mostafa Shourian (659), Guilan University, Rasht, Iran Gavin C. Snow  (291), University of the Witwatersrand, Johannesburg, South Africa Shashanka Sonowal (171), Biological Sciences Division, CSIR-North East Institute of Science and Technology, Branch Laboratory; Academy of Scientific and Innovative Research (AcSIR), CSIR-NEIST, Jorhat, India N.R. Souza  (561), Brain Chemistry Labs, Jackson, WY, United States

Giuseppe Torzillo  (367), IBE (Institute for the Bioeconomy), National Research Council (CNR), Florence, Italy Vitor Vasconcelos (147), CIIMAR, Interdisciplinary Centre of Marine and Environmental Research; Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal Marcelo Gomes Marçal Vieira Vaz  (245), Federal University of Viçosa, Viçosa, Brazil Natarajan Velmurugan  (171), Biological Sciences Division, CSIR-North East Institute of Science and Technology, Branch Laboratory; Academy of Scientific and Innovative Research (AcSIR), CSIR-NEIST, Jorhat, India Teresa Vilariño (675), University of Coruna, Coruna, Spain Pramod P. Wangikar (127), Indian Institute of Technology Bombay, Mumbai, India Christian Wilhelm (217), University of Leipzig, Leipzig, Germany Naicheng Wu (291), Kiel University, Kiel, Germany; Xi’an Jiaotong-Liverpool University, Suzhou, China Inga Zinicovscaia  (629), Joint Institute for Nuclear Research, Dubna, Russia; Horia Hulubei National Institute for R&D in Physics and Nuclear Engineering, Bucharest, Romania; Institute of Chemistry, Chisinau, Republic of Moldova Hussein Znad  (303), Curtin University, Perth, WA, Australia Hamidreza Zohoorian  (659), Guilan University, Rasht, Iran

Editor biography Ozcan Konur, as both a bioscientist and social scientist by training, has focused on the bibliometric evaluation of the research in several interdisciplinary innovative high-priority research areas at the level of the researchers, journals, institutions, countries, and research areas as well as the social implications of the research in these areas. He has also had extensive research interests in the development of social policies for disadvantaged people based on disability, age, religious beliefs, race, gender, and sexuality at the interface of science and policy. His current focus is on the algae, bioenergy and biofuels, and nanomedicine. He has published more than 100 journal papers, book chapters, and conference papers.

xxi

Preface Our society currently faces insurmountable challenges in the areas of energy (Abas et al., 2015; Hook and Tang, 2013; Hubbert, 1949) and healthcare (Anderson et al., 2003; Daniels, 2001; Leape et al., 2009) in the face of increasing global population (Keilman, 1998; Lutz and Samir, 2010; Raftery et al., 2012) and diminishing natural resources (Chisti, 2007, 2008) as well as the growing environmental and ecological concerns such as global warming (Dai, 2011; Root et al., 2003; Vitousek, 1994) greenhouse gas emissions (Johnson et al., 2007; Lashof and Ahuja, 1990; Riahi et al., 2011), and climate change (Karl and Trenberth, 2003; Oreskes, 2004; Thomas et al., 2004). Algae offer a way to deal these challenges and concerns for both sustainable and environment friendly bioenergy production (Chisti, 2007, 2008; Li et al., 2008) and for healthcare (Lee and Mooney, 2012; Rowley et al., 1999; Tonnesen and Karlsen, 2002) through the development of crucial biotechnology. This book provides a concise introduction to the science, technology, and medicine of algae, structured based on the major research fronts of the last five decades such as algal structures and properties, algal genomics, algal photosystems and photosynthesis, algal ecology, algal bioenergy and biofuels, algal biomedicine, algal foods, algal toxicology, and algal bioremediation. All these chapters were authored by the leading researchers in their respective research fields. This book fills a significant market opening for the handbook of the science, technology, and medicine of the algae, covering all types of algae such as macroalgae, microalgae, diatoms, coccolithophores, cyanobacteria, and dinoflagellates as well as their derivatives such as alginates, carrageenans, fucoidans, agars, agaroses, channelrhodopsins, microcystins, nodularins, and cyanovirins. This book would be a key reference in one volume for all the stakeholders such as policy makers, industrial leaders, professionals, academicians, and graduate students engaged in these major research streams of algae. It is a must interdisciplinary introduction and handbook for all the stakeholders engaged in science, technology, and medicine of algae in one volume. It covers the major research streams of the last five decades ranging from algal structures to algal bioremediation and algal biomedicine in one volume. It provides easy access to the leading scientists in their respective fields in one volume. The primary audience for this book would be all the stakeholders such as policy makers, industrial leaders, academicians, professionals, and graduate students engaged in these major research streams of algae covering the academic disciplines of ‘Marine Freshwater Biology’, ‘Plant Sciences’, ‘Biotechnology Applied Microbiology’, ‘Biochemistry Molecular Biology’, ‘Environmental Sciences’, ‘Microbiology’, ‘Ecology’, ‘Oceanography’, ‘Food Science Technology’, ‘Energy Fuels’, ‘Pharmacology Pharmacy’, ‘Toxicology’, ‘Chemistry Applied’, ‘Chemistry Physical’, ‘Cell Biology’, ‘Biophysics’, ‘Chemistry Multidisciplinary’, ‘Engineering Chemical’, ‘Multidisciplinary Sciences’, ‘Polymer Science’, ‘Engineering Environmental’, ‘Water Resources’, ‘Agricultural Engineering’, ‘Chemistry Organic’, ‘Fisheries’, ‘Chemistry Analytical’, ‘Biochemical Research Methods’, ‘Chemistry Medicinal’, ‘Genetics Heredity’, ‘Geosciences Multidisciplinary’, ‘Biology’, ‘Materials Science Multidisciplinary’, ‘Materials Science Biomaterials’, ‘Engineering Biomedical’, ‘Evolutionary Biology’, ‘Immunology’, ‘Nutrition Dietetics’, ‘Biodiversity Conservation’, ‘Paleontology’, ‘Nanoscience Nanotechnology’, ‘Physics Applied’, ‘Zoology’, ‘Optics’, ‘Medicine Research Experimental’, ‘Agriculture Multidisciplinary’, ‘Geography Physical’, ‘Green Sustainable Science Technology’, ‘Physiology’, ‘Electrochemistry’, ‘Oncology’, ‘Neurosciences’, ‘Physics Multidisciplinary’, ‘Agronomy’, ‘Soil Science’, ‘Physics Condensed Matter’, ‘Agriculture Dairy Animal Science’, ‘Public Environmental Occupational Health’, ‘Infectious Diseases’, ‘Endocrinology Metabolism’, ‘Virology’, ‘Surgery’, ‘Cell Tissue Engineering’, ‘Integrative Complementary Medicine’, ‘Pathology’, ‘Hematology’, ‘Medical Laboratory Technology’, ‘Dentistry Oral Surgery Medicine’, ‘Transplantation’, ‘Medicine General Internal’, and ‘Clinical Neurology’ among others. These stakeholders would form an informed opinion on the algae with an efficient access to all the major research themes of the last five decades authored by the leading scientists for each theme in one volume.

xxiii

xxiv  Preface

There are 42 chapter titles under 10 parts: Introduction, algal structures, algal genomics, algal photosystems and photosynthesis, algal ecology, algal bioenergy and biofuels, algal biomedicine, algal foods, algal toxicology, and algal biomedicine. The first section of the handbook provides an introduction to algal science, technology, and medicine. Konur (2020a) carries out a scientometric study of the research on the algal science, technology, and medicine based on the whole population of the research in this field while Konur (2020b) focuses on the pioneering research in the algal science, technology, and medicine. These studies at the macro level lay out the context for the handbook covering all major research fronts from toxicology to bioenergy and biofuels. The second section of the handbook provides the information on the structure and properties of the algae as key background materials on algae. Konur (2020c) carries out a scientometric study of the research on the algal structures. Barsanti and Gualtieri (2020) presents a case study of the macroalgal structures focusing on the anatomy of Euglena gracilis. Mathieson and Dawes (2020a) present a study on the biology and ecology of Northwest Atlantic seaweeds while Mathieson and Dawes (2020b) study autecology of Northwest Atlantic fucoid algae. These studies provide background information on the structure and properties of algae relevant for the following chapters in the handbook. These studies show that ‘phylogeny’ and ‘taxonomy’ of the algae as well as the ‘cell biology’ and ‘physiology’ of algae have been primary prolific research fronts (Falkowski et al., 2004; Littler and Littler, 1980; Menden-Deuer and Lessard, 2000). The third section of the handbook deals with the genomics of algae. Konur (2020d) presents a scientometric study of the research on the algal genomics. Arora et al. (2020) study the metabolic engineering of cyanobacteria for production of platform chemicals through a synthetic biology approach while Moreira et al. (2020) provide a study on the genomics perspectives on cyanobacteria research. O’Neill (2020) provides a case study of the macroalgal genomics on Euglena while Deka et al. (2020) explore omics approaches in diatoms and haptophytes for understanding the essence of stress phenomena in these algae. These studies show that ‘cyanobacterial genomics’ and ‘microalgal genomic studies’ have been the most prolific research fronts (Derelle et al., 2016; Ishiura et al., 1998; Kaneko et al., 2001). The fourth section of the handbook deals with algal photosystems and photosynthesis. Konur (2020e) provides a scientometric study of the research on the algal photosystems and photosynthesis. Goss et al. (2020) presents a study of the photosynthesis in diatoms while Konur (2020f) provides a study of the pioneering research on the cyanobacterial photosystems and photosynthesis. Finally, Vaz et al. (2020) study carbon and nitrogen metabolism in cyanobacteria focusing on the basic traits, regulation and biotechnological applications. These studies show that the most-prolific research fronts have been ‘cyanobacterial photosynthesis’ and ‘cyanobacterial photosystems’ (Ferreira et al., 2004; Guskov et al., 2009; Jordan et al., 2001). The fifth section of the handbook deals with the interaction of the algae with the ecology and environment. Konur (2020g) provides a scientometric study of the research on the algal ecology while Boudouresque et al. (2020) study the macroalgae as a tool for coastal management in the Mediterranean Sea. Next, Dalu et al. (2020) presents a study linking phytoplankton community structure to aquatic ecosystem functioning while Znad (2020) focuses on the microalgae culture technology for carbon dioxide biomitigation. These studies show that the most-prolific research fronts have been ‘ecology of phytoplankton’, ‘ecology of cyanobacteria’, ‘ecology of algae’, and ‘ecology of macroalgae’ (Boyd et al., 2000; Coale et al., 1996; Hecky and Kilham, 1988). The sixth section deals with algal biofuels and bioenergy. Konur (2020h) provides a scientometric analysis of the research on the algal bioenergy and biofuels. While Markou (2020) provides an overview of microalgal cultivation, biomass processing and application. Makareviciene and Sendzikiene (2020) present a study on the microalgal biodiesel production, Faraloni and Torzillo (2020) provides a study on the microalgal hydrogen production. Finally, Saba and Christy (2020) presents as study of the bioelectricity generation in algal microbial fuel cells while Konur (2020i) provides a scientometric study of the research on the bioethanol production from green macroalgae. These studies show that ‘microalgal bioenergy and biofuels’ has been the most prolific research front (Chisti, 2007, 2008; Brennan and Owende, 2010). The seventh section of the handbook deals with the applications of algae in healthcare. Konur (2020j) provides a scientometric study of the research on the algal biomedicine while Costa et al. (2020) focuses on the microalgal biotechnology applied in biomedicine. Next, Nowruzi et al. (2020) provide a study on the applications of cyanobacteria in biomedicine. While Qin (2020) focuses on the health benefits of bioactive seaweed substances. Finally, Konur (2020k) presents a study on the pioneering research on the wound care by alginates. These studies show that the field of ‘alginates’ has been the most prolific research front (Lee and Mooney, 2012; Rowley et al., 1999; Tonnesen and Karlsen, 2002). The eight section of the handbook focuses on the algal foods. Konur (2020l) provides a scientometric study of the research on the algal foods while Colla et al. (2020) focuses on the use of microalgae as a food source. These studies show that ‘macroalgal foods’ and ‘cyanobacterial foods’ have been the most prolific research fronts (Ciferri, 1983; Duan et al., 2006; Fleurence, 1999).

Preface  xxv

The ninth section of the handbook deals with toxicity of algae to both the environment and humans as well as harmful algal blooms. Konur (2020m) provides a scientometric study of the research on the algal toxicology, while Matsuyama and Oda (2020) present a study on the toxic effects of harmful algal blooms on finfish and shellfish. Next, Souza and Metcalf (2020) study the cyanobacterial toxins and their effects on human and animal health while Noguchi et al. (2020) present a study on the different intoxication mechanism between paralytic shellfish toxin (PST)- and/or tetrodotoxin-contaminated xanthid crabs and PST-contaminated edible shore crabs in Japan and their food poisonings. Finally, Esterhuizen-Londt and Pflugmacher (2020) present a study on the microcystins as environmental and human health hazards. These studies show that ‘algal toxins’ and ‘harmful algal blooms’ have been the most-prolific research fronts (Anderson et al., 2002; Carmichael, 1994; Codd et al., 2005). The last section of the handbook deals with the removal of heavy metals, dyes, and other toxic materials available in the environment such as wastewaters by algae. This section can also be considered as a special sub-set of algal ecology. Konur (2020n) provides a scientometric study of the research on the algal bioremediation while Cepoi and Zinicovscaia (2020) take Spirulina platensis as a model object for the environment bioremediation studies. Next, Alcantara et al. (2020) explore the potential of microalgae for the bioremediation of agro-industrial wastewaters and Zohoorian et al. (2020) provide a study on the microalgal bioremediation of heavy metals and dyes while Sastre de Vicente et al. (2020) present a study on the biosorption of chemical species by Sargassum algal biomass. These studies show that field of ‘macroalgal heavy metal bioremediation’ has been the most prolific research front while the other key research fronts have been ‘microalgal wastewater bioremediation’, ‘microalgal CO2 bioremediation’, and ‘microalgal heavy metal bioremediation’ (Aksu, 2002; Cai et al., 2013; Davis et al., 2003).

References Abas, N., Kalair, A., Khan, N., 2015. Review of fossil fuels and future energy technologies. Futures 69, 31–49. Aksu, Z., 2002. Determination of the equilibrium, kinetic and thermodynamic parameters of the batch biosorption of nickel(II) ions onto Chlorella vulgaris. Process Biochem. 38 (1), 89–99. Alcantara, C., Acien-Fernandez, F.G., Fernandez-Sevilla, J.M., Riano, B., Hernandez, D., Garcia-Gonzalez, M.C., et al., 2020. Exploring the potential of microalgae for the bioremediation of agro-industrial wastewaters. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25 (4B), 704–726. Anderson, L.M., Scrimshaw, S.C., Fullilove, M.T., Fielding, J.E., Normand, J., Task Force on Community Preventive Services, 2003. Culturally competent healthcare systems: a systematic review. Am. J. Prev. Med. 24 (3), 68–79. Arora, N., Jaiswal, D., Sengupta, S., Wangikar, P.P., 2020. Metabolic engineering of cyanobacteria for production of platform chemicals: a synthetic biology approach. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Barsanti, L., Gualtieri, P., 2020. Anatomy of Euglena gracilis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Boudouresque, C.F., Blanfune, A., Ruitton, S., Thibaut, T., 2020. Macroalgae as a tool for coastal management in the Mediterranean Sea. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Boyd, P.W., Watson, A.J., Law, C.S., Abraham, E.R., Trull, T., Murdoch, R., et al., 2000. A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization. Nature 407 (6805), 695–702. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sust. Energ. Rev. 14 (2), 557–577. Cai, T., Park, S.Y., Li, Y.B., 2013. Nutrient recovery from wastewater streams by microalgae: status and prospects. Renew. Sust. Energ. Rev. 19, 360–369. Carmichael, W.W., 1994. The toxins of cyanobacteria. Sci. Am. 270 (1), 78–86. Cepoi, L., Zinicovscaia, I., 2020. Spirulina platensis as a model object for the environment bioremediation studies. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26 (3), 126–131. Ciferri, O., 1983. Spirulina, the edible microorganism. Microbiol. Rev. 47 (4), 551–578. Coale, K.H., Johnson, K.S., Fitzwater, S.E., Gordon, R.M., Tanner, S., Chavez, F.P., et  al., 1996. A massive phytoplankton bloom induced by an ­ecosystem-scale iron fertilization experiment in the equatorial Pacific Ocean. Nature 383 (6600), 495–501. Codd, G.A., Morrison, L.F., Metcalf, J.S., 2005. Cyanobacterial toxins: risk management for health protection. Toxicol. Appl. Pharmacol. 203 (3), 264–272. Colla, E., Lupatini Menegotto, A.L., Kalschne, D.L., da Silva-Buzanello, R.A., Canan, C., Drunkler, D.A., 2020. Microalgae: a new and promising source of food. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Costa, J.A.V., Moreira, J.B., Fanka, L.S., da Costa Kosinski, R., de Morais, M.G., 2020. Microalgal biotechnology applied in biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London.

xxvi  Preface

Dai, A., 2011. Drought under global warming: a review. Wiley Interdiscip. Rev. Clim. Chang. 2 (1), 45–65. Dalu, T., Lemley, D.A., Snow, G.C., Wu, N.C., 2020. Linking phytoplankton community structure to aquatic ecosystem functioning: a mini-review of the current status and future directions. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Daniels, N., 2001. Justice, health, and healthcare. Am. J. Bioeth. 1 (2), 2–16. Davis, T.A., Volesky, B., Mucci, A., 2003. A review of the biochemistry of heavy metal biosorption by brown algae. Water Res. 37 (18), 4311–4330. Deka, D., Sonowal, S., Chikkaputtaiah, C., Velmurugan, N., 2020. Exploring ‘omics’ approaches: towards understanding the essence of stress phenomena in diatoms and haptophytes. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Derelle, E., Ferraz, C., Rombauts, S., Rouze, P., Worden, A.Z., Robbens, S., et al., 2016. Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc. Natl. Acad. Sci. U.S.A. 103 (31), 11647–11652. Duan, X.J., Zhang, W.W., Li, X.M., Wang, B.G., 2006. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem. 95 (1), 37–43. Esterhuizen-Londt, M., Pflugmacher, S., 2020. Microcystins as environmental and human health hazards. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Falkowski, P.G., Katz, M.E., Knoll, A.H., Quigg, A., Raven, J.A., Schofield, O., et al., 2004. The evolution of modern eukaryotic phytoplankton. Science 305 (5682), 354–360. Faraloni, C., Torzillo, G., 2020. Microalgal hydrogen production in the framework of bioeconomy. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Academic Press. Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., Iwata, S., 2004. Architecture of the photosynthetic oxygen-evolving center. Science 303 (5665), 1831–1838. Fleurence, J., 1999. Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Sci. Technol. 10 (1), 25–28. Goss, R., Wilhelm, C., Jakob, T., 2020. Photosynthesis in diatoms. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., Saenger, W., 2009. Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride. Nat. Struct. Mol. Biol. 16 (3), 334–342. Hecky, R.E., Kilham, P., 1988. Nutrient limitation of phytoplankton in fresh-water and marine environments—a review of recent evidence on the effects of enrichment. Limnol. Oceanogr. 33 (4), 796–822. Hook, M., Tang, X., 2013. Depletion of fossil fuels and anthropogenic climate change—a review. Energy Policy 52, 797–809. Hubbert, M.K., 1949. Energy from fossil fuels. Science 109 (2823), 103–109. Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., et al., 1998. Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. Science 281 (5382), 1519–1523. Johnson, J.M.F., Franzluebbers, A.J., Weyers, S.L., Reicosky, D.C., 2007. Agricultural opportunities to mitigate greenhouse gas emissions. Environ. Pollut. 150 (1), 107–124. Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W., Krauss, N., 2001. Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature 411 (6840), 909–917. Kaneko, T., Nakamura, Y., Wolk, C.P., Kuritz, T., Sasamoto, S., Watanabe, A., et al., 2001. Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res. 8 (5), 205–213. Karl, T.R., Trenberth, K.E., 2003. Modern global climate change. Science 302 (5651), 1719–1723. Keilman, N., 1998. How accurate are the United Nations world population projections? Popul. Dev. Rev. 24, 15–41. Konur, O., 2020a. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020b. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020c. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020d. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020e. The scientometric analysis of the research on the algal photosystems and phtosythesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020f. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020i. The scientometric analysis of the research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020j. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London.

Preface  xxvii

Konur, O., 2020k. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020l. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020m. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Konur, O., 2020n. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Lashof, D.A., Ahuja, D.R., 1990. Relative contributions of greenhouse gas emissions to global warming. Nature 344 (6266), 529–531. Leape, L., Berwick, D., Clancy, C., Conway, J., Gluck, P., Guest, J., et al., 2009. Transforming healthcare: a safety imperative. BMJ Qual. Saf. 18 (6), 424–428. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Li, Y., Horsman, M., Wu, N., Lan, C.Q., Dubois‐Calero, N., 2008. Biofuels from microalgae. Biotechnol. Prog. 24 (4), 815–820. Littler, M.M., Littler, D.S., 1980. The evolution of thallus form and survival strategies in benthic marine macroalgae field and laboratory tests of a functional form model. Am. Nat. 116 (1), 25–44. Lutz, W., Samir, K.C., 2010. Dimensions of global population projections: what do we know about future population trends and structures? Philos. Trans. R. Soc. Lond. B Biol. Sci. 365 (1554), 2779–2791. Makareviciene, V., Sendzikiene, E., 2020. Application of microalgae for the production of biodiesel fuel. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Markou, G., 2020. Overview of microalgal cultivation, biomass processing and application. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Mathieson, A.C., Dawes, C.J., 2020a. Biology and ecology of Northwest Atlantic seaweeds. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Mathieson, A.C., Dawes, C.J., 2020b. Autecology of Northwest Atlantic fucoid algae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Matsuyama, Y., Oda, T., 2020. Toxic effects of harmful algal blooms on finfish and shellfish. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Menden-Deuer, S., Lessard, E.J., 2000. Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnol. Oceanogr. 45 (3), 569–579. Moreira, C., Martins, J., Vasconcelos, V., Antunes, A., 2020. Genomics perspectives on cyanobacteria research. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Noguchi, T., Arakawa, O., Daigo, K., Oikawa, H., Asakawa, M., Miyazawa, K., 2020. Different intoxication mechanism between paralytic shellfish toxin (PST)- and/or tetrodotoxin-contaminated xanthid crabs and PST-contaminated edible shore crabs in Japan and their food poisonings. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Nowruzi, B., Sarvar, G., Blanco, S., 2020. Applications of cyanobacteria in biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. O’Neill, E., 2020. Using new techniques to study old favourites: a case study of Euglena. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Oreskes, N., 2004. The scientific consensus on climate change. Science 306 (5702), 1686. Qin, Y.M., 2020. Health benefits of bioactive seaweed substances. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Raftery, A.E., Li, N., Sevcikova, H., Gerland, P., Heilig, G.K., 2012. Bayesian probabilistic population projections for all countries. Proc. Natl. Acad. Sci. U.S.A. 109 (35), 13915–13921. Riahi, K., Rao, S., Krey, V., Cho, C., Chirkov, V., Fischer, G., et al., 2011. RCP 8.5—a scenario of comparatively high greenhouse gas emissions. Clim. Chang. 109 (1–2), 33. Root, T.L., Price, J.T., Hall, K.R., Schneider, S.H., Rosenzweig, C., Pounds, J.A., 2003. Fingerprints of global warming on wild animals and plants. Nature 421 (6918), 57–60. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Saba, B., Christy, A.D., 2020. Bioelectricity generation in algal microbial fuel cells. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Sastre de Vicente, M.E., Rodriguez-Barro, P., Herrero, R., Vilarino, T., Lodeiro, P., Barriada, J.L., 2020. Biosorption of chemical species by Sargassum algal biomass: equilibrium data, part I. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Souza, N.R., Metcalf, J.S., 2020. Cyanobacterial toxins and their effects on human and animal health. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Thomas, C.D., Cameron, A., Green, R.E., Bakkenes, M., Beaumont, L.J., Collingham, Y.C., et al., 2004. Extinction risk from climate change. Nature 427 (6970), 145–148. Tonnesen, H.H., Karlsen, J., 2002. Alginate in drug delivery systems. Drug Dev. Ind. Pharm. 28 (6), 621–630.

xxviii  Preface

Vaz, M.G.M.V., Almeida, A.V.M., de Castro, N.V., Nunes-Nesi, A., Araujo, W.L., 2020. Carbon and nitrogen metabolism in cyanobacteria: basic traits, regulation and biotechnological application. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Vitousek, P.M., 1994. Beyond global warming: ecology and global change. Ecology 75 (7), 1861–1876. Znad, H., 2020. Microalgae culture technology for carbon dioxide biomitigation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London. Zohoorian, H., Ahmadzadeh, H., Molazadeh, M., Shourian, M., Lyon, S., 2020. Microalgal bioremediation of heavy metals and dyes. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Academic Press, London.

Acknowledgments The Editor has been most grateful for the valuable contributions made by the contributing authors. He has also been most grateful to the distinguished researchers who have substantially contributed to the development of their respective research fields in the broader area of algal science, technology, and medicine. Finally, the Editor has been most grateful to Ms. Linda Versteeg-Buschman, Mr. Sam W. Young, and Mr. Debasish Ghosh from Elsevier for their kind collaborations in the commissioning, executing, and copy-editing of the handbook. Thanks are extended to the publishers who have kindly allowed the publication of many figures in the handbook.

xxix

Chapter 1

The scientometric analysis of the research on the algal science, technology, and medicine Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

1.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007), contributing positively to the ecosystems (Charlson et al., 1987; Martin and Fitzwater, 1988) and bioremediating greenhouse gases, heavy metals, and wastewaters (Davis et al., 2003; Wang et al., 2008) with the increasing public awareness of the toxicological impact of the algae (Jochimsen et al., 1998; MacKintosh et al., 1990) as evidenced with over 150,000 papers published since 1980. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate, the stakeholders should have timely and thorough access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b, 2012a). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal science, technology, and medicine and the underlying research areas as in fields of the algal research (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012b,c,d,e,f,g,h,i,j,k, 2018a,b,c), energy and fuels (Konur, 2012l,m,n, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g, 2017b,c,d,e,f, 2019b), biomedicine (Konur, 2016h, 2018d), and social sciences (Konur, 2012o,p,q,r,s,t). Although there have been over 4500 literature reviews on the algal science, technology, and medicine, there have been no published scientometric studies covering the whole range of research fronts and whole range of algae. This is contrast to the many published scientometric studies on science, technology, and medicine at large (Chiu and Ho, 2007; de Bakker et al., 2005; Ding et al., 2001; Li et al., 2011; Nederhof, 2006). Therefore, this paper presents the first-ever scientometric study of the research in algal science, technology, and medicine covering the whole range of research fronts as well as the whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate.

1.2  Materials and methodology The search for the scientometric analysis of the literature on the algal science, technology, and medicine was carried out in February 2019 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI), and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, and author keywords in the abstract pages of the papers. The full keyword set is given in Appendix. These keyword sets have been devised in two major parts: the keywords related to the algae and the selected set of authors. There have been seven distinct keyword sets for the first part: keywords related to the algae in general, microalgae, macroalgae, dinoflagellates and coccolithophores, cyanobacteria, and diatoms, as well as the selected set of journals related to algae. Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00001-2 © 2020 Elsevier Inc. All rights reserved.

3

4  PART | I  Introduction to the algal science, technology, and medicine

Additionally, a selected set of keywords has been used to eliminate the papers unrelated to algal science, technology, and medicine. The papers located through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subjected to the quality control exercise to ensure that these references have been primarily related to the algal science, technology, and medicine. This refined list of papers has formed the core sample for the scientometric analysis of the literature on the algal science, technology, and medicine. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. Additionally, the search has been carried out for the papers with at least 100 citations and the relevant scientometric data has been collected. These papers have been termed as ‘influential papers’. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

1.3 Results 1.3.1  Documents and indexes The search has resulted in 169,549 papers where there have been 144,822 articles, 13,172 meeting abstracts, 4584 reviews, 3147 notes, 1172 editorial materials, 1104 corrections, 781 letters, 358 news items, 124 corrections additions, 123 biographical items, and 117 book reviews. In the first instance, the papers excluding items such as meeting abstracts, news items, and corrections have been selected resulting in 154,506 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 149,908 papers in English. The other major languages have been French, Japanese, Russian, Spanish, German, and Chinese. This set of 149,908 papers has formed the core sample for the scientometric analysis of the literature on the algal science, technology, and medicine. The articles have formed 93.4% of the final sample while reviews, notes, editorial matters, and letters have formed 3.0%, 2.0%, 1.0%, and 0.5% of this sample, respectively. Additionally, 3.6% of these papers have been ‘proceedings papers’ and there have been 16 ‘retracted papers’. On the other hand, 99.0% of these papers have been indexed by the SCI-E while only 0.3% of the papers have been indexed by the SSCI and A&HCI. Additionally, 0.9% of the papers have been indexed by the ESCI.

1.3.2 Keywords The most-prolific keywords used in algal science, technology, and medicine have been determined based on the influential papers to locate the hot topics and the primary research fronts in the algal science, technology, and medicine as well as searching for the relevant papers There have been a number of most-prolific keywords for the algal science, technology, and medicine: ‘alga, algae, *phytoplankton, phyco*, dinoflagellate*, chlamydomon*, “green-alga*”, chlorella, microalga*, alginate*, agar, carrageenan*, macroalga*, rhodophy*, seaweed*, “red alga*”, diatoms, *cyanobact*, *synechoc*’. The other prolific keywords for the algae have been ‘periphyton*, photobioreactor*, ciguat*, *coccolith*, dinophy*, “okadaic acid*”, alexandrium, emiliania, “red tide*”, chlorophyt*, chlorophyc*, euglen*, “micro-alga*”, chrysophy*, dunaliella, haematococcus, nannochloropsis, scenedesmus, “brown alga*”, agars, fucoid*, gracilar*, kelp*, phaeophy*, porphyra, ulva*, caulerpa*, corallina*, fucus, gigartina*, laminaria*, saccharina, sargassum, nitell*, bacillarioph*, diatoma*, diatomite, diatom, thalassiosira*, *nitzschia, phaeodactylum, *cylindrospermops*, *microcystis, *microcystin*, *phycocyanin*, “blue-green alga*”, *anabaen*, cyanophy*, *nostoc*, *oscillatoria*, spirul*, arthrospira’.

1.3.3 Authors There have been over roughly 200,000 authors contributing to the research on the algal science, technology, and medicine in total. Similarly, there have been over 14,500 authors publishing the influential papers. The information on the most-prolific and influential 20 authors is provided in Table 1.1: Authors’ names, gender, institutions, countries, primary



TABLE 1.1  The most-prolific and influential authors in algal science, technology, and medicine. Gender

Institution

Country

Research fronts

Algae

I-0

I-100

I-100%

1

Takeshi Yasumoto

M

Tohoku Univ.

Japan

Toxicology

Dinoflagellates

297

48

36.6

2

Sallie W Chisholm

F

Massachusetts Inst. Technol.

United States

Genomics

Algae

123

47

17.0

3

Paul G Falkowski

M

Brookhaven Natl. Lab.

United States

Photosynthesis

Algae

123

45

34.5

4

Hans W Paerl

M

Univ. N. Carolina

United States

Ecology

Algae

247

42

14.8

5

Wayne W Carmichael

M

Wright State Univ.

United States

Toxicology

Cyanobacteria

116

40

45.9

6

Donald M Anderson

M

Woods Hole Ocean. Inst.

United States

Ecology

Algae

237

35

17.3

7

Karl Deisseroth

M

Stanford Univ.

United States

Biomedicine

Microalgae

74

34

17.3

8

Ulf Riebesell

M

Helmholtz Ctr. Ocean. Res.

Germany

Photosynthesis

Algae

185

32

32.6

9

Kaarina Sivonen

F

Univ. Helsinki

Finland

Genomics

Cyanobacteria

185

32

18.6

10

Francois MM Morel

M

Princeton Univ.

United States

Ecology

Algae

92

30

13.8

11

Arthur R Grossman

M

Carnegie Inst.

United States

Photosynthesis

Algae

156

29

13.8

12

Geoffrey A Codd

M

Univ. Dundee

United Kingdom

Toxicology

Cyanobacteria

203

28

21.5

13

Paul J Harrison

M

Univ. Brit. Columbia

Canada

Ecology

Algae

189

26

23.9

14

John A Raven

M

Univ. Dundee

United Kingdom

Photosynthesis

Algae

121

26

27.8

15

Norio Murata

M

Natl. Inst. Bas. Biol.

Japan

Structures

Cyanobacteria

109

26

15.1

16

Daniel Vaulot

M

Univ. Paris 06

France

Ecology

Algae

90

25

21.1

17

Brett A Neilan

M

Univ. New S. Wales

Australia

Toxicology

Cyanobacteria

159

24

26.7

18

Jean-David D Rochaix

M

Univ. Geneva

Switzerland

Photosynthesis

Microalgae

109

23

31.9

19

George B Witman

M

Univ. Massachusetts

United States

Structures

Microalgae

86

23

36.6

20

MR Badger

M

Australian Natl. Univ.

Australia

Photosynthesis

Algae

69

22

17.0

Average

149

32

Total %

2.0

11.0

M, male; F, female; I-0, no. papers, the number of papers for at least 69 papers; I-100, the number of influential papers with at least 100 citations for at least 22 papers; I-100%, the percentage of the number of influential papers with relative to the number of all the papers published.

Research on the algal science, technology, and medicine  Chapter | 1  5

Author

6  PART | I  Introduction to the algal science, technology, and medicine

research fronts, number of papers produced by these authors, the number of influential papers with at least 100 citations received (I-100), and the percentage of the number of influential papers with relative to the number of all the papers published (I-100%). The data on these authors shows that the most-prolific author with the highest citation impact has been ‘Takeshi Yasumoto’ of the Tohoku University of Japan, working primarily on the ‘toxicology’ of the dinoflagellates, with 297 papers. His citation impact is the highest with 48 influential papers. The other most-prolific authors with the high citation impact have been ‘Sallie W Chisholm’, ‘Paul G Falkowski’, ‘Hans W Paerl’, ‘Wayne W Carmichael’, and ‘Donald M Anderson’ with at least 22 influential papers each. The United States has been the most-prolific country for these authors with nine authors while United Kingdom, Japan, and Australia have been the other prolific countries with two authors each. On the other hand, Europe has had only six authors as a whole. In total, these top authors have been from nine countries. There has been a significant gender deficit among these top prolific and influential authors as only two of them are females: ‘Sallie W Chisholm’ and ‘Kaarina Sivonen’. Similarly, the most-prolific institution has been ‘University of Dundee’ with two authors. In total, these top authors have been affiliated with 19 institutions. The most-prolific research fronts have been the ‘photosynthesis’, ‘ecology’, and ‘toxicology’ of algae with six, five, and four authors, respectively. The other prolific fronts have been ‘structures and phylogeny’ and ‘genomics’ of algae with two authors each. Similarly, the most prolific types of algae studied by these top authors have been ‘algae’ as 11 authors have studied more than one type of algae. The other prolific types of algae have been ‘cyanobacteria’ and ‘microalgae’ with five and three authors, respectively. The number of papers published by these authors have ranged from 29 to 297 with 149 papers on average. These mostprolific authors have also contributed to nearly 2.0% and 11.0% of all the papers and influential papers, respectively. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the influential papers with relative to the number of all the papers published for each author in the last column (I-100%). These data shows that ‘Wayne W Carmichael’, ‘George B Whitman’, ‘Takeshi Yasumoto’, ‘Paul G Falkowski’, and ‘Ulf Riebesell’ have been the top influential authors with at least 32.0% ratios each.

1.3.4 Countries Nearly 99.6% of the papers have had country information in their abstract pages and 197 countries and territories have contributed to these papers overall. Table 1.2 provides the information about the most-prolific and influential 20 countries. These 20 top countries have produced 101.5% and 124.1% of all the papers and influential papers, respectively, as a whole. The most-prolific and influential country has been the United States producing 23.8% and 43.1% of all the papers and influential papers, respectively. The other prolific and influential countries have been United Kingdom, Germany, Canada, Japan, and France producing 11.0%, 10.4%, 8.0%, 7.4%, and 7.2% of the influential papers, respectively. The European countries have been relatively dominant in the top 20 country list as they have produced 37.7% and 47.8% of all the papers and influential papers, respectively, as a whole, surpassing significantly the United States. Similarly, the Asian countries in this top 20 list, have produced 23.4% and 23.0% of all the papers and influential papers, respectively, as a whole.

1.3.5 Institutions Over 99.6% of the papers have had their institutions listed in their abstract pages. For these papers, 25,994 institutions have contributed to the research on the algal science, technology, and medicine in total. However, only 2601 of these institutions have contributed to the influential papers. The information about the 20 most-prolific and influential institutions is given in Table 1.3. The most-prolific and influential institution has been the ‘French National Scientific Research Center’ (CNRS) of France publishing 3.2% and 4.5% of the all and influential papers, respectively. The other prolific and influential institutions have been ‘Helmholtz Association’ of Germany, ‘Sorbonne University’ of France, ‘Woods Hole Oceanographic Institution’ of the United States, ‘University of California San Diego’, and ‘University of North Carolina’ of the United States with over 1.7% of the influential papers each. The most-prolific country for these institutions has been the United States with nine institutions producing 14.3% of the influential papers. Additionally, Canada, France, and Germany have had three, two, and two institutions, respectively. On the other hand, Europe has had six institutions as a whole, producing 13.7% of the influential papers.

Research on the algal science, technology, and medicine  Chapter | 1  7



TABLE 1.2  The most-prolific and influential countries in algal science, technology, and medicine. Country

I-0

I-0%

I-100

I-100%

Surplus%

Europe

56,568

37.7

2782

47.8

10.1

Asia

50,037

23.4

1337

23.0

−0.4

1

United States

35,686

23.8

2509

43.1

19.3

2

United Kingdom

10,445

7.0

639

11.0

4.0

3

Germany

11,632

7.8

607

10.4

2.6

4

Canada

7935

5.3

466

8.0

2.7

5

Japan

13,362

8.9

430

7.4

−1.5

6

France

8360

5.6

418

7.2

1.6

7

Australia

6722

4.5

389

6.7

2.2

8

China

15,604

10.4

229

3.9

−6.5

9

Netherlands

3411

2.3

222

3.8

1.5

11

Spain

7232

4.8

196

3.4

−1.4

10

Sweden

3077

2.1

137

2.4

0.3

12

Italy

4657

3.1

136

2.3

−0.8

13

Norway

2266

1.5

133

2.3

0.8

14

Israel

1895

1.3

121

2.1

0.8

15

Denmark

2004

1.3

107

1.8

0.5

16

Switzerland

1460

1.0

107

1.8

0.8

17

India

7165

4.8

105

1.8

−3.0

18

S. Korea

5227

3.5

95

1.6

−1.9

19

New Zealand

1957

1.3

89

1.5

0.2

20

Belgium

2024

1.4

80

1.4

0.0

Total

152,121

101.5

7215

124.1

22.6

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Europe and Asia values are for only the top 20 countries; Surplus%, I-100% − I-0%.

The contribution of these institutions has ranged from 0.3% to 3.2% for all the papers each and from 1.2% to 4.5% for the influential papers each. Overall, these 20 institutions have contributed to 18.8% and 34.7% of all the papers and influential papers, respectively.

1.3.6  Research funding bodies Only 42.6% of these papers have had declared any research funding in their abstract pages and overall, 58,594 funding bodies have funded these papers. The corresponding funding rate for the influential papers has been 20.7%. The most-prolific funding body has been the ‘National Natural Science Foundation of China’, funding 3.8% of the papers. The other prolific funding bodies have been ‘Natural Science Foundation’ of the United States, ‘National Institutes of Health’ of the United States, ‘Natural Environment Research Council’ of the United Kingdom, ‘Fundamental Research Funds for the Central Universities’ of China, and ‘Natural Science Foundation of China’.

1.3.7  Publication years Fig. 1.1 shows the number of papers on the algal science, technology, and medicine, published between 1980 and 2018 as of February 2019.

Institutions

Country

I-0

I-0%

I-00

I-100%

Surplus%

United States

7448

5.0

833

14.3

9.3

Europe

13,014

8.7

795

13.7

5.0

Asia

5225

3.5

160

2.8

−0.7

1

French. Natl. Sci. Res. Ctr.—CNRS

France

4783

3.2

260

4.5

1.3

2

Helmholtz Assoc.

Germany

2230

1.5

154

2.6

1.1

3

Sorbonne Univ.

France

1774

1.2

135

2.3

1.1

4

Woods Hole Oceanogr. Inst.

United States

778

0.5

122

2.1

1.6

5

Univ. Calif. San Diego

United States

1178

0.8

120

2.1

1.3

6

Univ. N Carolina

United States

1336

0.9

98

1.7

0.8

7

Massachusetts Inst. Technol.—MIT

United States

461

0.3

92

1.6

1.3

8

Natl. Ocean. Atmosph. Admin—NOAA

United States

1057

0.7

91

1.6

0.9

9

Fish. Ocean. Canada

Canada

794

0.5

87

1.5

1

10

Max Planck Soc.

Germany

985

0.7

86

1.5

0.8

11

Univ. Washington

United States

764

0.5

86

1.5

1

12

Spanish Natl. Res. Counc.—CSIC

Spain

2133

1.4

84

1.4

0

13

Chinese Acad. Sci.

China

4526

3.0

83

1.4

−1.6

14

Stanford Univ.

United States

483

0.3

80

1.4

1.1

15

Commonwealth Sci. Ind. Res. Org.—CSIRO

Australia

699

0.5

77

1.3

0.8

16

Natl. Env. Res. Counc.—NERC

United Kingdom

1109

0.7

76

1.3

0.6

17

Univ. Calif. Santa Barbara

United States

640

0.4

75

1.3

0.9

18

Univ. British Columbia

Canada

1009

0.7

73

1.3

0.6

19

Dalhousie Univ.

Canada

643

0.4

72

1.2

0.8

20

Oregon State Univ.

United States

751

0.5

69

1.2

0.7

28,133

18.8

2020

34.7

15.9

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. The United States, Europe, and Asia values are for only the top 20 institutions. Surplus%, I-100% − I-0%.

8  PART | I  Introduction to the algal science, technology, and medicine

TABLE 1.3  The most-prolific and influential institutions in algal science, technology, and medicine.



Research on the algal science, technology, and medicine  Chapter | 1  9

FIG. 1.1  The number of publications in the algal science, technology, and medicine between 1980 and 2018.

The data in this figure shows that the number of papers has risen from 1603 papers in 1980 to 9479 papers in 2018. The most prolific decade has been the 2010s with 44.9% of the papers. Additionally, 13.2%, 17.4%, and 24.5% of the papers have been published in the 1980s, 1990s, and 2000s, respectively. Thus, the figure shows that there has been a steadily increasing trend between 1980 and 2018, steeper in the 2010s.

1.3.8  Source titles Overall, these papers have been published in 6012 journals. However, only 828 of these journals have contributed to the influential papers. Table 1.4 provides the information on the 20 most-prolific and influential journals. These 20 journals have published 15.9% and 39.7% of all the papers and influential papers, respectively, in total. The most-prolific and influential journal has been ‘Limnology and Oceanography’ publishing 1.1% and 5.6% of all the papers and influential papers, respectively. ‘Marine Ecology Progress Series’, ‘Journal of Phycology’, ‘Proceedings of the National Academy of Sciences of the United States of America’, ‘Nature’, ‘Bioresource Technology’, and ‘Applied and Environmental Microbiology’ have followed the top journal with at least 2.0% of the influential papers each. The most-prolific subject categories for these journals have been ‘Marine Freshwater Biology’, ‘Multidisciplinary Sciences’, and ‘Biotechnology and Applied Microbiology’ with seven, four, and three journals, respectively. The other prolific subjects have been ‘Ecology’, ‘Engineering Environment’, ‘Oceanography’, ‘Environmental Sciences’, and ‘Plant Sciences’ with two journals each.

1.3.9  Subject categories These papers have been indexed by 224 subject categories where only 117 of them have had indexed the influential papers. The information about the 10 most-prolific and influential subject categories is given in Table 1.5. The most-prolific and influential subject categories have been ‘Marine Freshwater Biology’, ‘Oceanography’, and ‘Biochemistry and Molecular Biology’ indexing 21.6%, 15.0%, and 11.7% of the influential papers, respectively. The other prolific and influential subjects have been ‘Biotechnology Applied Microbiology’, ‘Ecology’, ‘Plant Sciences’, and ‘Multidisciplinary Sciences’ with at least 8.3% of the influential papers, respectively.

1.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers with at least 100 citations, 4.5% of the research sample of 149,908 papers, Table 1.6. The data are presented in this table using a type of algae versus research front matrix.

Journals

Abbr.

Subject

I-0

I-0%

I-100

I- 100%

Surplus%

1

Limnology and Oceanography

Limnol. Oceanogr.

Limnol., Oceanogr.

1622

1.1

323

5.6

4.5

2

Marine Ecology Progress Series

Mar. Ecol. Prog. Ser.

Ecol., Mar. Fresh. Biol., Oceanogr.

2213

1.5

224

3.9

2.4

3

Journal of Phycology

J. Phycol.

Plant Sci., Mar. Fresh. Biol.

3904

2.6

223

3.8

1.2

4

Proceedings of the National Academy of Sciences of the United States of America

P. Natl. Acad. Sci. USA

Mult. Sci.

704

0.5

182

3.1

2.6

5

Nature

Nature

Mult. Sci.

269

0.2

167

2.9

2.7

6

Bioresource Technology

Bioresource Technol.

Agr. Eng., Biot. Appl. Microb., Ener. Fuels

2305

1.5

139

2.4

0.9

7

Applied and Environmental Microbiology

Appl. Environ. Microb.

Biot. Appl. Microb., Microbiol.

916

0.6

117

2.0

1.4

8

Science

Science

Mult. Sci.

226

0.2

115

2.0

1.8

9

Marine Biology

Mar. Biol.

Mar. Fresh. Biol.

1187

0.8

106

1.8

1.0

10

Journal of Biological Chemistry

J. Biol. Chem.

Bioch. Mol. Biol.

906

0.6

83

1.4

0.8

11

Toxicon

Toxicon

Phar. Phar., Toxic.

1214

0.8

80

1.4

0.6

12

Journal of Applied Phycology

J. Appl. Phycol.

Biot. Appl. Microb., Mar. Fresh. Biol.

3075

2.1

76

1.3

−0.8

13

Biomaterials

Biomaterials

Eng. Biomed., Mats. Sci. Biomats.

205

0.1

65

1.1

1.0

14

Plant Physiology

Plant Physiol.

Plant Sci.

1063

0.7

63

1.1

0.4

15

Journal of Plankton Research

J. Plankton Res.

Mar. Fresh. Biol., Oceanogr.

1302

0.9

61

1.0

0.1

16

Journal of the American Chemical Society

J. Am. Chem. Soc.

Chem. Mult.

293

0.2

61

1.0

0.8

17

Journal of Cell Biology

J. Cell Biol.

Cell Biol.

214

0.1

58

1.0

0.9

18

Environmental Science & Technology

Environ. Sci. Technol.

Eng. Env., Env. Sci.

486

0.3

56

1.0

0.7

19

Journal of Experimental Marine Biology and Ecology

J. Exp. Mar. Biol. Ecol.

Ecol., Mar. Fresh. Biol.

1055

0.7

56

1.0

0.3

20

Water Research

Water Res.

Eng. Env., Env. Sci., Water Res.

667

0.5

55

0.9

0.4

23,826

15.9

2310

39.7

23.8

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Values for four subject categories sciences are only for the top 20 journals. Surplus%, I-100% − I-0%.

10  PART | I  Introduction to the algal science, technology, and medicine

TABLE 1.4  The most-prolific and influential journals in algal science, technology, and medicine.

Research on the algal science, technology, and medicine  Chapter | 1  11



TABLE 1.5  The most-prolific and influential subject categories in algal science, technology, and medicine. Subject categories

I-0 No. papers

I-0% Papers

I-100 No. papers

I-100% Papers

Surplus%

1

Marine Freshwater Biology

35,299

23.5

1258

21.6

−1.9

2

Oceanography

11,070

7.4

871

15.0

7.6

3

Biochemistry Molecular Biology

14,656

9.8

683

11.7

1.9

4

Biotechnology Applied Microbiology

16,074

10.7

631

10.8

0.1

5

Ecology

9769

6.5

626

10.8

4.3

6

Plant Sciences

23,033

15.4

606

10.4

−5.0

7

Multidisciplinary Sciences

4805

3.2

489

8.4

5.2

8

Microbiology

10,244

6.8

446

7.7

0.9

9

Environmental Sciences

12,430

8.3

394

6.8

−1.5

10

Limnology

4472

3.0

360

6.2

3.2

Total

141,852

94.6

6364

109.4

14.8

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers; Surplus%, I-100% − I-0%.

The data show that the field of ‘ecology’ of algae has been the most prolific research front with 30.1% of the influential papers. The other key research fronts have been ‘biomedicine’, ‘structures and phylogeny’, ‘toxicology’, ‘photosynthesis’, and ‘bioenergy and biofuels’ of algae with 14.6%, 12.0%, 11.9%, 9.8%, and 9.5% of the influential papers, respectively. The most-prolific types of the algae have been ‘cyanobacteria’, ‘macroalgae’, ‘microalgae’, ‘phytoplankton’, and ‘algae’ with 23.6%, 22.9%, 16.9%, 11.6%, and 10.3% of the influential papers, respectively. The close reading of the data in this table shows that ‘ecology of phytoplankton’ and ‘macroalgal biomedicine’ have been the most-prolific individual research fronts with 10.5% and 10.0% of the influential papers, respectively. The other individual most-prolific research fronts have been ‘microalgal bioenergy and biofuels’, ‘cyanobacterial toxicology’, ‘algal ecology’, ‘cyanobacterial photosynthesis’, ‘macroalgal ecology’, ‘toxicology of dinoflagellates’, ‘structures and phylogeny of cyanobacteria’, ‘cyanobacterial ecology’, and ‘ecology of diatoms’ with 6.4%, 6.4%, 5.7%, 4.9%, 4.3%, 4.0%, 4.1%, 3.6%, and 3.3% of the influential papers, respectively.

1.4 Discussion As there have been over 150,000 core papers related to the algal science, technology, and medicine, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts. The primary mode of scientific communication has been articles while reviews have formed 3.0% of the sample. The primary index has been SCI-E indexing more than 99.0% of the papers while only 0.3% of the papers have been indexed by the SSCI and A&HCI focusing on the societal aspects of algal science, technology, and medicine. These findings suggest that there is substantial room for the research in social and humanitarian aspects such as policy-related studies as well as scientometric and consumer studies in this field. The most-prolific keywords related to the algal science, technology, and medicine have been determined through the detailed examination of the over 4.5% of all the papers with at least 100 citations. A detailed keyword set has been devised for the search (given in Appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the science, technology, and medicine have been ‘alga, algae, *phytoplankton, phyco*, dinoflagellate*, chlamydomon*, “green-alga*”, chlorella, microalga*, alginate*, agar, carrageenan*, macroalga*, rhodophy*, seaweed*, “red alga*”, diatoms, *cyanobact*, *synechoc*’. These keywords have formed the primary research fronts for the algal science, technology, and medicine.

Research fronts

Algae

Phytoplankton

Microalgae

Cyanobacteria

Diatoms

Dinofs.*

Macroalgae

Total

Total %

Structures

66

30

167

279

98

94

74

808

12.0

Genomics

4

25

88

76

11

0

12

216

3.2

Photosynthesis

81

0

142

332

41

39

26

661

9.8

Ecology

387

711

82

245

223

87

292

2027

30.1

Bioenergy

84

3

432

54

28

2

36

639

9.5

Biomedicine

18

16

131

135

11

0

675

986

14.6

Foods

4

0

0

14

0

0

59

77

1.1

Toxicology

11

0

4

429

41

267

48

800

11.9

Bioremediation

30

0

65

15

19

9

178

316

4.7

Other fronts

12

0

26

15

17

0

144

214

3.2

Total

697

785

1137

1594

489

498

1544

6744

Total

10.3

11.6

16.9

23.6

7.3

7.4

22.9

Numbers, the number of influential papers for each research front and type of algae; %, the percentage of influential papers for each research front and type of algae; Dinofs.*, dinoflagellates and it also includes coccolithophores.

12  PART | I  Introduction to the algal science, technology, and medicine

TABLE 1.6  The most-prolific research fronts in algal science, technology, and medicine.



Research on the algal science, technology, and medicine  Chapter | 1  13

The findings show that although over 200,000 authors have contributed to the research, 20 most-prolific and influential authors have shaped the literature on the algal science, technology, and medicine publishing 2.0% and 11.0% of all the papers and the influential papers, respectively (Table 1.1). The success of these authors, their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988) rather than the ‘quality’ paradigm (Kostoff et al., 2008a,b; Moravcsik and Murugesan, 1975; Seglen, 1997). The data provides the evidence for the presence of the significant gender deficit among the most-prolific authors as only two top authors are female, respectively (Table 1.1) (Bordons et al., 2003). The data on the papers by the most-prolific and influential authors highlight the primary research fronts as the ‘photosynthesis’, ‘ecology’, ‘toxicology’, ‘structures and phylogeny’, and ‘genomics’ of the algae. It has been found during the search process that the author names with two or more forenames or surnames have been spelt differently by the databases with significant implications for the recovery of their papers. One classical example has been the case of ‘Emilio Molina-Grima’ whose papers have been cited as both ‘Grima EM’ and ‘Molina-Grima E’. Similar difficulties have also been observed for the common names such as ‘Wang Y’ or ‘Li Y’ for the recovery of their papers and the related analysis. The data shows that although nearly 200 countries and territories have contributed to the research in algal science, technology, and medicine, most-prolific 20 countries contributed to 101.5% and 124.1% of all the papers and the influential papers, respectively (Table 1.2). The most-prolific and influential producers of the research have been the United States, Europe, Canada, and Japan as these countries have had the ‘first-mover advantage’ over the other countries. It is notable that the citation impact of China has been relatively small in relation to other top producers as China has produced 10.4% and 3.5% of all the papers and influential papers, respectively (Guan and Ma, 2007). Similarly, Japan, South Korea, and India have also had the reduced citation impact. As in the case of countries, although over 26,000 institutions have contributed to the research in algal science, technology, and medicine, the 20 most-prolific institutions, having the first-mover advantages, have published more than 18.8% of all the papers and 34.7% of the influential papers, respectively (Table 1.3). As only 42.6% and 20.7% of all the papers and influential papers have declared a research funding, respectively, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). It is notable that there has been significant research funding opportunities in China and Latin America in relation to the United States and Europe. Thus, there is ample room to extend the research funding opportunities to support the research and development in algal science, technology, and medicine. The steady rise in the number of the publications in this field in the 2000s and 2010s (as of February 2019) provides the strong evidence for the increasing public importance of the algal science, technology, and medicine in recent years (Fig. 1.1). The annual number of publications have risen to over 9300 papers and it is expected that the number of papers would continue to rise in the next decade with at least another 150,000 papers, provided that the opportunities for research funding would increase significantly, corresponding to the increasing importance of the algal science, technology, and medicine to the global society at large. Although over 6000 journals have contributed to the research in algal science, technology, and medicine, the 20 mostprolific journals, having the first-mover advantages, have published over 15.9% and 30.7% of all the papers and influential papers, respectively (Table 1.4). This finding has been most relevant for the top journals. The data on the Web of Science subject categories suggests that the first seven categories have been the key pillars of the research in algal science, technology, and medicine, forming the scientific basis of the research in this field: ‘Marine Freshwater Biology’, ‘Oceanography’, ‘Biochemistry and Molecular Biology’, ‘Biotechnology Applied Microbiology’, ‘Ecology’, ‘Plant Sciences’, and ‘Multidisciplinary Sciences’ (Table 1.5). As the journals related to algae in the top 20 journal list have published only 4.7% and 5.1% of all the papers and influential papers, respectively, the broad search strategy, covering all subject categories and journals, developed for this study, has been justified. The data on the research fronts have confirmed that the major research fronts have been ‘ecology’, ‘biomedicine’, ‘structures and phylogeny’, ‘toxicology’, ‘photosynthesis’, and ‘bioenergy and biofuels’ (Table 1.6). The most-studied the types of algae have been ‘cyanobacteria’, ‘macroalgae’, ‘microalgae’, ‘phytoplankton’, and ‘algae’ in general. The close reading the data in this table shows that ‘ecology of phytoplankton’, ‘macroalgal biomedicine’, ‘microalgal bioenergy and biofuels’, ‘cyanobacterial toxicology’, ‘algal ecology’, ‘cyanobacterial photosynthesis’, ‘macroalgal ecology’, ‘toxicology of dinoflagellates’, ‘structures and phylogeny of cyanobacteria’, ‘cyanobacterial ecology’, and ‘ecology of diatoms’ have been the most-prolific individual research fronts.

14  PART | I  Introduction to the algal science, technology, and medicine

It appears that the structure-processing-property relationships form the basis of the research in algal science, technology, and medicine as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

1.5 Conclusion This analytical study of the research in algal science, technology, and medicine at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as algal research complementing over 4600 literature reviews. The data has shown that the annual number of papers in this field has risen to over 9300 papers while there have been over 150,000 papers over the study period from 1980 to 2018. It is further expected that over 150,000 papers would be published in the next decade, corresponding to the increasing public importance of the algal science, technology, and medicine to the global society at large. The provision of the adequate research funding is essential for the further development of the research and practice in this field as only over 42% and 20% of all the papers and influential papers have declared a research funding, respectively. The key research fronts have been ‘ecology’, ‘biomedicine’, ‘structures and phylogeny’, ‘toxicology’, ‘photosynthesis’, and ‘bioenergy and biofuels’ of algae. The most-studied the types of algae have been ‘cyanobacteria’, ‘macroalgae’, ‘microalgae’, ‘phytoplankton’, and ‘algae’ in general. The close reading the data in Table 1.6 shows that ‘ecology of phytoplankton’, ‘macroalgal biomedicine’, ‘microalgal bioenergy and biofuels’, ‘cyanobacterial toxicology’, ‘algal ecology’, ‘cyanobacterial photosynthesis’, ‘macroalgal ecology’, ‘toxicology of dinoflagellates’, ‘structures and phylogeny of cyanobacteria’, ‘cyanobacterial ecology’, and ‘ecology of diatoms’ have been the most-prolific individual research fronts. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. It has been found that the detailed keyword set provided in Appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts and types of algae. A study on the scientometric analysis of 100 citation classics has been presented in Konur (2020b), complementing this chapter.

Appendix. The keyword sets A.1. Algae TI = (algae or algal or *phytoplankton* or phyco* or periphyton* or photobioreactor* or alga or algicid* or chlorarachniophy* or ‘photo-bioreactor*’ or ‘open pond*’ or ‘raceway pond*’) OR A.2. Dinoflagellates and coccolithophores TI = (dinoflagellat* or ciguat* or *coccolith* or dinophy* or ‘okadaic acid*’ or alexandrium or emiliania or ‘red tide*’ or brevetox* or gambier* or *gonyau* or *gymnodini* or haptophyt* or palytoxin* or prorocentr* or prymnesi* or saxitoxin* or ‘shellfish toxin*’ or zooxanthella* or amphidin* or akashiwo or isochrysis or karenia* or phaeocystis or symbiodinium or chrysophyt* or chrysophyc* or maitotoxin* or raphidophy* or ochromonas or peridin* or pfiesteria or azaspiracid* or dinocyst* or noctiluca* or *pectenotoxin* or ‘shellfish poison*’ or *yessotoxin* or aureococcus* or *ceratium or *chattonella or cochlodinium or crypthecodinium or gyrodinium or hematodinium or heterocapsa* or heterosigma or karlodinium or lingulodinium or mallomonas or ostreopsis or oxyrrhis or pleurochrysis or pyrocystis or pyrodinium or scrippsiella or rhodomonas or vaucheria or Xanthophyc*) OR A.3. Microalgae TI = (chlamydomon* or ‘green alga*’ or chlorella or microalga* or chlorophyt* or chlorophyc* or euglen* or ‘microalga*’ or chrysophy* or dunaliella or haematococcus or nannochloropsis or scenedesmus or channelrhodopsin* or cryptophy* or porphyridium or volvoc* or acetabularia or botryococcus or chlorococc* or phormidium or prototheca or tetraselmis or volvox or prasinophy* or cryptomonad* or desmidia* or eustigmatophy* or selenastr* or streptophy* or trebouxiophy*



Research on the algal science, technology, and medicine  Chapter | 1  15

or ankistrodesmus or aurantiochytr* or chroomonas or coccomyxa or cosmarium or cyanidioschyzon or cyanidium or ­desmodesmus or galdieria or klebsormid* or micrasterias or micromonas or monoraphid* or nannochloris or neochloris or ostreococcus or pediastrum or platymonas or polytomella or *kirchneriella or pyramimonas or schizochytrium) OR A.4. Macroalgae TI = (alginate* or agar or agarose* or carrageenan* or macroalga* or rhodophy* or seaweed* or ‘red alga*’ or ‘brown alga*’ or agars or fucoid* or gracilar* or kelp* or phaeophy* or porphyra or ulva* or caulerpa* or corallina* or fucus or gigartina* or laminaria* or saccharina or sargassum or nitell* or alginic or characea* or charophyt* or dictyota* or enteromorpha or fucale* or fucoxanthin* or halocynthia* or laminarin* or phlorotannin* or zygnema* or ascophyllum or bangiales or chondrus or cladophor* or codium or cystoseira or ecklonia or gelidium or kappaphycus or laurencia* or macrocystis or *ectocarp* or ceramiale* or pyropia* or rhodomela* or spirogyra or undaria or agarase* or algin or ‘macro-alga*’ or ‘seaweed*’ or agarophyt* or bryopsidale* or cryptonemia* or florideophy* or gelidiale* or griffithsia or griffithsin or halimeda* or *fucan* or lessonia* or rhodymeniale* or sargassac* or ulvophyc* or wakame or bangiophy* or ‘chara vulgaris’ or asparagopsis or bifurcaria or bostrychia or bryopsis or ceramium or chaetomorpha or chondracanthus or chondria or cladosiphon or delesseria* or desmarestia* or dictyopteris or durvillaea or ‘eisenia bicyclis’ or eucheuma or grateloupia or hizikia or hypnea or ishige or lithophyllum or lobophora or lomentaria or monostroma or mougeotia or oedogonium or padina or palmaria or pelvetia or plocamium or polysiphonia or rhodymenia* or scytosiphon* or solieria* or turbinaria or phyllophora* or charales or streptophyt* or ochrophyt* or halymenia* or bonnemaisonia* or charophyc* or porphyran or fucacea*) OR A.5. Diatoms TI = (diatoms or bacillarioph* or diatoma* or diatomite or diatom or thalassiosira* or *nitzschia or phaeodactylum or domoic* or chaetoceros or navicula or skeletonema or cyclotell* or stephanodisc* or achnanth* or asterionell* or aulacoseira or cocconeis or coscinodisc* or cylindrotheca or cymbella* or didymosphenia or ditylum or eunotia* or fragilaria* or gomphonema* or haslea* or melosira* or rhizosolenia* or stephanodiscus or synedra) OR SO = (‘Diatom Research’) OR A.6. Cyanobacteria TI = (*cyanobact* or *synechoc* or *cylindrospermops* or *microcystis or *microcystin* or * phycocyanin* or ‘bluegreen alga*’ or *anabaen* or cyanophy* or *nostoc* or *oscillatoria* or spirul* or arthrospira or *lyngbya* or *anatoxin* or cyanophage* or cyanotox* or phycobiliprotein* or phycobilisome* or *phycoerythrin* or saxitoxin* or aphanizomenon or planktothrix or prochloro* or trichodesmium or ‘methylamino-l-alanine*’ or bmaa or aeruginosin* or calothrix* or chroococca* or cryptophycin* or cyanelle* or cyanobiont* or cyanovir* or hapalindole* or nodularin* or phycocyanobilin* or phycobilin* or teleocidin* or acaryochloris or aphanothece or cyanophora or cyanothece or fischerella or fremyella or gloeobacter or mastigocladus or microcoleus or nodularia or plectonema or scytonem* or tolypothrix) OR SO = (‘Algal Research*’ or ‘European Journal of Phycology’ or ‘Harmful Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘British Phycological Journal’ or ‘Diatom Research’ or ‘Phycological Research’ or Algae or ‘Cryptogamie Algologie’ or Fottea*) OR A.7. Authors AU = (‘yasumoto t’ or ‘paerl hw’ or ‘martin jh’ or ‘deisseroth k’ or ‘boyden es’ or ‘riebesell u’ or ‘bhattacharya d’ or ‘fromme p’ or ‘barber j’ or ‘witman gb’ or ‘saenger w*’ or ‘atsumi s’ or ‘behrenfeld mj’ or ‘shen jr’) AND TS = [Algae] (the whole of keywords for the algae) A.8. Excluding terms NOT TI = (shewanella or pelagia or chlorophytum or pseudomonas or azotobacter or ‘bacterial alginate*’ or diatomic* or atom* or *molecule* or polynesia or propanoic or tiahura or sponge or leuconostoc or algas or gaas or ‘microbial alginate*’)

Acknowledgments The significant contribution of the authors of the pioneering studies in algal science, technology, and medicine to the development of the research in in this field have been gratefully acknowledged. The authors listed as the ‘most-prolific and influential authors’ in Table 1.1 have published at least 65 papers and 7 influential papers with at least 100 citations.

References Abramo, G., d’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173.

16  PART | I  Introduction to the algal science, technology, and medicine

Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sustain. Energy Rev. 14 (2), 557–577. Charlson, R.J., Lovelock, J.E., Andreae, M.O., Warren, S.G., 1987. Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate. Nature 326 (6114), 655–661. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Chiu, W.T., Ho, Y.S., 2007. Bibliometric analysis of tsunami research. Scientometrics 73 (1), 3–17. Davis, T.A., Volesky, B., Mucci, A., 2003. A review of the biochemistry of heavy metal biosorption by brown algae. Water Res. 37 (18), 4311–4330. de Bakker, F.G., Groenewegen, P., den Hond, F., 2005. A bibliometric analysis of 30 years of research and theory on corporate social responsibility and corporate social performance. Bus. Soc. 44 (3), 283–317. Ding, Y., Chowdhury, G.G., Foo, S., 2001. Bibliometric cartography of information retrieval research by using co-word analysis. Inf. Process. Manage. 37 (6), 817–842. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Guan, J., Ma, N., 2007. China's emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Jochimsen, E.M., Carmichael, W.W., An, J.S., Cardo, D.M., Cookson, S.T., Holmes, C.E.M., et al., 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N. Engl. J. Med. 338 (13), 873–878. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books Ltd., Stoke-on-Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2012a. The policies and practices for the academic assessment of blind students in higher education and professions. Energ. Educ. Sci. Technol. B 4 (si1), 240–244. Konur, O., 2012b. Prof. Dr. Ayhan Demirbas’ scientometric biography. Energ. Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012c. The evaluation of the biogas research: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012d. The evaluation of the bio-oil research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 379–392. Konur, O., 2012e. The evaluation of the biorefinery research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 347–358. Konur, O., 2012f. The evaluation of the research on the biodiesel: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012g. The evaluation of the research on the bioethanol: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012h. The evaluation of the research on the biofuels: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012i. The evaluation of the research on the biohydrogen: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012j. The evaluation of the research on the microbial fuel cells: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012k. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012l. The evaluation of the global energy and fuels research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (1), 613–628. Konur, O., 2012m. 100 Citation classics in energy and fuels. Energ. Educ. Sci. Technol. A 30 (si1), 319–332. Konur, O., 2012n. What have we learned from the citation classics in energy and fuels: a mixed study. Energ. Educ. Sci. Technol. A 30 (si1), 255–268. Konur, O., 2012o. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1893–1908. Konur, O., 2012p. The evaluation of the research on the arts and humanities in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (3), 1603–1618. Konur, O., 2012q. The evaluation of the educational research: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012r. The scientometric evaluation of the research on the deaf students in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1573–1588. Konur, O., 2012s. The scientometric evaluation of the research on the students with ADHD in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2012t. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Energ. Educ. Sci. Technol. B 4 (si1), 250–257. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716.



Research on the algal science, technology, and medicine  Chapter | 1  17

Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019b. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

18  PART | I  Introduction to the algal science, technology, and medicine

Konur, O., 2020b. 100 Citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Kostoff, R.N., Barth, R.B., Lau, C.G., 2008a. Quality vs. quantity of publications in nanotechnology field from the People’s Republic of China. Chin. Sci. Bull. 53 (8), 1272–1280. Kostoff, R.N., Barth, R.B., Lau, C.G., 2008b. Relation of seminal nanotechnology document production to total nanotechnology document production— South Korea. Scientometrics 76 (1), 43–67. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Li, J.F., Wang, M.H., Ho, Y.S., 2011. Trends in research on global climate change: a Science Citation Index Expanded-based analysis. Global Planet. Change 77 (1–2), 13–20. Lieberman, M.B., Montgomery, D.B., 1988. First-mover advantages. Strateg. Manage. J. 9 (S1), 41–58. MacKintosh, C., Beattie, K.A., Klumpp, S., Cohen, P., Codd, G.A., 1990. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 264 (2), 187–192. Martin, J.H., Fitzwater, S.E., 1988. Iron-deficiency limits phytoplankton growth in the northeast pacific subarctic. Nature 331 (6154), 341–343. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Moravcsik, M.J., Murugesan, P., 1975. Some results on the function and quality of citations. Soc. Stud. Sci. 5 (1), 86–92. Nederhof, A.J., 2006. Bibliometric monitoring of research performance in the social sciences and the humanities: a review. Scientometrics 66 (1), 81–100. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes-towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Seglen, P.O., 1997. Citations and journal impact factors: questionable indicators of research quality. Allergy 52 (11), 1050–1056. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718.

Chapter 2

100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

2.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007), contributing positively to the ecosystems (Charlson et al., 1987; Martin and Fitzwater, 1988) and bioremediating greenhouse gases, heavy metals, and wastewaters (Davis et al., 2003; Wang et al., 2008) with the increasing public awareness of the toxicological impact of the algae (Jochimsen et al., 1998; MacKintosh et al., 1990) as evidenced with over 150,000 papers published since 1980. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate, the stakeholders should have timely and thorough access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b, 2012a). In this respect, the scientometric studies (Garfield, 1955, 1972, 1979, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal science, technology, and medicine and the underlying research areas as in fields of the algal research (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012b,c,d,e,f,g,h,i,j,k, 2018a,b,c), energy and fuels (Konur, 2012l,m,n, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g, 2017b,c,d,e,f, 2019b), biomedicine (Konur, 2016h, 2018d), and social sciences (Konur, 2012o,p,q,r,s,t). Although there have been over 4500 literature reviews on the algal science, technology, and medicine, there have been no published scientometric studies of 100 citation classics in this field covering the whole range of research fronts and whole range of algae. This is contrast to the many published scientometric studies of the citation classics in science, technology, and medicine at large (Baltussen and Kindler, 2004a,b; Dubin et al., 1993; Garfield, 1987; LeFaivre et al., 2011; Paladugu et al., 2002). Therefore, this paper presents the first-ever scientometric study of 100 citation classics in algal science, technology, and medicine covering the whole range of research fronts as well as the whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate. It complements the study on the scientometric analysis of the research on the algal science, technology, and medicine (Konur, 2020b).

2.2  Materials and methodology The search for the scientometric analysis of the literature on the algal science, technology, and medicine was carried out in February 2019 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI), and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, and author keywords in the abstract pages of the papers. The full keyword set is given in Appendix.

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00002-4 © 2020 Elsevier Inc. All rights reserved.

19

20  PART | I  Introduction to the algal science, technology, and medicine

These keyword sets have been devised in two major parts: the keywords related to the algae and the selected set of authors. There have been seven distinct keyword sets for the first part: keywords related to the algae in general, microalgae, macroalgae, dinoflagellates and coccolithophores, cyanobacteria, diatoms, as well as the selected set of journals related to algae. Additionally, a selected set of keywords has been used to eliminate the papers unrelated to algal science, technology, and medicine. The papers located through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subjected to the quality control exercise to ensure that these references have been primarily related to the algal science, technology, and medicine. This refined list of papers has formed the core sample for the scientometric analysis of the literature on the algal science, technology, and medicine. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. Additionally, the search has been carried out for the papers with at least 100 citations and the relevant scientometric data has been collected. These papers have been termed as ‘influential papers’. Furthermore, the top 100 papers were selected as the core sample for this study on the scientometric analysis of 100 citation classics in algal science, technology, and medicine. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

2.3 Results 2.3.1  Documents and indexes The ‘articles’ have formed 65% of the sample while ‘reviews’ and ‘editorial matters’ have formed 33% and 2% of this sample, respectively. Additionally, 1% of these papers have been ‘proceedings papers’ and there have been no ‘retracted papers’. These two ‘editorial matters’ have been regrouped as ‘articles’. Thus, the sample has consisted of 67 ‘articles’ and 33 ‘reviews’. On the other hand, all of these papers have been indexed by the SCI-E.

2.3.2 Keywords The most-prolific keywords used in algal science, technology, and medicine have been determined based on the influential papers to locate the hot topics and the primary research fronts in the algal science, technology, and medicine as well as searching for the relevant papers There have been a number of most-prolific keywords for the algal science, technology, and medicine: ‘algal, algae, *phytoplankton, phyco*, dinoflagellate*, chlamydomon*, “green-alga*”, chlorella, microalga*, alginate*, agar, agarose*, carrageenan*, macroalga*, rhodophy*, seaweed*, “red alga*”, diatoms, *cyanobact*, *synechoc*’. The other prolific keywords for the algae have been ‘periphyton*, photobioreactor*, ciguat*, *coccolith*, dinophy*, “okadaic acid*”, alexandrium, emiliania, “red tide*”, chlorophyt*, chlorophyc*, euglen*, “micro-alga*”, chrysophy*, dunaliella, haematococcus, nannochloropsis, scenedesmus, “brown alga*”, agars, fucoid*, gracilar*, kelp*, phaeophy*, porphyra, ulva*, caulerpa*, corallina*, fucus, gigartina*, laminaria*, saccharina, sargassum, nitell*, bacillarioph*, diatoma*, diatomite, diatom, thalassiosira*, *nitzschia, phaeodactylum, *cylindrospermops*, *microcystis, *microcystin*, *phycocyanin*, “blue-green alga*”, *anabaen*, cyanophy*, *nostoc*, *oscillatoria*, spirul*, arthrospira’.

2.3.3 Authors There have been over roughly 495 authors contributing to these citation classics in the algal science, technology, and medicine in total. The information on the most-prolific and influential 22 authors is provided in Table 2.1: Authors’ names, gender, institutions, countries, primary research fronts, the number of influential papers with at least 100 citations received (I-100), the number of citation classics (I-CC), the number of citations received by the authors in total for their citation classics (NCC), and the number of influential papers with at least 1000 citations (1–1000). The information has also been provided for the ‘2018 Highly Cited Researchers’ (Clarivate Analytics, 2018). The data on these authors shows that the most-prolific author with the highest citation impact has been ‘Yusuf Chisti’ of the Massey University of New Zealand, working primarily on the ‘microalgal bioenergy and biofuels’ with 3 citation classics receiving 6322 citations in total.



TABLE 2.1  The most-prolific and influential authors in 100 citation classics in the algal science, technology, and medicine. Gender

Institution

Country

1

Yusuf Chisti

M

Massey Univ.

New Zealand

2

Wolfram Saenger

M

Free Univ. Berlin

3

Paul G Falkowski

M

4

Ernst Bamberg

5

Research fronts

Algae

I-100

I-CC

N-CC

I-1000

Bioenergy Biofuels

Microalgae

16

3

6322

2

Germany

Photosynthesis

Cyanobacteria

9

4

5472

3

Brookhaven Natl. Lab.

United States

Photosynthesis

Algae

45

4

4644

3

M

Max Planck Inst.

Germany

Biomedicine

Microalgae

9

3

4286

2

Georg Nagel

M

Max Planck Inst.

Germany

Biomedicine

Microalgae

8

3

4286

2

6

Karl Deisseroth*

M

Stanford Univ.

United States

Biomedicine

Microalgae

34

3

3813

1

7

Jan Kern

M

Tech. Univ. Berlin

Germany

Photosynthesis

Cyanobacteria

6

3

3807

2

8

Athina Zouni

F

Tech Univ. Berlin

Germany

Photosynthesis

Cyanobacteria

5

3

3807

2

9

David J Mooney*

M

Univ. Michigan

United States

Biomedicine

Macroalgae

18

3

3738

2

10

John H Martin

M

Mass Landing Marine Labs.

United States

Ecology

Phytoplankton

10

3

3456

2

11

Philip Cohen

M

Univ. Dundee

United Kingdom

Toxicology

Dinoflagellates

6

3

3443

2

12

Chris Bowler*

M

CNRS

France

Genomics

Diatoms

17

3

3375

2

13

Daniel S Rokhsar*

M

Joint Genome Inst.

United States

Genomics

Diatoms

6

3

3375

2

14

Steve E Fitzwater

M

Monterey Bay Aq. Res. Inst.

United States

Ecology

Phytoplankton

8

3

3370

2

15

Petra Fromme

F

Tech. Univ. Berlin

Germany

Photosynthesis

Cyanobacteria

13

2

3238

2

16

Horst Tobias Witt

M

Tech. Univ. Berlin

Germany

Photosynthesis

Cyanobacteria

8

2

3238

2

17

Norbert Krauss

M

Free University Berlin

Germany

Photosynthesis

Cyanobacteria

8

2

3238

2

18

Feng Zhang

M

Stanford Univ.

United States

Biomedicine

Microalgae

14

2

3035

1

19

Nils Kroger

M

Univ. Regensburg

Germany

Genomics

Diatoms

15

3

2930

1

20

Sallie W Chisholm

F

Massachusetts Inst. Technol.

United States

Genomics

Algae

47

3

2515

0

21

Michael J Behrenfeld

M

Oregon State Univ.

United States

Photosynthesis

Algae

19

2

2436

2

22

R Fauzi C Mantoura

M

Inst. Mar. Env. Res.

United Kingdom

Ecology

Phytoplankton

7

3

2395

0

Total

302

58

75,388

37

Average

15.1

2.9

3769

12.9

M, male; F, female; I-100, the number of influential papers with at least 100 citations for at least five papers; I-CC, number of citation classics; *, the ‘2018 Highly Cited Researchers’; NCC, the number of citations received by the authors in total for their citation classics; I-1000, the number of influential papers with at least 1000 citations.

100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  21

Author

22  PART | I  Introduction to the algal science, technology, and medicine

The other most-prolific authors with the high citation impact have been ‘Wolfram Saenger’, ‘Paul G Falkowski’, ‘Ernst Bamberg’, and ‘Georg Nagel’ with at least 3 citation classics with over 4000 citations each in total. The United States and Germany have been the most-prolific countries for these authors with nine authors each while United Kingdom has been the other prolific country with two authors. On the other hand, Europe has had only 12 authors as a whole. In total, these top authors have been from five countries. There has been a significant gender deficit among these top prolific and influential authors as only three of them are females: ‘Sallie W Chisholm’, ‘Petra Fromme’, and ‘Athina Zouni’. Similarly, the most-prolific institution has been ‘Technical University of Berlin’ with four authors. The other prolific institutions have been ‘Free University of Berlin’, ‘Max Planck Institute’, and ‘Stanford University’ with two authors each. In total, these top authors have been affiliated with 16 institutions. The most-prolific research front has been the ‘photosynthesis’ of algae with eight authors. The other prolific research fronts have been ‘biomedicine’, ‘genomics’, and ‘ecology’ of algae with five, four, and three authors, respectively. There have been six research fronts in total. Similarly, the most prolific types of algae studied by these top authors have been ‘cyanobacteria’ and ‘microalgae’ with six and five authors, respectively. The other prolific types of algae have been ‘algae’ in general, ‘phytoplankton’, and ‘diatoms’ with three authors each. Additionally, there have been one author each for ‘dinoflagellates’ and ‘macroalgae’. The number of influential papers published (1–100) by these authors have ranged from 5 to 47 with 15.1 papers on average. Similarly, the number of citation classics (I-CC) has ranged from 2 to 4 with 2.9 papers on average. The number of papers with at least 1000 citations (I-1000) has ranged from 0 to 3 with 1.9 papers on average. These authors have received between 2395 and 6322 citations each with 3769.4 citations per author on average.

2.3.4 Countries Overall, 31 countries have contributed to these classical papers. Table 2.2 provides the information about the most-prolific and influential 10 countries. These 10 top countries have produced 124.1% and 153% of the influential and classical papers, respectively, as a whole.

TABLE 2.2  The most-prolific and influential countries in 100 citation classics in the algal science, technology, and medicine. Country

I-100

I-100%

I-CC

Europe

2782

47.8

67

Asia

1337

23.0

28

1

United States

2509

43.1

46

2

Germany

639

11.0

25

3

United Kingdom

607

10.4

23

4

Australia

466

8.0

14

5

Canada

430

7.4

12

6

Japan

418

7.2

9

7

France

389

6.7

8

8

Italy

229

3.9

6

9

Netherlands

222

3.8

5

11

New Zealand

196

3.4

5

Total

7215

124.1

153

I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Europe and Asia values are for only the top 20 countries. I-CC, number of citation classics.

100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  23



The most-prolific and influential country has been the United States producing 43.1% and 46.0% of influential and classical papers, respectively. The other prolific and influential countries have been Germany, United Kingdom, Australia, and Canada producing 25%, 23%, 14%, and 12% of the citation classics, respectively. The European countries have been relatively dominant in the top 20 country list as they have produced 47.8% and 67% of the influential and classical papers, respectively, as a whole, surpassing significantly the United States. Similarly, the Asian countries in this top 20 list, have produced 23% and 28% of the influential and classical papers, respectively, as a whole.

2.3.5 Institutions Overall, 288 institutions have contributed to the research on the algal science, technology, and medicine in total. The information about the 10 most-prolific and influential institutions is given in Table 2.3. The most-prolific and influential institution has been ‘Woods Hole Oceanographic Institution’ of the United States and ‘Commonwealth Scientific & Industrial Research Organization’—CSIRO of Australia publishing six citation classics each. The other prolific and influential institutions have been ‘French National Scientific Research Center’—CNRS, ‘Helmholtz Association’. ‘Max Planck Society’ of Germany, and ‘University of North Carolina’ with five citation classics each. The most-prolific country for these institutions has been the United States with five institutions producing 21% of the citation classics. Additionally, Germany, Australia, France, and United Kingdom have had two institutions. On the other hand, Europe has had four institutions as a whole, producing 20% of the classical papers. The contribution of these institutions to the citation classics has ranged from 5% to 63.2%. Overall, these 10 institutions have contributed to 34.7% and 52% of all the influential and classical papers, respectively.

2.3.6  Research funding bodies Only 19% of these papers have had declared any research funding in their abstract pages and overall, 33 funding bodies have funded these papers. The corresponding funding rate for the influential papers has been 20.7%. The most-prolific funding body has been the ‘National Institutes of Health’ of the United States and ‘Australian Research Council’ with three and two classical papers, respectively.

TABLE 2.3  The most-prolific and influential institutions in 100 citation classics in the algal science, technology, and medicine. Institutions

Country

I-100

I-100%

I-CC

United States

833

14.3

21

Europe

795

13.7

20

Asia

160

2.8

6

1

Woods Hole Oceanogr. Inst.

United States

122

2.1

6

2

Commonwealth Sci. Ind. Res. Org.-CSIRO

Australia

77

1.3

6

3

French. Natl. Sci. Res. Ctr.-CNRS

France

260

4.5

5

4

Helmholtz Assoc.

Germany

154

2.6

5

5

Univ. N Carolina

United States

98

1.7

5

6

Max Planck Soc.

Germany

86

1.5

5

7

Univ. Washington

United States

86

1.5

5

8

Stanford Univ.

United States

80

1.4

5

9

Univ. British Columbia

Canada

73

1.3

5

10

Univ. Dundee

United Kingdom

65

Total

2020

5 34.7

52

I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. The United States, Europe, and Asia values are for only the top 20 institutions. I-CC, number of citation classics.

24  PART | I  Introduction to the algal science, technology, and medicine

2.3.7  Publication years Fig. 2.1 shows the number of classical papers on the algal science, technology, and medicine, published between 1980 and 2018 as of February 2019. The data in this figure shows that the number of papers has risen from two papers in 1980 to nine papers in 2008. The most prolific decade has been the 2000s with 45% of the papers. Additionally, 20%, 23%, and 7% of the papers have been published in the 1980s, 1990s, and 2010s, respectively. Thus, the figure shows that there has been concentration in the 2000s and 2010s.

2.3.8  Source titles Overall, these papers have been published in 68 journals. Table 2.4 provides the information on the 10 most-prolific and influential journals. These 10 journals have published 39.7% and 52% of the influential and classical papers, respectively, in total. The most-prolific and influential journals have been ‘Nature’ and ‘Science’ publishing 18% and 12% of the classical papers, respectively. ‘Limnology and Oceanography’, ‘Proceedings of the National Academy of Sciences of the United States of America’, ‘Journal of Phycology’, and ‘Environmental Science & Technology’ have been the other prolific journals with four, four, three, and three papers, respectively. The most-prolific subject categories for these journals have been ‘Multidisciplinary Sciences’, ‘Marine Freshwater Biology’, and ‘Oceanography’ with three, two, and two journals, respectively. These journals have been indexed by 12 subject categories in total.

2.3.9  Subject categories These papers have been indexed by 36 subject categories. The information about the 10 most-prolific and influential subject categories is given in Table 2.5. The most-prolific and influential subject categories have been ‘Multidisciplinary Sciences’ with 34 classical papers. ‘Biochemistry and Molecular Biology’, ‘Biotechnology Applied Microbiology’, ‘Marine Freshwater Biology’, and ‘Oceanography’ have been the other prolific subject categories indexing 12, 12, 11, and 9 the classical papers, respectively.

2.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential and classical papers, Table 2.6. The data are presented in this table using a type of algae versus research front matrix.

FIG. 2.1  The number of classical papers in the algal science, technology, and medicine between 1980 and 2018.

100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  25



TABLE 2.4  The most-prolific and influential journals in 100 citation classics in the algal science, technology, and medicine. Journals

Abbr.

Subject

I-100

I-100%

I-CC

1

Nature

Nature

Mult. Sci.

167

2.9

18

2

Science

Science

Mult. Sci.

115

2.0

12

3

Limnology and Oceanography

Limnol. Oceanogr.

Limnol., Oceanogr.

323

5.6

4

4

Proceedings of the National Academy of Sciences of the United States of America

P. Natl. Acad. Sci. USA

Mult. Sci.

182

3.1

4

5

Journal of Phycology

J. Phycol.

Plant Sci., Mar. Fresh. Biol.

223

3.8

3

6

Environmental Science & Technology

Environ. Sci. Technol.

Eng. Env., Env. Sci.

56

1.0

3

7

Marine Ecology Progress Series

Mar. Ecol. Prog. Ser.

Ecol., Mar. Fresh. Biol., Oceanogr.

224

3.9

2

8

Bioresource Technology

Bioresource Technol.

Agr. Eng., Biot. Appl. Microb., Ener. Fuels

139

2.4

2

9

Biomaterials

Biomaterials

Eng. Biomed., Mats. Sci. Biomats.

65

1.1

2

10

Biotechnology Advances

Biotechnol. Adv.

Biot. Appl. Microb.

22

0.4

2

2310

39.7

52

Total

I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Values for four subject categories sciences are only for the top 20 journals. I-CC, number of citation classics.

TABLE 2.5  The most-prolific and influential subject categories in 100 citation classics in the algal science, technology, and medicine. Subject categories

I-100 No. papers

I-100% Papers

I-CC

1

Multidisciplinary Sciences

489

8.4

34

2

Biochemistry Molecular Biology

683

11.7

12

3

Biotechnology Applied Microbiology

631

10.8

12

4

Marine Freshwater Biology

1258

21.6

11

5

Oceanography

871

15.0

9

6

Environmental Sciences

394

6.8

8

7

Plant Sciences

606

10.4

6

8

Energy Fuels

238

9

Ecology

626

10.8

4

10

Limnology

360

6.2

4

Total

6364

109.4

105

5

I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers; I-CC, number of citation classics.

Research fronts

Algae

Phytoplankton

Microalgae

Cyanobacteria

Diatoms

Dinofs.*

Macroalgae

I-CC %

I-100%

Structures

0

1

0

1

2

1

0

5

12.0

Genomics

0

0

2

1

2

0

0

5

3.2

Photosynthesis

1

1

1

8

0

0

0

11

9.8

Ecology

3

18

4

4

2

1

2

34

30.1

Bioenergy

1

0

12

0

0

0

1

14

9.5

Biomedicine

1

0

6

0

0

0

11

18

14.6

Foods

0

0

1

0

0

0

0

1

1.1

Toxicology

2

0

0

5

0

4

0

11

11.9

Bioremediation

0

0

0

0

0

0

1

1

4.7

Other fronts

0

0

0

0

0

0

0

0

3.2

I-CC %

8,0

20,0

26,0

19,0

6,0

6,0

15,0

100

100

I-100%

10.3

11.6

16.9

23.6

7.3

7.4

22.9

100

100

Numbers, the number of influential papers for each research front and type of algae; %, the percentage of influential papers for each research front and type of algae; Dinofs.*, dinoflagellates and it also includes coccolithophores; I-100%, the percentages for the influential papers with at least 100 citations; I-CC, number of citation classics.

26  PART | I  Introduction to the algal science, technology, and medicine

TABLE 2.6  The most-prolific research fronts in 100 citation classics in the algal science, technology, and medicine.



100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  27

The data show that the field of ‘ecology’ of algae has been the most prolific research front with 30.1% and 34% of the influential and classical papers, respectively. The other key research fronts have been ‘biomedicine’, ‘bioenergy and biofuels’, ‘toxicology’, and ‘photosynthesis’ algae with 18%, 14%, 11%, and 11% of the classical papers, respectively. The most-prolific types of the algae have been ‘microalgae’, ‘phytoplankton’, ‘cyanobacteria’, and ‘macroalgae’ with 26%, 20%, 19%, and 18% of the classical papers, respectively. The close reading the data in this table shows that ‘ecology of phytoplankton’, ‘microalgal bioenergy and biofuels’, and ‘macroalgal biomedicine’ have been the most-prolific individual research fronts with 18%, 12%, and 11% of the classical papers, respectively. The other individual most-prolific research fronts have been ‘cyanobacterial photosynthesis’, ‘microalgal biomedicine’, and ‘cyanobacterial toxicology’ with 8%, 6%, and 5% of the classical papers, respectively.

2.3.11  Citation classics This section provides the information on both the scientometric analysis and brief content overview of the most-cited 20 papers in algal science, technology, and medicine. The information on these papers is given in Table 2.7: authors’ names, publication years, document type, number of authors per paper, lead authors’ names, gender, and paper numbers (lead authors were determined based on the number of influential papers produced in this field with at least 10 influential papers), journals, subject areas, research fronts, types of algae, academic focus of the papers, number of citations received, and the number of citations per year.

2.3.11.1  Scientometric overview of the citation classics These papers have been published between 1982 and 2012. The most-prolific decades have been the 2000s, and 1990s with 10 and 4 papers, respectively. Additionally, there have been three papers each in the 1980s and 2010s. The reviews have been over-represented in these classical papers as there have been 11 articles and 9 reviews. The number of the authors of these papers has ranged from 1 to 117 while the mean number of authors has been 9.5. There have been 16 authors with at least 10 influential papers as the lead authors of the citation classics. There has been a significant gender deficit among the lead authors of these classical papers as only two authors are female: ‘Petra Fromme’ and ‘Sabeeha S Merchant’. The most-prolific lead author has been ‘Petra Fromme’ with two papers. In total, these citation classics have been published by 15 journals. The most-prolific journals have been ‘Nature’, ‘Renewable & Sustainable Energy Reviews’, and ‘Science’ with four, two, and two papers, respectively. In total, these papers have been indexed by 102 subject categories. The most-prolific categories have been ‘Multidisciplinary Sciences’, ‘Biotechnology and Applied Microbiology’, and ‘Biochemistry Molecular Biology’ with six, three, and three papers, respectively. In total, there have been seven research fronts. The most-prolific research front has been ‘Bioenergy and Biofuels’, ‘Biomedicine’, and ‘Photosynthesis’ of algae with five, four, and four papers, respectively. The other prolific research front has been ‘Ecology’ and ‘Toxicology’ of algae with three and two papers, respectively. There have been five types of algae covered by these classical papers. The most-prolific type of algae has been ‘microalgae’ with nine papers. In addition, there have been four, three, two, and two papers related to ‘cyanobacteria’, ‘macroalgae’, ‘phytoplankton’, and ‘dinoflagellates’, respectively. The most-studied topics have been the ‘PSII structure', ‘biodiesel production’, and ‘lipid production’ with three, two, and two papers, respectively. These papers have received between 1377 and 4463 citations each, with a mean value of 1884 citations per paper. On the other hand, the number of citations per year has ranged from 48.9 to 405.7 with a mean value of 143.4 citations per year. The papers by Chisti (2007), Lee and Mooney (2012), Mata et al. (2010), and Brennan and Owende (2010) have been the most-cited papers on the basis of the number of citations on average, working on the biodiesel production, properties and biomedical applications of alginates, biodiesel production, and biofuel production, respectively.

2.3.11.2  Brief overview of the content of the citation classics In total, there have been seven research fronts. The most-prolific research front has been ‘Bioenergy and Biofuels’, ‘Biomedicine’, and ‘Photosynthesis’ of algae with five, four, and four papers, respectively. The other prolific research front has been ‘Ecology’ and ‘Toxicology’ of algae with three and two papers, respectively. Algal bioenergy and biofuels Chisti (2007) discusses the production of biodiesel from microalgae to meet the global demand for the transport fuels replacing petrodiesel and second generation biodiesel from oil crops and wastes in a seminal review paper with 4463

Authors

Year

Doc.

N auths.

Lead authors

Journal

Subject area

Res. fronts

Algae

Topic

Cits.

Av. cits

1

Chisti

2007

R

1

Y Chisti-16

Biotechnol. Adv.

Biot. Appl. Microb.

Bioenergy

Microalgae

Biodiesel production

4463

405.7

2

Charlson et al.

1987

R

4

Nature

Mult. Sci.

Ecology

Phytoplankton

Climate regulation

2642

85.4

3

Ferreira et al.

2004

A

5

Science

Mult. Sci.

Photosynthesis

Cyanobacteria

PSII structure

2416

172.6

4

Mata et al.

2010

R

3

Renew. Sust. Ener. Rev.

Green Sust. Sci. Technol., Ener. Fuels

Bioenergy

Microalgae

Biodiesel production

2193

274.1

5

Boyden et al.

2005

A

5

ES Boyden-13, F Zhang-14, K Deisseroth-34

Nat. Neurosci.

Neurosci.

Biomedicine

Microalgae

Neural activity control

2046

157.4

6

Hu et al.

2008

R

7

M Seibert-10

Plant J.

Plant Sci.

Bioenergy

Microalgae

Lipid production

1848

184.8

7

Brennan & Owende

2010

R

2

Renew. Sust. Ener. Rev.

Green Sust. Sci. Technol., Ener. Fuels

Bioenergy

Microalgae

Biofuel production

1803

225.4

8

Reed & Mann

1985

A

2

Nucleic Acids Res.

Bioch. Mol. Biol.

Biomedicine

Macroalgae

DNA transfer

1762

53.4

9

Benya & Shaffer

1982

A

2

Cell

Bioch. Mol. Biol., Cell Biol.

Biomedicine

Macroalgae

Chondrocyte phenotype modulation

1762

48.9

10

Hillebrand et al.

1999

A

5

J. Phycol.

Plant. Sci., Mar. Fresh. Biol.

Ecology

Microalgae

Biovolume calculation

1751

92.2

11

Jordan et al.

2001

A

6

P Fromme-13

Nature

Mult. Sci.

Photosynthesis

Cyanobacteria

PSI structure

1665

97.9

12

Lee & Mooney

2012

R

2

DJ Mooney-18

Prog. Polym. Sci.

Polym. Sci.

Biomedicine

Macroalgae

Alginates

1660

276.7

13

Spolaore et al.

2006

R

4

J. Biosci. Bioeng.

Biot. Appl. Microb. Food Sci. Tech.

Food

Microalgae

Food applications

1604

133.7

14

Zouni et al.

2001

A

7

Nature

Mult. Sci.

Photosynthesis

Cyanobacteria

PSII structure

1573

92.5

J Barber-14

P Fromme-13

28  PART | I  Introduction to the algal science, technology, and medicine

TABLE 2.7  The top citation classics in the algal science, technology, and medicine.



Hallegraeff

1993

R

1

16

Loll et al.

2005

A

5

17

Merchant et al.

2007

A

117

18

Cohen et al.

1990

R

3

19

Behrenfeld & Falkowski

1997

A

2

20

Rodolfi et al.

2009

A

7

Average

2001

9.5

GM Hallegraeff-16

AR Grossman-29, GB Witman-23, C Bowler-17, PA Lefebvre-12, SS Merchant-10

MJ Behrenfeld-19, PG Falkowski-45

Phycologia

Plant. Sci., Mar. Fresh. Biol.

Toxicology

Dinoflagellates

Harmful blooms

1473

58.9

Nature

Mult. Sci.

Photosynthesis

Cyanobacteria

PSII structure

1422

109.4

Science

Mult. Sci.

Genomics

Microalgae

Flagellar structures

1417

128.8

Trends Biochem. Sci.

Bioch. Mol. Biol.

Toxicology

dinoflagellates

Cellular regulation

1412

50.4

Limnol. Oceanogr.

Limnol., Oceanogr.

Ecology

Phytoplankton

Carbon fixation

1397

66.5

Biotechnol. Bioeng.

Biot. Appl. Microb.

Bioenergy

Microalgae

Lipid production

1377

153.0

1884

143.4

Doc., document; A, article; R, review; Gender, gender of lead authors—female authors in italic; N paper, for the authors with at least 10 influential papers—number after the author names; Subject, web of science subjects; Topic, primary topic of the papers; Algae, type of algae studied; Res. fronts, primary research fronts studied; Cits., number of citations received in total; Av. Cits., number of citations per year.

100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  29

15

30  PART | I  Introduction to the algal science, technology, and medicine

c­ itations. He focuses on the production of algal biomass in raceway ponds and photobioreactors and production of microalgal biodiesel. He advances three ways to reduce the cost of the microalgal biodiesel: production in a biorefinery context, benefiting from the metabolic engineering of microalgae, and photobioreactor engineering. He covers microalgae (e.g., Chlorella), diatoms (e.g., Phaeodactylum), and coccolithophores (e.g., Isochrysis). He points out that producing 100 t of microalgal biomass fixes 183 t of carbon dioxide, usually from the flue gases of power plants. The estimated cost of producing 1 kg of microalgal biomass is below $3 for photobioreactors, which can be reduced to below $0.50 at an industrial scale. For the biomass with 30% oil by weight, the cost of the biomass for producing 1 L of microalgal oil is below $3 for photobioreactors. However, he notes that the average price of petrodiesel was $0.50 in the United States in 2006. He advances the strategic target as the reduction of price of microalgal oil from $3 to $0.50 in the long run. He asserts that this cost reduction is feasible through the production at an industrial scale in a biorefinery context and through metabolic engineering of microalgae and advances in photobioreactor engineering. Mata et al. (2010) discuss microalgal biodiesel production with a focus on the cultivation, harvesting, and processing of microalgal biomass in a review paper with 2193 citations. They also cover the other issue such as CO2 bioremediation, wastewater treatment, and production of high-value products in the biorefinery context. Hu et al. (2008) discuss the biodiesel production from microalgal oils, triacylglycerols (TAG), in a review paper with 1848 citations. They focus on the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, genomics of TAG production, and microalgae-based biofuel research within the historical context. They cover microalgae (e.g., Parietochloris), cyanobacteria (e.g., Trichodesmium), diatoms (e.g., Phaeodactylum), and dinoflagellates (e.g., Prymnesium). They determine the key determinants of the TAG production such as nutrient deprivation, light intensity, and temperature. They recommend further research in a number of areas such as metabolic engineering of microalgae and development of large-scale production systems. Brennan and Owende (2010) discuss production of microalgal biofuels in a review paper with 1803 citations. They focus on the microalgal biomass production, harvesting, and conversion technologies. They also discuss the synergistic coupling of microalgal biomass production with CO2 bioremediation and wastewater treatment. They assert that microalgal biofuels could replace petrofuels to meet the energy demand. Rodolfi et al. (2009) study microalgal lipid production in a low-cost photobioreactor in paper with 1377 citations. They find that both lipid content and areal lipid productivity of Nannochloropsis increased through nitrogen and phosphorus nutrient deprivation in an outdoor algal culture. They obtain lipid productivity of 204 mg/L/day with an average biomass productivity of 0.30 g/L/day and more than 60% final lipid content in nitrogen deprived media. They assert that this microalga has the potential for an annual production of 20 and more than 30 tons of lipid per hectare in the Mediterranean climate and in sunny tropical areas, respectively. Algal biomedicine Boyden et al. (2005) study the genetically targeted optical control of neural activity in a paper with 2046 citations. They adapt channelrhodopsin-2 (ChR2), a rapidly gated light-sensitive cation channel, by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons. They show reliable, millisecond-timescale control of neuronal spiking, as well as control of excitatory and inhibitory synaptic transmission. Reed and Mann (1985) study the capillary DNA transfer from agarose gels to nylon membranes in a paper with 1762 citations. They find that the alkaline solvent induces covalent fixation of DNA to the membrane. They observe saving in time and materials, an improved resolution, and substantial increase in sensitivity of subsequent hybridization analyses. Benya and Shaffer (1982) study the evolution of the differentiated collagen phenotype in the dedifferentiated chondrocytes when cultured in agarose gels in a paper with 1762 citations. They show a complete return to the differentiated collagen phenotype. Their results emphasize the primary role of cell shape in the modulation of the chondrocyte phenotype and show a reversible system for the study of gene expression. Lee and Mooney (2012) review the properties and biomedical applications of alginates in a review paper with 1660 citations. They provide a comprehensive overview of general properties of alginate and its hydrogels, their biomedical applications, and propose new perspectives for future studies with these biopolymers. Algal photosynthesis Ferreira et  al. (2004) study the structure of PSII from Thermosynechococcus elongatus at 3.5 Å resolution to reveal its molecular structure in a seminal paper with 2416 citations. Based on their findings, they propose a structure of the oxygenevolving center (OEC) containing a cubane-like Mn3CaO4 cluster and they discuss the implications for a possible oxygenevolving mechanism in cyanobacteria.



100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  31

Jordan et al. (2001) study the crystal structure of PSI from Synechococcus elongatus at 2.5 Å resolution in a seminal paper with 1665 citations. Based on their findings, they argue that the structural information on the proteins and cofactors and their interactions provides a basis for understanding the high efficiency of PSI in both light capturing and electron transfer. Zouni et al. (2001) study the crystal structure of photosystem PSII from Synechococcus elongatus at 3.8 Å resolution in a seminal paper with 1573 citations. The structure shows how protein subunits and cofactors are spatially organized. They also provide the information on the position, size and shape of the manganese cluster. Loll et al. (2005) study the crystal structure of photosystem PSII at 3.0 Å resolution in a seminal paper with 1422 citations. They show locations of and interactions between 20 protein subunits and 77 cofactors per monomer and provide insights into electron and energy transfer and photoprotection mechanisms in the reaction center and antenna subunits. They propose a lipophilic pathway for the diffusion of secondary plastoquinone and provide information about the Mn4Ca cluster. Algal ecology Charlson et al. (1987) discuss the biological regulation of the climate by algae in a review paper with 2638 citations. They note that the major source of ‘cloud-condensation nuclei’ (CCN) over the oceans is dimethylsulfide, which is produced by planktonic algae in sea water and oxidizes in the atmosphere to form a sulfate aerosol. The biological regulation of the climate is possible through the effects of temperature and sunlight on phytoplankton population and dimethylsulfide production. They argue that to counteract the warming due to doubling of atmospheric CO2, an approximate doubling of CCN would be needed. Hillebrand et al. (1999) study the biovolume calculation for microalgae in a paper with 1751 citations. They present a set of geometric shapes and mathematical equations for calculating biovolumes of microalgae to minimize the effort of microscopic measurement. Behrenfeld and Falkowski (1997) study the depth-integrated phytoplankton carbon fixation derived from satellite-based chlorophyll concentration in a paper with 1397 citations. They develop a light-dependent, depth-resolved model for carbon fixation that partitions environmental factors affecting primary production into those that influence the relative vertical distribution of primary production and those that control the optimal assimilation efficiency of the productivity profile. Algal toxicology Hallegraeff (1993) reviews ‘harmful algal blooms’ (HABs) and their global rise in a review paper with 1473 citations. He argues that the prediction of the impact of global climate change on marine HABs is difficult. He asserts that increasing temperature, enhanced surface stratification, alteration of ocean currents, intensification or weakening of local nutrient upwelling, stimulation of photosynthesis by elevated CO2, reduced calcification through ocean acidification, and heavy precipitation and storm events causing changes in land runoff and micronutrient availability may all produce contradictory species- or even strain-specific responses. Cohen et al. (1990) review okadaic acid in a paper with 1412 citations. They note that the okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A and this toxin is extremely useful for identifying biological processes that are controlled through the reversible phosphorylation of proteins. Algal food Spolaore et al. (2006) discuss the commercial applications of the microalgae in food in a review paper with 1604 citations. He notes that microalgae can be used to enhance the nutritional value of food and animal feed, play a crucial role in aquaculture, can be incorporated into cosmetics and are cultivated as a source of highly valuable molecules. Furthermore, microalgal oils are added to infant formulas and nutritional supplements. They argue that future research should focus on the improvement of production systems and the genetic modification of microalgal strains. Algal genomics Merchant et al. (2007) sequence the nuclear genome of Chlamydomonas and carry out a comparative phylogenomic analysis in a seminal paper with 1417 citations. They identify genes encoding proteins related to the photosynthetic and flagellar functions. Thus, they establish links between ciliopathy and the composition and function of flagella.

2.4 Discussion As there have been 100 citation classics with citations ranging from 689 citations to 4463 citations related to the algal science, technology, and medicine, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts.

32  PART | I  Introduction to the algal science, technology, and medicine

The primary mode of scientific communication has been articles while reviews have formed 33% of the sample. The primary index has been SCI-E indexing all of these papers. There have been no papers indexed by the SSCI and A&HCI focusing on the societal and humanitarian aspects of algal science, technology, and medicine. These findings suggest that there is substantial room for the research in social and humanitarian aspects such as policy-related studies as well as scientometric and consumer studies in this field. The most-prolific keywords related to the algal science, technology, and medicine have been determined through the detailed examination of the influential papers with at least 100 citations. A detailed keyword set has been devised for the search (given in Appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the algal science, technology, and medicine have been ‘alga, algae, *phytoplankton, phyco*, dinoflagellate*, chlamydomon*, “green-alga*”, chlorella, microalga*, alginate*, agar, carrageenan*, macroalga*, rhodophy*, seaweed*, “red alga*”, diatoms, *cyanobact*, *synechoc*’. These keywords have formed the primary research fronts for the algal science, technology, and medicine. The findings show that although nearly 500 authors have contributed to the research, 20 most-prolific and influential authors have shaped the literature on the algal science, technology, and medicine publishing 60 citation classics (Table 2.1). The success of these authors, their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988) rather than the ‘quality’ paradigm (Kostoff et al., 2008a,b; Moravcsik and Murugesan, 1975; Seglen, 1997). The data provides the evidence for the presence of the significant gender deficit among the most-prolific authors as only two top authors are female, respectively (Table 2.1) (Bordons et al., 2003). The data on the papers by the most-prolific and influential authors highlight the primary research fronts as the ‘photosynthesis’, ‘biomedicine’, ‘ecology’, ‘genomics’, and ‘toxicology’ of algae. The research front of ‘biomedicine’ replaces the ‘structures and phylogeny’ in the list of the research fronts for the influential papers. The data shows that although over 30 countries have contributed to these classical papers in algal science, technology, and medicine, most-prolific 10 countries have contributed to 124.1% and 153% of all the influential papers and classical papers, respectively (Table 2.2). The most-prolific and influential producers of the research have been the United States, Europe, Canada, Japan, and Australia as these countries have had the ‘first-mover advantage’ over the other countries. It is notable that China has been under-represented in this sample as it has produced only three classical papers (Guan and Ma, 2007). As in the case of countries, although nearly 290 institutions have contributed to the research in algal science, technology, and medicine, 10 most-prolific institutions, having the first-mover advantages, have published 58 classical papers (Table 2.3). As only 20.7% of influential papers have declared a research funding, only 19 classical papers have declared any research funding by 33 research funding bodies. The role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). Thus, there is ample room to extend the research funding opportunities to support the research and development in algal science, technology, and medicine. The 45 classical papers published in the 2000s (as of February 2019) provides the strong evidence for the increasing public importance of the algal science, technology, and medicine in recent years (Fig. 2.1). There have been nine classical papers published in 2008. Although 68 journals have contributed to these classical papers in algal science, technology, and medicine, the 10 mostprolific journals, having the first-mover advantages, have published 52 classical papers (Table 2.4). It is notable that top 2 journals, ‘Nature’ and ‘Science’ have published 30 classical papers together. The data on the Web of Science subject categories suggests that the first five categories have been the key pillars of the research these classical papers in algal science, technology, and medicine, forming the scientific basis of the research in this field: ‘Multidisciplinary Sciences’, ‘Biochemistry and Molecular Biology’, ‘Biotechnology Applied Microbiology’, ‘Marine Freshwater Biology’, and ‘Oceanography’ (Table 2.5). It is notable that the 34 classical papers have been indexed by the subject category of ‘Multidisciplinary Sciences’. As the journals related to algae in the top 10 journal list have published only 3 classical papers, the broad search strategy, covering all subject categories and journals, developed for this study, has been justified. The data on the research fronts have confirmed that the major research fronts have been the ‘ecology’, ‘biomedicine’, ‘bioenergy and biofuels’, ‘toxicology’, and ‘photosynthesis’ of algae (Table 2.6). These have been the same research fronts covered by the influential papers with at least 100 citations with a slightly different order. The most-studied types of algae have been ‘microalgae’, ‘phytoplankton’, ‘cyanobacteria’, and ‘macroalgae’. These have been the same types of algae covered by the influential papers with at least 100 citations with a different order.



100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  33

The close reading the data in Table 2.6 shows that ‘ecology of phytoplankton’, ‘microalgal bioenergy and biofuels’, ‘macroalgal biomedicine’, ‘cyanobacterial photosynthesis’, ‘microalgal biomedicine’, and ‘cyanobacterial toxicology’ have been the most-prolific individual research fronts. These have been the similar individual research fronts covered by the influential papers with at least 100 citations with a slightly different order. The extensive data on the 20 top citation classics largely confirm the findings of the earlier sections based on over 100 classical papers (Table 2.7). There has been a significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely over-represented in the 20 citation classic sample as there have been 9 reviews. Similarly, the most-prolific research fronts have been ‘bioenergy and biofuels’, ‘biomedicine’, ‘photosynthesis’, ‘ecology’, and ‘toxicology’ of algae. The most prolific types of algae have been ‘microalgae’, ‘cyanobacteria’, ‘macroalgae’, ‘phytoplankton’, and ‘dinoflagellates’. The most-studied topics have been the ‘PSII structure’, ‘biodiesel production’, and ‘lipid production’. It appears that the structure-processing-property relationships form the basis of the research in algal science, technology, and medicine as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

2.5 Conclusion This analytical study of the 100 citation classics in algal science, technology, and medicine at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research in the top classical papers in this field as in the case of new emerging technologies and processes such as algal research complementing over 4600 literature reviews. The provision of the adequate research funding is essential for the further development of the research and practice in this field as only 19 classical papers have declared a research funding. This low funding rate has been similar to the low funding rate of 20.7% in the influential papers as well. The 45 classical papers published in the 2000s (as of February 2019) provides the strong evidence for the increasing public importance of the algal science, technology, and medicine in recent years (Fig. 2.1). The major research fronts have been the ‘ecology’, ‘biomedicine’, ‘bioenergy and biofuels’, ‘toxicology’, and ‘photosynthesis’ of algae (Table 2.6). These have been the same research fronts covered by the influential papers with at least 100 citations with a slightly different order. The most-studied the types of algae have been ‘microalgae’, ‘phytoplankton’, ‘cyanobacteria’, and ‘macroalgae’. These have been the same types of algae covered by the influential papers with at least 100 citations with a different order. The close reading the data in this table shows that ‘ecology of phytoplankton’, ‘microalgal bioenergy and biofuels’, ‘macroalgal biomedicine’, ‘cyanobacterial photosynthesis’, ‘microalgal biomedicine’, and ‘cyanobacterial toxicology’ have been the most-prolific individual research fronts. These have been the similar individual research fronts covered by the influential papers with at least 100 citations with a slightly different order. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. It has been found that the detailed keyword set provided in Appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts. A study on the scientometric analysis of the research on the algal science, technology, and medicine has been presented in Konur (2020b), complementing this chapter.

Appendix. The keyword sets A.1. Algae in general TI = (algae or algal or *phytoplankton* or phyco* or periphyton* or photobioreactor* or alga or algicid* or chlorarachniophy* or ‘photo-bioreactor*’ or ‘open pond*’ or ‘raceway pond*’) OR A.2. Dinoflagellates and coccolithophores TI = (dinoflagellat* or ciguat* or *coccolith* or dinophy* or ‘okadaic acid*’ or alexandrium or emiliania or ‘red tide*’ or brevetox* or gambier* or *gonyau* or *gymnodini* or haptophyt* or palytoxin* or prorocentr* or prymnesi*

34  PART | I  Introduction to the algal science, technology, and medicine

or saxitoxin* or ‘shellfish toxin*’ or zooxanthella* or amphidin* or akashiwo or isochrysis or karenia* or phaeocystis or symbiodinium or chrysophyt* or chrysophyc* or maitotoxin* or raphidophy* or ochromonas or peridin* or pfiesteria or azaspiracid* or dinocyst* or noctiluca* or *pectenotoxin* or ‘shellfish poison*’ or *yessotoxin* or aureococcus* or *ceratium or *chattonella or cochlodinium or crypthecodinium or gyrodinium or hematodinium or heterocapsa* or heterosigma or karlodinium or lingulodinium or mallomonas or ostreopsis or oxyrrhis or pleurochrysis or pyrocystis or pyrodinium or scrippsiella or rhodomonas or vaucheria or Xanthophyc*) OR A.3. Microalgae TI = (chlamydomon* or ‘green alga*’ or chlorella or microalga* or chlorophyt* or chlorophyc* or euglen* or ­‘micro-alga*’ or chrysophy* or dunaliella or haematococcus or nannochloropsis or scenedesmus or channelrhodopsin* or cryptophy* or porphyridium or volvoc* or acetabularia or botryococcus or chlorococc* or phormidium or prototheca or tetraselmis or volvox or prasinophy* or cryptomonad* or desmidia* or eustigmatophy* or selenastr* or streptophy* or trebouxiophy* or ankistrodesmus or aurantiochytr* or chroomonas or coccomyxa or cosmarium or cyanidioschyzon or cyanidium or desmodesmus or galdieria or klebsormid* or micrasterias or micromonas or monoraphid* or nannochloris or neochloris or ostreococcus or pediastrum or platymonas or polytomella or *kirchneriella or pyramimonas or schizochytrium) OR A.4. Macroalgae TI = (alginate* or agar or agarose* or carrageenan* or macroalga* or rhodophy* or seaweed* or ‘red alga*’ or ‘brown alga*’ or agars or fucoid* or gracilar* or kelp* or phaeophy* or porphyra or ulva* or caulerpa* or corallina* or fucus or gigartina* or laminaria* or saccharina or sargassum or nitell* or alginic or characea* or charophyt* or dictyota* or enteromorpha or fucale* or fucoxanthin* or halocynthia* or laminarin* or phlorotannin* or zygnema* or ascophyllum or bangia* or chondrus or cladophor* or codium or cystoseira or ecklonia or gelidium or kappaphycus or laurencia* or macrocystis or ectocarp* or ceramiale* or pyropia* or rhodomela* or spirogyra or undaria or agarase* or algin or ‘macro-alga*’ or ‘sea-weed*’ or agarophyt* or bryopsidale* or cryptonemia* or florideophy* or gelidiale* or griffithsia or griffithsin or halimeda* or *fucan* or lessonia* or rhodymeniale* or sargassac* or ulvophyc* or wakame or bangiophy* or ‘chara vulgaris’ or asparagopsis or bifurcaria or bostrychia or bryopsis or ceramium or chaetomorpha or chondracanthus or chondria or cladosiphon or delesseria* or desmarestia* or dictyopteris or durvillaea or ‘eisenia bicyclis’ or eucheuma or grateloupia or hizikia or hypnea or ishige or lithophyllum or lobophora or lomentaria or monostroma or mougeotia or oedogonium or padina or palmaria or pelvetia or plocamium or polysiphonia or rhodymenia* or scytosiphon* or solieria* or turbinaria or phyllophora* or charales or streptophyt* or ochrophyt* or halymenia* or bonnemaisonia* or charophyc* or porphyran or fucacea*) OR A.5. Diatoms TI = (diatoms or bacillarioph* or diatoma* or diatomite or diatom or thalassiosira* or *nitzschia or phaeodactylum or domoic* or chaetoceros or navicula or skeletonema or cyclotell* or stephanodisc* or achnanth* or asterionell* or aulacoseira or cocconeis or coscinodisc* or cylindrotheca or cymbella* or didymosphenia or ditylum or eunotia* or fragilaria* or gomphonema* or haslea* or melosira* or rhizosolenia* or stephanodiscus or synedra) OR A.6. Cyanobacteria TI = (*cyanobact* or *synechoc* or *cylindrospermops* or *microcystis or *microcystin* or *phycocyanin* or ‘bluegreen alga*’ or *anabaen* or cyanophy* or *nostoc* or *oscillatoria* or spirul* or arthrospira or *lyngbya* or *anatoxin* or cyanophage* or cyanotox* or phycobiliprotein* or phycobilisome* or *phycoerythrin* or saxitoxin* or aphanizomenon or planktothrix or prochloro* or trichodesmium or ‘methylamino-l-alanine*’ or bmaa or aeruginosin* or calothrix* or chroococca* or cryptophycin* or cyanelle* or cyanobiont* or cyanovir* or hapalindole* or nodularin* or phycocyanobilin* or phycobilin* or teleocidin* or acaryochloris or aphanothece or cyanophora or cyanothece or fischerella or fremyella or gloeobacter or mastigocladus or microcoleus or nodularia or plectonema or scytonem* or tolypothrix) OR A.7. Journals SO = (‘Algal Research*’ or ‘European Journal of Phycology’ or ‘Harmful Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘British Phycological Journal’ or ‘Diatom Research’ or ‘Phycological Research’ or Algae or ‘Cryptogamie Algologie’ or Fottea*) OR A.8. Authors AU = (‘yasumoto t’ or ‘paerl hw’ or ‘martin jh’ or ‘deisseroth k’ or ‘boyden es’ or ‘riebesell u’ or ‘bhattacharya d’ or ‘fromme p’ or ‘barber j’ or ‘witman gb’ or ‘saenger w*’ or ‘atsumi s’ or ‘behrenfeld mj’ or ‘shen jr’) AND TS = [Algae] (the whole set of keywords for algae) A.9. Excluding terms NOT TI = (shewanella or pelagia or chlorophytum or pseudomonas or azotobacter or ‘bacterial alginate*’ or diatomic* or atom* or *molecule* or polynesia or propanoic or tiahura or sponge or leuconostoc or algas or gaas or ‘microbial alginate*’)



100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  35

Acknowledgments The significant contribution of the authors of the pioneering studies in algal science, technology, and medicine to the development of the research in in this field have been gratefully acknowledged. The authors listed as the ‘most-prolific and influential authors’ in Table 2.1 have published at least five influential papers and two classical papers each. Similarly, the ‘lead authors’ in Table 2.7 have published at least 10 influential papers each.

References Abramo, G., D’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Baltussen, A., Kindler, C.H., 2004a. Citation classics in anesthetic journals. Anesth. Analg. 98 (2), 443–451. Baltussen, A., Kindler, C.H., 2004b. Citation classics in critical care medicine. Intensive Care Med. 30 (5), 902–910. Behrenfeld, M.J., Falkowski, P.G., 1997. Photosynthetic rates derived from satellite-based chlorophyll concentration. Limnol. Oceanogr. 42 (1), 1–20. Benya, P.D., Shaffer, J.D., 1982. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30 (1), 215–224. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K., 2005. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8 (9), 1263–1268. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sustain. Energy Rev. 14 (2), 557–577. Charlson, R.J., Lovelock, J.E., Andreae, M.O., Warren, S.G., 1987. Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate. Nature 326 (6114), 655–661. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Clarivate Analytics, 2018. Highly Cited Researchers 2018. Identifying Top Talent in the Sciences and Social Sciences. Clarivate Analytics, Philadelphia, PA. Cohen, P., Holmes, C.F.B., Tsukitani, Y., 1990. Okadaic acid—a new probe for the study of cellular-regulation. Trends Biochem. Sci. 15 (3), 98–102. Davis, T.A., Volesky, B., Mucci, A., 2003. A review of the biochemistry of heavy metal biosorption by brown algae. Water Res. 37 (18), 4311–4330. Dubin, D., Hafner, A.W., Arndt, K.A., 1993. Citation classics in clinical dermatologic journals: citation analysis, biomedical journals, and landmark articles, 1945-1990. Arch. Dermatol. 129 (9), 1121–1129. Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., Iwata, S., 2004. Architecture of the photosynthetic oxygen-evolving center. Science 303 (5665), 1831–1838. Garfield, E., 1955. Citation indexes for science; a new dimension in documentation through association of ideas. Science 122 (3159), 108–111. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 1979. Citation Indexing: Its Theory and Application in Science, Technology, and Humanities. Wiley, New York. Garfield, E., 1987. 100 Citation classics from the Journal of the American Medical Association. JAMA, J. Am. Med. Assoc. 257 (1), 52–59. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Guan, J., Ma, N., 2007. China's emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32 (2), 79–99. Hillebrand, H., Durselen, C.D., Kirschtel, D., Pollingher, U., Zohary, T., 1999. Biovolume calculation for pelagic and benthic microalgae. J. Phycol. 35 (2), 403–424. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., et al., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54 (4), 621–639. Jochimsen, E.M., Carmichael, W.W., An, J.S., Cardo, D.M., Cookson, S.T., Holmes, C.E.M., et al., 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N. Engl. J. Med. 338 (13), 873–878. Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W., Krauss, N., 2001. Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution. Nature 411 (6840), 909–917. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books Ltd., Stoke-on-Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540.

36  PART | I  Introduction to the algal science, technology, and medicine

Konur, O., 2012a. The policies and practices for the academic assessment of blind students in higher education and professions. Energ. Educ. Sci. Technol. B 4 (si1), 240–244. Konur, O., 2012b. Prof. Dr. Ayhan Demirbas’ scientometric biography. Energ. Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012c. The evaluation of the biogas research: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012d. The evaluation of the bio-oil research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 379–392. Konur, O., 2012e. The evaluation of the biorefinery research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 347–358. Konur, O., 2012f. The evaluation of the research on the biodiesel: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012g. The evaluation of the research on the bioethanol: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012h. The evaluation of the research on the biofuels: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012i. The evaluation of the research on the biohydrogen: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012j. The evaluation of the research on the microbial fuel cells: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012k. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012l. The evaluation of the global energy and fuels research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (1), 613–628. Konur, O., 2012m. 100 Citation classics in energy and fuels. Energ. Educ. Sci. Technol. A 30 (si1), 319–332. Konur, O., 2012n. What have we learned from the citation classics in energy and fuels: a mixed study. Energ. Educ. Sci. Technol. A 30 (si1), 255–268. Konur, O., 2012o. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1893–1908. Konur, O., 2012p. The evaluation of the research on the arts and humanities in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (3), 1603–1618. Konur, O., 2012q. The evaluation of the educational research: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012r. The scientometric evaluation of the research on the deaf students in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1573–1588. Konur, O., 2012s. The scientometric evaluation of the research on the students with ADHD in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2012t. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Energ. Educ. Sci. Technol. B 4 (si1), 250–257. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453.



100 Citation classics in the algal science, technology, and medicine: A scientometric analysis Chapter | 2  37

Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019b. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Kostoff, R.N., Barth, R.B., Lau, C.G., 2008a. Quality vs. quantity of publications in nanotechnology field from the People’s Republic of China. Chin. Sci. Bull. 53 (8), 1272–1280. Kostoff, R.N., Barth, R.B., Lau, C.G., 2008b. Relation of seminal nanotechnology document production to total nanotechnology document productionSouth Korea. Scientometrics 76 (1), 43–67.

38  PART | I  Introduction to the algal science, technology, and medicine

Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. LeFaivre, K.A., Shadgan, B., O’Brien, P.J., 2011. 100 Most cited articles in orthopaedic surgery. Clin. Orthop. Relat. Res. 469 (5), 1487–1497. Lieberman, M.B., Montgomery, D.B., 1988. First-mover advantages. Strateg. Manag. J. 9 (S1), 41–58. Loll, B., Kern, J., Saenger, W., Zouni, A., Biesiadka, J., 2005. Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature 438 (7070), 1040–1044. MacKintosh, C., Beattie, K.A., Klumpp, S., Cohen, P., Codd, G.A., 1990. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 264 (2), 187–192. Martin, J.H., Fitzwater, S.E., 1988. Iron-deficiency limits phytoplankton growth in the northeast pacific subarctic. Nature 331 (6154), 341–343. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sustain. Energy Rev. 14 (1), 217–232. Merchant, S.S., Prochnik, S.E., Vallon, O., Harris, E.H., Karpowicz, S.J., Witman, G.B., et al., 2007. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318 (5848), 245–251. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Moravcsik, M.J., Murugesan, P., 1975. Some results on the function and quality of citations. Soc. Stud. Sci. 5 (1), 86–92. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Paladugu, R., Schein, M., Gardezi, S., Wise, L., 2002. One hundred citation classics in general surgical journals. World J. Surg. 26 (9), 1099–1105. Reed, K.C., Mann, D.A., 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13 (20), 7207–7221. Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., et al., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102 (1), 100–112. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes—towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Seglen, P.O., 1997. Citations and journal impact factors: questionable indicators of research quality. Allergy 52 (11), 1050–1056. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101 (2), 87–96. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Zouni, A., Witt, H.T., Kern, J., Fromme, P., Krauss, N., Saenger, W., et al., 2001. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409 (6821), 739–743.

Chapter 3

The scientometric analysis of the research on the algal structures Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

3.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 indexed-papers published since 1980. The field of algal structures, covering cell biology, phylogeny, and physiology of the algae, has been among the mostprolific research fronts over time as evidenced with over 30,500 papers, comprising over 20% of the algal research as a whole, published during the same study period, corresponding to the public concerns about the development of sustainable and environment-friendly bioproducts and bioprocesses at large. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate, the stakeholders should have timely and thorough access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b, 2012a). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal structures and the underlying research areas as in fields of the algal research (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012b,c,d,e,f,g,h,i,j,k, 2018a,b,c), energy and fuels (Konur, 2012l,m,n, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g, 2017b,c,d,e,f, 2019b), biomedicine (Konur, 2016h, 2018d), and social sciences (Konur, 2012o,p,q,r,s,t). Although there have been nearly 900 literature reviews on the algal structures, there have been no published scientometric studies. This is contrast to the many published scientometric studies on structure-related fields at large (Bansard et al., 2007; Li et al., 2009; Patra and Mishra, 2006; Tan et al., 2014). Therefore, this paper presents the first-ever scientometric study of the research in algal structures covering the whole range of research fronts as well as whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate.

3.2  Materials and methodology The search for the scientometric analysis of the literature on the algal structures was carried out in February 2019 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI), and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been developed by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in Appendix. These keyword sets have been devised in two major parts: the keywords related to structures and keywords related to the algae. There have been two distinct keyword sets for the first part: keywords related to the structures, and selected set of Web of Science subject categories primarily related to structures. On the other hand, the second part consists of the keywords related to the algae in general, dinoflagellates and coccolithophores, microalgae, macroalgae, diatoms, cyanobacteria, and a selected set of journal titles related to the algae. Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00003-6 © 2020 Elsevier Inc. All rights reserved.

41

42  PART | II  Algal structures

Additionally, a selected set of keywords and the Web of Science subject categories have been used to eliminate the papers unrelated to algal structures. The papers located through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subjected to the quality control exercise to ensure that these references have been primarily related to the algal structures. This refined list of papers has formed the core sample for the scientometric and content overview of the literature on the algal structures. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. Additionally, the search has been carried out for the papers with at least 100 citations and the relevant scientometric data have been collected. These papers have been termed as ‘influential papers’. Furthermore, the data on the scientometric analysis and brief content overview of 20 most-cited papers have also been provided focusing on the determination of the key research fronts for these citation classics, complementing sections on the keywords and research fronts. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

3.3 Results 3.3.1  Documents and indexes The search has resulted in 36,050 papers where there have been 30,171 articles, 3834 meeting abstracts, 929 reviews, 493 notes, 216 editorial materials, 250 corrections, and 87 letters. In the first instance, the papers excluding meeting abstracts and corrections have been selected resulting in 31,896 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 30,683 papers in English. The other major languages have been French, Russian, Japanese, Spanish, and German. This set of 30,683 papers has formed the core sample for the scientometric analysis of the literature on the algal structures. The articles have formed 94.6% of the final sample while reviews, notes, editorial matters, and letter have formed 2.9%, 1.5%, 0.7%, and 0.3% of this sample, respectively. Additionally, 2.8% of these papers have been ‘proceedings papers’ and there have been three ‘retracted papers’. On the other hand, 99.4% of these papers have been indexed by the SCI-E while only 0.1% of the papers have been indexed by the SSCI and A&HCI. Additionally, 0.5% of the papers have been indexed by the ESCI.

3.3.2 Keywords The most-prolific keywords used in algal structures have been determined based on the influential papers to locate the hot topics and the primary research fronts in the algal structures. There have been a number of most-prolific keywords for the first set of keywords for the structures: ‘origin*, evolution*, phyloge*, taxonom*, ancest*, plastid*, *diversity, *flagellar, cilia, tubulin, cell, physiol*, *nitrogen*, temperature, salinity, “reactive oxygen species”, biogeo*, *silica*, composition, circadian’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, phytoplankton, macroalga*, rhodophyt*, seaweed*, bacillariophy*, diatom, diatoms, and cyanobacter*’. The other prolific keywords for the algae have been ‘coccolith*, dinophycea*, Alexandrium, chlorophycea*, chlorophyt*, “green alga*”, microalga*, “micro-alga*”, Chlamydomonas, *Chlorella, Dunaliella, Euglena, Scenedesmus, “brown alga*”, phaeophycea*, kelp*, phaeophyt*, “red alga*”, Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, Ulva, bacillariophyt*, “blue green alga*”, “blue-green alga*”, *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, Synechocystis’.

3.3.3 Authors There have been over 52,500 authors contributing to the research on the algal structures in total. The information on the most-prolific and influential 20 authors is provided in Table 3.1: Authors’ names, gender, institutions, countries, primary research fronts, number of papers produced by these authors (I-0), the number of influential papers with at least 100 citations received (I-100), and the percentage of the number of influential papers with relative to the number of all the papers published (I-100%).



TABLE 3.1  The most-prolific and influential authors in algal structures. Gender

Institution

Country

Research fronts

Algae

I-0

I-100

I-100%

1

Norio Murata

M

Natl. Inst. Bas. Biol.

Japan

Temperature

Cyanobacteria

73

21

40.0

2

Takao Kondo

M

Nagoya Univ.

Japan

Clocks

Cyanobacteria

59

19

17.7

3

Paul G Falkowski

M

Brookhaven Natl. Lab.

United States

Nitrogen fixation

Algae

45

18

35.6

4

Paul J Harrison

M

Univ. Brit. Columbia

Canada

Nitrogen fixation

Algae

96

17

29.4

5

George B Witman

M

Worcester Fed. Exp. Biol.

United States

Flagellar structures

Microalgae

45

16

19.7

6

Sallie W Chisholm

F

Massachusetts Inst. Technol.

United States

Phylogeny

Cyanobacteria

51

15

20.3

7

Donald M Anderson

M

Woods Hole Ocean. Inst.

United States

Phylogeny

Algae

66

13

26.0

8

Susan S Golden

F

Texas A&M Univ.

United States

Clocks

Cyanobacteria

64

13

31.7

9

Robert Haselkorn

M

Univ. Chicago

United States

Nitrogen fixation

Cyanobacteria

50

13

48.1

10

Jean-David Rochaix

M

Univ. Geneva

Switzerland

Photosynthesis

Microalgae

41

13

65.0

11

Nils Kroger

M

Univ. Regensburg

Germany

Silica structures

Diatoms

27

13

11.8

12

Gianni Piperno

M

Rockefeller Univ.

United States

Flagellar structures

Microalgae

20

13

30.0

13

Enrique Flores

M

Univ. Seville

Spain

Nitrogen fixation

Cyanobacteria

102

12

33.3

14

Joel L Rosenbaum

M

Yale Univ.

United States

Flagellar structures

Microalgae

40

12

16.4

15

Manfred Sumper

M

Univ. Regensburg

Germany

Silica structures

Diatoms

36

12

26.2

16

Debashis Bhattacharya

M

Univ. Cologne

Germany

Phylogeny

Algae

67

11

40.7

17

John A Raven

M

Univ. Dundee

United Kingdom

Nitrogen fixation

Algae

42

11

47.8

18

Hideo Iwasaki

M

Nagoya Univ.

Japan

Clocks

Cyanobacteria

27

11

12.3

19

Mark E Hay

M

Univ. N Carolina

United States

Chemical defense

Macroalgae

23

11

40.0

20

Antonia Herrero

F

Univ. Seville

Spain

Nitrogen fixation

Cyanobacteria

81

10

17.7

Average

52.8

13.8

Total %

3.5

20.5

M, male; F, female; I-0, no. papers, the number of papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers with relative to the number of all the papers published.

The scientometric analysis of the research on the algal structures Chapter | 3  43

Author

44  PART | II  Algal structures

The data on these authors show that the most-prolific author with the highest citation impact has been ‘Norio Murata’ of the National Institute for Basic Biology of Japan, working primarily on the ‘temperature response’ of cyanobacteria, with 73 papers. His citation impact is highest with 21 influential papers. The other most-prolific authors with the high citation impact have been ‘Takao Kondo’, ‘Paul G Falkowski’, ‘Paul J Harrison’, ‘George B Witman’, and ‘Sallie W Chisholm’ with at least 15 influential papers each. The United States has been the most-prolific country for these authors with nine authors while Germany, Japan, and Spain have been the other prolific countries with three, three, and two authors, respectively. On the other hand, Europe has had only seven authors as a whole. In total, these top authors have been from seven countries. There has been a significant gender deficit among these top prolific and influential authors as only three of them are females: ‘Antonia Herrero’, ‘Sallie W Chisholm’, and ‘Susan S Golden’. Similarly, the most-prolific institutions have been ‘Nagoya University’, ‘University of Regensburg’, and ‘University of Seville’ with two authors each. In total, these top authors have been affiliated with 17 institutions. The most-prolific research front has been the ‘nitrogen fixation’ by algae with six authors, followed by ‘circadian clocks’, ‘flagellar structures’, and ‘phylogeny’ of algae with three authors each. Additionally, two authors have studied ‘silica structures’ of diatoms. Similarly, the most prolific type of algae studied by these top authors has been ‘cyanobacteria’ with eight authors. The other prolific types of algae have been ‘algae’ in general covering more than one type of algae, ‘microalgae’, and ‘diatoms’ with five, four, and two authors, respectively. The number of papers published by these authors have ranged from 20 to 102 with 52.8 papers on average. These mostprolific authors have also contributed to nearly 3.5% and 20.5% of all the papers and influential papers, respectively. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the influential papers with relative to the number of all the papers published for each author in the last column (I-100%). These data shows that ‘Jean-David Rochaix’, ‘Robert Haselkorn’, ‘John A Raven’, ‘Debashis Bhattacharya’, ‘Mark E Hay’, and ‘Norio Murata’ have been the top influential authors with at least 40.0% ratios.

3.3.4 Countries Nearly 99.7% of the papers have had country information in their abstract pages and 150 countries and territories have contributed to these papers overall. Table 3.2 provides the information about the most-prolific and influential 20 countries. These 20 most-prolific countries have produced 106.7% and 136.2% of all the papers and influential papers, respectively, as a whole. The most-prolific and influential country has been the United States producing 26.5% and 49.9% of all the papers and influential papers, respectively. The other prolific and influential countries have been Germany, United Kingdom, Canada, Japan, France, and Australia producing at least 7.5% of the influential papers each. The European countries have been relatively dominant in the top 20 country list as they have produced 43.5% and 56.2% of all the papers and influential papers, respectively, as a whole, surpassing significantly the United States. Similarly, the Asian countries in this top 20 list, have produced 28.6% and 21.0% of all the papers and influential papers, respectively, as a whole.

3.3.5 Institutions Over 99.7% of the papers have had their institutions listed in their abstract pages. For these papers, over 7600 institutions have contributed to the research on the algal structures in total. The information about the 20 most-prolific and influential institutions is given in Table 3.3. The most-prolific and influential institution has been the ‘French National Scientific Research Center’ (CNRS) publishing 4.0% and 5.1% of the all and influential papers, respectively. The other prolific and influential institutions have been ‘Helmholtz Association’ of Germany, ‘University of British Columbia’ of Canada, ‘Sorbonne University’ of France, ‘Woods Hole Oceanographic Institute’ and ‘University of California San Diego’ of the United States, ‘Nagoya University’ of Japan, and ‘Natural Environment Research Council’ (NERC) of the United Kingdom with over 2.0% of the influential papers each. The most-prolific country for these institutions has been the United States with seven institutions producing 13.0% of the influential papers. Additionally, Japan, France, and Germany have had five, two, and two institutions, respectively. On the other hand, Europe has had six institutions as a whole, producing 18.0% of the influential papers.

The scientometric analysis of the research on the algal structures Chapter | 3  45



TABLE 3.2  The most-prolific and influential countries in algal structures. Country

I-0

I-0%

I-100

I-100%

Surplus%

Europe

13,774

43.5

751

56.2

12.7

Asia

8672

28.6

280

21.0

−7.6

1

United States

8023

26.5

666

49.9

23.4

2

Germany

3203

10.6

170

12.7

2.1

3

United Kingdom

2374

7.8

168

12.6

4.8

4

Canada

1862

6.1

122

9.1

3

5

Japan

3244

10.7

109

8.2

−2.5

6

France

1891

6.2

103

7.7

1.5

7

Australia

1543

5.1

100

7.5

2.4

8

Netherlands

816

2.7

51

3.8

1.1

9

Spain

1532

5.1

42

3.1

−2.0

11

Israel

466

1.5

37

2.8

1.3

10

Sweden

691

2.3

35

2.6

0.3

12

China

2563

8.5

33

2.5

−6.0

13

Denmark

461

1.5

29

2.2

0.7

14

Italy

834

2.8

28

2.1

−0.7

15

Switzerland

298

1.0

27

2.0

1.0

16

Norway

429

1.4

25

1.9

0.5

17

New Zealand

421

1.4

20

1.5

0.1

18

Finland

279

0.9

19

1.4

0.5

19

Russia

901

3.0

18

1.3

−1.7

20

United States

8023

26.5

666

49.9

23.4

Total

32,331

106.7

1819

136.2

29.5

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Europe and Asia values are for only the top 20 countries. Surplus%, I-100% − I-0%.

The contribution of these institutions has ranged from 0.3% to 4.0% for all the papers and from 1.5% to 5.1% for the influential papers. Overall, these 20 institutions have contributed to 23.1% and 43.4% of all the papers and influential papers, respectively.

3.3.6  Research funding bodies Only 41.1% of these papers have had declared any research funding in their abstract pages and overall, over 15,200 funding bodies have funded these papers. The corresponding funding rate for the influential papers has been 21.3%. The most-prolific funding body has been the ‘National Natural Science Foundation of China’, funding 3.2% of the papers. The other prolific funding bodies have been ‘Natural Science Foundation’ and ‘National Institute of Health’ of the United States, ‘Natural Environment Research Council’ of the United Kingdom, ‘Australian Research Council’, ‘German Research Foundation’, and ‘Natural Sciences and Engineering Research Council’ of Canada.

3.3.7  Publication years Fig. 3.1 shows the number of papers on the algal structures, published between 1980 and 2018 as of February 2019.

46  PART | II  Algal structures

TABLE 3.3  The most-prolific and influential institutions in algal structures. Institutions

Country

I-0

I-0%

I-00

I-100%

Surplus%

United States

1391

4.6

174

13.0

8.4

Europe

3775

12.5

240

18.0

5.5

Asia

1444

4.8

121

9.1

4.3

1

French Natl. Sci. Ctr.—CNRS

France

1244

4.0

68

5.1

1.1

2

Helmholtz Assoc.

Germany

645

2.1

45

3.4

1.3

3

University of British Columbia

Canada

379

1.3

45

3.4

2.1

4

Sorbonne Univ.

France

553

1.8

35

2.6

0.8

5

Woods Hole Ocean. Inst.

Unite States

212

0.7

35

2.6

1.9

6

Nagoya Univ.

Japan

241

0.8

30

2.3

1.5

7

Univ. Calif. San Diego

United States

268

0.9

27

2.0

1.1

8

Natrl. Env. Res. Counc.—NERC

United Kingdom

277

0.9

26

2.0

1.1

9

Univ. Calif. Santa Barbara

United States

163

0.5

25

1.9

1.4

10

Univ. N Carolina

United States

296

1.0

24

1.8

0.8

11

Univ. Tokyo

Japan

645

2.1

23

1.7

−0.4

12

Spanish Natl. Res. Counc.—CSIC

Spain

516

1.7

23

1.7

0

13

Max Planck Society

Germany

285

0.9

23

1.7

0.8

14

Natl. Inst. Natr. Sci.

Japan

177

0.6

23

1.7

1.1

15

Natl. Inst. Bas. Biol.

Japan

163

0.5

23

1.7

1.2

16

Texas A&M Univ.

Japan

218

0.7

22

1.6

0.9

17

Michigan State Univ.

United States

187

0.6

22

1.6

1

18

Rutgers State Univ. New Brunswick

United States

160

0.5

21

1.6

1.1

19

Univ. Copenhagen

Denmark

255

0.8

20

1.5

0.7

20

Univ. Chicago

United States

105

0.3

20

1.5

1.2

6989

23.1

580

43.4

20.3

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. The United States, Europe, and Asia values are for only the top 20 institutions. Surplus%, I-100% − I-0%.

The data in this figure shows that the number of papers has risen from 386 papers in 1980 to 1600 and 1549 papers 2017 and 2018, respectively. The most prolific decade has been the 2010s with 40.7% of the papers. Additionally, 16.4%, 18.9%, and 24.0% of the papers have been published in the 1980s, 1990s, and 2000s, respectively. Thus, the figure shows that there has been a steadily increasing trend between 1980 and 2018, steeper in the 2000s and 2010s.

3.3.8  Source titles Overall, these papers have been published in 1975 journals. Table 3.4 provides the information on the 20 most-prolific and influential journals. These 20 journals have published 20.6% and 54.2% of all the papers and influential papers, respectively, in total. The most-prolific and influential journal has been ‘Journal of Phycology’ publishing 5.7% and 6.8% of all the papers and influential papers, respectively. ‘Limnology and Oceanography’, ‘Proceedings of the National Academy of Sciences of the United States of America’, ‘Marine Ecology Progress Series’, ‘Journal of Cell Biology’, and ‘Nature’ have followed the top journal with at least 3.1% of the influential papers each.



The scientometric analysis of the research on the algal structures Chapter | 3  47

FIG. 3.1  The number of publications in the algal structures between 1980 and 2018.

The most-prolific subject categories for these journals have been ‘Marine Freshwater Biology’ and ‘Cell Biology’ with six and five journals, respectively. ‘Plant Sciences’, ‘Biochemistry and Molecular Biology’, ‘Multidisciplinary Sciences’, and ‘Oceanography’ have followed these top subjects with four, four, three, and three journals, respectively. The other prolific subjects have been ‘Biotechnology and Applied Microbiology’, ‘Microbiology’, and ‘Ecology’ with two papers each.

3.3.9  Subject categories These papers have been indexed by 122 subject categories. The information about the 10 most-prolific and influential subject categories are given in Table 3.5. The most-prolific and influential subject categories have been ‘Marine Freshwater Biology’, ‘Plant Sciences’, and ‘Biochemistry Molecular Biology’, ‘Cell Biology’, and ‘Oceanography’ indexing at least 14.7% of the influential papers each. Overall, 125.0 and 140.6% of all the papers and influential papers have been indexed by these top 10 subject categories, respectively.

3.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers with at least 100 citations, 4.4% of the research sample of 30,683 papers, Table 3.6. The data shows that the field of the structures of ‘cyanobacteria’ and ‘microalgae' have been the most prolific research fronts with 24.4% and 20.8% of the influential papers. The other key research fronts have been the structures of ‘phytoplankton’, ‘macroalgae’, and ‘diatoms’ with at least 10% of the influential papers each. The key topical research fronts have been ‘phylogeny’ and ‘taxonomy’ of the algae as well as the ‘cell biology’ and ‘physiology’ of algae. The ‘flagellar structures’, ‘silica structures', ‘circadian clocks’, nitrogen metabolism’, and ‘salt’ and ‘temperature’ response of algae have been other prolific research fronts.

3.3.11  Citation classics This section provides the information on both the scientometric analysis and brief content overview of the most-cited 20 papers in algal structures. The information on these papers is given in Table 3.7: authors’ names, publication years, document type, number of authors per paper, lead authors’ names, gender, and paper numbers (lead authors were determined based on the number of papers produced in this field with at least five influential papers), journals, subject areas, research fronts, types of algae, academic focus of the papers, number of citations received, and the number of citations per year.

Journals

Abbr.

Subject

I-0

I-0%

I-100

I- 100%

Surplus%

1

Journal of Phycology

J. Phycol.

Plant Sci., Mar. Fresh. Biol.

725

5.7

91

6.8

1.1

2

Limnology and Oceanography

Limnol. Oceanogr.

Limnol., Ocean.

433

1.4

81

6.1

4.7

3

Proceedings of the National Academy of Sciences of the United States of America

P. Natl. Acad. Sci. USA

Mult. Sci.

236

0.8

70

5.2

4.4

4

Marine Ecology Progress Series

Mar. Ecol. Prog. Ser.

Ecol., Mar. Fresh. Biol., Ocean.

510

1.7

54

4.0

2.3

5

Journal of Cell Biology

J. Cell. Biol.

Cell Biol.

195

0.6

50

3.7

3.1

6

Nature

Nature

Mult. Sci.

61

0.2

43

3.1

2.9

7

Science

Science

Mult. Sci.

67

0.2

37

2.8

2.6

8

Marine Biology

Mar. Biol.

Mar. Fresh. Biol.

353

1.2

36

2.7

1.5

9

Applied and Environmental Microbiology

Appl. Env. Microb.

Biot. Appl. Microb., Microbiol.

241

0.8

34

2.5

1.7

10

Plant cell

Plant Cell

Bioch. Mol. Biol., Plant Sci., Cell Biol.

204

0.7

33

2.5

1.8

11

EMBO Journal

EMBO J.

Bioch. Mol. Biol., Cell Biol.

77

0.3

28

2.1

1.8

12

Plant Physiology

Plant Physiol.

Plant Sci.

333

1.1

25

1.9

0.8

13

Bioresource Technology

Bioresource Technol.

Agr. Eng., Biot. Appl. Microb., Ener. Fuels

410

1.4

23

1.7

0.3

14

Journal of Plankton Research

J. Plankton Res.

Mar. Fresh. Biol., Ocean.

337

1.1

21

1.6

0.5

15

Phycologia

Phycologia

Plant Sci., Mar. Fresh. Biol.

776

2.6

18

1.3

−1.3

16

FEBS Letters

FEBS Lett.

Bioch. Mol. Biol., Biophys., Cell Biol.

509

1.7

17

1.3

−0.4

17

Journal of Experimental Marine Biology and Ecology

J. Exp. Mar. Biol. Ecol.

Ecol., Mar. Fresh. Biol.

315

1.0

17

1.3

0.3

18

Journal of Biological Chemistry

J. Biol. Chem.

Bioch. Mol. Biol.

145

0.5

17

1.3

0.8

Journal of Bacteriology

J. Bacteriol.

Microbiol.

285

0.9

15

1.1

0.2

Cell

Cell

Cell Biol.

33

0.1

14

1.0

0.9

6245

20.6

724

54.2

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Values for four subject categories sciences are only for the top 20 journals. Surplus%, I-100% − I-0%.

48  PART | II  Algal structures

TABLE 3.4  The most-prolific and influential journals in algal structures.

The scientometric analysis of the research on the algal structures Chapter | 3  49



TABLE 3.5  The most-prolific and influential subject categories in algal structures. Subject categories

I-0 No. papers

I-0% Papers

I-100 No. papers

I-100% Papers

Surplus%

1

Marine Freshwater Biology

9716

32.1

352

26.3

−5.8

2

Plant Sciences

8689

28.7

250

18.7

−10

3

Biochemistry Molecular Biology

3340

11.0

233

17.4

6.4

4

Cell Biology

3411

11.3

210

15.7

4.4

5

Oceanography

2387

7.9

196

14.7

6.8

6

Ecology

2424

8.0

154

11.5

3.5

7

Multidisciplinary Sciences

1199

4.0

152

11.4

7.4

8

Microbiology

2820

9.3

142

10.6

1.3

9

Biotechnology Applied Microbiology

2838

9.4

102

7.6

−1.8

10

Limnology

1021

3.4

88

6.6

3.2

Total

87,845

125.0

1879

140.6

15.6

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers; Surplus%, I-100% − I-0%.

TABLE 3.6  The most-prolific research fronts in algal structures. Research fronts

Algae

Phyto­ plankton

Microalgae

Cyano­ bacteria

Diatoms

Dino­ flagellates

Cocco­ lithophores

Macroalgae

Total

No.

200

245

400

470

205

165

30

210

1925

%

10.4

12.7

20.8

24.4

10.6

8.6

1.6

10.9

Numbers, the number of influential papers for each research front and type of algae; %, the percentage of influential papers for each research front and type of algae.

3.3.11.1  Scientometric overview of the citation classics These papers have been published between 1980 and 2009. The most-prolific decade has been the 2000s with 13 papers. There have also been three papers each in the 1980s and 1990s. The reviews have been over-represented in these classical papers as there have been 15 articles and 5 reviews. The number of the authors of these papers has ranged from 1 to 119 while the mean number of authors has been 16.3. There have been 47 authors with at least 5 influential papers as the lead authors of the citation classics. There has been a significant gender deficit among the lead authors of these classical papers as only 13 authors are female (female authors are shown in italic). The most-prolific and influential lead authors have been ‘Nils Kroger’ and ‘George J Pazour’, and ‘Chris Bowler’ with three citation classics each. Additionally, 15 authors have contributed to 2 papers each. In total, these citation classics have been published by 15 journals. The most-prolific journals have been ‘Science’ and ‘Journal of Cell Biology’ with five and two papers, respectively. In total, these papers have been indexed by 14 subject categories. The most-prolific categories have been ‘Multidisciplinary Sciences’, ‘Marine Freshwater Biology’, and ‘Cell Biology’ with seven, six, and three papers, respectively. There have also been two papers each in ‘Environmental Sciences’, ‘Ecology’, ‘Oceanography’, and ‘Limnology’. In total, there have been nine research fronts. The most-prolific research front has been ‘composition’ of algae with six papers. The other prolific research front has been ‘flagellar structures’, ‘phylogeny’, ‘evolution’, and ‘silica structures’ with three, three, two, and two papers, respectively.

Authors

Year

Doc.

N Auths.

1

Hillebrand et al.

1999

A

5

2

Merchant et al.

2007

A

119

3

MendenDeuer & Lessard

2000

A

2

4

Armbrust et al.

2004

A

45

5

Anderson et al.

2002

R

6

Kroger et al.

1999

7

Reynolds et al.

8

9

Lead authors

Journal

Subject area

Res. fronts

Algae

Cits.

Av. cits

J. Phycol.

Plant. Sci., Mar. Fresh. Biol.

composition

Microalgae

1757

92.5

Science

Mult. Sci.

Flagellar structures

Algae

1427

129.7

Limnol. Oceanogr.

Limnol., Oceanogr.

Composition

Algae

1283

71.3

EV Armbrust-8; JA Berges-7; C Bowler-6; BR Green-6; AE Allen-6; MA Brzezinski-7; N Kroger-13; S Lucas-6; M Obornik; GJ Pazour-6; TA Rynearson-6; K Valentin-6; A Vardi-5

Science

Mult. Sci.

Silica structures

Diatoms

1158

82.7

3

DM Anderson-13; PM Glibert-9

Estuaries

Env. Sci., Mar. Fresh. Biol.

Composition

Algae

1090

68.1

A

3

N Kroger-13; M Sumper-12

Science

Mult. Sci.

Silica structures

Diatoms

976

51.4

2002

R

5

CS Reynolds-6

J. Plankton Res.

Mar. Fresh. Biol.; Oceanogr.

Classification

Phytoplankton

849

53.0

Guskov et al.

2009

A

6

Nat. Struct. Mol. Biol.

Bioch. Mol. Biol., Biophys., Cell Biol.

PSII structure

Cyanobacteria

810

90

Bowler et al.

2008

A

77

Nature

Mult. Sci.

Phylogeny

Diatoms

808

80.8

SS Merchant-10; GB Witman-16; A Salamov-6; E Lindquist-5; J Grimwood-5; J Schmutz-5; SK Dutcher-6; E Fernandez-6; H Fukuzawa-6; PA Lefebvre-9; GJ Pazour-6; C Bowler-6; IV Grigoriev-6; AR Grossman-10

C Bowler-6; AE Allen-6; J Grimwood-5; A Salamov-6; T Mock-5; K Valentin-6; JA Berges-7; A Kaplan-8; N Kroger-13; J La Roche-8; E Lindquist-5; S Lucas-6; LK Medlin-10; M Obornik-5; N Poulsen-5, TA Rynearson-5; J Schmutz-5; A Vardi-5; W Vyverman-5; EV Armbrust-8, BR Green-6; IV Grigoriev-6

50  PART | II  Algal structures

TABLE 3.7  The citation classics in algal structures.



10

Steneck et al.

2002

R

7

Biodiv. Cons.; Env. Sci.

Biodiversity

Macroalgae

772

48.3

11

Falkowski et al.

2004

R

7

Science

Mult. Sci.

Phylogeny

Phytoplankton

713

50.9

12

Martin et al.

2002

A

10

P. Natl. Acad. Sci. USA

Mult. Sci.

Phylogeny

Cyanobacteria

711

44.4

13

Smetacek

1985

A

1

Mar. Biol.

Mar. Fresh. Biol.

Evolution

Diatoms

702

21.3

14

Volkman et al.

1989

A

5

JK Volkman-7

J. Exp. Mar. Biol. Ecol.

Ecol., Mar. Fresh. Biol.

Composition

Algae

671

23.1

15

Pazour et al.

2000

A

7

GJ Pazour-6; JL Rosenbaum-12; GB Witman-16

J. Cell Biol.

Cell Biol.

Flagellar structures

Microalgae

661

36.7

16

Littler & Littler

1980

A

2

Am. Nat.

Ecol.; Evol. Biol.

Evolution

Macroalgae

622

16.4

17

Geider & La Roche

2002

R

2

J La Roche-8; RJ Geider-9

Eur. J. Phycol.

Plant Sci.; Mar. Fresh. Biol.

Composition

Algae

617

38.6

18

Nakajima et al.

2005

A

8

M Nakajima-5; T Nishiwaki-8; H Iwasaki-11; T Kondo-19

Science

Mult. Sci.

Clocks

Cyanobacteria

576

44.3

19

Converti et al.

2009

A

5

Chem. Eng. Proccess.

Ener. Fuels, Eng. Chem.

Composition

Microalgae

575

63.9

20

Cole et al.

1998

A

6

J. Cell Biol.

Cell Biol.

Flagellar structures

Microalgae

569

28.5

Average

2000

867

56.8

16.3

A Quigg-5; JA Raven-11; FJR Taylor-8

JL Rosenbaum-12

Doc., document; A, article; R, review; Gender, gender of lead authors—female authors in italic; N paper, for the authors with at least five influential papers—number after the author names; Subject, Web of Science subjects; Topic, primary topic of the papers; Algae, type of algae studied; Res. fronts,: primary research fronts studied; Cits., number of citations received in total; Av. Cits., number of citations per year.

The scientometric analysis of the research on the algal structures Chapter | 3  51

Environ. Conserv.

52  PART | II  Algal structures

There have been six types of algae covered by these classical papers. The most prolific type of algae has been ‘algae’ with five papers, closely followed by ‘diatoms’ and ‘microalgae’ with four papers each. In addition, there have been three, two, and two papers related to ‘cyanobacteria’, ‘macroalgae’, and ‘phytoplankton’, respectively. These papers have received between 569 and 1757 citations each, with a mean value of 867 citations per paper. On the other hand, the number of citations per year has ranged from 16.4 to 129.7 with a mean value of 56.8 citations per year. The papers by Merchant et al. (2007), Hillebrand et al. (1999), and Guskov et al. (2009) have been the most-cited papers on the basis of the number of citations on average, working on the flagellar structures, composition, and PSII structure, respectively.

3.3.11.2  Brief overview of the content of the citation classics There have been six major classes of papers: ‘composition’, ‘flagellar structures’, ‘phylogeny’, ‘evolution’, ‘silica structures’ of algae, and ‘other research fronts’ with six, three, three, two, two, and three papers, respectively. Composition Hillebrand et al. (1999) study the biovolume calculation for microalgae with varying shape and size in a paper with 1757 citations. They develop a set of equations for biovolume calculations from microscopically measured linear dimensions with high taxonomic resolution, up to the species level. Menden-Deuer and Lessard (2000) study the carbon to volume (CV) and nitrogen to volume (NV) relationships for various types of algae including diatoms and dinoflagellates in a paper with 1283 citations. They find that dinoflagellates were significantly more C dense than diatoms and there were few significant differences between CV relationships of other types of algae. Therefore, they develop one CV relationship for algae excluding diatoms. They further find that carbon density decreased significantly with increasing cell volume. Anderson et al. (2002) discuss the composition of the harmful algal blooms focusing on the nutrient enrichment in these blooms in a review paper with 1090 citations. They note the bloom production upon nitrogen and phosphorus enrichment with harmful impact on the fisheries resources, ecosystems, and human health due to the changes in the species composition: rise in the ratio of dissolved organic carbon to dissolved organic nitrogen. However, they caution that nutrient enrichment has not been only contributing factor for the blooms. Volkman et al. (1989) study the composition of algae used in mariculture in a paper with 690 citations. They find that the fatty acid composition changed among species of algae where chlorophyll (Chl) a and total fatty-acid contents were not related to cell volume. Fatty acids were 4–6 times more abundant than Chl a in most species. On the other hand, they find that polar lipids were present in all species. Geider and La Roche (2002) discuss the composition of microalgae determining carbon‑nitrogen‑phosphorus (CNP) ratio in a review paper with 617 citations. They note that the N:P ratio of algae is very plastic in nutrient-limited cells with most observations below the Redfield ratio of 16 and the critical N:P ratio in the range between 15 and 30. Lowest values of N:P are associated with nitrate- and phosphate-replete conditions. The highest values of N:P are observed in oligotrophic waters. The C:N ratio is also plastic. The average C:N ratios of nutrient-replete phytoplankton cultures are slightly greater than the Redfield ratio of 6.6. They argue that the Redfield N:P ratio does not reflect a physiological or biochemical constraint on the elemental composition of primary production. Converti et al. (2009) study the composition of microalgae in a paper with 575 citations. They focus on the effects of temperature and nitrogen concentration on the lipid content of microalgae as well as various lipid extraction methods. They find that the lipid content of microalgae was strongly influenced by the variation of these parameters: an increase in temperature from 20 to 25 °C doubled the lipid content of Nannochloropsis. On the other hand, a 75% decrease of the nitrogen concentration increased the lipid fractions of this microalga. Flagellar structures Merchant et al. (2007) study the flagellar structures of Chlamydomonas reinhardtii through the detailed study of its genome, revealing the evolution of key animal and plant functions, in a paper with 1427 citations. They sequence the 120-­megabase nuclear genome of Chlamydomonas and perform comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Their findings reveal previously unknown genes associated with photosynthetic and flagellar functions and establish links between ciliopathy and the composition and function of flagella. Pazour et al. (2000) study the Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, in a paper with 661 citations. They clone and sequence a Chlamydomonas cDNA encoding the IFT88 subunit of the



The scientometric analysis of the research on the algal structures Chapter | 3  53

i­ntraflagellar transport (IFT) particle and identify a Chlamydomonas insertional mutant that is missing this gene. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. They show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. They argue that that IFT is important for primary cilia assembly in mammals. Cole et al. (1998) study the Chlamydomonas kinesin-II-dependent IFT in a paper with 561 citations. IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. They find that the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin and IFT particles are composed of 15 polypeptides comprising two large complexes. They further find that the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies and mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. They argue that the microtubule-dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia. Phylogeny Bowler et al. (2008) study the evolutionary history of diatom genomes through the analysis of the Phaeodactylum genome in a paper with 808 citations. They report the complete genome sequence of the Phaeodactylum tricornutum and compare it with that of Thalassiosira pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. They note that the genome structures of these diatoms are significantly different and a substantial fraction of genes are not shared by these species. They document the presence of hundreds of genes from bacteria as they find more than 300 of these gene transfers in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. Falkowski et al. (2004) discuss the evolution of eukaryotic phytoplankton in a review paper with 713 citations. They note that photosynthetic eukaryotes evolved more than 1.5 billion years ago in the Proterozoic oceans. However, it was not until the Mesozoic Era that the three principal phytoplankton clades rose to ecological prominence. In contrast to their pioneering predecessors, the dinoflagellates, coccolithophores, and diatoms all contain plastids derived from an ancestral red alga by secondary symbiosis. They examine the geological, geochemical, and biological processes that contributed to the rise of these three, distantly related, phytoplankton groups. Martin et al. (2002) study the evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes revealing plastid phylogeny and thousands of cyanobacterial genes in the nucleus in a paper with 711 citations. They compare 24,990 proteins encoded in the Arabidopsis genome to the proteins from three cyanobacterial genomes, 16 other prokaryotic reference genomes, and yeast. They find that nearly 4500 of Arabidopsis protein-coding genes were acquired from the cyanobacterial ancestor of plastids. These proteins encompass all functional classes, and the majority of them are targeted to cell compartments other than the chloroplast. A phylogeny of chloroplast genomes inferred from 41 proteins and 8303 amino acids sites indicates that at least two independent secondary endosymbiotic events have occurred involving red algae and that amino acid composition bias in chloroplast proteins strongly affects plastid genome phylogeny. Evolution Smetacek (1985) study the role of sinking in diatom life-history cycles with the emphasis on its ecological, evolutionary and geological significance in a paper with 702 citations. He argues that rapid mass sinking of cells following diatom blooms represents the transition from a growing to a resting stage in the life histories of diatoms. Mass sinking is of survival value in those bloom diatoms that retain viability over long periods in cold, dark water but not in warm, nutrient-depleted surface water. Littler and Littler (1980) study the evolution of thallus form and survival strategies in benthic marine macroalgae in a paper with 622 citations. They develop a synthetic ‘functional form’ paradigm concerning hypothetically important adaptive features of algal structure and function and test by a costs/benefits strategic approach. The selection in fluctuating environments has favored opportunistic species having high net productivity, while those species able to persist in benign predictable habitats do so at the cost of lower photosynthetic rates and presumably slower growth. Selection has tended to reduce nutritive content in climax algal forms and has differentially favored the evolution of antipredator defenses in environmentally constant macrophyte communities. On the other hand, selection in mature communities has tended to increase allocation of materials to nonpigmented supportive structure at the expense of photosynthetic tissue. Later successional macroalgae showed greater toughness as well as greater resistance to wave-shearing forces than the opportunistic species, thus implicating selection for persistence as opposed to rapid growth in climax communities.

54  PART | II  Algal structures

Silica structures in diatoms Armbrust et al. (2004) study the genome of the diatom Thalassiosira pseudonana with the emphasis on its ecology, evolution, and metabolism in a paper with 1158 citations. They report the 34 million-base pair draft nuclear genome of the T. pseudonana and its 129 thousand-base pair plastid and 44 thousand-base pair mitochondrial genomes. They find 24 ­diploid nuclear chromosomes and identify novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle. Kroger et al. (1999) study the polycationic peptides from diatom biosilica that direct silica nanosphere formation in a paper with 976 citations. They show that a set of polycationic peptides (silaffins) isolated from diatom cell walls generate networks of silica nanospheres within seconds when added to a solution of silicic acid. Silaffins contain covalently modified lysine-lysine elements. These modifications drastically influence the silica-precipitating activity of silaffins. Other fronts Steneck et al. (2002) discuss the biodiversity, stability, and resilience of kelp forest ecosystems in a review paper with 772 citations. They focus on both the forestation and deforestation of the kelps. Global distribution of kelp forests is physiologically constrained by light at high latitudes and by nutrients, warm temperatures and other macrophytes at low latitudes. The well-developed kelp forests are most threatened by herbivory, usually from sea urchins. The recent global expansion of sea urchin harvesting has led to the widespread extirpation of this herbivore, and kelp forests have returned in some locations. Large predatory crabs have recently filled this void and they have become the new apex predator in this system. Fishing impacts on kelp forest systems have been both profound and much longer in duration than previously thought. Archaeological data suggest that coastal peoples exploited kelp forest organisms for thousands of years, occasionally resulting in localized losses of apex predators, outbreaks of sea urchin populations and probably small-scale deforestation. Over the past two centuries, commercial exploitation for export led to the extirpation of sea urchin predators. The large-scale removal of predators for export markets increased sea urchin abundances and promoted the decline of kelp forests over vast areas. It is possible that functional redundancies among predators and herbivores make this most diverse system most stable. Such biodiverse kelp forests may also resist invasion from non-native species. Climate changes have had measurable impacts on kelp forest ecosystems. However, overfishing appears to be the greatest manageable threat to kelp forest ecosystems over the 2025 time horizon. Reynolds et al. (2002) discuss the functional classification of the freshwater phytoplankton assemblages in a review paper with 849 citations. They promote a scheme of ‘vegetation recognition’, based upon the functional associations of species represented in the plankton. These groups are often polyphyletic, recognizing commonly shared adaptive features, rather than common phylogeny, to be the key ecological driver. They outline 31 such associations and the basic pattern of their distinctive ecologies. Nakajima et al. (2005) study the circadian oscillation of cyanobacterial KaiC phosphorylation in vitro in a paper with 576 citations. They reconstitute the self-sustainable oscillation of KaiC phosphorylation in vitro by incubating KaiC with KaiA, KaiB, and adenosine triphosphate. They find that the period of the in vitro oscillation was stable despite temperature change (temperature compensation), and the circadian periods observed in vivo in KaiC mutant strains were consistent with those measured in vitro.

3.4 Discussion As there have been over 30,600 core papers related to the algal structures, comprising over 20% of the papers on the algae as a whole, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts. The primary mode of scientific communication has been articles while reviews have formed 2.9% of the sample. The primary index has been SCI-E indexing more than 99.4% of the papers while only 0.1% of the papers have been indexed by the SSCI and A&HCI focusing on the societal aspects of algal structures. These findings suggest that there is substantial room for the research in social and humanitarian aspects such as policy-related studies as well as scientometric studies in this field. The most-prolific keywords related to the algal structures have been determined through the detailed examination of the over 1900 influential papers with at least 100 citations. A detailed keyword set has been devised for the search (given in Appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the structures have been ‘origin*, evolution*, phyloge*, taxonom*, ancest*, plastid*, *diversity, *flagellar, cilia, tubulin, cell, physiol*, *nitrogen*, temperature, salinity, “reactive oxygen species”, biogeo*, *silica*, composition, circadian’.



The scientometric analysis of the research on the algal structures Chapter | 3  55

Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, phytoplankton, macroalga*, rhodophyt*, seaweed*, bacillariophy*, diatom, diatoms, cyanobacter*’. These keywords have formed the primary research fronts for the algal structures. The findings show that although over 52,500 authors have contributed to the research, 20 most-prolific and influential authors have shaped the literature on the algal structures publishing 3.5% and 20.5% of all the papers and the influential papers, respectively (Table 3.1). The success of these authors, their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988). The data provides the evidence for the presence of the significant gender deficit among both the most-prolific authors (Table  3.1) and the lead authors of the citation classics as only 3 and 13 of these top authors are female, respectively (Table 3.7) (Bordons et al., 2003). The data on the papers by the most-prolific and influential authors highlight the primary research fronts as the ‘flagellar structures’, ‘phylogeny’, ‘evolution’, and ‘silica structures’ of algae. It has been found during the search process that the author names with two or more forenames or surnames have been spelt differently by the databases with significant implications for the recovery of their papers. The classical examples have been ‘SS Merchant’ and ‘J LaRoche’. Similar difficulties have also been observed for the common names such as ‘Wang Y’ or ‘Li Y’ for the recovery of their papers and the related analysis. The data shows that although over 150 countries and territories have contributed to the research in algal structures, mostprolific 20 countries contributed to 106.7% and 136.2% of all the papers and the influential papers, respectively (Table 3.2). The major producers of the research have been the United States, Canada, Australia, Japan, and Europe as these countries have had the ‘first-mover advantage’ over the other countries. It is notable that the citation impact of China has been relatively small in relation to other top producers as China has produced 8.5% and 2.5% of all the papers and influential papers, respectively (Guan and Ma, 2007). Similarly, Japan, Spain, and Russia have also had the reduced citation impact. As in the case of countries, although over 7600 institutions have contributed to the research in algal structures, the 20 most-prolific institutions, having the first-mover advantages, have published more than 23.1% of all the papers and 43.4% of the influential papers (Table 3.3). As only 41.1% and 23.1% of all the papers and influential papers have declared a research funding, respectively, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). It is notable that there has been significant research funding opportunities in China in relation to the United States and Europe. Thus, there is ample room to extend the research funding opportunities to support the research and development in algal structures at the global scale. The steady rise in the number of the publications in this field in the 2000s and 2010s (as of February 2019) provides the strong evidence for the increasing public importance of the algal structures in recent years (Fig. 3.1). The annual number of publications have risen to over 1600 papers and it is expected that the number of papers would continue to rise in the next decade with at least another 30,000 papers, provided that the opportunities for research funding would increase significantly, corresponding to the increasing importance of the algal structures to the global society at large. Although nearly 2000 journals have contributed to the research in algal structures, the 20 most-prolific journals, having the first-mover advantages, have published over 20.6% and 54.2% of all the papers and influential papers, respectively (Table 3.4). This finding has been most relevant for the top journals. The data on the Web of Science subject categories suggests that the first five categories have been the key pillars of the research in algal structures, indexing together 90.1% and 92.8% of all the papers and influential papers, respectively, forming the scientific basis of the research in this field: ‘Marine Freshwater Biology’, ‘Plant Sciences’, ‘Biochemistry Molecular Biology’, ‘Cell Biology’, and ‘Oceanography’ (Table 3.5). As the journals related to algae in the top 20 journal list have published only 8.3% and 8.1% of all the papers and influential papers, respectively, the broad search strategy, covering all subject categories and journals, developed for this study, has been justified. Similarly, ‘Cell Biology’ subject category has indexed only 11.3% and 15.7% of all the papers and influential papers, respectively. The data on the research fronts have confirmed that the major research fronts have been ‘cyanobacterial structures’ and ‘microalgal structures’ (Table 3.6). The key topical research fronts have been ‘phylogeny’ and ‘taxonomy’ of the algae as well as the ‘cell biology’ and ‘physiology’ of algae. The ‘flagellar structures’, ‘silica structures’, ‘circadian clocks’, nitrogen metabolism’, and ‘salt’ and ‘temperature’ response of algae have been other prolific research fronts. The extensive data on the 20 citation classics largely confirm the findings of the earlier sections based on over 30,500 papers (Table 3.7). There has been a significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely over-represented in the citation classic sample as there have been five reviews.

56  PART | II  Algal structures

Similarly, the most-prolific research fronts have been ‘composition’ of algae, ‘flagellar structures’, ‘phylogeny’, ‘evolution’, and ‘silica structures’ of the algae while the most prolific types of algae have been ‘algae’ in general, ‘diatoms’, and ‘microalgae’. It appears that the structure-processing-property relationships form the basis of the research in algal structures as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

3.5 Conclusion This analytical study of the research in algal structures at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as algal research complementing nearly 900 literature reviews. The data has shown that the annual number of papers in this field has risen to over 1600 papers while there have been over 30,500 papers over the study period from 1980 to 2018. It is further expected that the size of the research would exceed 30,000 papers in the next decade, corresponding to the increasing public importance of the algal structures to the global society at large. The provision of the adequate research funding is essential for the further development of the research and practice in this field as only 41.1% and 23.1% of all the papers and influential papers have declared a research funding, respectively. The key research fronts have been ‘cyanobacterial structures’ and ‘microalgal structures’. The key topical research fronts have been ‘phylogeny’ and ‘taxonomy’ of the algae as well as the ‘cell biology’ and ‘physiology’ of algae. The ‘flagellar structures’, ‘silica structures', ‘circadian clocks’, 'nitrogen metabolism’, and ‘salt’ and ‘temperature’ response of algae have been other prolific research fronts. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. It has been found that the detailed keyword set provided in Appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on the major research fronts.

Appendix. The keyword sets A.1. Structures, phylogeny, cell biology-related keywords TI = (origin* or neoproterozoic or evolution* or phyloge* or taxonom* or ancest* or pleistocene or ancient or classification or systematics or barcod* or plastid* or *diversity or *flagellar or flagellum or flagella or cilium or cilia or tubulin or dynein or axonem* or centriole* or organelle* or microtubule* or ‘basal body’ or centrin or ‘radial spoke*’ or swim* or deflagella or gamete* or cell or physiol* or cytoplasm* or clock* or lineage* or circadian or kaic or *nitrogen* or ­‘n-2’ or heterocyst or nitrate or ntca or chilling or temperature or cold or heat or freezing or desiccation or salt or salinity or *osmotic or *volume or riboswitch* or codon or ‘chemical defen*’ or ‘carbonic anhydrase*’ or ‘reactive oxygen species’ or *spore* or morpholog* or biogeo* or *silica* or silicon or frustul*or embryogenesis or spectroscop* or composition or zygote* or morphogenesis or redfield or stoichiometr* or taxa or biomineralization or microscop*) or WC = (‘cell biology’ or physiology) A.2. Algae-related keywords TI = (algae or algal or *phytoplankton* or periphyton* or alga or chlorarachniophy* OR dinoflagellat* or *coccolith* or dinophy* or alexandrium or emiliania or gambier* or *gonyau* or haptophyt* or prorocentr* or prymnesi* or zooxanthella* or amphidin* or akashiwo or isochrysis or karenia* or phaeocystis or symbiodinium or chrysophyt* or chrysophyc* or raphidophy* or ochromonas or pfiesteria or noctiluca* or aureococcus* or *ceratium or *chattonella or cochlodinium or crypthecodinium or gyrodinium or hematodinium or heterocapsa* or heterosigma or karlodinium or lingulodinium or mallomonas or ostreopsis or oxyrrhis or pleurochrysis or pyrocystis or pyrodinium or scrippsiella or rhodomonas or vaucheria or xanthophyc* OR chlamydomon* or ‘green alga*’ or chlorella or microalga* or chlorophyt* or chlorophyc* or euglen* or ‘micro-alga*’ or chrysophy* or dunaliella or haematococcus or nannochloropsis or scenedesmus or cryptophy*



The scientometric analysis of the research on the algal structures Chapter | 3  57

or ­porphyridium or volvoc* or acetabularia or botryococcus or chlorococc* or phormidium or prototheca or tetraselmis or volvox or prasinophy* or cryptomonad* or desmidia* or eustigmatophy* or selenastr* or streptophy* or trebouxiophy* or ankistrodesmus or aurantiochytr* or chroomonas or coccomyxa or cosmarium or cyanidioschyzon or cyanidium or desmodesmus or galdieria or klebsormid* or micrasterias or micromonas or monoraphid* or nannochloris or neochloris or ostreococcus or pediastrum or platymonas or polytomella or *kirchneriella or pyramimonas or schizochytrium OR macroalga* or rhodophy* or seaweed* or ‘red alga*’ or ‘brown alga*’ or fucoid* or gracilar* or kelp* or phaeophy* or porphyra or ulva* or caulerpa* or corallina* or fucus or gigartina* or laminaria* or saccharina or sargassum or nitell* or characea* or charophyt* or dictyota* or enteromorpha or fucale* or halocynthia* or laminarin* or zygnema* or ascophyllum or bangia* or chondrus or cladophor* or codium or cystoseira or ecklonia or gelidium or kappaphycus or laurencia* or macrocystis or ectocarp* or ceramiale* or pyropia* or rhodomela* or spirogyra or undaria or ‘macro-alga*’ or ‘sea-weed*’ or agarophyt* or bryopsidale* or cryptonemia* or florideophy* or gelidiale* or griffithsia or halimeda* lessonia* or rhodymeniale* or sargassac* or ulvophyc* or wakame or bangiophy* or ‘Chara vulgaris’ or asparagopsis or bifurcaria or bostrychia or bryopsis or ceramium or chaetomorpha or chondracanthus or chondria or cladosiphon or delesseria* or desmarestia* or dictyopteris or durvillaea or ‘eisenia bicyclis’ or eucheuma or grateloupia or hizikia or hypnea or ishige or lithophyllum or lobophora or lomentaria or monostroma or mougeotia or oedogonium or padina or palmaria or pelvetia or plocamium or polysiphonia or rhodymenia* or scytosiphon* or solieria* or turbinaria or phyllophora* or charales or streptophyt* or ochrophyt* or halymenia* or bonnemaisonia* or charophyc* or fucacea* OR diatoms or bacillarioph* or diatoma* or diatomite or diatom or thalassiosira* or *nitzschia or phaeodactylum or chaetoceros or navicula or skeletonema or cyclotell* or stephanodisc* or achnanth* or asterionell* or aulacoseira or cocconeis or coscinodisc* or cylindrotheca or cymbella* or didymosphenia or ditylum or eunotia* or fragilaria* or gomphonema* or haslea* or melosira* or rhizosolenia* or stephanodiscus or synedra OR *cyanobact* or *synechoc* or *cylindrospermops* or *microcystis or ‘blue-green alga*’ or *anabaen* or cyanophy* or *nostoc* or *oscillatoria* or spirul* or arthrospira or *lyngbya* or cyanophage* or cyanotox* or aphanizomenon or planktothrix or prochloro* or trichodesmium or calothrix* or chroococca* or cyanelle* or cyanobiont* or acaryochloris or aphanothece or cyanophora or cyanothece or fischerella or fremyella or gloeobacter or mastigocladus or microcoleus or nodularia or plectonema or scytonem* or tolypothrix) OR SO = (‘Algal Research*’ or ‘European Journal of Phycology’ or ‘Harmful Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘British Phycological Journal’ or ‘Diatom Research’ or ‘Phycological Research’ or Algae or ‘Cryptogamie Algologie’ or Fottea*)) A.3. Excluding keywords NOT TI = (shewanella or pelagia or chlorophytum or diatomic* or atom* or *molecule* or polynesia or propanoic or tiahura or sponge or leuconostoc or algas or gaas) NOT WC = (public* or integrative* or neurosci* or oncol* or med* or virol* or anatom* or hematol* or entomol* or ‘cell tissue’ or endocr* or pathol* or veterin* or dermat* or ‘physics atom*’ or ‘chemistry med*’ or food* or pharm* or developmental* or nutr* or immunol* or ‘materials science bio*’ or ‘engineering bio*’ or obstet* or cardiac* or infect* or periph*)

Acknowledgments The significant contribution of the authors of the pioneering studies in algal structures to the development of the research in in this field have been gratefully acknowledged. The authors listed as the ‘most-prolific and influential authors’ in Table 3.1 have published at least 10 influential papers and authors listed as the ‘lead authors’ in Table 3.7 have published at least 5 influential papers in algal structures.

References Abramo, G., D’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25, 704–726. 4B. Armbrust, E.V., Berges, J.A., Bowler, C., Green, B.R., Martinez, D., Putnam, N.H., et al., 2004. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306 (5693), 79–86. Bansard, J.Y., Rebholz-Schuhmann, D., Cameron, G., Clark, D., Van Mulligen, E., Beltrame, F., et al., 2007. Medical informatics and bioinformatics: a bibliometric study. IEEE Trans. Inf. Technol. Biomed. 11 (3), 237–243. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., et al., 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456 (7219), 239–244. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sustain. Energy Rev. 14 (2), 557–577.

58  PART | II  Algal structures

Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Cole, D.G., Diener, D.R., Himelblau, A.L., Beech, P.L., Fuster, J.C., Rosenbaum, J.L., 1998. Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J. Cell Biol. 141 (4), 993–1008. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P., del Borghi, M., 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem. Eng. Process. 48 (6), 1146–1151. Falkowski, P.G., Katz, M.E., Knoll, A.H., Quigg, A., Raven, J.A., Schofield, O., et al., 2004. The evolution of modern eukaryotic phytoplankton. Science 305 (5682), 354–360. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Geider, R.J., La Roche, J., 2002. Redfield revisited: variability of C:N:P in marine microalgae and its biochemical basis. Eur. J. Phycol. 37 (1), 1–17. Guan, J., Ma, N., 2007. China’s emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., Saenger, W., 2009. Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride. Nat. Struct. Mol. Biol. 16 (3), 334–342. Hillebrand, H., Durselen, C.D., Kirschtel, D., Pollingher, U., Zohary, T., 1999. Biovolume calculation for pelagic and benthic microalgae. J. Phycol. 35 (2), 403–424. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol. Adv. 29 (2), 189–198. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books Ltd., Stoke on Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2012a. The policies and practices for the academic assessment of blind students in higher education and professions. Energ. Educ. Sci. Technol. B 4 (si1), 240–244. Konur, O., 2012b. Prof. Dr. Ayhan Demirbas’ scientometric biography. Energ. Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012c. The evaluation of the biogas research: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012d. The evaluation of the bio-oil research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 379–392. Konur, O., 2012e. The evaluation of the biorefinery research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 347–358. Konur, O., 2012f. The evaluation of the research on the biodiesel: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012g. The evaluation of the research on the bioethanol: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012h. The evaluation of the research on the biofuels: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012i. The evaluation of the research on the biohydrogen: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012j. The evaluation of the research on the microbial fuel cells: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012k. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012l. The evaluation of the global energy and fuels research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (1), 613–628. Konur, O., 2012m. 100 Citation classics in energy and fuels. Energ. Educ. Sci. Technol. A 30 (si1), 319–332. Konur, O., 2012n. What have we learned from the citation classics in energy and fuels: a mixed study. Energ. Educ. Sci. Technol. A 30 (si1), 255–268. Konur, O., 2012o. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1893–1908. Konur, O., 2012p. The evaluation of the research on the arts and humanities in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (3), 1603–1618. Konur, O., 2012q. The evaluation of the educational research: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012r. The scientometric evaluation of the research on the deaf students in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1573–1588. Konur, O., 2012s. The scientometric evaluation of the research on the students with ADHD in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2012t. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Energ. Educ. Sci. Technol. B 4 (si1), 250–257. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716.



The scientometric analysis of the research on the algal structures Chapter | 3  59

Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019b. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

60  PART | II  Algal structures

Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020c. 100 Citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Kroger, N., Deutzmann, R., Sumper, M., 1999. Polycationic peptides from diatom biosilica that direct silica nanosphere formation. Science 286 (5442), 1129–1132. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Li, L.L., Ding, G.H., Feng, N., Wang, M.H., Ho, Y.S., 2009. Global stem cell research trend: bibliometric analysis as a tool for mapping of trends from 1991 to 2006. Scientometrics 80 (1), 39–58. Lieberman, M.B., Montgomery, D.B., 1988. First-mover advantages. Strateg. Manage. J. 9 (S1), 41–58. Littler, M.M., Littler, D.S., 1980. The evolution of thallus form and survival strategies in benthic marine macroalgae—field and laboratory tests of a functional form model. Am. Nat. 116 (1), 25–44. Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S., Lins, T., et al., 2002. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. U. S. A. 99 (19), 12246–12251. Menden-Deuer, S., Lessard, E.J., 2000. Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnol. Oceanogr. 45 (3), 569–579. Merchant, S.S., Prochnik, S.E., Vallon, O., Harris, E.H., Karpowicz, S.J., Witman, G.B., et al., 2007. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318 (5848), 245–251. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., et al., 2005. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 308 (5720), 414–415. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Patra, S.K., Mishra, S., 2006. Bibliometric study of bioinformatics literature. Scientometrics 67 (3), 477–489. Pazour, G.J., Dickert, B.L., Vucica, Y., Seeley, E.S., Rosenbaum, J.L., Witman, G.B., et al., 2000. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151 (3), 709–718. Reynolds, C.S., Huszar, V., Kruk, C., Naselli-Flores, L., Melo, S., 2002. Towards a functional classification of the freshwater phytoplankton. J. Plankton Res. 24 (5), 417–428. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes—towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Smetacek, V.S., 1985. Role of sinking in diatom life-history cycles: ecological, evolutionary and geological significance. Mar. Biol. 84 (3), 239–251. Steneck, R.S., Graham, M.H., Bourque, B.J., Corbett, D., Erlandson, J.M., Estes, J.A., et al., 2002. Kelp forest ecosystems: biodiversity, stability, resilience and future. Environ. Conserv. 29 (4), 436–459. Tan, J., Fu, H.Z., Ho, Y.S., 2014. A bibliometric analysis of research on proteomics in Science Citation Index Expanded. Scientometrics 98 (2), 1473–1490. Volkman, J.K., Jeffrey, S.W., Nichols, P.D., Rogers, G.I., Garland, C.D., 1989. Fatty acid and lipid composition of 10 species of microalgae used in mariculture. J. Exp. Mar. Biol. Ecol. 128 (3), 219–240. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718.

Chapter 4

Anatomy of Euglena gracilis Laura Barsanti, Paolo Gualtieri National Council of Research (CNR), Pisa, Italy

4.1  General overview Euglena gracilis (Fig.  4.1) belongs to the phylum Euglenozoa, (class Euglenophyceae, order Euglenales, family Euglenaceae) a prominent group of free-living aquatic flagellates, showing a complex spectrum of nutritional strategies from photoautotrophy to heterotrophy (Barsanti and Gualtieri, 2014; Leander et al., 2017). E. gracilis is an obligate mixotrophic alga, i.e., its primary mode of nutrition is phototrophy, but osmotrophy provides B12 vitamin essential for its growth. The cell, about 50 μm in length and with a diameter of about 10 μm, is anisokont, i.e., it possesses two flagella, dorsal and ventral, different for length and structure. Only the longer (dorsal) emerges from the cell. The diameter of the dorsal flagellum is increased by the paraxonemal rod, attached to the axoneme. As other members of its class, E. gracilis is characterized by a peculiar cell surface architecture called pellicle, which allows the cell to exhibit a variety of motility behavior. Euglena cells have an apical or more frequently sub-apical invagination divided into two distinct but connected regions: the canal and the reservoir. These two regions differ for the surface organization, since the pellicle continue into the canal, whereas the reservoir surface is smooth and lined only by plasma membrane. The photosynthetic machinery of E. gracilis has been acquired via a secondary endosymbiosis involving a prasynophyte green alga; as a consequence, Euglena possesses only chlorophylls a and b and carotenoids and the chloroplasts are surrounded by three membranes, and have no pyrenoids. The mitochondrion is characterized by paddle-shaped cristae. Euglena responds to the direction and intensity of light using a photoreceptor located inside the dorsal flagellum and a shading device named eyespot located inside the cytoplasm at the level of the photoreceptor. The chromosomes retain their condensed condition throughout the interphase, appearing as granular or filamentous threads. A conspicuous nucleolus is always present. Cytokinesis involves a longitudinal cleavage furrow. The main storage polymer is paramylon, a β-1,3-glucan synthesized in granular form inside the cytoplasm.

4.2  Pellicle and metaboly Euglena gracilis, similarly to other members of the class Euglenophyceae, is capable of performing large amplitude coordinate cell body deformations, referred to as metaboly. This peculiar motility depends on the features of the cell membrane complex called pellicle, characterized by a semi-continuous proteic layer made up of overlapping strips (Vismara et al., 2000; Leander et al., 2001). These strips or striae are long ribbons, usually arising in the flagellar canal, which extend from the cell apex to the posterior in a very elastic arrangement. Each strip is curved at both its edges, and in transverse section it shows a notch, an arched or slightly concave ridge, a convex groove and a heel region where adjacent strips interlock and articulate. E. gracilis is considered a highly metabolic species; its pellicle possesses one of the finest striation among Euglena species, the width of the strip (groove to groove distance) being only 240 nm. During metaboly, the pellicle strips slide relative to each other without changing their width or length. The cell shape excursions include peristaltic motion, bending, twisting, rounding and elongating. The ultrastructure of the pellicular complex shows three different structural levels (Fig. 4.2). The first level is the external surface of the cell. It consists of a continuous plasma membrane covering the ridges and grooves all over the cell, coated by a glycoproteic mucous secreted by the mucilage bodies present beneath the strips, arranged in helical rows. These bodies are peripheral compartments of the endoplasmic reticulum and discharge their content to the exterior of the cell via canals, which open into the grooves between the strips (Vismara et al., 2000).

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00004-8 © 2020 Elsevier Inc. All rights reserved.

61

62  PART | II  Algal structures

FIG. 4.1  Optical microscopy image of E. gracilis. ch, chloroplast; e, eyespot; f, flagellum; n, nucleus; ph, photoreceptor; r, reservoir. Bar: 10 μm.

FIG. 4.2  Transmission electron microscopy image of the surface of E. gracilis in transverse section, showing the three different structural levels of the pellicle. Asterisks point to the first level (mucus coating); a square bracket localizes the second level (ridges and grooves); arrowheads point the third level (microtubules). Scale bar 3 μm.

The second level is an electron-opaque peripheral cytoplasmic layer organized in ridges and grooves. It consists of roughly twisted proteic fibers belonging to a family of proteins called ‘articulins’. The overall structure gives the pellicle a very high resistance to tearing forces and higher flexibility, thanks also to the transversal fibers connecting the two longitudinal edges of the ridge of each strip. E. gracilis can resist to a pressure of more than 2000 psi (about 150 bar) before its pellicle breaks. The third level consists of the microtubular system associating and connecting each pellicular strip to the microtubules lining the flagellar canal and the reservoir.

4.3  Flagellar apparatus The flagellar apparatus of Euglena gracilis consists of two flagella of different length and structure (dorsal and ventral) inserted at the bottom of the reservoir onto their corresponding basal bodies, which are located inside the cytoplasm and connected with the flagellar root system (Gualtieri et al., 1990). The dorsal flagellum (Fig. 4.3A) is the locomotory flagellum of the cell, and it is the only emerging from the cell, while the ventral flagellum remains confined in the reservoir region and does not enter the canal. The surface of the dorsal flagellum is extensively coated by a rich assemblage of long and short thin, non-tubular hairs. Long hairs consist of single filament 3–4 μm long, with a diameter of 10 nm, arranged in a single row. Short hairs, precisely positioned, with respect to each other and to axonemal components, consist of a sheath about 240–300 nm in length, formed by loops, side arms and filaments (Fig. 4.3B). Short hairs lie parallel to each other in the longitudinal direction of the flagellum; two groups of short hairs are arranged helically on each narrow side of the flagellum, separated by each other by two membrane areas without hair attachments. In addition to the 9 + 2 axoneme, the dorsal flagellum contains a paraxial or paraflagellar rod (PFR), a complex, highly organized lattice-like structure that runs parallel to the axoneme (Fig. 4.3C). It is located latero-ventrally with respect to the axoneme and the cell body and extends from just above the transition region to the flagellum tip without reaching it. The PFR is a hollow structure with an outer diameter of 90 nm, made up of seven coiled filaments, with a diameter of 22 nm, forming a left-handed helix with a pitch of 45 degrees and a periodicity of 54 nm. A series of goblet-like projections extend from the surface of the rod forming the point of attachment between the rod and one of the axonemal doublet microtubules (Rosati et al., 1991; Verni et al., 1992). The PFR does not assume any consistent orientation with respect to the centralpair microtubules of the dorsal flagellum. Inside the membrane of the dorsal flagellum, connected to the axoneme and the paraxial rod, there is also the photoreceptor, aka flagellar swelling (Fig. 4.1). This organelle is a 3D-ordered assemblage of stacked membranes formed by 2D crystals of the photoreceptive proteins. The ventral flagellum is reduced to a short stub (Gualtieri et al., 1990), and its distal end approaches the locomotory flagellum at the level of the photoreceptor.



Anatomy of Euglena gracilis Chapter | 4  63

FIG. 4.3  (A) Schematic drawing of the locomotory flagellum of E. gracilis in longitudinal section showing its different components. (B) Schematical drawing of the short hairs. (C) Transmission electron microscopy image of the portion of the PFR showing the coiled filaments, scale bar 0.10 μm. (D) Transmission electron microscopy of the type II transition region, scale bar 0.20 μm.

Between the axonemes and the basal bodies there is the so called type II transition region (Fig. 4.3D). This region, devoid of the central microtubular elements, is characterized by a strong dilatation of the nine outer doublets, which leaves room for a fibrillar spiral suspended on the doublets by short spokes. Each outer doublet is associated via other spokes with a thickening of the membrane, whose foldings form a star with nine characteristics arms. The two basal bodies have a cylindrical form, with an average diameter of 0.2 μm, and an average length of 0.5 μm, and are positioned in an almost parallel configuration. The wall of the cylinder is discontinuous, and consists of nine microtubular triplets tilted to the radii at an angle of 130 degrees, and interconnected by transverse desmosomes. In each triplet the complete tubule A consists of 13 protofilaments, whereas the incomplete tubules B and C have 10 protofilaments. The proximal part of the basal body contain a fibro-granular structure termed the cartwheel, composed of a longitudinal central tubule and nine series of spokes joined to the triplets. The flagellar root system consists of three unequal microtubular roots termed dorsal, intermediate, and ventral. The dorsal root is anchored to the dorsal basal body, while the intermediated and ventral are associated with the ventral basal body. These roots play an important role in maintaining the cell shape, since they extend into the cytoplasm from the basal bodies along the cell periphery and toward the nucleus, lining also the reservoir and projecting into the cell, to contact the other organelles.

4.4 Mitochondrion A single mitochondrion per cell appear to be the rule in Euglena gracilis (Vickerman et al., 1991); it is a double-membranebound structure, the inner membrane protruding into the central matrix in the form of paddle-shaped cristae, called ‘discoidal’ cristae, homologous to the discoidal cristae of kinetoplastids (Fig. 4.4). This large reticulated mitochondrial network does not divide into fragments at any stage in the cell cycle, and divides in two only during cytokinesis. The mitochondrion contains DNA nucleoids, about 300 per cell, in form of circular molecules attached to the inner membrane, with a contour length as small as 1 μm. Euglena mitochondrion is interesting for its biochemical features, since this alga belongs to the eukaryotes with facultatively anaerobic mitochondria, i.e., it synthesizes ATP anaerobically in mitochondria (Zimorski et al., 2017). Under aerobic conditions either pyruvate or malate and lactate converted to pyruvate enter the mitochondrion (Zimorski et al., 2017). Pyruvate undergoes oxidative decarboxylation via pyruvate dehydrogenase yielding acetyl-CoA that enters a modified Krebs cycle in which alpha-ketoglutarate decarboxylase and succinate semialdehyde dehydrogenase replace the alpha-ketoglutarate dehydrogenase and succinate thiokinase of the classical Krebs cycle. Electrons are transferred via a branched respiratory chain to the electron sink, i.e., the oxygen, to generate the proton gradient for ATP synthesis.

64  PART | II  Algal structures

FIG. 4.4  Transmission electron microscopy image of a transverse section of E. gracilis showing the mitocondrion, scale bar 1 μm.

Under anaerobic growth condition, E. gracilis uses acetyl-CoA produced by pyruvate-NADP-oxidoreductase as the terminal electron acceptor, leading to the formation of an unusual end product among eukaryotes: wax esters. Mitochondrial wax ester fermentation includes anaerobic fumarate respiration and the same propionyl-CoA formation pathway as that present in mitochondria of animals that possess oxygen-independent energy metabolism in some stages of their lifecycle, such as the oyster Mytilus (Mollusca), the peanut worm Sipunculus (Sipuncula) or the polychaete worm Arenicola (Annelida) and parasites like Fasciola (Platyhelminthes) and Ascaris (Nematoda) (Mentel and Martin, 2010).

4.5 Chloroplasts The chloroplast of Euglena gracilis is surrounded by a 35–45 nm envelope, which consists of three membranes instead of the two present in other taxa. The third membrane represents the remnant of the acquisition of plastids from a prasinophyte green alga by secondary endosymbiosis, i.e., an ancient protozoan host engulfed and surrounded a primary plastid-­ containing endosymbiont (Leander et al., 2017). The inner membrane of the chloroplast traverses the colorless plastid matrix (the stroma), parallel to the plane of the maximum chloroplast cross-section, and forms sacs, about 24 nm thick, with a lumen of about 10 nm, arranged in stacks of three, to form lamellae. The girdle lamella, i.e., a lamella encircling the rim of the chloroplast and generally lying parallel to the chloroplast envelope, is absent in Euglena (Fig. 4.5). The chloroplasts are photosynthetically competent containing all of the pigments and proteins needed for CO2 fixation (Vickerman et al., 1991); they contain also their own nucleic acids and ribosomes. DNA occurs as tiny granules throughout the whole stroma. Chloroplasts are semiautonomous organelles that replicate their own DNA, and this replication is not linked to the division of the organelle. Synthesis of RNA and proteins is possible inside the chloroplasts, though they are not

FIG. 4.5  Transmission electron microscopy image of a chloroplast of E. gracilis in longitudinal section, scale bar 3 μm.



Anatomy of Euglena gracilis Chapter | 4  65

FIG. 4.6  Absorption spectrum of a chloroplast of Euglena gracilis (magenta line); chloroplyll a (dark green line); chlorophyll b (light green line), and carotenoids (orange line).

strictly autonomous from the nuclear genome. The plastid genes are transcribed and translated within the plastids that code for, and synthesize, some proteins of the photosystems, in particular subunits of the photosynthetic enzymes. The missing subunits of these complexes are coded in the nucleus and must be imported from the cytoplasm. Chloroplast development and division (self-replication) is coordinated with that of the cell since upon cell division the chloroplast complement is apportioned approximately equally to the two daughter cells. Once cell division is completed each chloroplast divides to restore the original plastid complement; this division requires a high level of pigment and protein synthesis to maintain photosynthetic competence in the newly divided chloroplasts. Light grown Euglena contains approximately 10 chloroplasts with an extensive thylakoid network. The photosynthetic compartment contains the pigments for absorbing light and channeling the energy of the excited pigment molecules into a series of photochemical and enzymatic reactions. The pigments are organized in proteic complexes embedded in the membrane of the lamellae. Only chlorophylls a and b are present, with echinenone as main accessory carotenoid (Barsanti et al., 2007, Fig. 4.6).

4.6 Nucleus Euglena gracilis is a uninucleate species characterized by an interphase spherical nucleus, about 5 μm in diameter, positioned in the central region of the cell (Fig. 4.7) (Leander et al., 2017). The nucleus is surrounded by a two-membrane envelope, each membrane about 7 nm thick. The envelope is perforated by pores 80–100 nm in diameter regularly disposed at 130 nm interval in a honeycomb hexagonal arrangement (Fig. 4.7, arrowheads). The total number of pores in the entire nucleus varies from 5000 to 8000. The nucleus contains a relatively large central nucleolus (about 0.5 m in diameter) associated with satellite nucleoli, and large amounts of permanently condensed chromatin, since the chromosomes retain their condensed condition throughout the interphase, appearing as granular or filamentous threads. There are 42–45 chromosomes in the nucleus and this high number indicates a possible polyploidy (Barsanti and Gualtieri, 2014). Only asexual reproduction is known in Euglena, with mitosis followed by cytokinesis. The mitosis is closed and characterized by an intranuclear spindle. The onset of division is indicated by movement of the nucleus to the anterior end of the body until it is closely pressed against the reservoir. During this migration, the basal bodies replicate and the two pairs separate across the anterior part of the cell. Prophase begins with the appearance in the nucleus of microtubules oriented across the cell. The nucleolus starts to elongate along the same axis as the nuclear microtubules, while the filamentous chromosomes become more condensed. At metaphase the nucleus is nearly cylindrical and the nucleolus is more drawn out with the two chromatids of the chromosomes oriented mainly along the division figure; there is no distinct equatorial plate. During the early-to-late metaphase, the locomotor apparatus (flagella, photoreceptor, and eyespot) replicates and the

66  PART | II  Algal structures

FIG. 4.7  Transmission electron microscopy image of E. gracilis nucleus, showing the nucleoulus, the satellite nucleoli and the nuclear membrane pores (arrowhead). Scale bar 0.3 μm.

reservoir divides. The daughter reservoirs open into the still single canal, but each now has its own contractile vacuole, eyespot, and flagella. At anaphase the chromatids separate and gradually migrate to the poles. Lastly the nucleolus and then the entire nucleus divide into two parts. Separation, segregation and anaphasic movements of the chromatids are irregular, and this, coupled with a very low chromatid velocity, results in an extremely long anaphase. The end of the anaphase is marked by a sudden flowing of the central region of the elongated nucleoulus to the poles; the persistent nuclear envelope seals around the groups of chromatids and the daughter nucleoli to form the telophase nuclei. Once telophase is established, with separate daughter nuclei, one of the two flagella in each daughter reservoir grows to emerge as a locomotory flagellum. A cleavage line is initiated between the now distinct daughter canals and progresses helically backward to separate the daughter cells (Buetow, 1968; Vickerman et al., 1991). Prior to cytokinesis, the number of pellicle strips around the cell periphery doubles. Each daughter cell (usually) inherits the same number of pellicle strips as the parent cell in a semiconservative manner. During strip doubling, new strips emerge within the articulation zones between mature strips. In the photoautotrophic euglenids, the newly produced pellicle strips do not extend to the posterior tip of the cell and consequently form whorled surface patterns of strip termination.

4.7 Locomotion Euglena gracilis possesses ellipsoid cells about 50 μm long, with a maximum diameter of about 10 μm. It navigates its environment exhibiting two different movements: helical swimming and local spinning (Tsang et al., 2018). Since light intensity determines swimming speed, rolling frequency, and sideways turning, at low light intensities (overcast daylight, or less than 1 W m− 2) cells swim along helical paths, whereas at high light intensities (skylight, or greater than 10 W m− 2) cells spin locally. As already explained Euglena senses the light signals via the photoreceptor located inside the dorsal flagellum and periodically shaded by the eyespot. This signal is converted into different three-dimensional (3D) flagellar beating patterns. These motions change the cell orientation and position in 3D space, which in turn affects the detected light signal. During helical swimming the velocity ranges from about 50 to 100 μm s− 1, and the cells rotate around their long axis anticlockwise at a frequency of about 1–2 Hz. Their dorsal flagellum beats at 20–40 Hz. During swimming this flagellum trails beside the cell body and performs helical waves, generated in a base-to-tip mode, twisting into two loops that are distributed on both sides of the cell. The thrust of the flagellum against the surrounding water is increased by 3–4 μm long hairs arranged in tufts of three to four, forming a single row that runs along the flagellum in a spiral pattern (Fig. 4.3A). The frequencies of body rolling and helical swimming are coupled, identical and phase locked. For high light intensities, when the light sensor saturates, cells spin, i.e., they do not rotate along their long axis but along their short axis in a plane perpendicular to the electrical vector of the light, in either a clockwise or anticlockwise direction. The flagellum twists into one loop extending toward the front of the cell and bends to the side opposite to the turning direction. The rotation is more or less continuous and the cell position is unaffected. For intermediate light intensities a polygonal swimming behaviors emerges from periodic switching



Anatomy of Euglena gracilis Chapter | 4  67

between two beat patterns that resemble those of helical swimming and spinning, which coincide with the swimming and turning phases of the polygon. Hence, the number of spinning beats determines the turning angle and hence the order of the polygon. These behaviors combine to cope with abrupt changes in the light environment. When cells coming from low light encounter a higher light intensity the polygonal swimming or the localized spinning occur, making the cells turn around to avoid the light barrier. When cells previously being at low light, experience a suddenly strong spatially homogeneous light, they initially spin and then swim in polygons of increasing order (eventually intermittently skipping the turning phases) and thereby increase their ‘search radius’ before ultimately escaping into darker regions and switching back to pure helical motion. When cells under low light are suddenly exposed to a spatial light gradient, they alternate between spinning, polygonal swimming and helical swimming, thereby executing a biased random walk down the light gradient in a ‘run-and-tumble’ way.

4.8  Photoreceptor system According to the definition of Gehring (2005), the prototypical eye, i.e., the common ancestor of all eyes, is a combination of a photoreceptor cell and a pigment cell, which achieves some directional selectivity by using screening pigment to block light coming from certain directions. The photoreceptor cell is located close to the effector, and transmits the information conveyed by the light directly to it (without an intervening information processing organelle). Euglena can be considered an example of this prototypical eye, since it possesses all the components described by Gehring (2005). The photoreceptor of Euglena gracilis is located inside the membrane of the dorsal flagellum, connected to its axoneme by the paraxial rod. It consists of a 3D ordered assemblage of about 50 stacked membranes formed by 2D crystals of about 106 photoreceptive proteins. Electron micrograph of negatively stained layers, obtained by ionically induced uncoupling of the 3D compact structure of the photoreceptor, reveals stain excluding units protruding from the surface of the layer, arranged into a regular mesh (Fig. 4.8A). After Fourier analysis (Fig. 4.8B), this mesh shows that the ordered patches are formed by the oligomers of the photoreceptive membrane spanning protein assembled in a hexagonal lattice as visible in the grayscale contour plot (Fig. 4.8C), that is a magnification of the central zone of Fig. 4.8A. The membrane layers of the photoreceptor are characterized by in-plane hydrophobic interactions, while their closely stacked disposition is due to the interlayer interactions between charged protein extramembrane domains and the membrane in adjacent layers through charge density matching (Barsanti et al., 2008). The photoreceptive proteins are characterized by optical bistability, i.e., they possess two isomeric forms A and B, which interconvert along a photocycling path photochemically but not thermically (Barsanti et  al., 1997). The spectral properties of the photoreceptor support the presence of rhodopsin-like proteins. The absorption spectrum of A state has a band centered at 498 nm (from now addressed as A498) (Fig. 4.9, green line); this is the dominant form in the photoreceptor under physiological conditions. The absorption spectrum of B state has a band centered at 462 nm (from now addressed as B462), (Fig. 4.9, blue line). A498 is the non-fluorescent form of the protein, i.e., under physiological conditions the photoreceptor does not fluoresce (Evangelista et al., 2003). B462 is the fluorescent form, energetically lower, which can be considered the signaling state of the protein. The eyespot consists of a loose collection of globules situated on the dorsal side of the reservoir, always in front of the photoreceptor. The pigments present in the eyespot globules are carotenoids such as β-carotene, diatoxanthin, and ­diadinoxanthin

FIG. 4.8  (A) Transmission electron micrograph of a surface of a lamella showing the ordered pattern of protein oligomers of the photoreceptor. Scale bar: 10 nm. (B) Fourier transform of the surface. (C) Grayscale contour plot of the central hexagon of the surface after Fourier filter application.

68  PART | II  Algal structures

FIG. 4.9  Absorption spectrum of the eyespot of Euglena gracilis (orange line); absorption spectra of the A498 isomer (green line); and of B462 isomer (blue line).

(Barsanti et al., 2009). The absorption spectrum of the eyespot shows a unique and large band centered at 460 nm (Fig. 4.9, orange line). The absorption spectrum of the eyespot perfectly matches the absorption spectrum of the B462 form.

4.9 Photoreception Euglena gracilis exhibits several forms of taxis in response to stimuli such as light, gravity, and oxygen. In the following we will focus only on the responses of E. gracilis to light. The cells normally swim by rotating along a helicoidally path; during this motion the photoreceptor proteins are in a photodynamic equilibrium in which A498 is the dominant isomer. The equilibrium is interrupted when the eyespot comes between the incoming light and the photoreceptor, thus screening the organelle (Gualtieri, 1993; Walne et al., 1998; Barsanti et al., 2012). Due to the superimposition of the absorption spectra of the eyespot and the isomer form B462, the photoisomerizable isomeric form A498 undergoes an intramolecular photoswitch, and converts to B462 (Mercatelli et al., 2009). When the photoreceptor is screened by the eyespot a change of the electrostatic field occurs. Since the photoreceptor and the paraxial rod are a structural unit (Verni et al., 1992), a photoelectric signal propagates through the paraxial rod filaments modifying the pitch of its helix, which in turn modifies the distribution of the mass of the rod along the axoneme. This leads to a change in the motion wave running along the flagellum, and eventually to a change in the swimming direction. The generalized receptor law equation by Giometto et al. (2015) can be considered the best model for interpreting the photobehavior of Euglena gracilis and its phototaxis, i.e., a widespread case of directed gradient-driven locomotion. This equation describes the photototactic potential (ϕ(I)) curve varying the light intensity: ϕ(I) = I * (Ic − I)/(Ir + I). These authors investigated Euglena accumulation pattern at 475 and 627 nm only. They determined that the maxim of the ϕ(I) curve was obtained at an intensity of 5.5 W m− 2 at 475 nm, while there was no cell response at 627 nm. The photoresponses of E. gracilis were described more than 100 years ago, but only recently the phototactic ability of this cell was exploited to direct cell migration. Ooka et al. (2014), measured the so called optimal light intensity curve and determined that 530 nm light has the greatest potential for promoting the aggregation of E. gracilis cells, in agreement with the spectroscopic (Gualtieri et al., 1989; Gualtieri, 1991; Barsanti et al., 2009) and biochemical data (Gualtieri et al., 1992; Barsanti et al., 2000b) already cited.

4.10  Practical importance Euglena gracilis can synthetized and store important functional and nutraceutical compounds such as vitamins A, C, and E (Takeyama et al., 1997; Kusmic et al., 1998); polyunsaturated fatty acids (Barsanti et al., 2000a); β-glucans (Barsanti et al., 2001); and wax esters (Dasgupta et al., 2012).



Anatomy of Euglena gracilis Chapter | 4  69

FIG. 4.10  Optical microscopy image of the non-photosynthetic WSLZ mutant of E. gracilis showing the paramylon granules filling up the cell body.

The key product of Euglena, currently under investigation worldwide for its immunopotentiating and immunostimulanting activities, is the reserve polysaccharide β-glucan aka Paramylon (Vismara et al., 2004; Russo et al., 2017; Scartazza et al., 2017; Kusmic et al., 2018; Barsanti et al., 2019; Barsanti and Gualtieri, 2019). Paramylon is already present in many over-the-counter products containing on E. gracilis and sold worldwide; companies such as Algaeon (www.algaeon-inc. com) and Euglena (www.euglena.jp) commercialize products containing Euglena gracilis for health and personal care (vitamins, PUFA, and β-glucans) or biofuel (wax esters). Euglena gracilis WT stores paramylon as large and small ellipsoid granules less than twice as long as wide, ranging from 1 to 2 μm and composed of concentric segments of unbranched linear β-(1,3)-d-glucan chains. The active form of paramylon consists of linear glucan nanofibrils of 4–10 nm, capable of interacting with the Dectin receptors present on the membranes of the immune system cells (Barsanti et al., 2011). To guaranty high commercial yield and purity necessary to obtain nanofibrils without any other cell components contamination, the non-photosynthetic WSLZ mutant of Euglena gracilis is used (Rosati et  al., 1996), since this cell can accumulate paramylon granules up to 95% of the cell dry weight (Fig. 4.10). For these characteristics, the WSLZ mutant can be considered the main alternative source of β-glucan to Saccharomyces cerevisiae (baker’s yeast) and other algae and bacteria used worldwide. Euglena gracilis can grow autotrophically or heterotrophically under many different conditions and carbon sources, tolerating a broad range of pH, and high concentrations of inorganic contaminant such as heavy metals and high nutrient discharge such as agriculture runoff and dairy wastewater.

References Barsanti, L., Gualtieri, P., 2014. Algae: Anatomy, Biochemistry, and Biotechnology, second ed. CRC Press, Boca Raton, FL. Barsanti, L., Gualtieri, P., 2019. Paramylon, a potent immunomodulator frm WZSL mutant of Euglena gracilis. Molecules 24, 3114–3130. https://doi. org/10.3390/molecules24173114. Barsanti, L., Passarelli, V., Walne, P.L., Gualtieri, P., 1997. In vivo photocycle of the Euglena gracilis photoreceptor. Biophys. J. 72 (2), 545–553. Barsanti, L., Bastianini, A., Passarelli, V., Tredici, M.R., Gualtieri, P., 2000a. Fatty acid content in wild type and WZSL mutant of Euglena gracilis. J. Appl. Phycol. 12 (3–5), 515–520. Barsanti, L., Passarelli, V., Walne, P.L., Gualtieri, P., 2000b. The photoreceptor protein of Euglena gracilis. FEBS Lett. 482 (3), 247–251. Barsanti, L., Vismara, R., Passarelli, V., Gualtieri, P., 2001. Paramylon (β-1,3-glucan) content in wild type and WZSL mutant of Euglena gracilis. Effects of growth conditions. J. Appl. Phycol. 13 (1), 59–65. Barsanti, L., Evangelista, V., Frassanito, A.M., Vesentini, N., Passarelli, V., Gualtieri, P., 2007. Absorption microspectroscopy, theory and applications in the case of the photosynthetic compartment. Micron 38 (3), 197–213. Barsanti, L., Coltelli, P., Evangelista, V., Passarelli, V., Frassanito, A.M., Gualtieri, P., Vesentini, N., et al., 2008. Low-resolution characterization of the 3D structure of the Euglena gracilis photoreceptor. Biochem. Biophys. Res. Commun. 375 (3), 471–476. Barsanti, L., Coltelli, P., Evangelista, V., Passarelli, V., Frassanito, A.M., Gualtieri, P., Vesentini, N., et al., 2009. In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle. Photochem. Photobiol. 85 (1), 304–312. Barsanti, L., Passarelli, V., Evangelista, V., Frassanito, A.M., Gualtieri, P., 2011. Chemistry, physico-chemistry and applications linked to biological activities of β-glucans. Nat. Prod. Rep. 28 (3), 457–466.

70  PART | II  Algal structures

Barsanti, L., Evangelista, V., Passarelli, V., Frassanito, A.M., Gualtieri, P., 2012. Fundamental questions and concepts about photoreception and the case of Euglena gracilis. Integr. Biol. 4 (1), 22–36. Barsanti, L., Coltelli, P., Gualtieri, P., 2019. Paramylon treatment improves quality profile and drought resistance in Solanum lycopersicum L. cv. MicroTom. Agronomy 9, 394–410. Buetow, D.E., 1968. The Biology of Euglena. Volume I. General Biology and Ultrastructure. Academic Press, N.Y. Dasgupta, S., Fang, J.S., Brake, S.S., Hasiotis, S.T., Zhang, L., 2012. Biosynthesis of sterols and wax esters by Euglena of acid mine drainage biofilms: implications for eukaryotic evolution and the early Earth. Chem. Geol. 306-307, 139–145. Evangelista, V., Passarelli, V., Barsanti, L., Gualtieri, P., 2003. Fluorescence behavior of Euglena photoreceptor. Photochem. Photobiol. 78 (1), 93–97. Gehring, W.J., 2005. New perspectives on eye development and the evolution of eyes and photoreceptors. J. Hered. 96 (3), 171–184. Giometto, A., Altermatt, F., Maritan, A., Stocker, R., Rinaldo, A., 2015. Generalized receptor law governs phototaxis in the phytoplankton Euglena gracilis. Proc. Natl. Acad. Sci. U. S. A. 112 (22), 7045–7050. Gualtieri, P., 1991. Microspectroscopy of photoreceptor pigments in flagellated algae. Crit. Rev. Plant Sci. 9 (6), 474–495. Gualtieri, P., 1993. Euglena gracilis: is the photoreception enigma solved? J. Photochem. Photobiol. B 19 (1), 3–14. Gualtieri, P., Barsanti, L., Passarelli, V., 1989. Absorption spectrum of a single isolated paraflagellar swelling of Euglena gracilis. BBA-Gen. Subjects 993 (2–3), 293–296. Gualtieri, P., Barsanti, L., Passarelli, V., Verni, F., Rosati, G., 1990. A look into the reservoir of Euglena gracilis: SEM investigation of the flagellar apparatus. Micron Microsc. Acta 21 (3), 131–138. Gualtieri, P., Pelosi, P., Passarelli, V., Barsanti, L., 1992. Identification of a rhodopsin photoreceptor in Euglena gracilis. BBA-Gen. Subjects 1117 (1), 55–59. Kusmic, C., Barsacchi, L., Barsanti, L., Gualtieri, P., Passarelli, V., 1998. Euglena gracilis as source of the antioxidant vitamin E. Effects of culture conditions in the wild strain and in the natural mutant WZSL. J. Appl. Phycol. 10 (6), 555–559. Kusmic, C., Barsanti, L., di Lascio, N., Faita, F., Evangelista, V., Gualtieri, P., 2018. Anti-fibrotic effect of paramylon nanofibers from the WZSL mutant of Euglena gracilis on liver damage induced by CCl4 in mice. J. Funct. Foods 46, 538–545. Leander, B.S., Witek, R.P., Farmer, M.A., 2001. Trends in the evolution of the euglenid pellicle. Evolution 55 (11), 2115–2135. Leander, B.S., Lax, G., Karnkowska, A., Simpson, A.G.B., 2017. Euglenida. In: Archibald, J.M., Simpson, A.G.B., Slamovits, C.H., Margulis, L., Melkonian, M., Chapman, D.J., et al. (Eds.), Handbook of the Protists. Springer, Cham, pp. 1–42. Mentel, M., Martin, W., 2010. Anaerobic animals from an ancient, anoxic ecological niche. BMC Biol. 8, 32. Mercatelli, R., Quercioli, F., Barsanti, L., Evangelista, V., Coltelli, P., Gualtieri, P., Passarelli, V., et al., 2009. Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photoreceptive processes: the case of Euglena gracilis. Biochem. Biophys. Res. Commun. 385 (2), 176–180. Ooka, H., Ishii, T., Hashimoto, K., Nakamura, R., 2014. Light-induced cell aggregation of Euglena gracilis towards economically feasible biofuel production. RSC Adv. 4 (40), 20693–20698. Rosati, G., Verni, F., Barsanti, L., Passarelli, V., Gualtieri, P., 1991. Ultrastructure of the apical zone of Euglena gracilis: photoreceptors and motor apparatus. Electron Microsc. Rev. 4 (2), 319–342. Rosati, G., Barsanti, L., Passarelli, V., Giambelluca, A., Gualtieri, P., 1996. Ultrastructure of a novel non-photosynthetic Euglena mutant. Micron 27 (5), 367–373. Russo, R., Barsanti, L., Evangelista, V., Frassanito, A.M., Longo, V., Gualtieri, P., Pucci, L., et al., 2017. Euglena gracilis paramylon activates human lymphocytes by upregulating pro-inflammatory factors. Food Sci. Nutr. 5 (2), 205–214. Scartazza, A., Picciarelli, P., Mariotti, L., Curadi, M., Barsanti, L., Gualtieri, P., 2017. The role of Euglena gracilis paramylon in modulating xylem hormone levels, photosynthesis and water-use efficiency in Solanum lycopersicum L. Physiol. Plant. 161 (4), 486–501. Takeyama, H., Kanamaru, A., Yoshino, Y., Kakuta, H., Kawamura, Y., Matsunaga, T., 1997. Production of antioxidant vitamins β-carotene, vitamin C, and vitamin E, by two-step culture of Euglena gracilis Z. Biotechnol. Bioeng. 53 (2), 185–190. Tsang, A.C.H., Lam, A.T., Riedel-Kruse, I.H., 2018. Polygonal motion and adaptable phototaxis via flagellar beat switching in the microswimmer Euglena gracilis. Nat. Phys. 14 (12), 1216–1222. Verni, F., Rosati, G., Lenzi, P., Barsanti, L., Passarelli, V., Gualtieri, P., 1992. Morphological relationship between paraflagellar swelling and paraxial rod in Euglena gracilis. Micron Microsc. Acta 23 (1–2), 37–44. Vickerman, K., Brugerolle, G., Mignot, J.P., 1991. Mastigophora. In: Harrison, F.W., Corliss, J.O. (Eds.), Microscopic Anatomy of Invertebrates. Vol. 1: Protozoa. Wiley-Liss, New York, pp. 13–159. Vismara, R., Barsanti, L., Lupetti, P., Passarelli, V., Mercati, D., Gualtieri, P., Dallai, R., et al., 2000. Ultrastructure of the pellicle of Euglena gracilis. Tissue Cell 32 (6), 451–456. Vismara, R., Vestri, S., Frassanito, A.M., Barsanti, L., Gualtieri, P., 2004. Stress resistance induced by paramylon treatment in Artemia sp. J. Appl. Phycol. 16 (1), 61–67. Walne, P.L., Passarelli, V., Barsanti, L., Gualtieri, P., 1998. Rhodopsin: a photopigment for phototaxis in Euglena gracilis. Crit. Rev. Plant Sci. 17 (5), 559–572. Zimorski, V., Rauch, C., van Hellemond, J.J., Tielens, A.G.M., Martin, W.F., 2017. The mitochondrion of Euglena gracilis. In: Schwartzbach, S.D., Shigeoka, S. (Eds.), Euglena: Biochemistry, Cell and Molecular Biology. Springer, Cham, pp. 19–37.

Chapter 5

Biology and ecology of Northwest Atlantic seaweeds Arthur C. Mathieson, Clinton J. Dawes University of New Hampshire, Durham, NH, United States

5.1 Introduction During the past several decades a variety of floristic, phenological and biogeograhical investigations of seaweeds from the Northwest Atlantic (i.e., Eastern Canadian Arctic to Maryland) have been conducted by phycologists at the University of New Hampshire (Durham, NH, USA), with particular emphasis upon shorelines in New Hampshire and southern Maine (Figs. 5.1 and 5.2). Such information should be increasingly important in the future, because of the many conflicting needs and uses that may diminish and/or alter these valuable resources and habitats, including the potential impacts of global warming (Barnhardt et al., 1995; Walsh, 2012). In the present synopsis we include information regarding Northwest Atlantic seaweed populations, with particular emphasis on New England and the Canadian Maritime Provinces. Comparative information for some other geographic areas is also given, including the Northeast Atlantic that has strong floristic affinities and evolutionary histories. South (1987) suggests that a close relationship exists between the Arctic, Northwest and Northeast Atlantic floras and that a single flora existed during the early Oligocene.

5.2  General seaweed biology 5.2.1  What are algae? Trainor (1978) defined algae as photosynthetic nonvascular plants that contain chlorophyll “a” and have simple reproductive structures. Patterson (2000) considered seaweeds or benthic macroscopic marine algae (singular = alga) to be protists and not quite plants or animals. Algae lack true roots, stems, leaves, and the vascular tissue (xylem and phloem) found in flowering plants. Unlike mosses and vascular plants, algae have sex organs that are usually one-celled; if multicellular, all cells function in reproduction. Balduf (2003) recommended that the phyla Chlorophyta (green algae) and Rhodophyta (red algae) be placed in the Kingdom Plantae and the brown algae (class Phaeophyceae) in the Kingdom Chromista and Phylum Ochrophyta. Presently the estimated global numbers of marine red, brown, and green algal species are 8000, 1972, and 13,000 (Guiry and Guiry, 2017).

5.2.2  Types of seaweeds and their habitats Seaweeds exhibit diverse morphologies and affinities (Mathieson and Dawes, 2017). They may be lithophytic, psammophytic, epiphytic, endophytic, or parasitic. In the Northwest Atlantic many seaweeds (e.g., Laminaria digitata) grow attached to hard substrata (rocks, outcrops, shells) and are lithophytic. Kelps (brown seaweeds) and Codium (a green seaweed), if attached to mussels or shells, can become dislodged and thrown ashore on beaches (Witman, 1987). Psammophytic seaweeds such as the “wire-weed” red alga Ahnfeltia plicata and the multiseriate filamentous brown alga Protohaloperis radicans grow on/in unconsolidated sediments or on rocky substrata impacted by sand scouring. In tropical areas psammophytic species are more common than in temperate or boreal areas. Excessive epiphytic/epizoic biomass may render host seaweeds vulnerable to drag (Wahl, 1997), as in the case of blue mussels (Mytilus edulis) that seek refuge from predation by recruiting onto the holdfasts of the sugar kelp Saccharina latissima. A few red seaweeds, such as Bostrychia radicans,

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00005-X © 2020 Elsevier Inc. All rights reserved.

71

72  PART | II  Algal structures

71° 40¢

71° 30¢ Kennebunk

Cape Elizabeth (7 miles ) Cape Arundel

N

NH

MAINE Cape Neddick

43° 10¢

York River Pi

sc a Ri taq ve ua r

Portsmouth

Great Bay Estuary System Isles of Shoals

NH Open Coast (Nearshore)

Seabrook

Gulf of Maine

Hampton-Seabrook Estuary System

MASS Salisbury

42° 50¢

Merrimack River

Plum I 1 mile Cape Ann FIG. 5.1  The New England coastline between Cape Arundel, Maine and Cape Ann, Massachusetts, including four major coastal-estuarine area in New Hampshire, the Isles of Shoals, Great Bay Estuary System, NH open coast (nearshore), and the Hampton-Seabrook Estuary System (Mathieson, 1989).

Caloglossa leprieurii, and Polysiphonia subtilissima, colonize mudflats and marshes, while Sargassum fluitans and S. ­natans are only found detached and floating. Epiphytic algae (e.g., Ulva, Ectocarpus, and Ceramium) grow on other algae and seagrasses and are often most abundant during late summer (Brady-Campbell et al., 1984). Endophytic algae grow within various seaweed hosts (e.g., Sphaceloderma caespitula within Chondrus crispus). Some brown algal endophytes may be gametophytes of other macroalgae (Hubbard et al., 2004; Peters, 2003) or independent species. Parasitic seaweeds, including members of the Chlorophyceae, Phaeophyceae, and Rhodophyceae, may cause etiolation, hyperplasia, and necrosis of various hosts (Apt, 1988). Several parasitic red algae are alloparasites and not closely related to their host. For example, the red alga Vertebrata lanosa grows abundantly on the brown alga Ascophyllum nodosum (Fig. 5.3), and it has been described as an obligate epiphyte (Rindi and Guiry, 2004), a hemiparasite or partial parasite (Ciciotte and Thomas, 1997; Garbary et al., 2005), or a true parasite (Penot et al., 1993). The rhizoids of V. lanosa can digest host cells and they have protoplasmic connections with Ascophyllum cells (Levin and Mathieson, 1991; Rawlence, 1972; Rawlence and Taylor, 1972). Parasitisum in advanced red algae occurs in ca. 8% of the described genera; it apparently evolved independently several times (Blouin and Lane, 2012). The majority of red algal parasites are adelphoparasites that

Biology and ecology of Northwest Atlantic seaweeds Chapter | 5  73



Isles of Shoals

Maine

Duck Dover Pt. 4300

43° 05¢ Great Bay

Great Bay Estuary System

Isles of Shoals

New Hampshire

Atlantic Ocean

42° 55¢ Hampton Seabrook Estuary System 70° 55¢

Appledore

Maine

Jaffrey Pt.

Smuttynose Malaga

N.H.

Gosport Hbr. Cedar Lunging

White 70 38

Star

42 58 70 36

1 Mile

70° 45¢

FIG. 5.2  The New Hampshire coastal zone, including the nearshore open coast, the Great Bay Estuary System, the Hampton-Seabrook Estuary System, and the Isles of Shoals. The Shoals archipelago occurs within New Hampshire and Maine and consists of eight islands (see enlargement on right) (Mathieson, 1989).

share a recent common ancestor with their free-living host species (Hancock and Lane, 2010). Clement et al. (2014) note that red algae are prone to parasitism from congeners, seemingly due to their characteristic pit plugs by which parasites deposit their cellular components into the host. Goff (1997) states that some parasites can inject nuclei into their host and genetically transform them into a parasite. Salomaki and Lane (2014) found that red algal parasites often do not maintain their own plastid and instead hijack and maintain a photosynthetically inactive plastid from their host. In typical eukaryotic parasites non-essential genes are lost, as they rely on their host for energy and nutrition. As noted by Hull (1997), the complexity of algal habitats is due to small-scale variations in shape, size, and texture (Gee and Warwick, 1994). Higher population densities of macro- and meio-fauna are often correlated with enhanced morphological features of algae (Hicks, 1980). Thus, increased living space may be available for other organisms, as well as shelter from predation (Coull and Wells, 1983), protection from desiccation and wave action (Whatley and Wall, 1975), and differential food availability and sediment load (Hicks, 1980).

5.2.3  Anatomy and morphology Morphological and anatomical features are useful in identifying seaweeds (Mathieson and Dawes, 2017), including thallus construction, branching patterns, presence of specialized cells, modes of growth (apical, intercalary, or diffuse), and reproductive features. In addition, the types of pigments, reserve products, cell wall chemistry, and molecular characteristic are also important. Seaweed morphologies include: (1) uniseriate filaments consisting of a single row of cells that are unbranched (Chaetomorpha spp.) or branched (Cladophora spp.); (2) filaments with two (Percursaria) or more rows of cells (some tubular Ulva spp.); (3) intertwined filaments that form siphonous axes (Codium spp.) or multicellular filaments (Chordaria flagelliformis); (4) solid (Hypnea musciformis) or hollow axes (Champia farlowii); and (5) cylindrical (Chorda filum) or foliose to blade-like algae (Ulva lactuca). Cylindrical or bladed species may have a dense central filamentous medulla (Laminaria and Saccharina), a pseudoparenchymatous core with spherical or cube-like cells (Hummia onusta), or

74  PART | II  Algal structures

FIG. 5.3  Morphological and reproductive variability of Ascophyllum nodosum populations growing within different habitats in southern Maine and New Hampshire, USA. (A) Stunted fertile frond from the exposed eastern shoreline of Smuttynose Island, Isle of Shoals, Maine; (B,C) stunted and highly parasitized fronds of A. nodosum with abundant parasitic Vertebrata lanosa from a semi-exposed open coastal site at Fort Stark, Newcastle, New Hampshire; (D) portion of a fertile non-parasitized frond from a semi-exposed open coastal site at Fort Stark, New Hampshire; (E) portion of a fertile frond from an inner estuarine site within Great Bay, New Hampshire; (F) a fertile terete fragment from Cedar Point, Little Bay, NH (spring): note initiation of some proliferous branches; (G) A. nodosum f. scorpioides collected at Cedar Point, Little Bay, NH (summer). Scalar 3 cm (Mathieson and Guo, 1992).

r­emain mostly hollow with a few central medullary filaments (Agardhiella subulata). Axes can also be parenchymatous, with closely compacted isodiametric cells (Pogotrichum filiforme). Brown algae that form fleshy crusts are often overlooked (Davis and Wilce, 1987) such as those at Halibut Point, MA, which have 14 calcareous and fleshy crustose species. Sears and Wilce (1975) described these cobble communities as modified turfs with closely adhering upright filaments. Crusts are tolerant of strong wave action and movement of loose cobbles (Davis and Wilce, 1987). Some red algae, mostly in the order Corallinales, contain calcium carbonate (calcite; CaCO3) and either form crusts or erect axes that are rigid. Variable growth patterns may occur in seaweeds (Mathieson and Dawes, 2017). Apical growth can occur by division of a single cell (Sphacelaria, Polysiphonia) or by a group of apical cells (Grateloupia). Growth by cell division may be diffuse and often near the bases of branches (Pylaiella), or it is restricted to an intercalary meristem (Laminaria). Trichothallic division is a form of intercalary growth where cell divisions produce apical hairs or filaments in addition to new branch tissue (Hincksia). Hairs may be ephemeral or long lasting.



Biology and ecology of Northwest Atlantic seaweeds Chapter | 5  75

The size and morphology of many algal species can be altered in unique habitats (Morales et al., 1998). Seaweeds growing in protected regions such as the Bay of Fundy, NS (Edelstein et al., 1970), Passamaquoddy Bay, NB (South et al., 1988), and Cobscook Bay, ME (Mathieson et al., 2010) tend to be larger than those on contiguous exposed open coasts. Saccharina latissima has pronounced morphological variability, ranging from broad to narrow fronds on the open coast, to narrow and elongated fronds in reversing fall sites near the Bay of Fundy (Fig. 5.4A–G). Recently, another very narrow and elongated taxon S. angustissimum (Fig. 5.4H–J) was genetically delineated (Augyte et al., 2018); it is endemic to mid-coastal Maine and restricted to very exposed low intertidal habitats (Mathieson and Dawes, 2017; Mathieson et al., 2008a). Some of the longest fronds of the sugar kelp Saccharina latissimia occur within Cobscook Bay and are 3–5 m long (Fig. 5.4E–G). South et al. (1988) noted that this kelp had very delicate blades in sheltered subtidal sites in Passamaquoddy Bay. In the Gulf of Maine most kelps are 1.75 m/s), particularly within the ebb dominated parts of the channel (i.e., pilings 4–8) versus more quiescent flood areas (i.e., pilings 1–3).

6.7  Ice damage Fig. 6.7 illustrates the effects of ice damage on estuarine fucoid algae during a relatively mild winter (1987–1988) like those common during the 1990s and 2000s. A wide range of biomass values were documented in 10 representative intertidal blocks, which were thawed and analyzed; these ranged from ~0.1 to 1.8 g dry weight/m2 with a mean of 3.2 g/m2. Most ice-rafted material was Ascophyllum nodosum (88%), with smaller amounts of A. nodosum f. s­ corpioides, Fucus

Average length of fronds (cm)

98  PART | II  Algal structures

45 Ascophyllum

35 25 15

Fucus 5

Velocity (m sec–1)

2.5 2.0 13 hours 1.5 1.0 0.5

1

2

3

4

5

6

7

8

Pilings FIG. 6.7  A synopsis of biomass and species composition from thawed estuarine ice-rafted material in ice blocks numbered 1–10 from Great Bay, New Hampshire during a relatively mild winter of 1987–1988 (Mathieson, 1989).

vesiculosus, Spartina alterniflora and terrestrial remnants of leaves and twigs. Periodic freezing and thawing during such mild winters can cause substantial pruning and morphological damage to A. nodosum (Chock and Mathieson, 1976; Hardwick-Witman, 1985; Mathieson et al., 1982). In addition, Ascophyllum fragments rafted in ice and subsequently deposited into salt marshes are a major source of A. n. f. scorpioides (Chock and Mathieson, 1976, 1983). In comparing the extreme winter of 1980–1981 with the mild winter of 1987–1988, approximately 300 times more A. nodosum was ice-rafted (i.e., 1.0 kg dry weight/m2) during the earlier period. In addition, the former event caused the demise of nearly 50% of the alga’s large fall standing crop of approximately 136 tons dry weight. As noted previously much of this material enters the detrital pool and is of major significance to estuarine productivity (Josselyn and Mathieson, 1980; Norton and Mathieson, 1983).

6.8  Functional roles of fucoids in estuarine habitats If detached populations of Ascophyllum nodosum and Fucus spp. are abundant in salt marsh habitats they can contribute 1–3 times as much detrital material as vascular plants and 50 times more than other open coastal seaweeds (Brinkhuis, 1976, 1977; Chock and Mathieson, 1983; Josselyn and Mathieson, 1980; Mathieson and Dawes, 2001). In addition, fucoid algae decompose 3–10 times faster than salt marsh vascular plants, releasing nitrogen and phosphorus. Gerard (1999) found that removal of A. nodosum f. scorpioides from a marsh on Long Island, NY caused a significant reduction of vascular plant biomass, presumably due to a loss of fucoid tissue nutrients.

Autecology of Northwest Atlantic fucoid algae Chapter | 6  99



Zn

102 101

ppm Cd

a

18 16 14 12 10 8 6 4 2 0

a

Dyer

Goose

Falls

ppm Cu

103

Cu

200 150 100 50 0

Clean

Cd

Dyer

Goose

a

50

a

a

Falls

Clean

b

b

Falls

Clean

Pb

40 ppm Pb

ppm Zn

104

Dyer

Goose

a

a

Falls

Clean

30 20

ab

10 0

Dyer

Goose

FIG. 6.8  Seasonal variation of (A) dissolved inorganic N (μg-at/L) and Ascophyllum nodosum tissue composition; (B–D) expressed as ash-free dry weight and based upon the mean values ± SE from seven estuarine study sites within the Great Bay Estuary System, NH/ME; (B) % N; (C)% C; (D) C/N values; (E) % ash (Hardwick-Witman and Mathieson, 1986).

Fig. 6.8 summarizes the mean monthly tissue nitrogen and carbon variations in estuarine population of Ascophyllum nodosum from the Great Bay Estuary System, NH/MA (Hardwick-Witman and Mathieson, 1986). Overall, tissue N followed the cycle of ambient N, with the lowest tissue N (1.8%) occurring in July and highest values in February (3.6%). Tissue N levels remained high in spring, while ambient water N declined rapidly. The period of rapid ambient nutrient depletion represented a period of maximum growth for A. nodosum, while minimal growth occurred in the winter when nitrogenous nutrients were maximal (cf. David, 1943; Mathieson et al., 1976; Vadas et al., 1976). Although tissue N in A. nodosum generally coincides with ambient dissolved nitrogen content, growth does not. Kornfeldt (1982) emphasized that several perennial seaweeds accumulate nitrates to sustain growth when ambient nutrients were low (cf. Black and Dewar, 1949; Chapman et al., 1978; Wallentinus, 1979). Thus, there can be a lag in the decrease of tissue N as compared with ambient N in seawater. Seasonal values for tissue carbon showed no conspicuous seasonal trend, with values only varying from 44.6% to 48.9%. The C/N ratios were highest in summer and early fall, and they declined to low values in the winter and early spring; these ratios primarily reflected variations in tissue N, since the % C was relatively constant. No seasonal pattern in percent ash was evident and the range of mean ash content was  0.05) (Medeiros et al., 2017).

Acknowledgments We thank several colleagues and former students (undergraduate and graduate) at the University of New Hampshire for their help with various field investigations. Our studies were supported by funds from Maine-New Hampshire Sea Grant (NOAA), the New Hampshire Agricultural Experiment Station, and the Leslie Hubbard Marine Endowment Fund. The paper is published as Jackson Estuarine Laboratory Contribution Number 571. Lastly we acknowledge the encouragement and help of our wives, Myla Mathieson and Kathy Dawes, who were involved with several of the field studies, particularly those in Maine.

References Baardseth, E., 1970. Synopsis of Biological Data on Knobbed Wrack Ascophyllum nodosum (L.) Le Jolis. Food and Agriculture Organization, Rome. Black, W.A.P., Dewar, E.T., 1949. Correlation of some of the physical and chemical properties of the sea with the chemical constitution of the algae. J. Mar. Biol. Assoc. UK 28 (3), 673–699.



Autecology of Northwest Atlantic fucoid algae Chapter | 6  101

Brinkhuis, B.H., 1976. The ecology of temperate salt-marsh fucoids. I. Occurrence and distribution of Ascophyllum nodosum ecads. Mar. Biol. 34 (4), 325–338. Brinkhuis, B.H., 1977. Comparisons of salt-marsh fucoid production estimated from three different indices. J. Phycol. 13 (4), 328–335. Chapman, A.R.O., 1995. Functional ecology of fucoid algae: twenty-three years of progress. Phycologia 34 (1), 1–32. Chapman, A.R.O., Markham, J.W., Luning, K., 1978. Effects of nitrate concentration on the growth and physiology of Laminaria saccharina (Phaeophyta) in culture. J. Phycol. 14 (2), 195–198. Chock, J.S., Mathieson, A.C., 1976. Ecological studies of the salt marsh ecad scorpioides (Hornemann) Hauck of Ascophyllum nodosum (L.) Le Jolis. J. Exp. Mar. Biol. Ecol. 23 (2), 171–190. Chock, J.S., Mathieson, A.C., 1983. Variations of New England estuarine seaweed biomass. Bot. Mar. 26 (2), 87–97. David, H.M., 1943. Studies in the autecology of Ascophyllum nodosum Le Jol. J. Ecol. 31 (2), 178–199. Dawes, C.J., 1998. Marine Botany, second ed. John Wiley and Sons, Chichester. Edelstein, T., McLachlan, J., 1975. Autecology of Fucus distichus spp. distichus (Phaeophyceae, Fucales) in Nova Scotia, Canada. Mar. Biol. 30 (49), 305–324. Fritsch, F.E., 1959. The Structure and Reproduction of the Algae, Volume 2. Foreword, Phaeophyceae, Rhodophyceae, Myxophyceae. Cambridge Univ. Press, Cambridge. Gerard, V.A., 1999. Positive interactions between cordgrass, Spartina alterniflora and the brown alga, Ascophyllum nodosum ecad scorpioides, in a midAtlantic coast salt marsh. J. Exp. Mar. Biol. Ecol. 239 (1), 157–164. Gibb, D.C., 1957. The free-living forms of Ascophyllum nodosum (L.) Le Jol. J. Ecol. 45 (1), 49–83. Hallfors, G., 1976. The plant cover of some littoral biotopes at Krunnit (NE Bothnian Bay). Acta Univ. Oul. A 42, 87–95. Hallfors, G., Kangas, P., Lappalainen, A., 1975. Littoral benthos of the northern Baltic Sea. III. Macrobenthos of the hydrolittoral belt of filamentous algae on rocky shores in Tvarminne. Int. Rev. Hydrobiol. 60 (3), 313–333. Hallfors, G., Niemi, A., Ackefors, H., Lassig, J., Leppakoski, E., 1981. Biological oceanography. Elsevier Oceanogr. Ser. 30, 219–274. Hardwick-Witman, M.N., 1985. Biological consequences of ice rafting in a New England salt marsh community. J. Exp. Mar. Biol. Ecol. 87 (3), 283–298. Hardwick-Witman, M.N., Mathieson, A.C., 1986. Tissue nitrogen and carbon variations in New England estuarine Ascophyllum nodosum (L.) Le Jolis populations (Fucales, Phaeophyta). Estuaries 9 (1), 43–48. Haupt, A.W., 1953. Plant Morphology. McGraw Hill, New York, NY. Josselyn, M.N., Mathieson, A.C., 1978. Contributions of receptacles from the fucoid Ascopyllum nodosum to the detrital pool of a north temperate estuary. Estuaries 1 (4), 258–261. Josselyn, M.N., Mathieson, A.C., 1980. Seasonal influx and decomposition of autochthonous macrophyte litter in a north temperate estuary. Hydrobiologia 71 (3), 197–208. Knight, M., Parke, M., 1950. A biological study of Fucus vesiculosus L. and F. serratus L. J. Mar. Biol. Assoc. UK 29 (2), 439–514. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 Citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

102  PART | II  Algal structures

Kornfeldt, R.A., 1982. Relation between nitrogen and phosphorus content of macroalgae and the waters of Northern Oresund. Bot. Mar. 25 (4), 197–201. Levin, P.S., Mathieson, A.C., 1991. Variation in a host-epiphyte relationship along a wave exposure gradient. Mar. Ecol. Prog. Ser. 77 (2–3), 271–278. Lewis, J.R., 1964. The Ecology of Rocky Shores. English University Press, London. Lubchenco, J., 1980. Algal zonation in the New England rocky intertidal community: experimental analysis. Ecology 61 (2), 333–344. MacFarlane, C., 1932. Observations on the annual growth of Ascophyllum nodosum. Proc. N.S. Inst. Sci. 18 (2), 37–52. Mathieson, A.C., 1989. Phenological patterns of northern New England seaweeds. Bot. Mar. 32 (5), 419–438. Mathieson, A.C., Dawes, C.J., 2001. A muscoides-like Fucus from a Maine salt marsh: its origin, ecology, and taxonomic implications. Rhodora 103 (914), 172–201. Mathieson, A.C., Dawes, C.J., 2011. A floristic comparison of benthic “marine” algae in Bras d’Or Lake, Nova Scotia with five other Northwest Atlantic embayments and the Baltic Sea in northern Europe. Rhodora 113 (955), 300–350. Mathieson, A.C., Dawes, C.J., 2017. Seaweeds of the Northwest Atlantic. Univ. Mass. Press, Amherst, MA. Mathieson, A.C., Guo, Z.Y., 1992. Patterns of fucoid reproductive biomass allocation. Br. Phycol. J. 27 (3), 271–292. Mathieson, A.C., Hehre, E.J., 1986. A synopsis of New Hampshire seaweeds. Rhodora 88 (853), 1–139. Mathieson, A.C., Shipman, J.W., O’Shea, J.R., Hasevlat, R.C., 1976. Seasonal growth and reproduction of estuarine fucoid algae in New England. J. Exp. Mar. Biol. Ecol. 25 (3), 273–284. Mathieson, A.C., Penniman, C.A., Busse, P.K., Tveter-Gallagher, E., 1982. The effect of ice on Ascophyllum nodosum within the Great Bay Estuary System of New Hampshire-Maine. J. Phycol. 18 (3), 331–336. Mathieson, A.C., Dawes, C.J., Wallace, A.L., Klein, A.S., 2006. Distribution, morphology, and genetic affinities of dwarf embedded Fucus populations from the Northwest Atlantic. Bot. Mar. 49 (4), 283–303. McCourt, R.M., 1984a. Niche differences between sympatric Sargassum species in the northern Gulf of California. Mar. Ecol. Prog. Ser. 18 (1–2), 139–148. McCourt, R.M., 1984b. Seasonal patterns of abundance, distributions, and phenology in relation to growth strategies of three Sargassum species. J. Exp. Mar. Biol. Ecol. 74 (2), 141–156. Medeiros, I.D., Mathieson, A.C., Rajakaruna, N., 2017. Heavy metals in seaweeds from a polluted estuary in coastal Maine. Rhodora 119 (979), 201–211. Moss, B., 1970. Meristems and growth control in Ascophyllum nodosum (L.) Le Jol. New Phytol. 69 (2), 253–260. Niemeck, R.A., Mathieson, A.C., 1976. An ecological study of Fucus spiralis L. J. Exp. Mar. Biol. Ecol. 24 (1), 33–48. Norton, T.A., Mathieson, A.C., 1983. The biology of unattached seaweeds. In: Round, F.E., Chapman, D.J. (Eds.), Progress in Phycological Research. Elsevier, Amsterdam, pp. 333–386. Norton, T.A., Mathieson, A.C., Neushul, M., 1981. Morphology and environment. In: Lobban, C.S., Wynne, M.J. (Eds.), The Biology of Seaweeds. Blackwell Scientific, Oxford, pp. 421–451. Perkins, E.J., 1974. The Biology of Estuaries and Coastal Waters. Academic Press, London. Petrie, B., Bugden, G., 2002. The physical oceanography of the Bras d’Or Lakes: general introduction. Proc. N.S. Inst. Sci. 42 (1), 9–36. Pringle, J.D., Mathieson, A.C., 1987. Chondrus crispus Stackhouse. Food and Agriculture Organization, Rome, pp. 50–118. Rawlence, D.J., 1972. An ultrastructural study of the relationships between rhizoids of Polysiphonia lanosa (L.) Tandy (Rhodophyceae) and tissues of Ascophyllum nodosum (L.) Le Jolis. Phycologia 11 (3–4), 279–290. Rawlence, D.J., Taylor, A.R.A., 1972. A light and electron microscopic study of rhizoid development in Polysiphonia lanosa (L.) Tandy. J. Phycol. 8 (1), 15–24. Robertson, B.L., 1987. Reproductive ecology and canopy structure of Fucus spiralis L. Bot. Mar. 30 (6), 475–482. Schramm, W., 1996. The Baltic Sea and its transition zones. In: Schramm, W., Nienhuis, P.H. (Eds.), Marine Benthic Vegetation: Recent Changes and Effects of Eutrophication. Springer, Berlin, pp. 131–165. Sideman, E.J., Mathieson, A.C., 1983a. The growth, reproductive phenology and longevity of non-tide pool Fucus distichus (L.) Powell in New England. J. Exp. Mar. Biol. Ecol. 68 (2), 111–127. Sideman, E.J., Mathieson, A.C., 1983b. Ecological and genecological distinctions of a high intertidal dwarf form of Fucus distichus (L.) Powell in New England. J. Exp. Mar. Biol. Ecol. 72 (2), 171–188. Smith, G.M., 1955. Cryptogamic Botany. Vol. 1. Algae and Fungi. McGraw-Hill, New York, NY. South, G.R., Hill, R.D., 1970. Studies on marine algae of Newfoundland. I. Occurrence and distribution of free-living Ascophyllum nodosum in Newfoundland. Can. J. Bot. 48 (10), 1697–1701. South, G.R., Hooper, R.G., 1980. A Catalogue and Atlas of the Benthic Marine Algae of the Island of Newfoundland. Memorial University of Newfoundland, St. Johns, Newfoundland. Strain, P.M., Yeats, P.A., 2002. The chemical oceanography of the Bras d’Or Lakes. Proc. N.S. Inst. Sci. 42 (1), 37–64. Subrahmanyan, R., 1961. Ecological studies on Fucales II. Fucus spiralis L. J. Indian Bot. Soc. 40, 335–354. Sze, P., 1986. A Biology of Algae. Wm. C. Brown Publishers, Dubuque, IA. Taylor, W.R., 1960. Marine Algae of the Eastern Tropical and Subtropical Coasts of the Americas. University of Michigan Press, Ann Arbor, MI. Vadas, R.L., Keser, M., Rusanowski, P.C., 1976. Influence of thermal loading on the ecology of intertidal algae. In: Esch, G.W., MacFarlane, R.W. (Eds.), Thermal Ecology Symposium, Augusta, Georgia, 2–5 April, 1975, pp. 202–212. Wallentinus, I., 1979. Environmental Influences on Benthic Microvegetation in the Trosa-Asko Area, Northern Baltic Proper. II. On the Significance of Chemical Constituents in Some Macroalgal Species (Ph.D.), Stockholm.

Chapter 7

The scientometric analysis of the research on the algal genomics Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

7.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 papers published since 1980. The algal genomics have been among the most-prolific research fronts over time as evidenced with nearly 16,000 papers published during the same study period, corresponding to the public concerns about the development of sustainable and environment-friendly bioproducts. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field, the stakeholders should have timely and thorough access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal genomics and the underlying research areas as in fields of the algal research (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a,b,d, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012a,b,c,d,e,f,g,h,i,j, 2018a,b), nanobiomaterials (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e, 2019c), biomedicine (Konur, 2016i, 2018c), and social sciences (Konur, 2012k,l,m,n,o,p,q,r,s,t,u,v,y,z,aa,ab,ac,ad,ae,af). Although there have been 510 literature reviews on the algal genomics, there has been no published scientometric studies in the journal literature. However, there have been a limited number of scientometric studies as book chapters (Konur, 2016a, 2019b). Therefore, this paper presents the scientometric study of the research in algal genomics covering the whole range of research fronts as well as whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field.

7.2  Materials and methodology The search for the scientometric analysis of the literature on the algal genomics was carried out in December 2018 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI), and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in Appendix. These keyword sets have been devised in two major parts: the keywords related to genomics and keywords related to the algae. There have been two distinct keyword sets for the first part: the set of core journal titles related to genomics as well as the journals indexed by the subject category of ‘Genetics and Heredity’ and keywords related to the genomics. The keywords related to proteomics, transcriptomics, lipidomics, and metabolomics have also been included. On the other hand, the second part consists of the keywords related to the algae in general, dinoflagellates and coccolithophores, microalgae, macroalgae, diatoms, cyanobacteria, and journal titles related to the algae.

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00007-3 © 2020 Elsevier Inc. All rights reserved.

105

106  PART | III  Algal genomics

The papers found through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subject to the quality control exercise to ensure that these references have been primarily related to the algal genomics. This refined list of papers have formed the sample for the scientometric and content overview of the literature on the algal genomics. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. The data on the scientometric analysis and content overview of 20 most-cited papers have also been provided focusing on the determination of the key research fronts for these citation classics, complementing sections on the keywords and research fronts. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

7.3 Results 7.3.1  Documents and indexes The search has resulted in 18,270 papers where there have been 15,011 articles, 2031 meeting abstracts, 515 reviews, 328 notes, 166 corrections, 104 editorial materials, 55 letters, and 33 corrections and additions. In the first instance, the papers excluding meeting abstracts and corrections have been selected resulting in 16,103 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 15,433 papers in English. The other major languages have been Russian, French, Japanese, and Chinese. This set of 15,433 papers has formed the sample for the scientometric analysis of the literature on the algal genomics. The articles have formed 93.8% of the final sample while reviews, notes, editorial matters, and letters have formed 3.2%, 2.0%, 0.6%, and 0.3% of this sample, respectively. Additionally, 2.1% of these papers have been ‘proceedings papers’ and three papers have been ‘retracted papers’. On the other hand, 99.6% of these papers have been indexed by the SCI-E while only six papers have been indexed by the SSCI focusing on the societal aspects of algal genomics. There have been no papers indexed by the A&HCI. Additionally, 0.4% of the papers have been indexed by the ESCI.

7.3.2 Keywords The most-prolific keywords used in algal genomics have been determined to assess the hot topics and the primary research fronts in the algal genomics. There have been a number most-prolific keywords for the first set of keywords for the genomics: ‘*dna, gene, *genetic*, *genom*, phylogen*, sequenc*, *transcript*’. The other prolific key words have been ‘cloning, codon, mutant*, *nucleot*, proteom*, *rna’. On the other hand, the most-prolific journals related to genomics have been ‘Current Genetics’, ‘Gene’, ‘BMC Genomics’, ‘Molecular Biology and Evolution’, and ‘Molecular General Genetics’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, and algal, dinoflagellate*, macroalga*, rhodophyt*, and seaweed*, bacillariophycea*, diatom, diatoms, and cyanobacter*’. The other prolific keywords for the algae have been ‘coccolith*, dinophycea*, Alexandrium, chlorophycea*, chlorophyt*, “green alga*”, microalga*, “micro-alga*”, Chlamydomonas, *Chlorella, Dunaliella, Euglena, Scenedesmus, “brown alga*”, phaeophycea*, kelp*, phaeophyt*, “red alga*”, Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, Ulva, bacillariophyt*, “blue green alga*”, “blue-green alga*”, *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, Synechocystis’. On the other hand, the most-prolific journals related solely to the algal research have been ‘Algal Research*’, ‘European Journal of Phycology’, ‘Harmful Algae’, ‘Journal of Applied Phycology’, ‘Journal of Phycology’, and ‘Phycologia’.

7.3.3 Authors There have been 30,088 authors contributing to the research on the algal genomics in total. The information on the mostprolific and influential 20 authors is provided in Table 7.1: Authors’ names, gender, institutions, countries, primary research fronts, number of papers produced by these authors, the number of influential papers with at least 100 citations received (I100), and the percentage of the number of influential papers with relative to the number of all the papers published (I-100%).



TABLE 7.1  The most-prolific and influential authors in algal genomics. Gender

Institution

Country

Research fronts

I-0

I-100

I-100%

1

Jean-David Rochaix

M

Univ. Geneva

Switzerland

Microalgal photosynthesis

89

19

21

2

Robert Haselkorn

M

Univ. Chicago

United States

Cyanobacterial nitrogen fixation and toxicology

63

18

29

3

Arthur R Grossman

M

Carnegie Inst. Wash.

United States

Microalgal photosynthesis and ecology

89

16

18

4

Sallie W Chisholm

F

Massachusetts Inst. Technol.

United States

Cyanobacterial phylogeny, photosynthesis, and ecology

42

16

38

5

Debashis Bhattacharya

M

Rutgers St. Univ.

United States

Phylogeny of dinoflagellates

98

15

15

6

Chris Bowler

M

CNRS

France

Phylogeny and structures of diatoms

54

12

22

7

Brett A Neilan

M

Univ. New S. Wales

Australia

Cyanobacterial toxicology

61

11

18

8

Linda K Medlin

F

Alfred Wegener Inst. Polar Mar. Res.

Germany

Phylogeny of diatoms

57

11

14

9

C Peter Wolk

M

Michigan St. Univ.

United States

Cyanobacterial nitrogen fixation

51

10

20

10

Kaarina Sivonen

F

Univ. Helsinki

Finland

Cyanobacterial toxicology and phylogeny

48

10

21

11

Satoshi Tabata

M

Kazusa DNA Res. Inst.

Japan

Cyanobacterial structures

44

10

23

12

Igor V Grigoriev

M

Jt. Genom. Inst.

United States

Microalgal phylogeny

na

10

67

13

Asaf Salamov

M

Jt. Genom. Inst.

United States

Microalgal phylogeny

na

10

67

14

Susan S Golden

F

Univ. Calif. San Diego

United States

Cyanobacterial circadian clocks

59

9

15

15

Nicole Tandeau de Marsak

F

CNRS

France

Cyanobacterial genomics

58

9

16

16

Norio Murata

M

Natl. Inst. Bas. Biol.

Japan

Cyanobacterial photosynthesis

48

9

19

17

David J Scanlan

M

Univ. Warwick

United Kingdom

Cyanobacterial ecology

38

9

24

18

Hideya Fukuzawa

M

Univ. Tokyo

Japan

Cyanobacterial and microalgal photosynthesis

34

9

26

19

Frederic Partensky

M

CNRS

France

Cyanobacterial photosynthesis

28

9

32

20

Yves van de Peer

M

Univ. Ghent

Belgium

Algal phylogeny

18

9

50

Average

57

12

27

Total

1028

231

555

Total %

6.5

31.6

M, male; F, female; No. papers, the number of papers for at least 16 papers; I-0, the number of the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers with relative to the total number of papers published.

The scientometric analysis of the research on the algal genomics Chapter | 7  107

Author

108  PART | III  Algal genomics

The data on these authors shows that the most-prolific author with the highest citation impact has been ‘Jean-David Rochaix’ of the University of Geneva, working primarily on the microalgal photosynthesis, with 89 papers. His citation impact is highest with 19 influential papers. The other most-prolific authors with the high citation impact have been ‘Robert Haselkorn’, ‘Arthur R Grossman’, ‘Sallie W Chisholm’, and ‘Debashis Bhattacharya’ with 15 or more influential papers each. It is notable that only five of these authors are female. The United States has been the most-prolific country for these authors with eight authors while France and Japan followed the United States with three authors each. On the other hand, Europe has had eight authors as a whole. Similarly, the most-prolific institutions have been ‘Centre National de la Recherche Scientifique’ (CNRS) and ‘Joint Genome Institution’ with three and two papers, respectively. The most-prolific research fields have been the ‘algal phylogeny’, ‘algal photosynthesis’, ‘algal ecology’, ‘algal toxicology’, and ‘algal structures’ with six, five, three, and three authors, respectively. On the other hand, the most-studied algae have been ‘cyanobacteria’, ‘microalgae’, and ‘diatoms’ with 12, 5, and 2 authors, respectively. The number of papers published by these authors have ranged from 18 to 98. These most-prolific authors have contributed to nearly 6.5% and 31.6% of all the papers and influential papers, respectively. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the influential papers with relative to the number of all the papers published for each author in the last column (I-100%). These data shows that ‘Igor V Grigoriev’ and ‘Asaf Salamov’ have been the top influential authors with 67% ratio each. The other most-influential authors have been ‘Yves van de Peer’, ‘Sallie W Chisholm’, ‘Frederic Partensky’, ‘Robert Haselkorn’, and ‘Hideya Fukuzawa’ with 50%, 38% 32%, 19%, and 26% ratios, respectively.

7.3.4 Countries Nearly 99.8% of the papers have had country information in their abstract pages and 137 countries and territories have contributed to these papers overall. Table 7.2 provides the information about the most-prolific and influential 20 countries. These 20 most-prolific countries have produced 111.8% and 148.4% of all the papers and influential papers, respectively as a whole. The most-prolific and influential country has been the United States producing 30.4% and 50.7% of all the papers and influential papers, respectively. Additionally, Germany and Japan have emerged as the second and third most-prolific countries following the United States producing 11.5% and 13.8% of all the papers, respectively. These counties have also produced 17.8% and 14.8% of the influential papers, respectively. France, United Kingdom, Canada, Australia, France, and Switzerland have been the other prolific and influential countries. On the other hand, China has produced 11.6% and 1.8% of all the papers and influential papers, respectively. The European countries have been dominant in the top 20 country list as they have produced 48.3% and 66.4% of all the papers and influential papers, respectively, as a whole, surpassing significantly both the United States, Japan, and Canada.

7.3.5 Institutions Over 99.8% of the papers have had their institutions listed in their abstract pages. For these papers, 4640 institutions have contributed to the research on the algal genomics in total. The information about the 20 most-prolific and influential institutions is given in Table 7.3. The most-prolific and influential institution has been the ‘CNRS’ publishing 7.8% and 5.4% of the influential and all papers, respectively. ‘Sorbonne University’, ‘Helmholtz Association’, and ‘University of Geneva’ have been the other influential institutions as they have produced each 3.1% of the influential papers. The most-prolific country for these institutions has been the United States with eight institutions. On the other hand, France, Germany, and Japan have had three institutions each and Europe has had seven institutions as a whole. The contribution of these institutions has ranged from 0.3% to 5.4% for all the papers and from 1.9% to 7.8% for the influential papers. Overall, these 20 institutions have contributed to 28.4% and 55.4% of these papers, respectively.

7.3.6  Research funding bodies Only 47.5% of these papers have had declared any research funding in their abstract pages and overall, 9051 funding bodies have funded these papers.

The scientometric analysis of the research on the algal genomics Chapter | 7  109



TABLE 7.2  The most-prolific and influential countries in algal genomics. Country

I-0

I-0%

I-100

I-100%

Europe

7651

48.3

486

66.4

1

United States

4819

30.4

371

50.7

2

Germany

1820

11.5

130

17.8

3

Japan

2189

13.8

108

14.8

4

France

1267

8.0

88

12.0

5

United Kingdom

981

6.2

68

9.3

6

Canada

927

5.9

67

9.2

7

Australia

717

4.5

55

7.5

8

Switzerland

251

1.6

31

4.2

9

Netherlands

316

2.0

21

2.9

11

Spain

632

4.0

19

2.6

10

Israel

248

1.6

19

2.6

12

Belgium

293

1.9

18

1.5

13

Italy

349

2.2

16

2.2

14

Finland

167

1.1

14

1.9

15

China

1751

11.1

13

1.8

16

Denmark

157

1.0

11

1.5

17

Sweden

312

2.0

10

1.4

18

Czech Rep.

159

1.0

10

1.4

19

Norway

175

1.1

9

1.2

20

New Zealand

178

1.1

8

1.1

Total

17,708

111.8

1086

148.4

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers.

The most-prolific funding bodies have been the ‘National Institute of General Medical Sciences’ of the United States, and ‘National Natural Science Foundation of China’ funding 4.4% and 4.1% of the papers, respectively. The other prolific funding bodies have been the ‘National Science Foundation’, ‘Gordon and Betty Moore Foundation’, ‘Natural Environment Research Council’, ‘Natural Sciences and Engineering Research Council of Canada’, ‘Deutsche Forschungsgemeinschaft’, ‘Australian Research Council’, and ‘National Institutes of Health’ with at least 0.5% of the papers each.

7.3.7  Publication years Fig. 7.1 shows the number of papers on the algal genomics, published between 1980 and 2018 as of December 2018. The data in this figure shows that the number of papers has risen from 147 papers in 1980 to 889 papers in 2016. The most prolific decade has been the 2010s with 42.5% of the papers. On the other hand, 9.9%, 21.9%, and 26.7% of the papers have been published in the 1980s, 1990s, and 2000s, respectively. Thus, the figure shows that there has been a steadily increasing trend between 1980 and 2018.

7.3.8  Source titles Overall, these papers have been published in 1408 journals. Table 7.4 provides the information on the 20 most-prolific and influential journals. These 20 journals have published 23.2% and 55.3% of all the papers and influential papers, respectively, in total.

110  PART | III  Algal genomics

TABLE 7.3  The most-prolific and influential institutions in algal genomics. Institutions

Country

I-0

I-0%

I-00

I-100% papers

1

Ctr. Natl. Rec. Sci.-CNRS

France

849

5.4

57

7.8

2

Sorbonne Univ.

France

413

2.6

23

3.1

3

Helmholtz Assoc.

Germany

225

1.4

23

3.1

4

Univ. Geneva

Switzerland

115

0.7

23

3.1

5

Nagoya Univ.

Japan

176

1.1

21

2.9

6

Univ. Chicago

United States

116

0.7

21

2.9

7

Michigan St. Univ.

United States

148

0.9

20

2.7

8

Univ. Tokyo

Japan

414

2.6

19

2.6

9

Woods Hole Ocean. Inst.

United States

94

0.6

19

2.6

10

Univ. Brit. Columbia

Canada

157

1.0

18

2.6

11

Massachusetts Inst. Technol.

United States

85

0.5

18

2.5

12

Kyoto Univ.

Japan

185

1.2

17

2.3

13

Jt. Genom. Inst.

United States

48

0.3

17

2.3

14

Univ. Freiburg

Germany

133

0.8

16

2.2

15

Carnegie Inst. Sci.

United States

109

0.7

16

2.2

16

Humboldt Univ. Berlin

Germany

101

0.6

16

2.2

17

Dalhousie Univ.

Canada

165

1.0

15

2.0

18

Univ. Calif. San Diego

United States

156

1.0

15

2.0

19

Jt. Bioenerg. Inst.

United States

47

0.3

15

2.0

20

Univ. Paris Saclay

France

194

1.2

14

1.9

3930

24.8

403

55.1

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers.

1000 900

Number of papers

800 700 600 500 400 300 200 100

FIG. 7.1  The number of publications in the algal genomics between 1980 and 2018.

18

16

20

14

20

12

20

10

20

08

20

06

20

04

Publication years

20

02

20

00

20

98

20

96

19

94

19

93

19

91

19

89

19

87

19

85

19

80

19

19

19

80

0



TABLE 7.4  The most-prolific and influential journals in algal genomics. Abbr.

Subject

I-0

I-0%

I-100

I- 100%

1

Proceedings of the National Academy of Sciences of the United States of America

P. Natl. Acad. Sci. USA

Mult. Sci.

218

1.4

75

10.2

2

Journal of Phycology

J. Phycol.

Plant Sci.; Mar. Fresh. Biol.

833

5.3

44

6.0

3

Applied and Environmental Microbiology

Appl. Environ. Microb.

Biot. Appl. Microb.; Microbiol.

267

1.7

31

4.2

4

Journal of Bacteriology

J. Bacteriol.

Microbiol.

353

2.2

26

3.6

5

Plant Physiology

Plant Physiol.

Plant Sci.

276

1.7

24

3.3

6

Nature

Nature

Mult. Sci.

40

0.3

24

3.3

7

Journal of Biological Chemistry

J. Biol. Chem.

Bioch. Mol. Biol.

225

1.4

19

2.6

8

Plant Cell

Plant Cell

Bioch. Mol. Biol.; Plant Sci.; Cell Biol.

87

0.6

19

2.6

9

Science

Science

Mult. Sci.

33

0.2

18

2.5

10

Molecular Biology and Evolution

Mol. Biol. Evol.

Bioch. Mol. Biol.; Evol. Biol.; Gen. Hered.

142

0.9

17

2.3

11

EMBO Journal

EMBO J.

Bioch. Mol. Biol.; Cell Biol.

45

0.3

16

2.2

12

Journal of Cell Biology

J. Cell Biol.

Cell Biol.

49

0.3

15

2.1

13

Journal of Molecular Evolution

J. Mol. Evol.

Bioch. Mol. Biol.; Evol. Biol.; Gen. Hered.

111

0.7

13

1.8

14

Nucleic Acids Research

Nucleic Acids Res.

Bioch. Mol. Biol.

231

1.5

12

1.6

15

Plant Journal

Plant J.

Plant Sci.

86

0.5

10

1.4

16

Plant Molecular Biology

Plant Mol. Biol.

Bioch. Mol. Biol.; Plant Sci.

285

1.8

9

1.2

17

Biochemistry

Biochemistry

Bioch. Mol. Biol.

74

0.5

9

1.2

18

Molecular & General Genetics

Mol. Gen. Genet.

Bioch. Mol. Biol.; Gen. Hered.

132

0.8

8

1.1

19

Molecular Phylogenetics and Evolution

Mol. Phylogenet. Evol.

Bioch. Mol. Biol.; Evol. Biol.; Gen Hered.

107

0.7

8

1.1

20

Molecular Microbiology

Mol. Microbiol.

Bioch. Mol. Biol.; Microbiol.

84

0.5

8

1.1

3678

23.2

405

55.3

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers.

The scientometric analysis of the research on the algal genomics Chapter | 7  111

Journals

112  PART | III  Algal genomics

The most-prolific and influential journal has been ‘Proceedings of the National Academy of Sciences of the United States of America’ publishing 1.4% and 10.2% of all the papers and influential papers, respectively. The ‘Journal of Phycology’, ‘Applied and Environmental Microbiology’, ‘Journal of Bacteriology’, ‘Plant Physiology’, and ‘Nature’ have followed this top journal as the most prolific and influential journals. The most-prolific subject category for these journals has been ‘Biochemistry and Molecular Biology’ with 11 journals, followed by ‘Plant Sciences’ with 6 journals. The other prolific subjects have been ‘Cell Biology’, ‘Genetics and Heredity’, and ‘Microbiology’ with four journals each and ‘Evolutionary Biology’ and ‘Multidisciplinary Sciences’ with two journals each. It is notable that ‘Journal of Phycology’ has been the only journal related to the algae in this top 20 journal list. Similarly, ‘Molecular Phylogenetics and Evolution’, ‘Journal of Molecular Evolution’, ‘Molecular Biology and Evolution’, and ‘Molecular & General Genetics’ have been the only journals indexed by the subject category of ‘Genetics and Heredity’ in this top list.

7.3.9  Subject categories The information about the 10 most-prolific and influential subject categories are given in Table 7.5. As expected, the mostprolific subject category has been ‘Biochemistry & Molecular Biology’ indexing 28.3% and 29.2% of all the papers and influential papers, respectively. The subject category of ‘Plant Sciences’ has closely followed the top subject indexing 23.9% and 20.5% of all the papers and influential papers, respectively. The other prolific and influential subjects have been ‘Multidisciplinary Sciences’, ‘Microbiology’, ‘Genetics Heredity’, ‘Cell Biology’, and ‘Marine Freshwater Biology’ with at least 10.8% of the influential papers each. Thus, the first-seven categories have been the key pillars of the research in algal genomics, indexing together 120.2% of the influential papers. Thus, the research in algal genomics shows an interdisciplinary character covering both science and technology ranging from ‘Biotechnology’ and ‘Biochemistry & Molecular Biology’ to ‘Genetics and Heredity’.

7.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers, 4.6% of the research sample of 15,833 papers, Table 7.6. The records in this dataset has been refined from 732 papers to 718 papers to focus on the core papers for the field of algal genomics.

TABLE 7.5  The most-prolific and influential subject categories in algal genomics. Subject categories

I-0 No. papers

I-0% Papers

I-100 No. papers

I-100% Papers

1.

Biochemistry Molecular Biology

3457

28.3

214

29.2

1.1.

Plant Sciences

3783

23.9

150

20.5

1.2.

Multidisciplinary Sciences

917

5.8

125

17.0

1.3.

Microbiology

2254

14.2

114

15.6

1.4.

Genetics Heredity

2337

14.8

106

14.5

6

Cell Biology

1053

6.7

92

12.6

7

Marine Freshwater Biology

2940

18.6

79

10.8

8

Evolutionary Biology

854

5.4

56

7.7

9

Biotechnology Applied Microbiology

2036

12.9

54

7.4

10

Ecology

595

3.8

28

3.8

Total

20,226

128

1018

139

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers.

The scientometric analysis of the research on the algal genomics Chapter | 7  113



TABLE 7.6  The most-prolific research fronts in algal genomics. Research fronts

Algae

Microalgae

Cyano­ bacteria

Diatoms

Dino­ flagellates

Macroalgae

Total

1

Structures

0

28

6

10

2

0

46 (6.4%)

2

Phylogeny

15

29

28

17

58

39

186 (25.9%)

3

Genomic methodology

5

71

46

9

14

8

153 (21.3%)

4

Photosynthesis

1

33

73

1

0

0

108 (15.0%)

5

Ecology

6

23

64

3

8

3

107 (14.9%)

6

Biofuels

4

17

14

4

0

0

39 (5.4%)

7

Biomedicine

1

7

17

1

0

8

34 (4.7%)

8

Toxicology

0

0

40

1

3

0

44 (6.1%)

9

Other

0

0

0

1

0

0

1 (0.1%)

Total

32 (4.5%)

208 (29.0%)

288 (40.1%)

47 (6.5%)

85 (11.8%)

58 (8.1%)

718

Numbers, the number of influential papers for each research front and type of algae; %, the percentage of influential papers for each research front and type of algae; the number in bold: the most-prolific research fronts with at least 15 influential papers.

The data shows that the field of ‘cyanobacterial photosynthesis’ and ‘microalgal genomic studies’ have been the most prolific research fronts with 73 and 71 papers, forming 10.2% and 9.9% of these influential papers, respectively. The other key research fronts have been ‘cyanobacterial ecology’, ‘phylogeny of dinoflagellates and coccolithophores’, ‘cyanobacterial genomic studies’, ‘cyanobacterial toxicology’, and ‘macroalgal phylogeny’ with 8.9%, 8.1%, 6.4%, 5.6%, and 5.4% of the influential papers, respectively. The most-studied the types of algae have been ‘cyanobacteria’ and ‘microalgae’ with 40.1% and 29.0% of the influential papers, respectively. Additionally, the papers on the ‘dinoflagellates and coccolithophores’, ‘macroalgae’, ‘diatoms’, and ‘algae in general’ formed 11.8%, 8.1%, 6.5%, and 4.5% of these papers, respectively. On the other hand, the most-studied individual research fronts have been ‘phylogeny’ and ‘genomic methodology’ with 25.9% and 21.3% of these papers, respectively. Additionally, the papers related to ‘photosynthesis’, ‘ecology’, ‘structures’, ‘toxicology’, ‘biofuels’, and ‘biomedicine’ formed 15.0%, 14.9%, 6.4%, 6.1%, 5.4%, and 4.7% of these papers, respectively.

7.3.11  Citation classics This section provides the information on both the scientometric analysis and content overview of the most-cited 20 papers in algal genomics. The information on these papers is given in Table 7.7: authors’ names, publication years, document type, number of authors per paper, lead authors’ names, gender, and paper numbers (lead authors were determined based on the number of papers produced in this field), journals, subject areas, research fronts, types of algae, academic focus of the papers, number of citations received, and the number of citations per year.

7.3.11.1  Scientometric overview of the citation classics These papers have been published between 1988 and 2016. The most-prolific decades have been the 1990s and 2000s with seven and nine papers, respectively. Additionally, there have been two papers each published in the 1980s and 2010s. The articles have been over-represented in these classical papers as there have been 19 articles and only one review. The number of the authors of these papers has ranged from 1 to 117 while the mean number of authors has been 22.3. The most-prolific lead author has been ‘Chris Bowler’ with four citation classics. The other prolific lead authors have been ‘Kamel Jabbari’ and ‘Sylvia Lucas’ with three citation classics each. In total, 39 authors contributed to these citation classics. Additionally, ‘Andrew E Allen’, ‘E Virginia Armbrust’, ‘Sallie W Chisholm’, ‘Steven G Ball’, ‘Beverley R Green’, ‘Igor V Grigoriev’, ‘Jane Grimwood’, ‘Nils Kroger’, ‘Erika Lindquist’, ‘Jeffrey D Palmer’, ‘Yves van de Peer’, ‘Gabrielle

TABLE 7.7  The citation classics in algal genomics. Lead authors

Journal

Subject area

Res. fronts

Algae

Topic

Cits.

Av. cits

117

SS Merchant-6; O Valloni-5; EJH Harris-7; A Salamov-10; E Lindquist-8; S Lucas-8; J Grimwood-7; J Schmutz-7; E Fernandez-5; H Fukuzawa-9; M Hippler-6; K Jabbari-6; PA Lefebvre-7; IT Paulsen 8; M Elias 5; SG Ball-8; C Bowler-12; IV Grigoriev-10; AR Grossman-16

Science

Mult. Sci.

Phylogeny

MicroalgaeChlamydomonas

Comparative phylegenomic analysis

1385

126

A

45

EV Armbrust-8; C Bowler-12; BR Green-7; AE Allen-5; N Kroger-5; S Lucas-8; B Palenik-5; TA Rynearson-5; K Valentin-7

Science

Mult. Sci.

Phylogeny

DiatomsThalassiosira

Phylegenomic analysis

1138

81

1997

A

3

G Muyzer-5

Appl. Environ. Microb.

Biot. Appl. Microb.; Microbiol.

Genomics

Cyanobacteria

Gene amplification

801

42

Bowler et al.

2008

A

77

C Bowler-12; AE Allen-5; J Grimwood-7; K Jabbari-6; A Kuo-7; A Salamov-10; T Mock6; K Valentin-7; A Falciatore-5; A Kaplan-6; N Kroger-5; E Lindquist-8; S Lucas-8; LK Medlin-11; TA Rynearson-5; J Schmutz-7; EV Armbrust-8; BR Green-7; Y van de Peer-9; IV Grigoriev-10

Nature

Mult. Sci.

Phylogeny

DiatomsPhaeodactylum

Comparative phylegenomic analysis

780

78

5

Williams

1988

A

1

Method. Enzymol.

Bioch. Res. Meth.; Bioch. Mol. Biol.

Photosynthesis

CyanobacteriaSynechocystis

Genetic transformation of PSII

764

25

6

Rocap et al.

2003

A

24

Nature

Mult. Sci.

Ecology

CyanobacterşaProchlorococcus

Genome divergence in oceans

733

49

7

Martin et al.

2002

A

10

P. Natl. Acad. Sci. USA

Mult. Sci.

Phylogeny

Cyanobacteria

Comparative phylegenomic analysis

705

44

8

Kindle

1990

A

1

P. Natl. Acad. Sci. USA

Mult. Sci.

Genomics

MicroalgaeChlamydomonas

Nuclear transformation

682

24

Authors

Year

Doc.

N auths.

1

Merchant et al.

2007

A

2

Armbrust et al.

2004

3

Nubel et al.

4

G Rocap-6; FW Larimer-5; WR Hess-8; AF Post-6; MB Sullivan-6; SW Chisholm-16

9

Matsuzaki et al.

2004

A

42

10

Pazour et al.

2000

A

11

Boynton et al.

1988

12

Rubio et al.

13

T Kuroiwa-5; H Nozaki-5

Nature

Mult. Sci.

Genomics

MicroalgaeCyanidioschyzon

Genome sequence analysis

672

48

7

J. Cell Biol.

Cell Biol.

Structures

MicroalgaeChlamydomonas

Intraflagellar transport

651

36

A

11

Science

Mult. Sci.

Genomics

MicroalgaeChlamydomonas

Chloroplast transformation

600

20

2001

A

7

Gene. Dev.

Cell Biol., Dev. Biol., Gen. Hered.

Ecology

Microalgae

Phosphate starvation signaling pathway

559

33

Turner et al.

1999

A

4

JD Palmer-5

J Eukaryot. Microbiol.

Microbiol.

Phylogeny

Cyanobacteria

Comparative phylegenomic analysis

539

28

14

Kohler et al.

1997

A

8

CF Delwiche-8; JD Palmer-5

Science

Mult. Sci.

Phylogeny

Microalgae

Comparative phylegenomic analysis

514

24

15

Kaneko et al.

2001

A

22

Y Nakamura-8; CP,Wolk-10; M Kohara-5; S Tabata-10

DNA Res.

Gen. Hered.

Ecology

CyanobacteriaAnabaena

Nitrogen fixation

513

30

16

Radakovits et al.

2010

R

4

MC Posewitz-6

Eukaryot. Cell

Microbiol., Mycol.

Biofuels

Algae

Genetic engineering

500

63

17

Moore et al.

1998

A

3

G Rocap-6; SW Chisholm-16

Nature

Mult. Sci.

Ecology

CyanobacteriaProchlorococcus

Genome divergence in oceans

498

25

18

Scholin et al.

1994

A

4

CA Scholin-5; DM Anderson-5

J. Phycol.

Mar. Fresh. Biol.; Plant Sci.

Phylogeny

DinoflagellatesAlexandrium

Genome divergence

490

20

19

Derelle et al.

2016

A

26

F Partensky-9; K Jabbari-6; C Bowler-12; SG Ball-8; Y van de Peer-9; H Moreau-5

P. Natl. Acad. Sci. USA

Mult. Sci.

Genomics

MicroalgaeOstreococcus

Genome sequencing

487

244

20

Ishiura et al.

1998

A

9

M Ishiura-5; SS Golden-9; T Kondo-5

Science

Mult. Sci.

Structures

Cyanobacteria

Circadian clocks

456

23

Total

425

13,467

1063

Average

20

673

53

Doc., document; A, article; R, review; Gender, gender of lead authors—female authors in italic; N paper, for the authors with at least 16 papers with 0 citations and with at least 5 influential papers—number after the author names; Subject, Web of Science subjects; Topic, primary topic of the papers; Algae, type of algae studied; Res. fronts, primary research fronts studied; Cits., number of citations received in total; Av. Cits., number of citations per year.

116  PART | III  Algal genomics

Rocap’, ‘Tatiana A Rynearson’, ‘Asaf Salamov’, ‘Jeremy Schmutz’, and ‘Klaus Valentin’ have contributed two citation classics each. There has been a significant gender deficit among the lead authors of these classical papers as only 20 out of 78 lead authors listed in Table 7.7 are female. In total, these citation classics have been published by only 11 journals. The most-prolific journals have been ‘Science’, ‘Nature’, and ‘Proceedings of the National Academy of Sciences of the United States of America’ with five, four, and three papers, respectively. In total, these papers have been indexed by nine subject categories. The most-prolific category has been ‘Multidisciplinary Sciences’ with 12 papers indexing 3 most prolific journals. The fields of ‘Microbiology’, ‘Cell Biology’, and ‘Genetics and Heredity’ followed this top subject with three, three, and two papers, respectively. In total, there have been six research fronts. The most-prolific research fronts have been ‘phylogeny of algae’, ‘genomic methodology of algae’, and ‘ecology of algae’ with seven, five, and four papers, respectively. Additionally, there have been two, one, and one papers related to the research fronts of ‘algal structures’, ‘algal biofuels’, and ‘algal photosynthesis’, respectively. There have been no papers related to ‘algal toxicology’ and ‘algal biomedicine’. There have been five types of algae covered by these classical papers. The most prolific types of algae have been ‘microalgae’ and ‘cyanobacteria’ with eight papers each. Additionally, there have been two, one, and one papers related to ‘diatoms’, ‘dinoflagellates’, and ‘algae in general’, respectively. There have been no papers related to ‘macroalgae’. On the other hand, the most-studied type of algae on the individual basis has been ‘Chlamydomonas’ with four papers. The other types of algae have been ‘Alexandrium, Anabaena, Cyanidioschyzon, Ostreococcus, Phaeodactylum, Prochlorococcus, Synechocystis, Thalassiosira’. The most-studied topics have included ‘genomic divergence of algae’, ‘comparative phylogenomic analysis of algae’, and ‘genome sequencing of algae’. These papers have received between 456 and 1385 citations each, totaling in 13,467 citations with a mean value of 673 citations. On the other hand, the number of citations per year has ranged from 20 to 244 with a mean value of 53 citations per year.

7.3.11.2  Brief overview of the content of the citation classics There have been four major classes of papers: ‘phylogeny of algae’, ‘genomic methodology of algae’, ‘ecology of algae’, and ‘other research fronts’ with seven, five, four, and four papers, respectively. In the final section, there have been two, one, and one papers related to the research fronts of ‘algal structures’, ‘algal biofuels’, and ‘algal photosynthesis’, respectively. Phylogeny of algae Microalgae  Merchant et al. (2007) sequence the nuclear genome of Chlamydomonas and carry out a comparative phylogenomic analysis in a seminal paper with 1385 citations. They identify genes encoding proteins related to the photosynthetic and flagellar functions. Thus, they establish links between ciliopathy and the composition and function of flagella. Kohler et al. (1997) study the plastid of probable green algal origin in apicomplexan parasites in a paper with 514 citations. They localize the DNA of Toxoplasma gondii to a discrete organelle surrounded by four membranes. Phylogenetic analysis of the tufA gene encoded by the genomes of T. gondii, Eimeria tenella, and Plasmodium falciparum grouped this organellar genome with cyanobacteria and plastids, showing consistent clustering with green algal plastids. They argue that the Apicomplexa acquired a plastid by secondary endosymbiosis, probably from a green alga. Diatoms  Armbrust et al. (2004) study the nuclear genome of the Thalassiosira pseudonana together with plastids and mitochondrial genomes in a paper with 1138 citations. They identify diploid nuclear chromosomes and genes for silicic acid transport and formation of silica-based cell walls and high-affinity iron uptake among others. Bowler et al. (2008) study the complete genome sequence of the Phaeodactylum tricornutum and carry out a comparative phylegenomic analysis in relation to T. pseudonana to evaluate evolutionary history of diatom genomes in a paper with 780 citations. They find that their genome structures are dramatically different. They additionally find the presence of hundreds of genes from bacteria. Cyanobacteria  Martin et al. (2002) carry out a comparative phylogenomic analysis of cyanobacteria and compare proteins encoded in the Arabidopsis genome to the proteins from 3 cyanobacterial genomes, other prokaryotic reference genomes, and yeast in a paper with 705 citations. They estimate that 18% of Arabidopsis protein-coding genes were acquired from the cyanobacterial ancestor of plastids. They further find that at least two independent secondary endosymbiotic events had occurred involving red algae and amino acid composition bias in chloroplast proteins strongly affects plastid genome phylogeny.



The scientometric analysis of the research on the algal genomics Chapter | 7  117

Turner et  al. (1999) study the phylogenetic relationships among cyanobacteria and plastids by small-subunit rRNA sequence analysis in a paper with 539 citations. They find that their results are in agreement with earlier studies in the assignment of individual taxa to specific sequence groups. They further find that all plastids cluster as a strongly supported monophyletic group arising near the root of the cyanobacterial line of descent. Dinoflagellates  Scholin et  al. (1994) identify group- and strain-specific genetic markers for globally distributed Alexandrium focusing on the sequence analysis of a fragment of the small-subunit rRNA genes of various strains of A. in a paper with 490 citations. They identify major classes of sequences, indicative of both species- and strain-specific genetic markers as their sequences provide finer-scale species and population resolution. They argue that human-assisted dispersal of dinoflagellates has been a way for inoculating a region with diverse representatives of the dinoflagellate complex from geographically and genetically distinct source populations. Genomic methodology of algae Cyanobacteria  Nubel et al. (1997) amplify 16S rRNA genes from cyanobacteria and plastids using a set of oligonucleotide primers in a paper with 801 citations. Their procedure allows rapid and phylogenetically meaningful identification without pure cultures or molecular cloning. Microalgae  Kindle (1990) studies the high-frequency nuclear transformation of Chlamydomonas reinhardtii in a paper with 682 citations. She achieves nuclear transformation rates of approximately 103 transformants per micrograms of plasmid DNA. She argues that the availability of efficient nuclear and chloroplast transformation in C. provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclear-chloroplast genome interactions. Matsuzaki et al. (2004) sequence the genome of the Cyanidioschyzon merolae 10D in a paper with 672 citations. They identify 5331 genes. They determine the unique characteristics of this genomic structure as a lack of introns, only three copies of rDNA units that maintain the nucleolus, and two dynamin genes that are involved only in the division of mitochondria and plastids. Their findings support the hypothesis of the existence of single primary plastid endosymbiosis. Boynton et al. (1988) study the chloroplast transformation in Chlamydomonas with high velocity microprojectiles in a paper with 600 citations. They find that the bombardment of three mutants of the chloroplast atpB gene with high-velocity tungsten microprojectiles that were coated with cloned chloroplast DNA carrying the wild-type gene permanently restored the photosynthetic capacity of the algae. The restored wild-type atpB gene remains in all transformants as an integral part of the chloroplast genome and is expressed and inherited normally. Derelle et al. (2016) carry out a genome analysis of the Ostreococcus tauri in a paper with 487 citations. They find that the nuclear genome has an extremely high gene density and it is structurally complex. They argue that the complete genome sequence, unusual features, and downsized gene families, make this microalgae an ideal model system for research on eukaryotic genome evolution, including chromosome specialization and green lineage ancestry. Ecology of algae Cyanobacteria  Rocap et al. (2003) compare the genomes of two Prochlorococcus strains with the largest evolutionary distance and with different light intensities for growth in a paper with 733 citations. They find that the high-light-adapted strains have the smallest genome while low-light-adapted strains have larger genomes. They further find that these two strains have large number of shared genes. Kaneko et al. (2001) determine the nucleotide sequence of the entire genome of nitrogen-fixing Anabaena sp. strain PCC 7120 in a paper with 513 citations. They find that the genome of A. consisted of a single chromosome and six plasmids. The chromosome bears potential protein-encoding genes, sets of rRNA genes, tRNA genes, and genes for small structural RNAs. They assign more than 60 genes involved in various processes of heterocyst formation and nitrogen fixation to the chromosome. They identify 195 genes coding for components of two-component signal transduction systems on the chromosome. Moore et al. (1998) isolate and analyze distinct co-occurring populations of Prochlorococcus at two locations in the North Atlantic in a paper with 498 citations. They find that co-isolates from the same water sample have very different light-dependent physiologies, one growing maximally at light intensities at which the other is completely photoinhibited. Despite this ecotypic differentiation, they further find that the co-isolates have 97% similarity in their 16S rRNA sequences. Microalgae  Rubio et al. (2001) compare PHR1 (Phosphate Starvation Response 1) gene of Arabidopsis thaliana with PSR1 (Phosphorus Starvation Response 1) gene from Chlamydomonas reinhardtii in a paper with 559 citations. They find

118  PART | III  Algal genomics

that PHR1, PSR1, and other members of the protein family share a MYB (myeloblastosis) domain and a predicted coiledcoil (CC) domain, defining a subtype within the MYB superfamily, the MYB-CC family. They further find that PHR1binding sequences are present in the promoter of Pi (phosphate) starvation-responsive structural genes and argue that this protein acts downstream in the Pi starvation signaling pathway. Other research fronts Algal structures  Microalgae: Pazour et al. (2000) study the Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737 in a paper with 651 citations. They clone and sequence a C. cDNA encoding the IFT88 subunit of the IFT (Intraflagellar transport) particle and identify a C. insertional mutant that is missing this gene. They show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal and argue that IFT is important for primary cilia assembly in mammals. Cyanobacteria: Ishiura et al. (1998) study the expression of a gene cluster kaiABC as a circadian feedback process in Synechococcus in a paper with 456 citations. They find that a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in this cyanobacterium, and KaiA sustains the oscillation by enhancing kaiC expression. Algal biofuels  Radakovits et al. (2010) discuss the genetic engineering of algae for optimized biofuel production in a review paper with 500 citations. They focus on the potential paths of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biofuels. Algal photosynthesis  Williams (1988) carries out the molecular analysis of electron transport in the photosystem II reaction center (PSII) of Synechocystis 6803 in a paper with 764 citations. He outlines a procedure for deleting PSII genes from this cyanobacterium to create a PSII mutant, replacing the deleted genes to restore photosynthetic function as well as the properties of the genetic transformation system in this cyanobacterium.

7.4 Discussion As there have been nearly 16,000 core papers related to the algal genomics, comprising more than 10% of the papers on the algae as a whole, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts (Konur, 2011, 2012a,b,c,d,e,f,g,h,i,j, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a,b,c,d,e,f,g,h,i, 2017a,b,c,d,e, 2018a,b,c, 2019a,b,d, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). The primary mode of scientific communication has been articles while reviews have formed 3.2% of the sample. The primary index has been SCI-E indexing more than 99.6% of the papers while only six papers have been indexed by the SSCI focusing on the societal aspects of bioenergy and biofuels. These findings suggest that there is substantial room for the research on the societal and humanitarian aspects of algal genomics such as consumer studies in in this field as well as scientometric studies in genomics (Konur, 2016a, 2019b). The most-prolific keywords related to the algal genomics have been determined through the detailed examination of the influential papers with at least 100 citations. A detailed keyword set has been devised for the search (given in Appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the genomics have been ‘*dna, gene, *genetic*, *genom*, phylogen*, sequenc*, *transcript*’. The other prolific key words have been ‘cloning, codon, mutant*, *nucleot*, proteom*, *rna’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, and algal, dinoflagellate*, macroalga*, rhodophyt*, and seaweed*, bacillariophycea*, diatom, diatoms, and cyanobacter*’. These keywords have formed the primary research fronts for the algal genomics. The findings show that although over 30,000 authors have contributed to the research, 20 most-prolific authors have shaped the literature on the algal genomics publishing 6.5% and 31.6% of all the papers and the influential papers, respectively (Table 7.1) (Konur, 2016a, 2019b). The success of these authors and their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988). The data provides the evidence for the presence of the significant gender deficit among both the lead authors (Table 7.1) and the authors of the citation classics (Table 7.7) (Bordons et al., 2003; Konur, 2016a, 2019b). The data on the papers by the most-prolific authors highlight the primary research fronts as the ‘algal phylogeny’, ‘algal photosynthesis’, ‘algal ecology’, ‘algal toxicology’, and ‘algal structures’. The data in Table 7.1 provides information on the most-prolific and influential authors, institutions, countries, journals, topics, and their citation impact in terms of the I-100 and I-100% by these authors.



The scientometric analysis of the research on the algal genomics Chapter | 7  119

It has been found during the search process that the author names with two or more forenames or surnames have been spelt differently by the databases with significant implications for the recovery of their papers. For example, there have been 33 and 29 papers for ‘Van Etten JL’, and ‘Vanetten JL’, respectively. As another example, there have been 25 and 17 papers for ‘Merchant SS’ and ‘Merchant S’, respectively. Other similar difficulties have arisen for ‘Medlin LK’, ‘de Marsac NT’, ‘van de Peer Y’, ‘Ball SG’, ‘Paulsen IT’, ‘Larimer FW’, ‘Buikema WJ’, and ‘Garcia-Pichel F’. Similar difficulties have also been observed for the common names such as ‘Wang Y’ or ‘Li Y’ for the recovery of their papers and the related analysis. The data shows that although 137 countries and territories have contributed to the research in algal genomics, mostprolific 20 countries contributed to 111.8% and 148.4% of all the papers and the influential papers, respectively (Table 7.2). The major producers of the research have been the United States, Japan, and Europe as these countries have had the ‘firstmover advantage’ over the other countries. It is notable that the citation impact of China has been small in relation to these top producers as China has produced 11.6% and 1.8% of all the papers and influential papers, respectively (Guan and Ma, 2007; Konur, 2016a, 2019b). As in the case of countries, although over 4600 institutions have contributed to the research in algal genomics, the 20 most-prolific institutions from the United States, Japan, and Europe, having the first-mover advantages, have published more than 28% of all the papers and 55% of the influential papers, respectively (Table 7.3). As over 47% of the papers have declared a research funding, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009, Konur, 2016a, 2019b). It is notable that there has been significant research funding opportunities in China in relation to the United States and Europe. The steady rise in the number of the publications in this field in the 2000s and 2010s (as of December 2018) provides the strong evidence for the increasing public importance of the algal genomics in recent years (Fig. 7.1). The annual number of publications have risen to nearly 900 papers and it is expected that the number of papers would exceed 15,000 papers in the next decade, corresponding to the increasing importance of the algal genomics to the global society at large. Although over 1400 journals have contributed to the research in algal genomics, the 20 most-prolific journals, having the first-mover advantages, have published over 23% and 55% of all the papers and influential papers, respectively (Table 7.4). This finding has been most relevant for ‘Proceedings of the National Academy of Sciences of the United States of America’ publishing 1.4% and 10.2% of all the papers and influential papers, respectively. The data on the Web of Science subject categories suggests that the first seven subjects, covering over 120% of the influential papers have formed the scientific basis of the research in this field: ‘Biochemistry & Molecular Biology’, ‘Plant Sciences’, ‘Multidisciplinary Sciences’, ‘Microbiology’, ‘Genetics Heredity’, ‘Cell Biology’, and ‘Marine Freshwater Biology’ (Table 7.5). As the journals indexed by the subject category of ‘Genetics & Heredity’ have published only over 14% of both all the papers and influential papers, the broad search strategy, covering all subject categories, developed for this study, has been justified. The data on the research fronts have confirmed that the major research fronts have been ‘cyanobacterial photosynthesis’ and ‘microalgal genomic studies’. The other key research fronts have been ‘cyanobacterial ecology’, ‘phylogeny of dinoflagellates and coccolithophores’, ‘cyanobacterial genomic studies’, ‘cyanobacterial toxicology’, and ‘macroalgal phylogeny’. The most-studied the types of algae has been ‘cyanobacteria’ and ‘microalgae’. On the other hand, the most-studied individual research fronts have been ‘algal phylogeny’, ‘algal genomic methodology’, ‘algal photosynthesis’, and ‘algal ecology’ with 25.9%, 21.3%, 15%, and 14.9% of these influential papers, respectively. The extensive data on the 20 citation classics largely confirm the findings of the earlier sections based on the 15,833 papers (Table 7.7). There has been significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely under-represented in the citation classic sample as there has been only one review. Similarly, the most-prolific research fronts have been ‘algal phylogeny’, ‘genomic methodology of algae’, and ‘algal ecology’ with seven, five, and four papers, respectively. The most prolific type of algae have been ‘microalgae’ and ‘cyanobacteria’ with eight papers each. The most-studied type of algae on the individual basis has been ‘Chlamydomonas’ with four papers. The most-studied topics have included ‘genomic divergence of algae’, ‘comparative phylogenomic analysis of algae’, and ‘genome sequencing of algae’. It appears that the structure-processing-property relationships form the basis of the research in algal genomics as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

120  PART | III  Algal genomics

7.5 Conclusion This analytical study of the research in algal genomics at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the development of the research and practice in this field. Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as bioenergy and biofuels (Konur, 2012a,b,c,d,e,f,g,h,i,j, 2018a,b), nanobiomaterials (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e, 2019c), biomedicine (Konur, 2016i, 2018c), complementing 510 literature reviews. The data has shown that the annual number of papers in this field has risen to nearly 900 papers while there have been over 15,800 papers over the study period from 1980 to 2018. It is further expected that the size of the research output would continue to increase in the incoming years and decades with another estimated 15,800 papers in the next decade in the first instance, corresponding to the increasing public importance of the algal genomics to the global society at large. The key research fronts have been cyanobacterial photosynthesis’, ‘microalgal genomic studies’, ‘cyanobacterial ecology’, ‘phylogeny of dinoflagellates and coccolithophores’, ‘cyanobacterial genomic studies’, ‘cyanobacterial toxicology’, and ‘macroalgal phylogeny’. The most-studied the types of algae has been ‘cyanobacteria’ and ‘microalgae’. On the other hand, the most-studied individual research fronts have been ‘algal phylogeny’, ‘algal genomic methodology’, ‘algal photosynthesis’, and ‘algal ecology’. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies (Konur, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). It has been found that the detailed keyword set provided in Appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts.

Appendix. The keyword sets used for the search of the literature on the algal genomics A.1  Genomics related keywords (TI = (clade* or ‘chloroplast transformation’ or chromosom* or cloning or codon or cytogen* or *dna or dnab or epigen* or gene or genes or *genetic* or *genom* or genotyp* or intron* or lipidom* or ‘metabolic eng*’ or metabolom* or ‘molecular evolution’ or mutagen* or mutant* or mutation*or ‘nuclear transformation’ or *nucleot* or omics or pcr or phycodna* or phylogen* or proteom* or ribosom* or *rna or sequenc* or ‘synthetic biology’ or ‘systems biology’ or ‘transcribed spacer*’ or *transcript* or ‘trans-splic*’ or transsplic* or transgen* or transposon) OR WC = (genetic*) or SO = (‘Acs Synthetic Bio*’ or ‘Bmc Systems Bio*’ or ‘Iet Systems Bio*’ or ‘Metabolic Eng*’ or ‘Molecular Systems Bio*’ or ‘Clinical Proteomics’ or ‘Comparative Biochemistry and Physiology D-Genomics*’ or ‘Current Proteomics’ or ‘Expert Review of Proteomics’ or ‘Journal of Proteomics’ or ‘Molecular & Cellular Proteomics’ or Proteomics* or ‘Current Metabolomics’ or Metabolomics or ‘Molecular Omics’ or Omics* or ‘Integrative Biology’ or ‘Biochimica et Biophysica Acta-Gene*’ or ‘Development Genes*’)) NOT TI = (clone* or ftir* or ergenom* or optogen* or neuron*)

A.2  Algae-related keywords A.2.1  Algae general TI = (alga or algae or algal or chlorarachn* or Bigelowiella or chromophyt* or phycolog* or phycodna*) OR SO = (Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘Cryptogamie Algologie’ or ‘European Journal of Phycology’ or Fottea* or ‘Harmful Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Research’).

A.2.2  Dinoflagellates and coccolithophores TI = (azaspiracid* or brevetoxin* or chrysophycea* or chlorococcales or chrysophyt* or ciguatera or *ciguatoxin* or *coccolith* or coccolithophore* or dinocyst* or dinoflagell* or dinophyceae or dinophyt* or haptophyt* or maitotoxin* or palytoxin* or *pectenotoxin* or peridiniales or picophytoplankton or prymnesiophycea* or raphidophycea* or raphidophyt* or saxitoxin* or ‘shellfish poison*’ or ‘shellfish toxin*’ or *yessotoxin* or zooxanthella* or Akashiwo or Alexandrium or Amphidinium or Aureococcus or *Ceratium or *Chattonella or Cochlodinium or Crypthecodinium or Dinophysis



The scientometric analysis of the research on the algal genomics Chapter | 7  121

or Emiliania or Gambierdiscus or *Gonyaulax or *Gymnodinium or Gyrodinium or Hematodinium or Heterocapsa or Heterosigma or Isochrysis or Karenia* or Karlodinium or Mallomonas or Noctiluca or Ochromonas or Ostreopsis or Oxyrrhis or Peridinium or Pfiesteria or phaeocystis or Prorocentrum or Prymnesium or Scrippsiella or Symbiodinium or Vaucheria).

A.2.3 Microalgae TI = (chlorophycea* or chlorophyt* or cryptomonad* or cryptophycea* or cryptophyt* or euglen* or eustigmatophycea* or ‘green alga*’ or microalga* or ‘micro-alga*’ or *prasinophycea* or * streptophyt* or tbrebouxiophycea* or volvocales or Acetabularia or Ankistrodesmus or Asteromonas or Aurantiochytrium or Botryococcus or Chaetoceros or Chlamydomonas or *Chlorella or *Chlorococcum or Coccomyxa or Cyanidioschyzon or Cyanidium or Desmodesmus or Dunaliella or Euglena or Galdieria or Haematococcus or Micrasterias or Micromonas or Monoraphidium or Nannochloropsis or Neochloris or Ostreococcus or Pediastrum or Phormidium or Platymonas or Polytomella or Porphyridium or Prototheca or Pseudokirchneriella or Pyramimonas or Scenedesmus or Schizochytrium or Selenastrum or Tetraselmis or Trebouxia or Volvox).

A.2.4 Macroalgae TI = (‘brown alga*’ or carrageenan* or characea* or charophyt* or cladophorales or cryptonemiales or dictyotales or florideophycea* or fucale* or *fucan* or fucoid* or fucoidan* or fucoxanthin or gelidiales or gigartinale* or gracilariales or kelp or laminariale* or laminarin or macroalga* or ‘macro-alga*’ or phaeophycea* or phaeophyt* or phlorotannin* or ‘red alga*’ or rhodophyceae or rhodophyt* or seaweed* or ‘sulfated polysaccharide*’ or ulvale* or ulvan* or ulvophycea* or zygnematophycea* or ‘Chara vulgaris’ or Alaria or Ascophyllum or Asparagopsis or Asparagopsis or Bangia* or Bostrychia or Bryopsis or Catenella or Caulerpa or Ceramium or Chaetomorpha or Chondrus or Cladophora or Codium or Corallina or Cystoseira or Ecklonia or Ectocarpus or ‘Eisenia bicyclis’ or Enteromorpha or Eucheuma or Fucus or Gelidium or Gigartina* or Gracilaria or Gracilariopsis or Grateloupia or Halimeda or Halocynthia or Hizikia or Kappaphycus or Klebsormidium or Laminaria or Laurencia* or Lessonia or Lomentaria or Macrocystis or Monostroma or Mougeotia or Nitella or Nitellopsis or Oedogonium or Padina or Palmaria or Pelvetia or Plocamium or Polysiphonia or Porphyra or Pyropia or Saccharina or Sargassum or Scytosiphon or Solieria or Spirogyra or Turbinaria or Ulva or Undaria).

A.2.5 Diatoms (TI = (bacillariophycea* or bacillariophyt* or diatom or diatomaceous or diatomite or diatoms or ‘domoic acid*’ Asterionella or Chaetoceros or Coscinodiscus or Cyclotella or Cylindrotheca or Didymosphenia or Navicula or Nitzschia or Skeletonema or Stephanodiscus or Thalassiosira) OR SO = (‘Diatom Research’)) NOT (TI = (diatomic* or atom* or diatomyid*) OR SO = (‘Journal of Chemical Physics’))

A.2.6 Cyanobacteria TI = (allophycocyanin* or anabaen* or anatoxin* or ‘blue green alga*’ or ‘bluegreen alga*’ or ‘blue-green alga*’ or bmaa or ‘C-phycocyanin’ or cryptophycin* or cyanelle* or cyanobacter* or cyanophage* or cyanophycin* or cyanophyt* or cyanophyceae or cyanotoxin* or cyanovirin* or cylindrospermopsin* or cylindrospermopsin* or *lyngbya or ‘methylaminol-alanine*’ or *microcystin* or nodularin* or nostocales or oscillatoriales or phycobiliprotein* or phycobilisome* or phycocyanobilin* or phycobilin* or *phycoerythrin* or phycocyanin* or picocyanobacter* or *saxitoxin* or *teleocidin* or Acaryochloris or *Anabaena or Anacystis or Aphanizomenon or Aphanothece or Arthrospira or Calothrix or Cyanophora or Cyanothece or Cylindrospermopsis or *Lyngbya* or Mastigocladus or Microcoleus or Microcystis or Moorea or Nodularia or Nostoc or Oscillatoria or Planktothrix or Plectonema or Prochlorococcus or Prochloron or Prochlorothrix or Scytonema or Spirulina or *Synechococcus or Synechocystis or Tolypothrix or Trichodesmium) NOT TI = (leuconostoc).

A.2.7  Algae-related journals SO = (Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘Cryptogamie Algologie’ or ‘Diatom Research’ or ‘European Journal of Phycology’ or Fottea* or ‘Harmful Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Bulletin’ or ‘Phycological Research’).

Acknowledgments The significant contribution of the authors of the pioneering studies in algal genomics (authors listed as the ‘most-prolific and influential authors’ in Table 7.1 and authors listed as the ‘lead authors’ in Table 7.7) to the development of the research in in this field have been gratefully acknowledged. These authors have published at least 16 papers and 5 influential papers with at least 100 citations, respectively in algal genomics.

122  PART | III  Algal genomics

References Abramo, G., D’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Armbrust, E.V., Berges, J.A., Bowler, C., Green, B.R., Martinez, D., Putnam, N.H., et al., 2004. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306 (5693), 79–86. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., et al., 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456 (7219), 239–244. Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., et al., 1988. Chloroplast transformation in Chlamydomonas with high-velocity microprojectiles. Science 240 (4858), 1534–1538. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sustain. Energy Rev. 14 (2), 557–577. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Derelle, E., Ferraz, C., Rombauts, S., Rouze, P., Worden, A.Z., Robbens, S., et al., 2016. Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc. Natl. Acad. Sci. U. S. A. 103 (31), 11647–11652. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Guan, J., Ma, N., 2007. China’s emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol. Adv. 29 (2), 189–198. Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., et al., 1998. Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. Science 281 (5382), 1519–1523. Kaneko, T., Nakamura, Y., Wolk, C.P., Kuritz, T., Sasamoto, S., Watanabe, A., et al., 2001. Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res. 8 (5), 205–213. Kindle, K.L., 1990. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U. S. A. 87 (3), 1228–1232. Kohler, S., Delwiche, C.F., Denny, P.W., Tilney, L.G., Webster, P., Wilson, R.J.M., et al., 1997. A plastid of probable green algal origin in apicomplexan parasites. Science 275 (5305), 1485–1489. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books Ltd., Stoke on Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2012a. Prof. Dr. Ayhan Demirbas’ scientometric biography. Energ. Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012b. The evaluation of the biogas research: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012c. The evaluation of the bio-oil research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (1), 379–392. Konur, O., 2012d. The evaluation of the biorefinery research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (1), 347–358. Konur, O., 2012e. The evaluation of the research on the biodiesel: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012f. The evaluation of the research on the bioethanol: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012g. The evaluation of the research on the biofuels: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012h. The evaluation of the research on the biohydrogen: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012i. The evaluation of the research on the microbial fuel cells: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012j. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012k. The evaluation of the global research on the education: a scientometric approach. Procedia Soc. Behav. Sci. 47, 1363–1367. Konur, O., 2012l. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1893–1908. Konur, O., 2012m. The evaluation of the educational research: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012n. The evaluation of the interdisciplinary research on the terrorism: a scientometric approach. Soc. Polit. Econ. Cult. Res. 4 (3–4), 113–144. Konur, O., 2012o. The evaluation of the research on the Arts and Humanities in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (3), 1603–1618.



The scientometric analysis of the research on the algal genomics Chapter | 7  123

Konur, O., 2012p. The evaluation of the research on the doctoral education: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (si1), 593–600. Konur, O., 2012q. The gradual improvement of disability rights for the disabled tenants in the UK: the promising road is still ahead. Soc. Polit. Econ. Cult. Res. 4 (3), 71–112. Konur, O., 2012r. The scientometric evaluation of the institutional research: the Karadeniz Universities—part 3. Energ. Educ. Sci. Technol. B 4 (si1), 850–856. Konur, O., 2012s. The scientometric evaluation of the institutional research: the Karadeniz Universities—part 2. Energ. Educ. Sci. Technol. B 4 (si1), 844–849. Konur, O., 2012t. The scientometric evaluation of the institutional research: the Karadeniz Universities—part 1. Energ. Educ. Sci. Technol. B 4 (si1), 836–843. Konur, O., 2012u. The scientometric evaluation of the institutional research: the Eastern Anatolian Universities—part 2. Energ. Educ. Sci. Technol. B 4 (si1), 813–819. Konur, O., 2012v. The scientometric evaluation of the institutional research: the Eastern Anatolian Universities—part 1. Energ. Educ. Sci. Technol. B 4 (si1), 794–802. Konur, O., 2012y. The scientometric evaluation of the institutional research: the Southeastern Anatolian Universities—part 2. Energ. Educ. Sci. Technol. B 4 (si1), 776–781. Konur, O., 2012z. The scientometric evaluation of the institutional research: the Southeastern Anatolian Universities—part 1. Energ. Educ. Sci. Technol. B 4 (si1), 763–769. Konur, O., 2012. The scientometric evaluation of the institutional research: the 2008 Universities—Part 2. Energ. Educ. Sci. Technol. B 4 (si1), 568–574. Konur, O., 2012. The scientometric evaluation of the institutional research: the 2008 Universities—Part 1. Energ. Educ. Sci. Technol. B 4 (si1), 547–553. Konur, O., 2012. The scientometric evaluation of the institutional research: the Sirnak University case. Energ. Educ. Sci. Technol. B 4 (si1), 521–529. Konur, O., 2012. The scientometric evaluation of the research on the students with learning disabilities in higher education. Soc. Polit. Econ. Cult. Res. 4 (1–2), 1–69. Konur, O., 2012. The scientometric evaluation of the research on the deaf students in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1573–1588. Konur, O., 2012. The scientometric evaluation of the research on the students with ADHD in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–626. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–682. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–108. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–556. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–422. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–302. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–370. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2016c. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016d. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016f. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016g. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486.

124  PART | III  Algal genomics

Konur, O., 2016h. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016i. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Algal drugs: the state of the research. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019b. Algal genomics. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019c. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O., 2019d. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O. (Ed.), 2020a. The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), The Handbook of Algal Science, Microbiology, Technology, and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Lieberman, M.B., Montgomery, D.B., 1988. First-mover advantages. Strateg. Manage. J. 9 (S1), 41–58. Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S., Lins, T., et al., 2002. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. U. S. A. 99 (19), 12246–12251. Matsuzaki, M., Misumi, O., Shin-I, T., Maruyama, S., Takahara, M., Miyagishima, S.Y., et al., 2004. Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428 (6983), 653–657.



The scientometric analysis of the research on the algal genomics Chapter | 7  125

Merchant, S.S., Prochnik, S.E., Vallon, O., Harris, E.H., Karpowicz, S.J., Witman, G.B., et al., 2007. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318 (5848), 245–251. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Moore, L.R., Rocap, G., Chisholm, S.W., 1998. Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature 393 (6684), 464–467. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Nubel, U., Garcia-Pichel, F., Muyzer, G., 1997. PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl. Environ. Microbiol. 63 (8), 3327–3332. Pazour, G.J., Dickert, B.L., Vucica, Y., Seeley, E.S., Rosenbaum, J.L., Witman, G.B., et al., 2000. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151 (3), 709–718. Radakovits, R., Jinkerson, R.E., Darzins, A., Posewitz, M.C., 2010. Genetic engineering of algae for enhanced biofuel production. Eukaryot. Cell 9 (4), 486–501. Rocap, G., Larimer, F.W., Lamerdin, J., Malfatti, S., Chain, P., Ahlgren, N.A., et al., 2003. Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424 (6952), 1042–1047. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Rubio, V., Linhares, F., Solano, R., Martin, A.C., Iglesias, J., et al., 2001. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev. 15 (16), 2122–2133. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes-towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Scholin, C.A., Herzog, M., Sogin, M., Anderson, D.M., 1994. Identification of group- and strain-specific genetic-markers for globally distributed Alexandrium (dinophyceae). II. Sequence analysis of a fragment of the LSU rRNA gene. J. Phycol. 30 (6), 999–1011. Turner, S., Pryer, K.M., Miao, V.P.W., Palmer, J.D., 1999. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J. Eukaryot. Microbiol. 46 (4), 327–338. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Williams, J.G.K., 1988. Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol. 167, 766–778.

Chapter 8

Metabolic engineering of cyanobacteria for production of platform chemicals: A synthetic biology approach Neha Arora, Damini Jaiswal, Shinjinee Sengupta, Pramod P. Wangikar Indian Institute of Technology Bombay, Mumbai, India

Abbreviations 2,3-DHIV 2,3-dihydoxyisovalerate 2-KB 2-ketobutyrate 2KV 2-ketovalerate 2-PGA 3-phosphoglyceric acid ackA acetate kinase adc acetoacetate decarboxylase adh secondary alcohol dehydrogenase adhE2 aldehyde-alcohol dehydrogenase ADP adenosine diphosphate ALE adaptive laboratory evolution alsD 2-acetolactate decarboxylase alsS acetolactate synthase AR acetoin reductase aspC aspartate aminotransferase atoB thiolase atoDA acetyl-CoA acyltransferase ATP adenosine triphosphate BDDH butanediol dehydrogenase buk butyrate kinase CDP-ME 4-diphosphocytidyl 2-C-methyl-d-erythriol CDP-MEP 4-diphosphocytidyl 2-C-methyl-d-erythriol phosphate cimA citramalate synthase crt crotonase CYTB6F cytochrome b6-f complex DhaB glycerol dehydratase DHAP dihydroxyacetone phosphatase DMAPP dimethylallyl diphosphate DXP 1-deoxy-d-xylulose 5-phosphate DXS 1-deoxy-d-xylulose 5-phosphate synthase efe ethylene forming enzyme Fdx 2Fe-2S ferredoxin FNR ferredoxin—NADP(+) reductase FPP farnesyl diphosphate fucO 1,2-propanediol reductase Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00008-5 © 2020 Elsevier Inc. All rights reserved.

127

128  PART | III  Algal genomics

G3P glyceraldehyde 3-phosphate gabD NADP-dependent succinate semialdehyde dehydrogenase galP galactose permease gldA glycerol dehydrogenase glgC glucose 1phosphate adenyl transferase gloAB glyoxalase gltA citrate synthase glyDH glycerol dehydrogenase GPD1 glycerol-3-phosphate dehydrogenase hbd 3-hydroxy butyryl-CoA dehydrogenase HMBPP 4-hydoxy-3-methylbut-2enyl-diphosphate HMG 3-hydroxy-3-methyl-glutary HmgR Hmg-CoA reductase HmgS Hmg-CoA synthase HOR2 glycerol-3-phosphatase idi isopentyl diphosphate isomerase ilvC acetohydroxyacid isomeroreductase ilvD dihydroxyacid dehydratase IPP isopentenyl diphosphate ispA farnesyl diphosphate synthase ispG hydroxylmethylbutenyl diphosphate reductase isps isoprene synthase kgd 2-keto glutarate decarboxylase kgtP alpha-ketoglutarate permease KIC ketoisocaproate KIV ketoisovalerate kivd ketoacid decarboxylase KMV 2-keto-3-methyl valerate ldhA lactate dehydrogenase leuBCD 3-isopropylmalate dehydrogenase lldP lactate/H+ symporter Mcr malonyl-CoA reductase MECPP 2-C methyl-d-erythritol-4-diphosphate MEP 2-C methyl-d-erythritol-4-phosphate MG methylglyoxal mgsA methylglyoxal synthase Mk mevalonic acid kinase Msr malonate semialdehyde reductase NADPH nicotinamide adenine dinucleotide phosphate nphT7 acetoacetyl-CoA synthase ogdc 2-OG decarboxylase PduP CoA-acylating propionaldehyde dehydrogenase phaB acetoacetyl-CoA reductase PhaCE PHB synthase phaJ R-specific crotonase PHB polyhydroxybutyrate PMD mevalonic acid 5-diphosphate decarboxylase PMK mevalonic acid 5-phosphate kinase ppc phosphoenolpyruvate carboxylase PQ plastoquinone PSI photosystem I PSII photosystem II pta phosphotransacetylase ptb phosphotransbutyrylase



Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  129

PYR pyruvate RuBP ribulose bisphosphate sadh primary-secondary alcohol dehydrogenase shc squalene hopene cyclase sigE sigma factor E skpyd4 β-alanine aminotransferase S-LG S-lactoylglutathione sqs squalene synthase ssdh succinate semialdehyde dehydrogenase sth soluble transhydrogenase ter trans-2-enoyl-CoA reductase Thl thiolase tpiA triosephosphate isomerase udhA uronate dehydrogenase xpkA xylulose-5-phosphate phosphoketolase xylEAB xylose isomerase and xylulokinase yqhD aldehyde reductase

8.1 Introduction The dependence of our modern society on fossil fuel-based energy and carbon-based chemicals since the 19th century has led to rapid depletion of non-renewable resources, escalation of greenhouse gas emissions and environment deterioration (Bornke and Broer, 2010; Mika et al., 2018; Sun et al., 2018). This scenario demands sustainable technologies for the production of biofuels and platform chemicals particularly from renewable and carbon-neutral resources (Geilen et al., 2010). Between the two, platform chemicals are the building blocks for an array of chemical intermediates and polymers (Jang et al., 2012). The bio-based platform chemicals can be categorized into four major groups depending on its carbon moieties, i.e., C3, C4, C5 and C6, respectively (MRF, 2018). Among these categories, C3 (glycerol and 3-hydroxypropinoic acid) contributes to the maximum share of 60.78%, in terms of volume. The US Department of Energy (DOE), in the year 2004, published a comprehensive report which identified top 12 building blocks, derived from biomass from over 300 possible platform chemicals capable of strengthening the bio-based economy (Werpy and Petersen, 2004). To this end, extensive efforts have been devoted towards the development and production of platform chemicals from bio-based raw materials including lignocellulosic biomass, plants and microorganism (natural/engineered). In addition, the global market for bio-based platform chemicals is expected to increase to $18.8 billion by 2021 with an annual growth of 8.3% (MRF, 2018). Bridging the gap between the production of renewable platform chemicals from low-titer and high priced, to a high-titer and low priced compounds can be met by addressing certain key factors which includes: (1) generation of engineered microorganisms for efficient production; (2) optimization of bioprocess and downstream parameters for increasing the yields of the product; (3) cost-effective and copious feedstocks for cultivation of microorganism; (4) compatibility with existing industrial bioprocess systems (Angermayr et al., 2015; Jang et al., 2012). Certain strains of microorganisms have become the natural choice for the production of platform chemicals (Lee et al., 2011). The traditional microbial platforms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae are the most promising candidates for renewable platform chemicals (Buschke et al., 2013). However, all of these bio factories rely on glucose or sugar-based feedstocks which increases the cost of production of the desired chemical (Hirokawa et al., 2015). In this regard, photosynthetic microorganisms particularly cyanobacteria offer an advantage as compared to the heterotrophic microorganism, due to their inherent capability to utilize sunlight and fix atmospheric CO2 for the generation of biomass (Desai and Atsumi, 2013). The key attributes that make cyanobacteria ideal hosts for chemical production include their high growth rate (compared to plants and algae), survival under edaphic environments, low cost minimal nutrient requirement and ease of genetic engineering (compared to algae) (Heimann, 2016). To date, various cyanobacteria have been engineered to produce an array of chemicals including isobutyraldehyde, isobutanol, 1-butanol, ethylene, isoprene, acetone, fatty acids, fatty alcohols, etc. by engineering exogenous pathways (Fig. 8.1). The present chapter summarizes the pathway engineered for the synthesis of different platform chemicals from cyanobacteria. The recent advancements and tools in the field of synthetic biology to enhance the production of chemical in cyanobacterial host has also been reviewed. Thereafter, an outlook on the challenges, limitations and future developments of tailoring cyanobacteria metabolism has been discussed.

130  PART | III  Algal genomics

4H+ ATP

ADP+Pi

FNR

NADPH

Fdx

PSII

PQ pool

Cytb6F PSI

2H2O O2 + 4H+

4H+ CO2

DXP

ATPase

NADPH ATP

Acehol

methylglyoxal

MEP

CDP-MEP

RuBP

MECPP

Glycerol DHAP

G3P

IPP DMAPP

FPP

Squalene

PYR

3-hydroxypropionaldehyde

Lactate

1, 3 propanediol 2-acetolactate

AcetyICoA

acetoacetate

citraconate

Isobutanol

2,3-butanediol

Crotonyl-CoA Butyryl-CoA

2-ketovalerate

1-butanol

Isopropanol

N-butyraldehyde

2-aceto-2-hydroxy butanoate 2,3-dihyroxy-3methylvalerate

isobutyraldehyde

3-hydroxybutyryl-CoA

acetate

b-methyl-D-malate

2, 3-dihydroxy isovalerate

acetonin

acetoacetyl-CoA

citrimalate

2-ketobutyrate

lactaldehyde

Glycerol-3-P

CBB cycle

HMBPP

Isoprene

1,2 propanediol

3-PGA

CDP-ME

1-butanol Citrate Oxaloacetate Isocitrate

2-methyl-1-butanol Malate

TCA cycle a ketoglutarate

Fumarate

Ethylene

Succinate semialdehyde Succinate

FIG. 8.1  Overview of platform chemicals from cyanobacteria based on the precursor molecules. Products derived from pyruvate, acetyl-CoA/TCA, and dihydroxyacetone phosphate are indicated in green and diagonal lines, pink and dotted, and blue and horizontal lines, respectively. An abbreviated Calvin Benson cycle (CBB) and tricarboxylic acid cycle (TCA) are shown.

8.2  Cyanobacteria: A chassis for platform chemicals Cyanobacteria (blue-green algae) are photosynthetic prokaryotes that are primary producers and progenitors to eukaryotic algae and plants (Stanier and Cohen-Bazire, 1977). They encompass distinct group of gram-negative prokaryotes which differ in morphology, physiology and reproduction (Rippka et al., 2009). Cyanobacteria capture energy from sunlight to synthesize high-energy intermediates particularly adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). These then drive the endergonic reactions of Calvin Benson-Bassham (CBB) cycle which fixes CO2 into organic carbon (Stanier and Cohen-Bazire, 1977). Depending on the product of interest, the cyanobacterial metabolism could be rewired by introducing heterologous enzymes via genetic engineering (Angermayr et al., 2015; Knoot et al., 2018; Oliver and Atsumi, 2014; Sengupta et al., 2018). An array of industrially important products can be produced from the cyanobacterial cell factories such as biomass, biofuels, bioplastics and platform chemicals (Heidorn et al., 2011). These sun-driven cell factories can also be utilized for bioremediation and production of biologically active compounds and as biofertilizers (Heidorn et al., 2011). Given the tremendous potential of cyanobacteria in the field of biotechnology and environment, to date more than 300 cyanobacterial genomes have been sequenced, annotated and publicly available (Hagemann and Hess, 2018; Shih et al., 2013).



Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  131

Among the cyanobacterial strains, Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 (hereafter referred to as PCC 7942, PCC 6803 and PCC 7002, respectively) have emerged as model strains for metabolic engineering. The strain PCC 7942 has been used since the early 1970s after it was shown to be naturally transformed (Shestakov and Khyen, 1970; Triana et al., 2014). It is an obligate phototroph, has a rod-like shape and the ability to survive in fresh water under low nutrients (JGI, 2018; Triana et al., 2014). The genome size of this cyanobacterium is ~2.7 Mb with two plasmids (~8 and 46 kb) and has a doubling time of 8.5 h (in BG-11 at 38 °C, 500 μmol photons m−2 s−1 and 3% CO2) (Yu et al., 2015). This cyanobacterium has also emerged as the prokaryotic model for studying the circadian rhythm (Holtman et al., 2005; Welkie et al., 2018). The manually curated genome scale metabolic model, iSyf715 of PCC 7942 was completed in the year 2014, which can provide guidance to engineer strains for the production of various platform chemicals (Table 8.1). iSyf715 contains 715 genes, 851 metabolic reactions, 838 metabolites, 530 enzymes, and 79 multimeric enzymes and enzymatic complexes (Triana et al., 2014). Synechocystis sp. PCC 6803 (hereafter PCC 6803) is an extensively studied, unicellular, non‑nitrogen fixing fresh water cyanobacterium. This is also the first cyanobacterium whose genome (~3.57 Mb) was completely sequenced (Fu, 2009; Kaneko et al., 1996). The doubling time of the cyanobacterium is 6.6 h when cultivated at 30 °C, 300 μmol photons m−2 s−1 and 3% CO2 in BG-11 (Yu et al., 2015). The updated genomic scale metabolic model of PCC 6803, iSyn731, comprising of 731 genes, 511 proteins, 1156 reactions, and 996 metabolites. This coupled with the detailed knowledge of its physiology have enabled the engineering of this cyanobacterium for production of various chemicals as listed in Table 8.2 (Knoop et al., 2010; Saha et al., 2012). Synechococcus sp. PCC 7002 is a fast-growing marine cyanobacterium (doubling time of 4.1 h when cultivated at 38 °C, 500 μmol photons m−2 s−1 and 3% CO2 and 2.6 h in ammonia or urea supplemented media) (Hendry et  al., 2016, 2017; Jaiswal et al., 2018a; Yu et al., 2015). The cyanobacterium has a genome size of ~3.4 Mb with six plasmids and is capable of growing under high salinity and high light intensity (Hendry et al., 2016). The genome scale model of PCC 7002 is iSyp708. The platform chemicals synthesized from PCC 7002 have been listed in Table 8.3. Indeed, these three model strains are capable of growing under various cultivation modes such as photoautotrophy, mixotrophy and heterotrophy and can efficiently take up foreign DNA either naturally or by conjugation. Furthermore, genetic markers, synthetic biology tools (neutral sites, promoters, ribosome binding sites and terminators) and facile replica plating methods are being established for these strains at a rapid pace (Fu, 2009; Holtman et al., 2005). The above properties make these strains attractive for the synthesis for platform chemicals. In the section below, the platform chemicals synthesized from these model cyanobacterial strains have been discussed to illustrate the push and pull strategy of genetic engineering for enhancing the titers of the target product.

8.3  Strategies for enhancing the titers of platform chemicals in cyanobacterial strains In general, the main engineering strategies for the production of platform chemicals include: 1. Selection of a metabolic pathway and the relevant enzymes from different biological sources, codon optimization and expression. It is desired that the chosen enzymes possess high specific activity and the ability to remain active under oxygenic environment. 2. Utilization of strong native promoters (cpcB560, psbA2), optimization of the ribosomal binding sites (RBS) of cyanobacteria, use of high copy number plasmids resulting in optimal expression of target genes. 3. Knocking down the competing pathways such as polyhydroxybutyrate, glycogen, acetate, lactate, etc. to enhance the carbon flux toward precursor molecules. 4. Adaptive laboratory evolution (ALE) to improve the tolerance toward the products of interest. Table 8.4 shows that many of the platform chemicals show growth retardation at fairly low concentrations and ALE of the strains would be necessary. However, to date, only a limited number of ALE studies have been carried out for improving the tolerance toward the target product. For example, the butanol tolerance in PCC 6803 was increased from 0.2% (v/v) to 0.5% (v/v) while the isobutanol tolerance was increased from 2 to 5 g/L, respectively (Matsusako et al., 2017; Wang et al., 2014). Further, evolution of strains for improved tolerance toward intermediates metabolites in the pathway may also lead to improved fitness of the cyanobacteria. 5. Overexpressing transporters for higher efflux rates such as lactate transporters which enhanced the lactate titers. 6. Introducing transhydrogenase for the interconversion of NADPH and NAD+, thereby increasing the pool of NADH. 7. Cultivating the engineered strain under photomixotrophic mode which has an advantage of overcoming the stagnant growth of cyanobacteria in dense cultures due to light limitation or under dark conditions as the additional carbon source in the media could be then be utilized by the cells (Kanno and Atsumi, 2017).

132  PART | III  Algal genomics

TABLE 8.1  List of platform chemicals, genes expressed/deleted, promoter utilized, titers attained and cultivation time in Synechococcus elongatus PCC 7942. Platform chemical

Genes (overexpressed/ deleted)

Promoter

Titer (g/L)

Cultivation time (days)

Reference

2,3-Butanediol

alsS, alsD, adh

PLlacO1

2.38

21

Oliver et al. (2013)

0.5

3

Oliver et al. (2014)

1.1

4

McEwen et al. (2016)

alsS, alsD, adh, galP

1

4

alsD, alsS, adh, glpFKD

0.6

2

alsD, alsS, adh, glpFKD,tpiA

0.5

2

0.02

5

Chwa et al. (2016)

alsS, alsD, adh alsS, alsD, adh, xylEAB

PLlacO1, Ptrc

Acetone

nphT7, atoDA, adc, xpkA, pta

Isopropanol

Thl, atoAD, adc, sadh, pta

0.03

14

Hirokawa et al. (2017a)

Thl, atoA, atoD, adc

0.03

9

Kusakabe et al. (2013)

Thl, atoA, atoD, adc, sadh

0.2

15

Hirokawa et al. (2015)

PLlacO1, Ptrc

1.22

14

Hirokawa et al. (2017c)

PLlacO1

0.28

14

Hirokawa et al. (2016)

1,3-Propanediol

GPD1, HOR2, DhaB, yqhD

PLlacO1

Kanno and Atsumi (2017)

1,2-Propanediol

mgsA, yqhD, gldA, sadh

Ptrc

0.15

10

Li and Liao (2013)

1-Butanol

atoB, adhE2, ter, crt, hbd

PLlacO1, Ptrc

0.02

7

Lan and Liao (2011)

nphT7, phaB, phaJ, ter, PduP, YqhD

PLlacO1

0.4

12

Lan et al. (2013)

nphT7, bldh, yqhD, phaJ, phaB, ter

PLlacO1, Ptrc

0.03

20

Lan and Liao (2011)

1.1

30

Lai and Lan (2018)

0.6

8

Li et al. (2014)

0.45

6

Atsumi et al. (2009)

0.3

12

Shen and Liao (2012)

nphT7, phaB, phaJ, ter, ptb, buk Isobutanol

ΔglgC, kivd, yqhD, ilvCD

PLlacO1

alsS, ilvC, ilvD, kivd, yqhD 25

2-Methyl-1-butanol

Kivd, yqhD, cimAΔ2 , leuBCD

Ptrc

3-Hydroxy propionic acid

Mcr, Msr

0.65

16

Lan et al. (2015)

Mcr, Msr, ppc, aspC, adc, Skpyd4

0.35

16

Lan et al. (2015)

Succinate

gabD, kgd, gltA, ppc

0.4

8

Lan and Wei (2016)

Δglgc, gltA, ppc

0.43 mg/L

2

Li et al. (2016)

Δglgc, sdhB

Psmt

0.63 mg/L

2

Huang et al. (2016)

mgsA, lldP, gloAB

PLlacO1

1.23

24

Hirokawa et al. (2017b)

ldhA, lldP, udhA

Ptrc

0.2

4

Niederholtmeyer et al. (2010)

ldhDnARsdR, lldP

0.83

10

Li et al. (2015)

Isobutyraldehyde

alsS, ilvC, ilvD

1.1

12

Atsumi et al. (2009)

Squalene

Dxs, idi, ispA, sqs(II)

PLlacO1

0.02

10

Choi et al. (2017)

PcpcB1

0.007

Dxs, idi, ispA, sqs

PLlacO1

4.83 mg/L/ OD730



Choi et al. (2016)

Idi, isps, dxs, ispG

Ptrc

1.26

21

Gao et al. (2016)

d-Lactate

Isoprene

Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  133



TABLE 8.2  Details of platform chemicals derived from Synechocystis sp. PCC 6803 along with genes expressed/deleted, promoter utilized, titers attained and cultivation time information. Platform chemical

Genes (overexpressed/ deleted)

Promoter

Titer (g/L)

Cultivation time (days)

Reference

d-Lactate

Gld A101-syn, sth

Ptrc

1.14

12

Varman et al. (2013b)

ldh, ΔphaCE, Δpta

Pcpc560

1.06

7

Zhou et al. (2014)

ldh, sth

Ptrc

0.2

15

Angermayr et al. (2012)

Gld A101-syn, sth + anaerobic digestion wastewater

1.26

20

Hollinshead et al. (2014)

ldhB, ldhL

0.02

18

Joseph et al. (2014)

ldh-syn

0.12

10

Angermayr and Hellingwerf (2013)

l-Lactate

2,3-Butanediol

Als, aldc, ar

Ptrc

0.6

29

Savakis and Hellingwerf (2015)

Isobutanol

Kivd, adh

Plac

0.3

21

Varman et al. (2013a)

1-Butanol

phaA, phaB, phaJ, ter, adh2, ΔphaCE

PpsbA2, Ptrc

0.02

8

Anfelt et al. (2015)

Isoprene

IpsS

PpsbA2

50 μg/DCW/D



Lindberg et al. (2010)

250 μg/DCW/D



Bentley et al. (2014)

Fni, IpsS, mk, pmk, pmd, Hmgs, Hmg-CoA, AtoB, ΔglgX/ΔglgA IpsS, dxs

PrbcL

1.16 ng/mL h



Pade et al. (2016)

IpsS, fni, Δcpc operon

PpsbA2

400 μg/L

3

Chaves et al. (2016)

2.5 mg/L

3

Chaves et al. (2017)

Cpcb*L7*IpsS Squalene

Δshc



0.67 mg/OD750/L



Bergquist et al. (2014)

Acetone

Adc, cftAB, Δpta, ΔphaCE

Pcpc, PrbcL

0.036

4

Zhou et al. (2012)

Succinate

ΔackA, sigE

PpsbaII

0.004

3

Osanai et al. (2015)

ppc

Ptrc

0.192

3

Hasunuma et al. (2016)

1.8

3

Hasunuma et al. (2018)

2.3

25

Ungerer et al. (2012)

718 μL/L/h



Xiong et al. (2015)

ppc Ethylene

efe

PpsbA

efe

1,2-Propanediol

Δogdc, Δssdh, 3Xefe, kgtP

PpsbA2

1.17

6

Zhu et al. (2015)

ΔglgC, efe

Ptrc

718 μL/L/h



Veetil et al. (2017)

1

12

David et al. (2018a)

72 μg/L

7

Ziegler et al. (2014)

msgA, yqhD, adh gldA, yqhD, mgsA, fucO



8.4  Engineering model cyanobacterial strains for platform chemicals The platform chemicals synthesized from cyanobacteria can be broadly categorized depending on the precursor molecule as illustrated in Fig. 8.1. As depicted in Fig. 8.1, the three major precursor molecules include; pyruvate, acetyl-CoA and dihydroxyacetone phosphate (DHAP). Indeed, a particular platform chemical can be synthesized from two or more completely different pathways for example 1-butanol can be synthesized from pyruvate as well as acetyl-coA (Fig. 8.1). An overview of the engineered cyanobacterial strains for respective platform chemicals is summarized in the below sections.

134  PART | III  Algal genomics

TABLE 8.3  Production of platform chemicals along with the genes expressed/deleted, promoter utilized, titers attained, and cultivation time from Synechococcus sp. PCC 7002. Platform chemical

Genes (overexpressed/deleted)

Promoter

Titer (g/L)

Cultivation time (days)

Reference

Lactate

ldh, ΔglnA

Plac

0.92

4

Gordon et al. (2016)

2,3-Butanediol

alsD, adh

1.6

16

Nozzi et al. (2017)

1,2-Propanediol

gldA, yqhD, mgsA, fucO



0.2 mg/L

7

Ziegler et al. (2014)

1,3-Propanediol

DAR1, GPP2, dhaB1-3, orf2, orf2b, yqhD



1 mg/L

7

Chin et al. (2015)

TABLE 8.4  The initial tolerance (g/L) of model cyanobacterial strains toward toxic platform chemicals. Product

Cyanobacterial strain

Initial tolerance (g/L)

Reference

Isobutanol

PCC 7942

0.75

Atsumi et al. (2009)

PCC 6803

2

Matsusako et al. (2017)

PCC 7942

8

Dexter and Fu (2009)

PCC 6803

12

Matsusako et al. (2017)

Isopropanol

PCC 7942

5

Arai et al. (2017)

2,3-BD

PCC 7942

20

Oliver et al. (2013)

2-Pentanol

PCC 6803

0.8

Matsusako et al. (2017)

1-Butanol

PCC 6803

1

Matsusako et al. (2017)

Ethanol

8.4.1  Pyruvate derived products The pyruvate derived platform chemicals production pathways include lactate, 2,3-butanediol, isobutyraldehyde, isobutanol, 1-butanol, 2-methyl-1-butanol, isoprene, and squalene.

8.4.1.1 Lactate Lactate (both l and d forms) is an essential substrate for the production of biodegradable plastics (polylactic acid) and also widely utilized in food additives, cosmetics and various pharmaceutical compounds (Hirokawa et al., 2017b). It has been reported that ~450 million kg of polylactic acid (PLA) is produced annually and is expected to increase in the coming years (Zhou et al., 2014). Lactate can either be chemically produced by hydrolysis of lactonitrile (byproduct of acrylomitrile) or via fermentation of sugars derived from food crops (Hirokawa et al., 2017b; Zhou et al., 2014). The former relies on non-renewable petroleum resource while the latter may compete with food supplies. This necessitates the development of production strategies from inexpensive and renewable feedstocks. In this regard, production of lactate from cyanobacteria has been demonstrated in all the three model cyanobacterial strains (PCC 7942, PCC 6803, and PCC 7002) by heterologous expression of lactate dehydrogenase (ldh) gene that catalyzes the conversion of pyruvate to lactate (Fig. 8.2A). An alternate strategy was reported that made use of glycerol dehydrogenease (GlyDH) from Bacillus coagulans in place of ldh in PCC 6803. Additionally, soluble transhydrogenase (sth) gene from Pseudomonas aeruginosa was expressed, which replenishes the pool for NADH required by GlyDH, resulting in a lactate productivity of 0.1 g/L/d (Varman et al., 2013b). The authors further tried to improve the lactate titers by cultivating the engineered strain in acetate supplemented media (15 mM) and attained 2.17 g/L of lactate in 30 days. Addition of acetate in the growth medium, provides the required acetyl-CoA pool for the synthesis of biomass components thereby increasing the pyruvate flux toward the lactate synthesis (Varman et al., 2013b). Further, the above engineered strain was cultivated in anaerobic digestion wastewater (20% + 80% BG-11) containing high amounts of acetate and resulted in 1.26 g/L d-lactate in 20 days (Hollinshead et al., 2014). Another interesting

Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  135

FIG. 8.2  Outline of metabolic pathways of platform chemicals derived from pyruvate engineered in model cyanobacterial strains. MEP: Non-mevalonate pathway and MVA : Mevalonate pathway

136  PART | III  Algal genomics

strategy deployed for lactate production (1.23 g/L) in PCC 7942 was conversion of DHAP to lactate using methylglyoxal synthase (msgsA), glyoxalase (gloA and gloB), and lactate transporter (lldP) genes along with maintaining pH of the growth media neutral as illustrated in Fig. 8.2A (Hirokawa et al., 2017b).

8.4.1.2 2,3-Butanediol Various industrially relevant products including plasticizers, inks, fungicides, polymers (1,3-butadiene), explosives, liquid fuel additives (methyl ethyl ketone) can be derived from 2,3-butanediol (2,3-BD) (Nozzi and Atsumi, 2015; Oliver et  al., 2013). The demand for bio-based 2,3-BD is ~74 kilotons and expected to grow at a rate of 3.2% (Savakis and Hellingwerf, 2015). To this end, model cyanobacterial strains have been engineered for the production of 2,3-BD (Table 8.3 and Fig. 8.2B). In cyanobacteria, 2,3-BD can be synthesized by introducing three genes; als (acetolactate synthase) which converts pyruvate to acetolactate, alsdc (acetolactate decarboxylase) catalyzing the conversion of acetolactate to acetoin and ar (acetoin reductase) or bddh (butanediol dehydrogenase) converting acetoin to 2,3-BD (Fig. 8.2B). The maximum productivity of 2,3-BD (0.3 g/L/d) has been produced in PCC 7942 under photomixotrophic cultivation mode using glycerol (5 g/L) as carbon source (Kanno and Atsumi, 2017). However, PCC 7942 can’t utilize glycerol as carbon source, hence respiratory pathway for glycerol uptake and metabolism was constructed in the cyanobacteria along with 2,3-BD pathway. The authors heterologously expressed glycerol facilitator (GlpF), glycerol kinase (GlpK) and membrane-associated GY3P dehydrogenase (GlpD) genes for the uptake and conversion of glycerol to DHA and DHAP (Kanno and Atsumi, 2017). Utilization of glycerol as carbon source not only enhanced the 2,3-BD production but is also a cheap feedstock being the by-product of biodiesel production. Oliver et al. (2014) optimized the ribosomal binding sites (RBS) for enhancing the 2,3-BD production in PCC 7942 (Oliver et al., 2014). The authors tested various combination of RBS (ranging from strongest to weakest) using the als, alsD and adh genes construct under Plac promoter and produced 0.16 g/L/d of 2,3-BD with intermediate strength RBS for alsS and alsD gene while the weakest RBS for adh gene which was ~2-folds higher than non-optimized RBS construct (Oliver et al., 2014).

8.4.1.3  Isobutylaldehyde, isobutanol, 1-butanol and 2-methyl-1-butanol Isobutylaldehyde is an important platform chemical for the production of plasticizers, isobutyric acid and isobutanol (Rodriguez and Atsumi, 2012). It is currently being synthesized from propylene, carbon monoxide and hydrogen obtained from petro-fuels (Rodriguez and Atsumi, 2012). However, due to its low boiling point (63 °C) and high vapor pressure (66 mmHg) it can be easily stripped from the microbial cultures making its extraction feasible (Atsumi et al., 2009). To date, isobutylaldehyde has been produced only in PCC 7942 by introducing four genes namely; acetolactate synthase (alsS), ­acetohydroxy-acid isomoreductase (ilvC), dihydroxy-acid dehydratase (ilvD), and 2-ketoacid deceaboxylase (kdc) as depicted in Fig. 8.2C. A maximum productivity of 0.14 g/L/d was achieved in PCC 7942 under Ptrc promoter and supplementation of 50 mM sodium bicarbonate in the growth medium (Atsumi et al., 2009). Recently, the above strain has been re-engineered to overexpress the pyruvate kinase (pk), malic enzymes (ME), and malate dehydrogenase (MDH) which enhanced the productivity to 0.05 mmol gDW−1 h−1 (Jazmin et al., 2017). Upregulation of these genes relieves the metabolic bottleneck and aids in channeling pyruvate flux toward isobutylaldehyde synthesis. High chain alcohol such as isobutanol which can be substituted for ethanol as biofuel and also can be dehydrated to form butenes which is a bulk chemical for synthetic rubber (Shi et al., 2013). Utilizing the above synthetic metabolic pathway, isobutylaldehyde can be converted to isobutanol by expressing alcohol dehydrogenase (yqhD) (Fig. 8.2C). A mutant strain of PCC 7942 was engineered which expressed the isobutanol metabolic pathway and lacked glucose-1-phosphate adenyltransferase (glgC) that lead to inhibition of glycogen synthesis (Li et al., 2014). The authors reported no growth defect in the glycogen mutant strain along with 0.06 mg/L/d isobutanol production. 1-Butanol is a key chemical feedstock for various solvents, polymers and plasticizers (Lan et al., 2013). Approximately, 2.9 million metric tons of 1-butanol is consumed world-wide with a $ 5.7 billion market and expected to grow at rate of 4.7% per year (Lan et al., 2013). 1-Butanol can be synthesized utilizing two pathways; coenzyme (CoA)-dependent pathway and the keto-acid pathway (Lan and Liao, 2011). The CoA-dependent pathway involves reverse β oxidation (native fermentation pathway in Clostridium sp.) and involves six enzymes catalyzing the formation of 1-butanol from acetyl-CoA which has been engineered in cyanobacteria for producing 1-butanol (Tables 8.1, 8.2 and Fig. 8.2D). This pathway has been modified to replace the Bcd/EcfAB complex (catalyzes the hydrogenation of crotonyl-CoA to butyryl-CoA) with trans-2enoyl-CoA reductase (Ter) as the complex is difficult to express in recombinant systems (Lan et al., 2013). However, the maximum 1-butanol (36 mg/L/d) was recorded in PCC 7942, by engineering the complete ATP driven butanol pathway (Fig 8.2D) along with phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), which remove CoA moiety from butyryl-CoA (Lai and Lan, 2018).



Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  137

Parallel to the above higher alcohols, 2-methyl-1-butanol (2-MB) can also be utilized as a potential substitute for next generation gasoline, fuel blends and chemical intermediate (Shen and Liao, 2012). To date, 2-MB has been produced only in PCC 7942, with a maximum productivity of 20.8 mg/L/d by introducing isoleucine pathway genes (leuABCD, kivd, yqhD) and the citramalate synthase gene (cimA) as shown in Fig. 8.2E (Shen and Liao, 2012).

8.4.1.4  Isoprene and squalene Isoprene (C5H16) is a volatile hydrocarbon, primarily utilized as a substrate for rubber and isoprenoids (vitamin A and steroids hormones) production (Pade et al., 2016). Plants produce and release isoprene under environmental stress conditions such as heat stress, however sequestration and harvesting of isoprene from plants is unfeasible and thus microbial hosts are ideal for its production (Lindberg et al., 2010). Isoprene can be produced in microbial hosts via two biosynthetic pathways: the mevalonic acid pathway (MAV) present in the eukaryotes and archaea and the methyl-erythritol-4-phosphate (MEP) pathway, existing in the prokaryotic and plant plastids as depicted in Fig. 8.2F (Lindberg et al., 2010). Cyanobacteria have the MEP pathway and introduction of a single gene (isoprene synthase) from Pueraria montana (codon optimized for cyanobacteria—SkIpsS) in PCC 7942 and PCC 6803 led to isoprene production (Gao et al., 2016; Lindberg et al., 2010). The maximum production of isoprene (0.8 mg/L/d) was attained in PCC 6803 by fusing the cpcB gene with IpsS gene using linker of 7 bp (L7) (Chaves et al., 2017). The fusion of IpsS gene with highly expressed native protein (cpcB) alleviated the low Kcat of IpsS, by overproduction of the enzyme. On the other hand, introduction of MAV in glycogen mutant strains of PCC 6803 resulted in 120–250 μg isoprene/g/DCW in 196 h, respectively (Bentley et al., 2014). Squalene (C30H50) is a long chain triterpene which is widely utilized in food, personal care and medicinal industries (Choi et al., 2017). Squalene can be produced in cyanobacteria via MEP catalyzed by squalene synthase (sqs) enzyme (Fig. 8.2G). The maximum productivity (4. 98 mg/L/OD730) has been attained in PCC 7942 by engineering dxs, dxr, idi, ispA, and sqs genes (Choi et al., 2016). Further, deletion of squalene hopene cyclase (shc) gene in PCC 6803, an increase in the squalene production was recorded as compared to wild type which produced negligible chemical (Bergquist et al., 2014).

8.4.2  Acetyl-CoA derived products Typically, the titers of the acetyl-CoA derived products are comparatively lower compared to those derived from pyruvate due to the low carbon flux toward acetyl-CoA and TCA cycle (Knoot et al., 2018). Indeed, increasing the acetyl-CoA pool is one of the straight forward method for enhancing the titer of the desired product. This can be achieved by cultivating the cyanobacterial cells under dark, anaerobic conditions that trigger glycolysis (degradation of the stored glycogen) or via nitrogen and phosphorous deprivation (Knoot et al., 2018). Both of these strategies have been deployed for increasing the titers of acetone, isopropanol and succinate (Kusakabe et al., 2013; Zhou et al., 2012). However, nutrient deprivation results in low biomass accumulation leading to overall low titers of the product. The main platforms chemicals derived from acetyl-CoA include, acetate, isopropanol, succinate and ethylene, respectively.

8.4.2.1  Acetone and isopropanol The simplest ketone, acetone is flammable chemical extensively utilized as the solvent, precursor for isopropanol and intermediate for polymers (Chwa et al., 2016). Commercially, acetone is derived from propylene via Cumene process while fermentation of Clostridia using glucose as feedstocks results in 50% acetone conversion while the other half of the carbon is released as CO2 (Zhou et al., 2012). Acetyl-coA is generated from fatty acid β oxidation and glycogenolysis which can be directed toward acetone production. The maximum acetone productivity (9 mg/L/d) was achieved in PCC 6803 by introducing coenzyme A transferase (cftAB), acetoacetate decarboxylase (adc) genes and deletion of acetate synthesis gene (phosphotransacetylase) and PHB synthesis (phaCE) under dark and anaerobic conditions as shown in Fig. 8.3A (Zhou et al., 2012). Isopropanol is an important secondary alcohol and can be dehydrated to produce propylene; an industrially relevant material (Kusakabe et al., 2013). The acetone biosynthetic pathway can be engineered to produce isopropanol by introducing primary-secondary alcohol dehydrogenase (sadh) (Fig. 8.3A). The maximum productivity (13.31 mg/L/d) of isopropanol has been produced in PCC 7942 by introduction of thiolase (thl), acetyl-CoA acyltransferase (atoDA), acetoacetate decarboxylase (adc), and sadh genes under dark, anaerobic, nitrogen and phosphorous limited conditions (Hirokawa et al., 2015).

8.4.3  Tricarboxylic acid cycle derived products 8.4.3.1 Succinate Succinate (1,2-ethane dicarboxylic acid) has the potential to be converted to an array of valuable chemicals such as tetrahydrofuran (polymer precursor), γ-butyrolactone (for medicinal use), 1,4-butanediol (for biodegradable plastics),

138  PART | III  Algal genomics

FIG. 8.3  Details of engineered pathways in cyanobacteria for the production of platform chemicals derived from acetyl-CoA, TCA(Tricarboxylic acid) cycle, and DHAP(Dihydroxyacetone phosphate).

N-methylpyyrolidone (polymer precursor), diethyl succinate (a green solvent), adipic acid (nylon precursor), etc. (Hasunuma et  al., 2016; McKinlay et  al., 2007). These chemicals find their application in various industries including surfactant, bioplastics, food, pharmaceutical, agricultural and ion chelators (Hasunuma et al., 2018; Wang et al., 2009). The current market for succinic acid is 30,000–50,000 tons per year and is expected to increase to 700,000 tons per year by 2020 (Hasunuma et al., 2018). Succinate is naturally produced by cyanobacteria in the oxidative branch of TCA cycle where α-ketoglutarate is decarboxylated to succinate semialdehyde by α-ketoglutarate decarboxylase (kgd), followed by oxidation to succinate catalyzed by succinate semi aldehyde dehydrogenase (gabD) as shown in Fig. 8.3B (Lan and Wei, 2016). Further, overexpression of citrate synthase (gltA), along with NADP-dependent succinate semialdehyde (gabD), 2-keto glutarate decarboxylase (kgd) and ppc genes resulted in 53.7 mg/L/d of succinate production in PCC 7942 (Lan and Wei, 2016). However, on the other hand the maximum titer productivity of 0.6 g/L/d was achieved in PCC 6803 overexpressing only phosphoenolpyruvate carboxylase (ppc) under dark, anaerobic conditions at a temperature of 37 °C and with 25 g-DCW/L cells (Hasunuma et al., 2018).

8.4.3.2 Ethylene Ethylene serves as a raw material for a vast number of products ranging from plastics (polyethylene, polystyrene, and polyvinyl chloride), and fibers quintessential for chemical industries (Ungerer et  al., 2012; Zhu et  al., 2015). It is also used as a gaseous plant hormone in agriculture to promote growth and control ripening of the fruits (Zhu et al., 2015). The global production of ethylene exceeds 140 million tons and is expected to grow at a rate of 4% per year (Veetil et al., 2017). Currently, ethylene is produced by steam cracking of long chain hydrocarbons obtained from petroleum or ethane resulting in the release of ~1.5–3 tons of CO2 per ton of ethylene (Ungerer et al., 2012). The three biological pathways known for synthesis of ethylene include; (1) synthesis from methionine via 1-aminocyclopropane-1-carboxylic acid (ACC) catalyzed by ACC synthase and ACC oxidase which is present in plants (2) from methionine via 2-keto-4-methyl-thiobutyric acid (KMBA) using NADH:Fe (III) EDTA oxidoreductase in prokaryotes and (3) in few plant pathogens via ethylene forming enzyme (EFE) complex using 2-oxoglutatrate (2-OG), arginine and dioxygen as substrates (Guerrero et al., 2012). The latter pathway has been exploited in both PCC 7942 and PCC 6803 for the production of ethylene (Fig. 8.3C). The ethylene maximum productivity of 295 mg/L/d reported in mutant strain of PCC 6803 which has deletion of 2-OG decarboxylase (OGDC) and succinic semialdehyde dehydrogenases (SSDH) along with heterologous expression of 2-OG permease (kgtP) and EFE (three copies) cultivated in semi-continuous mode with 1 mM of 2-OG feeding every 3 days (Zhu et al., 2015). Deletion of OGDC and SSDH along with insertion of EFE gene in neutral sites as well as knock down sites lead to routing of 2-OG efflux toward ethylene (Zhu et al., 2015). Further, the expression of kgtP (required for the extrinsic 2-OG uptake) and the addition of 2-OG (the substrate for EFE) into the growth media enhanced the ethylene production (Zhu et al., 2015).



Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  139

8.4.4  DHAP derived products The two main products derived from DHAP include 1,2-propanediol and 1,3-propanediol and have been reported to have comparatively higher titers as to acetyl-CoA derived platform chemicals (Knoot et al., 2018). Indeed, the titers depend on a number of factors such as length of the pathway, availability of the precursors and reducing equivalents, toxicity of the product or intermediates, respectively.

8.4.4.1  1,2-Propanediol and 1,3-propanediol 1,2-Propanediol (commonly known as propylene glycol) is bifunctional organic compound and can be utilized to form antifreeze, heat transfer fluids, plasticizers, cosmetics, food and pharmaceuticals (Biebl et al., 1999). Currently, 1,2-­propanediol (1,2-PD) is produced from petroleum sources and has a market share of 2.18 million (David et al., 2018b). It can be synthesized by engineering, three-step biosynthetic pathway involving the conversion of DHAP to methylglyoxal or acetol followed by its subsequent conversion to 1,2-PD (Fig. 8.3D). The maximum productivity of ~79 mg/L/d has been attained in PCC 6803 by heterologous expression of methylglyoxal synthase (mgsA), alcohol dehydrogenase (yqhD) and NADPH dependent glycerol dehydrogenases (adh) (David et al., 2018a). Interestingly, glycogen mutants (Δglgc) lead to a 30% reduction in 1,2-PD production indicating channeling of glycogen flux toward its production (David et al., 2018b). 1,3-Propanediol (1,3-PD) has been extensively utilized for the production of polytrimethylene terephthalate (PTT), an essential polymer in carpet and textile industries (Hirokawa et al., 2017c). The four gene pathway of 1,3-PD has been engineered in all the three model cyanobacterial strains (Fig. 8.3E). The maximum production (87 mg/L/d) has been achieved in PCC 7942 utilizing a combination of promoters (Plac and Ptrc) for expressing the genes glycerol-3-phosphatase dehydrogenase (gpd1) and glycerol-3-phosphatase (hor2) under the control of Plac while glycerol dehydratase (DhaB) and yqhD regulated by Ptrc (Hirokawa et al., 2017c).

8.5  Synthetic biology tools for enhancing the yields in model cyanobacterial strains Cyanobacteria are amenable to genetic engineering, but a deficiency of synthetic biology tools as compared to other model organisms particularly E. coli and yeast has impeded the implementation of these organisms for the synthesis of platform chemicals on large scale (Zess et al., 2016). Indeed, the development of synthetic biology tool-box will facilitate not only in-depth understanding of the biology of cyanobacteria but also fast-track the production of platform chemicals (Sengupta et al., 2018). The presence of oligoploidy/polyploidy, circadian rhythms, restriction/modification systems, codon usage, etc. make them different from E. coli, thus necessitating the construction of essential synthetic biology tools including promoters, transcriptional terminators, and ribosome binding sites specific to cyanobacteria for efficient expression of heterologous gene (Taton et al., 2014). Indeed, the lack of predictive tools for identification and characterization of cyanobacterial synthetic biology parts has significantly impeded its commercial realization. The main elements of the synthetic tool box have been described in brief in the sections below.

8.5.1 Promoters The major difference between the cellular machinery of E. coli and cyanobacteria is the RNA polymerase (RNAP) and the associated σ factors (Berla et al., 2013). In cyanobacterial the β′ subunit of RNAP is split into two subunits namely RpoC1 (γ) and RpoC2 (β′), while the C-terminal of RpoC2 has a large insertion domain which is DNA binding domain (Heidorn et al., 2011). Moreover, the σ factors in cyanobacteria belong to the σ70 family while the common bacterial σ54 factors are absent (Heidorn et al., 2011). Cyanobacteria possess three distinct family of σ70 factors that bind to different promoter sequences namely Type 1, Type II, and Type III (Ramey et al., 2015). Type I represent RpoD consensus promoter similar to bacterial systems, i.e., −35 and −10 elements, which are mainly responsible for expression of housekeeping genes while Type II promoter sequences are associated with −10 hexamer/enhancer motif and Type III identify is still unknown (Ramey et al., 2015). Indeed, both Type II and III are reported to be utilized by cyanobacteria for the expression of stress related genes such as heat, osmotic, salt, etc., respectively (Ramey et al., 2015). Given the differences in the central transcription machinery, the well-established promoters in E. coli including Plac, Ptrc, λPR have yielded low to medium (13–18-folds) induction in PCC 6803 and PCC 7942 (Zess et al., 2016). Further, these heterotrophic inducible promoters are modulated using chemical inducers such as IPTG or anhydrotetracycline (aTc) (Lin et al., 2017; Oliver et al., 2013). However, these approaches are not economically viable during large scale cultivation. Moreover, some of the inducers like aTc are sensitive to light hence restricted under outdoor light condition (Liu and Pakrasi, 2018). Thus, there’s a need for the characterization of existing native promoters of cyanobacteria which are derived from essential genes such as photosynthesis including

140  PART | III  Algal genomics

Photosystem I (PSI; PpsaA, PpsaD), PSII (PpsbAI, PpsbA2), photosynthesis antenna proteins (Pcpc), CO2 fixation (PrbcL, Pcmp, Psbt) (Wang et al., 2012). Although, these promoters show relatively high levels of gene expression particularly Pcpc, these are light induced, show little or no expression in dark (Ramey et al., 2015). To this end, metal induced promoters have been developed for model cyanobacterial strains (PCC 6803 and PCC 7942) including PcoaA (cobalt induced), PcoaT (cobalt, zinc induced), PziaA (cadmium and zinc induced), Psmt (zinc induced), PnrsB (nickel induced), PisiAB (iron repressed) (Berla et al., 2013). However, these promoters are difficult to use due to operational issues (glass wares have to thoroughly washed to remove trace metals) and cells have to be starved for extensive periods remove metals leading to stress response, making their usage in industries difficult (Berla et al., 2013). Further, native promoter library has been built for all the three model strains (Englund et al., 2016; Huang and Lindblad, 2013; Liu and Pakrasi, 2018; Ng et al., 2015; Zess et al., 2016), respectively. Recently, a robust promoter identification tool was developed, bTSSfinder which predicted promoters pertaining to different classes of σ factors (σA, σC, σH, σG, σF) with high accuracy, however, the characterization of the predicted promoters needs to be further evaluated to understand gene expression levels (Shahmuradov et al., 2017). Thus, identification and characterization of novel promoters and establishment of promoter library unique to cyanobacteria are quintessential to increase the titters of the target product.

8.5.2  Transcriptional terminators Cyanobacteria (6903 and PCC 7942) generally possesses the Rho independent transcription termination (Ramey et  al., 2015). The introduction of the terminator sequence downstream to the heterologous gene prevents the expression of adjacent genes while placing it upstream, avoids background expression transcription effect on the upstream genes (Wang et al., 2012). To date, only a few terminators have been reported in cyanobacteria including E. coli TrrnB, cyanobacterial TRBCL, bacteriophage TT7 and rrnBT1-T7TE (combination of rrnB and T7) (Heidorn et al., 2011; Wang et al., 2012). Recently, native terminator library was constructed for PCC 6803 which constituted of seven terminators corresponding to the photosynthesis genes (Liu and Pakrasi, 2018). The authors reported that although two terminators (TatpC and TpsbC) showed 10-fold difference in expression but were unable to completely repress the expression of PRtrc10 promoter. Thus, a little information is available on the native terminators in cyanobacteria and warrants further investigation.

8.5.3  Ribosomal binding sites The initiation of translation in prokaryotes begin by binding of a ribosome to the mRNA at the ribosome binding site (RBS), containing the core Shine-Dalgarno (SD) sequence (Heidorn et al., 2011). Interestingly, in PCC 6803 only 26% of the total genes have the core SD sequence as compared to 57% of the genes in E. coli (Heidorn et al., 2011). The construction of native RBS library in cyanobacteria can significantly improve the rate of translation of the heterologous genes particularly when multiple genes are expressed as a part of an operon (Liu and Pakrasi, 2018). There are various tools available for the prediction of RBS sites including Ribosome Binding site Calculator, UTR designer and RBS designer (Immethun and Moon, 2018). The RBS library for PCC 7002 comprises of an 8-member library, predicted using Ribosome Binding site Calculator which showed 143-fold range of translation initiation rates (TIR) (Markley et al., 2015). Another library for PCC 6803 comprising of 08 RBS obtained from BioBrick Registry of standard biological parts, 02 native derived from RBS upstream of the psbA2 and rbcL genes while 01 being synthetic RBS was generated (Englund et al., 2018). This library was further enriched by construction of native RBS library comprising of a total of 20 RBS with exactly 22 bp upstream of the translation start codon, corresponding to the photosynthetic genes (Liu and Pakrasi, 2018). The authors selected RBS-ndhJ and RBS-psaF as these showed maximum TIR. Indeed, optimization of RBS plays a vital role in fine tuning the expression of genes in cyanobacteria and warrants further investigation.

8.6  Future outlook and challenges The consistent research efforts toward the development of cyanobacteria as a green mine for the production of platform chemicals in the past two decades has expanded our understanding of the metabolic pathways and biology of these microorganism. However, to unlock their potential and make them competitive with E. coli and yeast, it is imperative to increase the titers, productivity and stability of the engineered cyanobacteria in an industrial setting. Indeed, this requires bioprospection of fast growing cyanobacterial capable of growing in high light, temperatures, salinity, pH, and wastewaters which are key attributes for making a strain commercially viable. To this end, robust and fast-growing cyanobacterial strains such as Synechococcus elongatus UTEX 2973 and Synechococcus elongatus PCC 11801 have shown potential for production of platform chemicals (Jaiswal et al., 2018b; Yu et al., 2015). Since, most of the studies to date have synthesized the platforms



Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  141

chemicals in continuous light, it will be of utmost importance to get insights into the production of these chemicals under diurnal cycle which plays an important role in carbon partitioning particularly during night time. Extensive work is needed in terms of characterization of synthetic biology parts, particularly promoter engineering that can be regulated using environmental cues such as diurnal cycle, light intensity, CO2 capture and utilization which can expedite the scale-up of the engineered cyanobacterial strains in open systems. The advancements in the cyanobacterial omics including transcriptomics, proteomics, metabolomics, and fluxomics have envisaged the creation of efficient genetic components library. Further, well-defined synthetic tools can provide in-depth understanding on the complex biology of cyanobacteria aiding custom tailoring the cell for enhanced target product. In addition, as the precursors of the platform’s chemicals belong to central carbon pathway, constraint-based metabolic models such as flux-based models can act as blue print for identifying the bottleneck of the engineered pathway thereby enabling routing of carbon flux toward target product. Moreover, recent exploitation of advanced trans-gene editing tools including CRISPR-Cas9 in cyanobacteria has unlocked the potential to fine tune the expression of the heterologous genes and increase the transformation efficiency. Parallel to the above stated points, development of low cost photobioreactors, optimization of harvesting, extraction and downstream processing of the platform chemical are quintessential. Ultimately, the formulation of regulatory policies for large-scale cultivation of engineered cyanobacteria would play an important role in the eventual commercialization of these processes. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c, d,e,f,g,h,i,j,k,l,m,n,o).

Acknowledgments The authors acknowledge grant from Department of Biotechnology (DBT), Government of India, awarded to P.P.W. toward DBT-PAN IIT Center for Bioenergy (Grant No.: BT/EB/PAN IIT/2012) and the Indo-US Science and Technology Forum (IUSSTF) grant awarded to P.P.W. (Grant No.: IUSSTF/JCERDC-SGB/IUABC-IITB/2016). N.A. acknowledges Indian Institute of Technology, Bombay for providing the Institute postdoctoral fellowship. D.J. is thankful to the Council of Scientific and Industrial Research (CSIR), India for the research fellowship.

References Anfelt, J., Kaczmarzyk, D., Shabestary, K., Renberg, B., Rockberg, J., Nielsen, J., et al., 2015. Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production. Microb. Cell Fact. 14, 167. Angermayr, S.A., Hellingwerf, K.J., 2013. On the use of metabolic control analysis in the optimization of cyanobacterial biosolar cell factories. J. Phys. Chem. B 117 (38), 11169–11175. Angermayr, S.A., Paszota, M., Hellingwerf, K.J., 2012. Engineering a cyanobacterial cell factory for production of lactic acid. Appl. Environ. Microbiol. 78 (19), 7098–7106. Angermayr, S.A., Rovira, A.G., Hellingwerf, K.J., 2015. Metabolic engineering of cyanobacteria for the synthesis of commodity products. Trends Biotechnol. 33 (6), 352–361. Arai, S., Hayashihara, K., Kanamoto, Y., Shimizu, K., Hirokawa, Y., Hanai, T., et al., 2017. Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system. Biotechnol. Bioeng. 114 (8), 1771–1778. Atsumi, S., Higashide, W., Liao, J.C., 2009. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat. Biotechnol. 27 (12), 1177–1180. Bentley, F.K., Zurbriggen, A., Melis, A., 2014. Heterologous expression of the mevalonic acid pathway in cyanobacteria enhances endogenous carbon partitioning to isoprene. Mol. Plant 7 (1), 71–86. Bergquist, J., Englund, E., Pattanaik, B., Ubhayasekera, S.J.K., Stensjo, K., Lindberg, P., 2014. Production of squalene in Synechocystis sp. PCC 6803. PLoS One 9 (3), e90270. Berla, B.M., Saha, R., Immethun, C.M., Maranas, C.D., Moon, T.S., Pakrasi, H.B., 2013. Synthetic biology of cyanobacteria: unique challenges and opportunities. Front. Microbiol. 4, 246. Biebl, M.H., Menzel, K., Zeng, A.P., Deckwer, W.D., 1999. Microbial production of 1,3-propanediol. Appl. Microbiol. Biotechnol. 52 (3), 289–297. Bornke, F., Broer, I., 2010. Tailoring plant metabolism for the production of novel polymers and platform chemicals. Curr. Opin. Plant Biol. 13 (3), 354–362. Buschke, N., Schafer, R., Becker, J., Wittmann, C., 2013. Metabolic engineering of industrial platform microorganisms for biorefinery applications—­ optimization of substrate spectrum and process robustness by rational and evolutive strategies. Bioresour. Technol. 135, 544–554. Chaves, J.E., Romero, P.R., Kirst, H., Melis, A., 2016. Role of isopentenyl-diphosphate isomerase in heterologous cyanobacterial (Synechocystis) isoprene production. Photosynth. Res. 130 (1–3), 517–527. Chaves, J.E., Rueda-Romero, P., Kirst, H., Melis, A., 2017. Engineering isoprene synthase expression and activity in cyanobacteria. ACS Synth. Biol. 6 (12), 2281–2292. Chin, J.W., Anderson, M.A., Jianping, C.U.I., Spieker, M., 2015. U.S. Patent Application No. 14/681,462. Choi, S.Y., Lee, H.J., Choi, J.Y., Kim, J., Sim, S.J., Um, Y.S., et al., 2016. Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria. Biotechnol. Biofuels 9, 202.

142  PART | III  Algal genomics

Choi, S.Y., Wang, J.Y., Kwak, H.S., Lee, S.M., Um, Y.S., Kim, Y., et al., 2017. Improvement of squalene production from CO2 in Synechococcus elongatus PCC 7942 by metabolic engineering and scalable production in a photobioreactor. ACS Synth. Biol. 6 (7), 1289–1295. Chwa, J.W., Kim, W.J., Sim, S.J., Um, Y.S., Woo, H.M., 2016. Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition. Plant Biotechnol. J. 14 (8), 1768–1776. David, C., Schmid, A., Adrian, L., Wilde, A., Buhler, K., 2018a. Production of 1,2-propanediol in photoautotrophic Synechocystis is linked to glycogen turn-over. Biotechnol. Bioeng. 115 (2), 300–311. David, C., Schmid, A., Buhler, K., 2018b. Cellular physiology controls photoautotrophic production of 1,2-propanediol from pools of CO2 and glycogen. Biotechnol. Bioeng. https://doi.org/10.1002/bit.26883. Desai, S.H., Atsumi, S., 2013. Photosynthetic approaches to chemical biotechnology. Curr. Opin. Biotechnol. 24 (6), 1031–1036. Dexter, J., Fu, P.C., 2009. Metabolic engineering of cyanobacteria for ethanol production. Energ. Environ. Sci. 2 (8), 857–864. Englund, E., Liang, F.Y., Lindberg, P., 2016. Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Sci. Rep. 6, 36640. Englund, E., Shabestary, K., Hudson, E.P., Lindberg, P., 2018. Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound. Metab. Eng. 49, 164–177. Fu, P.C., 2009. Genome‐scale modeling of Synechocystis sp. PCC 6803 and prediction of pathway insertion. J. Chem. Technol. Biotechnol. 84 (4), 473–483. Gao, X.Y., Gao, F., Liu, D., Zhang, H., Nie, X.Q., Yang, C., 2016. Engineering the methylerythritol phosphate pathway in cyanobacteria for photosynthetic isoprene production from CO2. Energ. Environ. Sci. 9 (4), 1400–1411. Geilen, F.M.A., Engendahl, B., Harwardt, A., Marquardt, W., Klankermayer, J., Leitner, W., 2010. Selective and flexible transformation of biomassderived platform chemicals by a multifunctional catalytic system. Angew. Chem. Int. Ed. 49 (32), 5510–5514. Gordon, G.C., Korosh, T.C., Cameron, J.C., Markley, A.L., Begemann, M.B., Pfleger, B.F., 2016. CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab. Eng. 38, 170–179. Guerrero, F., Carbonell, V., Cossu, M., Correddu, D., Jones, P.R., 2012. Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803. PLoS One 7 (11), e50470. Hagemann, M., Hess, W.R., 2018. Systems and synthetic biology for the biotechnological application of cyanobacteria. Curr. Opin. Biotechnol. 49, 94–99. Hasunuma, T., Matsuda, M., Kondo, A., 2016. Improved sugar-free succinate production by Synechocystis sp. PCC 6803 following identification of the limiting steps in glycogen catabolism. Metab. Eng. Commun. 3, 130–141. Hasunuma, T., Matsuda, M., Kato, Y., Vavricka, C.J., Kondo, A., 2018. Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803. Metab. Eng. 48, 109–120. Heidorn, T., Camsund, D., Huang, H.H., Lindberg, P., Oliveira, P., Stensjo, K., et al., 2011. Synthetic biology in cyanobacteria: engineering and analyzing novel functions. Methods Enzymol. 497, 539–579. Heimann, K., 2016. Novel approaches to microalgal and cyanobacterial cultivation for bioenergy and biofuel production. Curr. Opin. Biotechnol. 38, 183–189. Hendry, J.I., Prasannan, C.B., Joshi, A., Dasgupta, S., Wangikar, P.P., 2016. Metabolic model of Synechococcus sp. PCC 7002: prediction of flux distribution and network modification for enhanced biofuel production. Bioresour. Technol. 213, 190–197. Hendry, J.I., Prasannan, C., Ma, F.F., Mollers, K.B., Jaiswal, D., Digmurti, M., et al., 2017. Rerouting of carbon flux in a glycogen mutant of cyanobacteria assessed via isotopically. Biotechnol. Bioeng. 114 (10), 2298–2308. Hirokawa, Y., Suzuki, I., Hanai, T., 2015. Optimization of isopropanol production by engineered cyanobacteria with a synthetic metabolic pathway. J. Biosci. Bioeng. 119 (5), 585–590. Hirokawa, Y., Maki, Y., Tatsuke, T., Hanai, T., 2016. Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway. Metab. Eng. 34, 97–103. Hirokawa, Y., Dempo, Y., Fukusaki, E., Hanai, T., 2017a. Metabolic engineering for isopropanol production by an engineered cyanobacterium, Synechococcus elongatus PCC 7942, under photosynthetic conditions. J. Biosci. Bioeng. 123 (1), 39–45. Hirokawa, Y., Goto, R., Umetani, Y., Hanai, T., 2017b. Construction of a novel d-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942. J. Biosci. Bioeng. 124 (1), 54–61. Hirokawa, Y., Maki, Y., Hanai, T., 2017c. Improvement of 1,3-propanediol production using an engineered cyanobacterium, Synechococcus elongatus by optimization of the gene expression level of a synthetic metabolic pathway and production conditions. Metab. Eng. 39, 192–199. Hollinshead, W.D., Varman, A.M., You, L., Hembree, Z., Tang, Y.J., 2014. Boosting d-lactate production in engineered cyanobacteria using sterilized anaerobic digestion effluents. Bioresour. Technol. 169, 462–467. Holtman, C.K., Chen, Y., Sandoval, P., Gonzales, A., Nalty, M.S., Thomas, T.L., et al., 2005. High-throughput functional analysis of the Synechococcus elongatus PCC 7942 genome. DNA Res. 12 (2), 103–115. Huang, H.H., Lindblad, P., 2013. Wide-dynamic-range promoters engineered for cyanobacteria. J. Biol. Eng. 7 (1), 10. Huang, C.H., Shen, C.R., Li, H., Sung, L.Y., Wu, M.Y., Hu, Y.C., 2016. CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942. Microb. Cell Fact. 15, 196. Immethun, C.M., Moon, T.S., 2018. Synthetic gene regulation in cyanobacteria. Adv. Exp. Med. Biol. 1080, 317–355. Jaiswal, D., Prasannan, C.B., Hendry, J.I., Wangikar, P.P., 2018a. SWATH tandem mass spectrometry workflow for quantification of mass isotopologue distribution of intracellular metabolites and fragments labeled with isotopic 13C carbon. Anal. Chem. 90 (11), 6486–6493. Jaiswal, D., Sengupta, A., Sohoni, S., Sengupta, S., Phadnavis, A.G., Pakrasi, H.B., et al., 2018b. Genome features and biochemical characteristics of a robust, fast growing and naturally transformable cyanobacterium Synechococcus elongatus PCC 11801 isolated from India. Sci. Rep. 8 (1), 16632.



Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  143

Jang, Y.S., Kim, B., Shin, J.H., Choi, Y.J., Choi, S., Song, C.W., et al., 2012. Bio-based production of C2-C6 platform chemicals. Biotechnol. Bioeng. 109 (10), 2437–2459. Jazmin, L.J., Xu, Y., Cheah, Y.E., Adebiyi, A.O., Johnson, C.H., Young, J.D., 2017. Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production. Metab. Eng. 42, 9–18. JGI, 2018. Synechococcus elongatus PCC 7942. Joint Genome Institute, Walnut Creek, CA. Joseph, A., Aikawa, S., Sasaki, K., Tsuge, Y., Matsuda, F., Tanaka, T., et al., 2014. Utilization of lactic acid bacterial genes in Synechocystis sp. PCC 6803 in the production of lactic acid. Biosci. Biotechnol. Biochem. 77 (5), 966–970. Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., et al., 1996. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3 (3), 109–136. Kanno, M., Atsumi, S., 2017. Engineering an obligate photoautotrophic cyanobacterium to utilize glycerol for growth and chemical production. ACS Synth. Biol. 6 (1), 69–75. Knoop, H., Zilliges, Y., Lockau, W., Steuer, R., 2010. The metabolic network of Synechocystis sp. PCC 6803: systemic properties of autotrophic growth. Plant Physiol. 154 (1), 410–422. Knoot, C.J., Ungerer, J., Wangikar, P.P., Pakrasi, H.B., 2018. Cyanobacteria: promising biocatalysts for sustainable chemical production. J. Biol. Chem. 293 (14), 5044–5052. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 Citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Kusakabe, T., Tatsuke, T., Tsuruno, K., Hirokawa, Y., Atsumi, S., Liao, J.C., et al., 2013. Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light. Metab. Eng. 20, 101–108. Lai, M.J., Lan, E.I., 2018. Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria. Biotechnol. Bioeng. https://doi.org/10.1002/ bit.26903. Lan, E.I., Liao, J.C., 2011. Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide. Metab. Eng. 13 (4), 353–363. Lan, E.I., Wei, C.T., 2016. Metabolic engineering of cyanobacteria for the photosynthetic production of succinate. Metab. Eng. 38, 483–493. Lan, E.I., Ro, S.Y., Liao, J.C., 2013. Oxygen-tolerant coenzyme A-acylating aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria. Energ. Environ. Sci. 6 (9), 2672–2681. Lan, E.I., Chuang, D.S., Shen, C.R., Lee, A.M., Ro, S.Y., Liao, J.C., 2015. Metabolic engineering of cyanobacteria for photosynthetic 3-hydroxypropionic acid production from CO2 using Synechococcus elongatus PCC 7942. Metab. Eng. 31, 163–170. Lee, J.W., Kim, H.U., Choi, S., Yi, J., Lee, S.Y., 2011. Microbial production of building block chemicals and polymers. Curr. Opin. Biotechnol. 22 (6), 758–767. Li, H., Liao, J.C., 2013. Engineering a cyanobacterium as the catalyst for the photosynthetic conversion of CO2 to 1,2-propanediol. Microb. Cell Fact. 12, 4.

144  PART | III  Algal genomics

Li, X., Shen, C.R., Liao, J.C., 2014. Isobutanol production as an alternative metabolic sink to rescue the growth deficiency of the glycogen mutant of Synechococcus elongatus PCC 7942. Photosynth. Res. 120 (3), 301–310. Li, C., Tao, F., Ni, J., Wang, Y., Yao, F., Xu, P., 2015. Enhancing the light-driven production of d-lactate by engineering cyanobacterium using a combinational strategy. Sci. Rep. 5, 9777. Li, H., Shen, C.R., Huang, C.H., Sung, L.Y., Wu, M.Y., Hu, Y.C., 2016. CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production. Metab. Eng. 38, 293–302. Lin, P.C., Saha, R., Zhang, F.H., Pakrasi, H.B., 2017. Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocystis sp. PCC 6803. Sci. Rep. 7, 17503. Lindberg, P., Park, S.S., Melis, A., 2010. Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metab. Eng. 12 (1), 70–79. Liu, D., Pakrasi, H.B., 2018. Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803. Microb. Cell Fact. 17 (1), 48. Markley, A.L., Begemann, M.B., Clarke, R.E., Gordon, G.C., Pfleger, B.F., 2015. Synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. ACS Synth. Biol. 4 (5), 595–603. Matsusako, T., Toya, Y., Yoshikawa, K., Shimizu, H., 2017. Identification of alcohol stress tolerance genes of Synechocystis sp. PCC 6803 using adaptive laboratory evolution. Biotechnol. Biofuels 10, 307. McEwen, J.T., Kanno, M., Atsumi, S., 2016. 2,3 Butanediol production in an obligate photoautotrophic cyanobacterium in dark conditions via diverse sugar consumption. Metab. Eng. 36, 28–36. McKinlay, J.B., Vieille, C., Zeikus, J.G., 2007. Prospects for a bio-based succinate industry. Appl. Microbiol. Biotechnol. 76 (4), 727–740. Mika, L.T., Csefalvay, E., Nemeth, A., 2018. Catalytic conversion of carbohydrates to initial platform chemicals: chemistry and sustainability. Chem. Rev. 118 (2), 505–613. MRF, 2018. Bio-Based Platform Chemicals Market Research Report-Global Forecast to 2023. Market Research Future, Maharashtra, India. Ng, A.H., Berla, B.M., Pakrasi, H.B., 2015. Fine-tuning of photoautotrophic protein production by combining promoters and neutral sites in the cyanobacterium Synechocystis sp. strain PCC 6803. Appl. Environ. Microbiol. 81 (19), 6857–6863. Niederholtmeyer, H., Wolfstadter, B.T., Savage, D.F., Silver, P.A., Way, J.C., 2010. Engineering cyanobacteria to synthesize and export hydrophilic products. Appl. Environ. Microbiol. 76 (11), 3462–3466. Nozzi, N.E., Atsumi, S., 2015. Genome engineering of the 2,3-butanediol biosynthetic pathway for tight regulation in cyanobacteria. ACS Synth. Biol. 4 (11), 1197–1204. Nozzi, N.E., Case, A.E., Carroll, A.L., Atsumi, S., 2017. Systematic approaches to efficiently produce 2,3-butanediol in a marine cyanobacterium. ACS Synth. Biol. 6 (11), 2136–2144. Oliver, J.W.K., Atsumi, S., 2014. Metabolic design for cyanobacterial chemical synthesis. Photosynth. Res. 120 (3), 249–261. Oliver, J.W.K., Machado, I.M.P., Yoneda, H., Atsumi, S., 2013. Cyanobacterial conversion of carbon dioxide to 2,3-butanediol. Proc. Natl. Acad. Sci. U. S. A. 110 (4), 1249–1254. Oliver, J.W.K., Machado, I.M.P., Yoneda, H., Atsumi, S., 2014. Combinatorial optimization of cyanobacterial 2,3-butanediol production. Metab. Eng. 22, 76–82. Osanai, T., Shirai, T., Iijima, H., Nakaya, Y., Okamoto, M., Kondo, A., et al., 2015. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium. Front. Microbiol. 6, 1064. Pade, N., Erdmann, S., Enke, H., Dethloff, F., Duhring, U., Georg, J., et al., 2016. Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803. Biotechnol. Biofuels 9, 89. Ramey, C.J., Baron-Sola, A., Aucoin, H.R., Boyle, N.R., 2015. Genome engineering in cyanobacteria: where we are and where we need to go. ACS Synth. Biol. 4 (11), 1186–1196. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 2009. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111, 1), 1–61. Rodriguez, G.M., Atsumi, S., 2012. Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity. Microb. Cell Fact. 11, 90. Saha, R., Verseput, A.T., Berla, B.M., Mueller, T.J., Pakrasi, H.B., Maranas, C.D., 2012. Reconstruction and comparison of the metabolic potential of cyanobacteria Cyanothece sp. ATCC 51142 and Synechocystis sp. PCC 6803. PLoS One 7 (10), e48285. Savakis, P., Hellingwerf, K.J., 2015. Engineering cyanobacteria for direct biofuel production from CO2. Curr. Opin. Biotechnol. 33, 8–14. Sengupta, A., Pakrasi, H.B., Wangikar, P.P., 2018. Recent advances in synthetic biology of cyanobacteria. Appl. Microbiol. Biotechnol. 102 (13), 5457–5471. Shahmuradov, I.A., Razali, R.M., Bougouffa, S., Radovanovic, A., Bajic, V.B., 2017. bTSSfinder: a novel tool for the prediction of promoters in cyanobacteria and Escherichia coli. Bioinformatics 33 (3), 334–340. Shen, C.R., Liao, J.C., 2012. Photosynthetic production of 2-methyl-1-butanol from CO2 in cyanobacterium Synechococcus elongatus PCC7942 and characterization of the native acetohydroxyacid synthase. Energ. Environ. Sci. 5, 9574–9583. Shestakov, S.V., Khyen, N.T., 1970. Evidence for genetic transformation in blue-green alga Anacystis nidulans. Mol. Gen. Genet. 107 (4), 372–375. Shi, A., Zhu, X., Lu, J., Zhang, X., Ma, Y., 2013. Activating transhydrogenase and NAD kinase in combination for improving isobutanol production. Metab. Eng. 16, 1–10. Shih, P.M., Wu, D.Y., Latifi, A., Axen, S.D., Fewer, D.P., Talla, E., 2013. Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc. Natl. Acad. Sci. U. S. A. 110 (3), 1053–1058.



Metabolic engineering of cyanobacteria for production of platform chemicals Chapter | 8  145

Stanier, R.Y., Cohen-Bazire, G., 1977. Phototrophic prokaryotes: the cyanobacteria. Annu. Rev. Microbiol. 31, 225–274. Sun, T., Li, S.B., Song, X.Y., Diao, J.J., Chen, L., Zhang, W.W., 2018. Toolboxes for cyanobacteria: recent advances and future direction. Biotechnol. Adv. 36 (4), 1293–1307. Taton, A., Unglaub, F., Wright, N.E., Zeng, W.Y., Paz-Yepes, J., Brahamsha, B., et al., 2014. Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Res. 42 (17), e136. Triana, J., Montagud, A., Siurana, M., Fuente, D., Urchueguia, A., Gamermann, D., et al., 2014. Generation and evaluation of a genome-scale metabolic network model of Synechococcus elongatus PCC7942. Metabolites 4 (3), 680–698. Ungerer, J., Tao, L., Davis, M., Ghirardi, M., Maness, P.C., Yu, J.P., 2012. Sustained photosynthetic conversion of CO2 to ethylene in recombinant Synechocystis sp. PCC 6803. Energ. Environ. Sci. 5 (10), 8998–9006. Varman, A.M., Xiao, Y., Pakrasi, H.B., Tang, Y.J., 2013a. Metabolic engineering of Synechocystis sp. strain PCC 6803 for isobutanol production. Appl. Environ. Microbiol. 79 (3), 908–914. Varman, A.M., Yu, Y., You, L., Tang, Y.J., 2013b. Photoautotrophic production of d-lactic acid in an engineered cyanobacterium. Microb. Cell Fact. 12, 117. Veetil, V.P., Angermayr, S.A., Hellingwerf, K.J., 2017. Ethylene production with engineered Synechocystis sp PCC 6803 strains. Microb. Cell Fact. 16, 34. Wang, D., Li, Q., Li, W.L., Xing, J.M., Su, Z.G., 2009. Enzyme and microbial technology improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli. Enzyme Microb. Technol. 45 (6–7), 491–497. Wang, B., Wang, J., Zhang, W., Meldrum, D.R., 2012. Application of synthetic biology in cyanobacteria and algae. Front. Microbiol. 3, 344. Wang, Y., Shi, M., Niu, X., Zhang, X., Gao, L., Chen, L., et al., 2014. Metabolomic basis of laboratory evolution of butanol tolerance in photosynthetic Synechocystis. Microb. Cell Fact. 13, 151. Welkie, D.G., Rubin, B.E., Diamond, S., Hood, R.D., Savage, D.F., Golden, S.S., 2018. A hard day’s night: cyanobacteria in diel cycles. Trends Microbiol. 27 (3), 231–242. Werpy, T.A., Petersen, G. (Eds.), 2004. Top Value Added Chemicals From Biomass: Volume I—Results of Screening for Potential Candidates From Sugars and Synthesis Gas. National Renewable Energy Laboratory, Golden, CO. Xiong, W., Morgan, J.A., Ungerer, J., Wang, B., Maness, P.C., Yu, J.P., 2015. The plasticity of cyanobacterial metabolism supports direct CO2 conversion to ethylene. Nat. Plants 1, 15053. Yu, J.J., Liberton, M., Cliften, P.F., Head, R.D., Jacobs, J.M., Smith, R.D., et al., 2015. Synechococcus elongatus UTEX 2973, a fast-growing cyanobacterial chassis for biosynthesis using light and CO2. Sci. Rep. 5, 8132. Zess, E.K., Begemann, M.B., Pfleger, B.F., 2016. Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. Biotechnol. Bioeng. 113 (2), 424–432. Zhou, J., Zhang, H., Zhang, Y., Li, Y., Ma, Y., 2012. Designing and creating a modularized synthetic pathway in cyanobacterium Synechocystis enables production of acetone from carbon dioxide. Metab. Eng. 14 (4), 394–400. Zhou, J., Zhang, H., Meng, H., Zhang, Y., Li, Y., 2014. Production of optically pure d-lactate from CO2 by blocking the PHB and acetate pathways and expressing d-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803. Process Biochem. 49 (12), 2071–2077. Zhu, T., Xie, X.M., Li, Z.M., Tan, X.M., Lu, X.F., 2015. Enhancing photosynthetic production of ethylene in genetically engineered Synechocystis sp. PCC 6803. Green Chem. 17 (1), 421–434. Ziegler, K., Weissert, C., Duehring, U., Chin, J.W., Anderson, M.A., Jianping, C.U.I., et al., 2014. U.S. Patent Application No. 14/057,014.

Chapter 9

Genomics perspectives on cyanobacteria research Cristiana Moreiraa,b, Joana Martinsa,b, Vitor Vasconcelosa,b, Agostinho Antunesa,b a

CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Porto, Portugal, bDepartment of Biology, Faculty of Sciences, University of Porto, Porto, Portugal

9.1  Cyanobacteria, their toxins and natural products Cyanobacteria are ancient living prokaryotic photosynthetic microorganisms that are widely distributed throughout the several biogeographical areas of the world. From hot deserts to cold polar regions cyanobacteria are natural inhabitants being also widely dispersed in the several aquatic and terrestrial environments also occurring in aerial conditions (Bahl et al., 2011; Jungblut et al., 2005). Simple in structure they can be presented in colonial or in filamentous forms in these latter specialized cells can be found such as akinetes and heterocysts with special functions, serving namely as resting cells or as fixators of nitrogen, respectively. Though cyanobacteria are important contributors of oxygen to Earth they carry negative impacts mainly in water quality and in human health (Fig. 9.1). In this sense under eutrophic conditions (excess of nutrients in the water) mass agglomerations can be formed designated as blooms that additionally carry another impact, the release of harmful toxins named cyanotoxins. These have been widely studied and are known in general to be hepatotoxic, neurotoxic, cytotoxic being also attributed a genotoxic and carcinogenic potential (Moreira et al., 2013a; van Apeldoorn et al., 2007). Overall cyanotoxins represent an increased risk to humans and also animal and plant life (Fig. 9.2). In fact the World Health Organization has demanded the surveillance of one of the most severe cyanotoxins the microcystins after an outbreak attributed to this cyanotoxin in a dialysis center in Caruaru (Brazil) that resulted in the loss of 60 patients (Jochimsen et al., 1998). Apart for this, some countries have regulated the surveillance of other cyanotoxins such as the case of the cytotoxin cylindrospermopsin (Australia, Brazil, New Zealand), the neurotoxins anatoxin-a (New Zealand, USA) and the saxitoxins (Australia, Brazil, New Zealand) (Burch, 2008). These overall present distinct modes of action being also chemically distinct. In addition to cyanotoxins, cyanobacteria have the ability to produce other natural products (NP) that present interesting bioactivities such as anticancer, multidrug-reversing, antibacterial, antifungal, antiviral, anti-inflammatory and potent enzyme inhibition, among others. A large proportion of cyanobacterial bioactive NP including cyanotoxins are mainly produced by polyketide synthase (PK), nonribosomal peptide synthetase (NRPS) or a mixture of both ((PKS)-NRPS) biosynthetic pathways (Welker and von Dohren, 2006). More recently, the ability of cyanobacteria to produce peptides through a biosynthetic pathway assigned as post-ribosomal peptide synthesis (PRPS) has been reported (Arnison et al., 2013). Marine cyanobacteria, particularly belonging to the orders Oscillatoriales and Nostocales, generally have a large number of NRPS and PKS biosynthetic pathways and are important contributors of NP to the pharmaceutical industry (Tidgewell et al., 2010). In spite of this, the bulk of NP described from cyanobacteria arise from several genera including the freshwater Microcystis and Nostoc, the terrestrial Hapalosiphon, and the marine Lyngbya (recently reclassified on the basis of genetic data as Moorea) (Dittmann et al., 2015). This last genus has already yielded more than 190 new NPs in the last 20 years, accounting for more than 40% of all reported marine NPs (Kleigrewe et al., 2016). The resulting products of the cyanobacterial biosynthetic pathways are mostly unknown being therefore expectable that novel structures and bioactivities will continuously arise from the study of these organisms. In the study of cyanobacterial NP analytical and molecular methods can be applied though the main focus of this chapter are the molecular methods.

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00009-7 © 2020 Elsevier Inc. All rights reserved.

147

148  PART | III  Algal genomics

FIG. 9.1  Bloom episode at River Tâmega (North of Portugal) in September of 2017.

Hepatotoxins D-Glu H N

CH3 HN O

H3C

OCH3

O

O CH2

CH3

H

O

H3C

CH3

H N

H N O

COOH

CH3

H3C

H

CH3

H

O O CH3 H

NH

Mdhb H

HN

O

CO2H

H

CH3 O

CH3

N

HN

H NH

HN

O

OCH3 H

CO2H

H

CH3

H

NH

NH H3C

Adda

H

O

Microcystin-LR NH

Arg

Masp

NH HN

NH2

Microcystin-LR

Nodularin

Neurotoxins

Cytotoxin OH

NH2

O NH

NH2

HN

O

CH3

N

N

Anatoxin-a

N OH H2N

NH

NH2

H

Saxitoxin

O

H

O O

HO

S

O

H H3C

H

H

N

NH NH

+

OH O HN

NH O

Cylindrospermopsin

FIG. 9.2  Chemical structure of the main cyanotoxins.

9.2  PCR based methods Molecular methods namely the ‘Polymerase Chain Reaction’ (PCR) arose in cyanobacterial diagnosis in the beginning of 1990s. This method consists in the amplification of a specific region of a gene of the cyanobacterial genome that allows in summary the identification of cyanobacterial presence (structural gene) (Table 9.1) or of cyanobacterial toxicity (gene clusters) (Table 9.2). Altogether these gene markers were developed and allow the detection of one genera (Microcystis), three species (Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Planktothrix agardhii) and of five main cyanotoxins (microcystins, nodularins, cylindrospermopsins, anatoxin-a, saxitoxins) in several matrices such as pure cultures, water and soil. In cyanobacterial studies normally isolates are obtained from any environmental system and characterized with the

Genomics perspectives on cyanobacteria research Chapter | 9  149



TABLE 9.1  List of primers for cyanobacteria genomic detection. Target

Primer

Primer sequence 5′-3′

Reference

16S rRNA

27F

AGAGTTTGATCCTGGCTCAG

(Neilan et al., 1997)

809R

GCTTCGGCACGGCTCGGGTCGATA

(Jungblut et al., 2005)

740F

GGCYRWAWCTGACACTSAGGGA

(Neilan et al., 1997)

1494R

TACGGTTACCTTGTTACGAC

PCβF

GGCTGCTTGTTTACGCGACA

PCβR

CCAGTACCACCAGCAACTAA

Cyl2

GGCATTCCTAGTTATATTGCCATACTA

Cyl4

GCCCGTTTTTGTCCCTTTCGTGC

rpoC1_Plank_F271

TGTTAAATCCAGGTAACTATGACGGCCTA

rpoC1_P_agardhii_R472

GCGTTTTTGTCCCTTAGCAACGG

gyrF

GGACGTTTACGAGAACTAGCCTA

gyrR

GGTCTTGGTTTGTCCCTCAA

Micr 184F

GCCGCRAGGTGAAAMCTAA

Micr 431R

AATCCAAARACCTTCCTCCC

Phycocyanin operon (PCβ)

C. raciborskii rpoC1

P. agardhii rpoC1

M. aeruginosa gyrB

Microcystis sp.

(Neilan et al., 1995)

(Wilson et al., 2000)

(Churro et al., 2012)

(Tanabe et al., 2007)

(Neilan et al., 1997)

TABLE 9.2  List of primers for cyanotoxins genomic detection. Target

Primer

Primer sequence 5′-3′

Reference

mcyA

mcyA-CD1F

AAAATTAAAAGCCGTATCAAA

(Hisbergues et al., 2003)

mcyA-CD1R

AAAAGTGTTTTATTAGCGGCTCAT

MSF

ATCCAGCAGTTGAGCAAGC

MSR

TGCAGATAACTCCGCAGTTG

mcyB 2156-F

ATCACTTCAATCTAACGACT

mcyB 3111-R

AGTTGCTGCTGTAAGAAA

PSCF1

GCAACATCCCAAGAGCAAAG

PSCR1

CCGACAACATCACAAAGGC

PKDF1

GACGCTCAAATGATGAAAC

PKDR1

GCAACCGATAAAAACTCCC

PKEF1

CGCAAACCCGATTTACAG

PKER1

CCCCTACCATCTTCATCTTC

PKGF1

ACTCTCAAGTTATCCTCCCTC

PKGR1

AATCGCTAAAACGCCACC

AMT Fw

ATTGTAAATAGCTGGAATGAGTGG

AMT Rev

TTAGGGAAGTAATCTTCACAG

M13

GGCAAATTGTGATAGCCACGAGC

M14

GATGGAACATCGCTCACTGGTG

Microcystis sp. mcyA

Microcystis sp. mcyB

Microcystis sp. mcyC

Microcystis sp. mcyD

Microcystis sp. mcyE

Microcystis sp. mcyG

cyrA

cyrB

(Tillett et al., 2001)

(Mikalsen et al., 2003)

(Ouahid et al., 2005)

(Kellmann et al., 2006)

(Schembri et al., 2001)

Continued

150  PART | III  Algal genomics

TABLE 9.2  List of primers for cyanotoxins genomic detection—cont’d Target

Primer

Primer sequence 5′-3′

Reference

cyrC

K18

CCTCGCACATAGCCATTTGC

(Fergusson and Saint, 2003)

M4

GAAGCTCTGGAATCCGGTAA

(Schembri et al., 2001)

cynsulfF

ACTTCTCTCCTTTCCCTATC

(Mihali et al., 2008)

cylnamR

GAGTGAAAATGCGTAGAACTTG

anaC-genF

TCTGGTATTCAGTCCCCTCTAT

anaC-genR

CCCAATAGCCTGTCATCAA

anaC-anabF

GCCCGATATTGAAACAAGT

anaC-anabR

CACCCTCTGGAGATTGTTTA

sxtA855_F

GACTCGGCTTGTTGCTTCCCC

sxtA1480_R

GCCAAACTCGCAACAGGAGAAGG

sxtG432_F

AATGGCAGATCGCAACCGCTAT

sxtG928_R

ACATTCAACCCTGCCCATTCACT

sxtI 682F

GGATCTCAAAGAAGATGGCA

sxtI 877R

GCCAAACGCAGTACCACTT

cyrJ

anaC

Dolichospermum sp. anaC

sxtA

sxtG

sxtI

(Rantala-Ylinen et al., 2011a)

(Rantala-Ylinen et al., 2011a)

(Savela et al., 2015)

(Lopes et al., 2012)

PCR technique in terms of toxicity. Other studies, however, use water samples collected from near shores of rivers, lakes, lagoons or reservoirs and assess this same toxicity applying the molecular markers developed for such. In contrast to the analytical methods that allow the enumeration of the cyanotoxins and establishment of the level of toxicity by comparing the obtained toxic value with the guideline established value adopted by legislation, molecular methods through the PCR technique permit the characterization of the genes associated with cyanotoxicity in the environment allowing to determine the toxic potential of that environment or sample. This means that if the gene is present then it is probable the onset of toxicity in that ecosystem since the toxic genetic information was previously detected and when blooming conditions become available cyanobacteria can proliferate massively and cause cyanotoxins outbreaks. Other methodologies based in the ‘Polymerase Chain Reaction’ namely the multiplex-PCR have been developed and applied in cyanobacterial diagnosis and consist in the amplification of more than one gene fragment where three to five gene amplifications in one single reaction can be performed. These multiple reactions permit the amplification of the species and associated toxicity as described by Wilson et al. (2000) and also the amplification of five genes involved in microcystins production specific of Microcystis genus as described by Ouahid et al. (2005). Another well applied technique based in the PCR method is the Real-time PCR that consists in the quantification of the number of gene copies that exist in a single sample allowing to determine the number of cells per mL in that sample of a given genus or of a cyanobacterial toxic gene. Real-time PCR available protocols in cyanobacteria diagnosis consists mainly in the quantification of the genera Microcystis (Martins et al., 2011) of the cylindrospermopsin producing species Cylindrospermopsis raciborskii (Moreira et al., 2011) or of the Planktothrix agardhii (Churro et al., 2012) in which the sensitivity assays developed allow the minimum estimation of 8.8, 258 cells per mL and 8 cells per μL, respectively. In real-time PCR multiple reactions can also be performed with a maximum of four gene copy numbers being quantified per sample (Al-Tebrineh et al., 2012). These methods though more sensible than the single or multiple PCR reactions rely mostly on the application of primers firstly developed for single PCR. Altogether PCR and Real-Time PCR reactions require the development of tools such as primers that are specific of key cyanobacterial genes. Nowadays the pursue in the improvement of these tools and their combination in multiple reactions both in PCR and Real-Time PCR is determinant in cyanobacteria genomic research contributing to a faster and reliable diagnosis in any type of environment or sample. All main cyanotoxins gene clusters have been already annotated and sequenced resulting in the development of tools (primers) in their detection from all types of matrices contributing with faster tools in its screening and diagnosis. Overall molecular methods namely those based in the PCR reaction are characterized as being faster and more specific providing a prompt response in a cyanobacterial toxicological scenario than for instance the analytical methods that besides requiring

Genomics perspectives on cyanobacteria research Chapter | 9  151



technical expertize are more laborious and time consuming than the molecular ones. Another important issue relates to the fact that with molecular methods it is possible to evaluate the co-occurrence of all the main cyanotoxins in the same sample in a faster manner than with the analytical methods where only so far three of the main cyanotoxins can be enumerated simultaneously (Zervou et al., 2017). This is important to assess particularly in water quality management and in bloom composition particularly if there is a mixture of strains in the same bloom event. Also and not less important is the application of DNA sequencing particularly when assessing the strain composition of a given sample or when it is intended to identify a certain cyanobacterial genus or species. This technique can further be used in phylogenetic and biogeographic inferences and in next-generation sequencing. These will be discussed in further sections of this chapter. With the development of the degenerated oligonucleotide PCR primers to amplify the regions encoding the ketosynthase (KS) and adenylation (A) domains from the PKS and NRPS gene clusters (Table 9.3), respectively (Moffitt and Neilan, 2001; Neilan et al., 1999), the screening for the bioactive potential of cyanobacterial strains and the evaluation of the variety of NP in individual and among different strains has become more efficient (Brito et al., 2015). The same has occurred with the development of primers to detect specific cyanobacteria NP such as the protease inhibitors aeruginosin (Ishida et al., 2009), cyanopeptolin (Dittmann et al., 2001) and microviridin (Murakami et al., 1997); the antifungal hassalidin (Vestola et al., 2014); the angiotensin-converting enzyme and leucine amino peptidases inhibitor microginin (Dittmann et al., 2001); the sunscreen scytonemin; and one of the most common compounds responsible for odor and taste in blooms episodes, the geosmin (Watson et al., 2008). In addition, primers for the detection of ribosomal peptides such as cyanobactins have been designed. It is estimated that 10–30% of all cyanobacteria may have the ability to produce cyanobactins that seem to be widespread among symbiotic and free-living cyanobacteria from terrestrial, freshwater, and marine environments (Martins and Vasconcelos, 2015).

TABLE 9.3  List of primers for cyanobacterial natural products genomic detection. Target

Primer

Primer sequence 5’ → 3’

Reference

PKS

DKF

GTGCCGGTNCCRTGNGYYTC

(Moffitt and Neilan, 2001)

DKR

GCGATGGAYCCNCARCARMG

MTF2

GCNGGYGGYGCNTAYGTNCC

MTR

CCNCGDATYTTNACYTG

aerA

aerA_F

GATAGCACCCAGAACGGAAGC

aerB

aerB_R

CGTTAAACGGATGGTTAGAGC

aerM

aep1F

GAAGCATTACAACCACAGCG

aerM

aep1R

GGTTTAACTGCGCTAACTGGTG

aerB

aep2F

GAAGTCAGACCAGATAGCTCCG

aerB

aep2R

GCGTGGTGCAGTAAAGTTGTCTG

aerG

aep6F

GTGTGCATCATATTCTGGCTG

aerG

aep6R

GCGCGATCGCTCAATTCCTG

aerI

aep7F

GCTGAACCTCCCAAGATTGG

aerI

aep7R

GGGTAACTCCACAGACATAG

aerI

aep68F

GCAACTGTACTGGGAGAATTAG

aerI

aep68R

GGCTAATCCTTGGGCGATCG

mcnC

mcnC_F

TAAGGATAATTTCTTTGAATTGGGAG

mcnE

mcnE_R

GGGAATAATCTCTAAATCAACAGC

NRPS

(Neilan et al., 1999)

Aeruginosin (Cadel-Six et al., 2008)

(Fewer et al., 2013)

Cyanopeptolin (Cadel-Six et al., 2008)

Continued

152  PART | III  Algal genomics

TABLE 9.3  List of primers for cyanobacterial natural products genomic detection—cont’d Primer

Primer sequence 5’ → 3’

Reference

mdnB

mdnBfw

TTGGCTGGTTTTTGGGATAG

(Ziemert et al., 2010)

mdnB

mdnBrv

CGATCGCATTGGAAATAGGT

mdnC

mdn mult fw

TCACTCGAAATTACCAGAGGAA

mdnC

mdn mult rv

CGGTGTAATCAAGAAAAGTGCT

mdnA

mdnC fw

GAAGGTTTGCAATTTTGTCCA

mdnA

mdnC rv

CGCCAACGGGATTAATTTCT

hasV

hasV-fw

TCTAGATGGTTGGAGTGTGGC

hasV

hasV-rev

AGGATGCGGTAGCTTTGAGGAGGCG

hasN

hasN-fw

GTAGATGCGGTGCCATTGAC

hasN

hasN-rev

GACTACCACTGATTGCTTCCAC

hasO

hasO-fw

GCCCAAGCATTAATCCAGTTAG

hasO

hasO-rev

GCATCTTCTGGTTGCTCTAC

hasN

AF

TGCGTCGTCAAGGTTGGATATTAAC

hasN

AR

CATCTAAATCTGAAGATAATTCCTC

Microginin

MgnDan_EF

Patent no. EP 05 026 396

MgnDan_AR

Patent no. EP 05 026 396

scyC

Forward primer

GTNTAYTTYCAYTGG

scyC

Reverse primer

ADCKYTTDATRTTCAT

geoA

Geo799F

GCCGCTAACCTCACTAACGA

geoA

Geo927R

AAGGAGAACATTCACACGCTCT

A

CBT_AF

TTVGGYTAYGAYTTYGG

A

CBT_AR

AGACCARGAACGRACTTC

Target Microviridin

Hassallidins (Vestola et al., 2014)

Kramer, unpublished (see e.g., Martins et al., 2009)

Scytonemin (Balskus et al., 2011)

Geosmin (John et al., 2018)

Cyanobactins (Martins et al., 2013)

9.3  Non-PCR based methods Molecular methods based in the PCR technique such as ‘denaturating gradient gel electrophoresis’ (DGGE), ‘restriction fragment length polymorphism’ (RFLP), and ‘random amplified polymorphic DNA’ (RAPD) have been applied to cyanobacteria and cyanotoxins research, allowing the profiling of cyanobacterial communities directly from the environment, where they have been commonly used (see Saker et al., 2009 for a review). Using these methods, through toxicological oriented studies it is possible to infer the diversity and successional patterns of a potentially toxic cyanobacterial population present in a certain community (Ye et al., 2009). DGGE, a technique for separation of PCR products, allows the sequence-dependent separation of small DNA fragments according to their melting proprieties (Nollau and Wagener, 1997). In cyanobacteria research this has been used to study the genetic diversity of cyanobacteria, including toxic cyanobacteria (Ramos et al., 2017; Jasser et al., 2013; Bukowska et al., 2014) as well monitoring the presence of toxigenic and non-toxigenic cyanobacteria in the environment (Fewer et al., 2009; Yen et al., 2012).



Genomics perspectives on cyanobacteria research Chapter | 9  153

In RFLP, restriction enzyme(s) digests PCR products of specific genes/loci prior to electrophoresis (Palinska et  al., 2011) whereas in RAPD, total genomic DNA is amplified by PCR with random short primers that present a high probability to anneal to the entire genome (Neilan et al., 1995). However, despite several studies have been performed using this method, the banding patterns are more complex than the ones with RFLP, and with a very low reproducibility yield, and so this method seems to be no longer recommended (Wilmotte et al., 2017). RFLP analysis allows to: provide signature profiles specific to a genus, species or strain, differentiate between distinct cyanotoxin producers without sequencing and characterize the (sub)population genetic diversity. RFLP of the 16S rRNA and ITS assays were used to construct a diagnostic key for cyanobacterial species identification, with the exception of few strains that could not be distinguished (Valerio et al., 2009). More recently genomic fingerprinting using highly repetitive sequences to differentiate close cyanobacterial strains has been achieved. Using 16S rRNA, ‘Enterobacterial repetitive intergenic consensus’ (ERIC), ‘Short Tandemly Repeated Repetitive’ (STRR1a) and ‘Highly Iterated Palindromes’ (HIP) primers it was demonstrated that each marker produced a unique and strain-specific banding pattern (Shokraei et al., 2019). Non-PCR based methods based on DNA probes, such as DNA microarrays and Fluorescence in situ hybridization (FISH) may be utilized in cyanobacteria and/or cyanotoxins research, although they are less commonly used than PCRbased techniques. DNA microarrays usually include sequences (probes) for only one or a few genes allowing a number of genera/species to be detected (Rantala-Ylinen et  al., 2017). cDNA microarrays are used for screening genome-wide changes in gene expression and include probes for all or most genes in one strain (Burja et al., 2003). They have been applied in gene expression in cyanobacterial species (Li et al., 2004) and in the genetic profiling of natural communities (Rudi et al., 2000). The application of this technology in cyanotoxins studies is more recent, and has been applied to validate the detection of microcystins (mcyE) and nodularin (ndaF) in cyanobacterial strains and in environmental samples (Rantala et al., 2008; Rantala-Ylinen et al., 2011b). More recently, a ‘reverse transcriptase (RT) microarray’—‘CYANO RT-Microarray’—was developed for the detection of very low expression of mRNA from cyanotoxin genes but further studies are needed to understand its efficacy (Medlin, 2018). PCR techniques in the detection of cyanobacterial DNA or cyanotoxins involve DNA extraction that destroys cells or colony organizations. Thus, a rapid assay that allows both the identity and activity determination of cyanotoxin producing species still remains a challenge. FISH studies have been useful in the identification of cyanobacteria in natural samples and also of the cyanotoxin microcystin (mcyA) in the cyanobacterium Microcystis aeruginosa (Metcalf et al., 2009). The combination of FISH with flow cytometry demonstrates to be a useful approach to discriminate between toxic and non-toxic Microcystis strains in environmental samples and laboratory-cultured strains (Gan et  al., 2010). More recently, a whole cell ‘Tyramid Signal Amplification-Fluorescent in situ Hybridization’ (TSA-FISH) assay was developed to the detection of MC-producing Microcystis strains, targeting mcyA mRNA transcription as a proxy of MC-synthetase production (Zeller et  al., 2016). Nevertheless, the measurement of microcystins by HPLC was not provided, and their in situ application was not demonstrated. Later, in order to provide the identity and activity of cyanotoxin producing species in freshwater lakes a TSA-FISH was applied. Results revealed that the assay allowed acquiring rapidly information of the presence and abundance of potentially toxic species, while identifying species actively producing MC-synthetase mRNA (Brient et al., 2017).

9.4  Next-generation sequencing Besides the PCR and non-PCR based methods ‘next-generation sequencing’ (NGS) has been recently applied in cyanobacterial genomics research. This method consists in the amplification of a wide region of the 16S rRNA gene allowing to evaluate the cyanobacterial community in a given type of ecosystem or sample. These studies are still scarce in cyanobacterial research and detain so far only two publications in which two distinct set of primers based in the 16S rRNA region were applied. Firstly Kleinteich et al. (2014) published a work where through a pyrosequencing approach the characterization of the cyanobacterial community in Antarctica cyanobacterial mats was achieved. In their study an evaluation on the cyanotoxicty through two separate techniques the liquid chromatography-mass spectrometry and the ‘Polymerase Chain Reaction’ through cyrAB and cyrJ genes was conducted. More recently Berry et al. (2017) studied the interaction between the bacterial community and the cyanobacterial community in Western Lake Erie. The fact that only two studies have been published leaves much to discover about the cyanobacterial diversity in many of the biogeographical areas of the world. Another important remark in these type of studies is the assessment of cyanotoxins occurrence either through the traditional analytical techniques or the more advanced molecular methods contributing to a more elaborated ecosystem characterization.

9.5  Phylogeny and biogeography Phylogenetic studies are applied in cyanobacteria research as a means to assess the genetic diversity, phylogeography or the taxonomic positioning of a given taxa (Table 9.4). These studies rely mainly in the amplification of the c­ yanobacterial

154  PART | III  Algal genomics

TABLE 9.4  Genetic markers used and their respective application in taxonomy, phylogeny, phylogeography, and biogeography studies on cyanobacteria. Genetic marker

Taxonomy

Phylogeny

Phylogeography

Biogeography









PC-IGS ftsZ



glnA



gltX



gyrB



pgi



recA



tpi



16S-23S ITS1-L







16S-23S ITS1-S rpoB



rpoC1



nifH



⃰ ⃰

⃰ ⃰

nifD



16 rRNA









16S-23S ITS









rbcLX



hetR



psbA



rbcL



rbcS



genetic information where several primers have already been applied. If PCR methods are used then the genetic information is required and in opposite if non-PCR methods are applied then fingerprinting profiles are necessary (see Section 9.3). Choosing the method to analyze the genetic relationships among strains carries distinct phylogenetic approaches. In this sense when applying PCR based methods algorithms such as neighbor-joining (NJ), maximum likelihood (ML) or Bayesian (BAY) inferences are those that are mostly applied. In contrast in the non-PCR based methods UPGMA (unweighted pair group method with arithmetic mean) and PHYLIP algorithms are the most frequently used. Phylogenetic inferences conducted on cyanobacterial strains permit the estimation of the number of genotypes of a given strain in a given environment. This enables to determine the genetic diversity of those taxa in that ecosystem. Genetic information can be based in solely one genetic marker or in a conjunction of more than one (concatenation). Also if strains are collected from distinct geographic locations a phylogeographic inference can be achieved. This is based in the geographic grouping of the strains or in a more general manner in the spatial arrangement of the chosen taxa. If the main aim is to assess the temporal and spatial grouping of the strains then biogeographic signatures or patterns can be inferred. In biogeography the concept that everything is everywhere but environment selects is well accepted and in cyanobacteria some studies have showed that for instance dispersion profiles can be determined if a phylogenetic analysis is conducted. This is particularly important if well-known invasive cyanobacteria strains such as Cylindrospermopsis raciborskii are present. Dispersion in the form of passive dispersal can be achieved through wellestablished vectors such as winds, river courses, fishes, birds, humans or scientific activities (Kristiansen, 1996). In taxonomic positioning some genetic markers have been applied and consist in the definition on the positioning of certain taxa namely isolated strains and determine their monophyletic or polyphyletic grouping. One of the studies that



Genomics perspectives on cyanobacteria research Chapter | 9  155

­demonstrate this belongs to Valerio et al. (2009) where a global phylogeny of six cyanobacterial orders the 16S rRNA marker showed that the Nostocales, Stigonematales and Pleurocapsales form monophyletic groups while the Chroococcales and Oscillatoriales are polyphyletic. These type of studies are important to assess the taxonomic resolution of a given taxa in comparison with others that are closely related. Most studies involving a phylogenetic approach are base in the clustering of one genetic marker whether in other studies a concatenation approach (multiple genes) as helped to unravel the genetic diversity of certain cyanobacterial taxa such as occurred in Cylindrospermopsis raciborskii or in the common Microcystis aeruginosa. In both studies strains collected from several culture collections around the world aided simultaneously in their origin and dispersal since early times. In fact when analyzing with the three main algorithms in phylogeny (NJ, ML, BAY) Moreira et al. (2013b) has shown that M. aeruginosa may have originated in Africa and then possibly through migratory bird routes may have colonized the European continent from where it then spread to the remaining continental groups (Moreira et al., 2014). Also other authors have demonstrated that in Microcystis aeruginosa a lack of phylogeographic structuring revealed its cosmopolitan nature (van Gremberghe et al., 2011). All these studies were based in the genetic information of the strains, i.e., PCR based methods (PCR and DGGE). Though PCR-based phylogenies are well over applied than the fingerprinting phylogenies the application of both in the same study has never been observed being individually applied. Therefore the criterion relies on the specificity of the method chosen (PCR or non-PCR) and consequently the respective phylogeny is produced and analyzed.

9.6  Biodiscovery of new compounds Genomic studies can aid in the prospection of new compounds if primers are developed. The cyanobacterial based degenerate primers developed by Neilan et al. (1999) and Moffitt and Neilan (2001) based in the polyketide synthase and nonribosomal peptide synthetase has helped in the bioprospection of new compounds in cyanobacterial strains. In fact Culture Collections are a rich source of cyanobacterial taxa and through a genomic approach can be characterized in terms of the diversity on natural products composition. Also the application of these and more specific primers (see Section 9.2) in complex matrices such as water, soil or cyanobacterial mats can allow to determine the natural compound composition and the availability of these molecules in environmental samples permitting to serve as a directional tool in chemical compound isolation. Genome and gene mining has significantly improved the discovery of natural products (e.g., Leao et al., 2017), especially as a complementary approach to the chemical methods. An example of this is the recent discover of the columbamides, from Moorea strains, through the combination of mass-spectrometry metabolic profiling with genomic analysis (Kleigrewe et al., 2015). Bioinformatics tools are essential for the identification and characterization of the biosynthetic gene clusters either from isolated strains, or metagenome analyses. The ‘Antibiotics & Secondary Metabolite Analysis Shell’ (antiSMASH) (Blin et al., 2013; Medema et al., 2011) which last version—antiSMASH2.0—allows the simultaneous processing of multiple contigs, analysis of protein sequences, and expanded structure prediction, has been commonly used with good results. However, some misidentification of certain domains may occur when compared with the DELTA-BLASTP ‘Domain-Enhanced Lookup Time Accelerated Basic Local Alignment’ Search Tool (NCBI) analysis (Moss et al., 2018). Other techniques like ‘Cluster Finder’ (Cimermancic et al., 2014), NapDoS (Ziemert et al., 2012), NRPS/PKS analysis (Bachmann and Ravel, 2009), among others are useful for the development of novel secondary metabolites. In addition, metagenomics combinatorial biosynthesis and synthetic biology techniques are also emerging strategies for the discovery of NP (Cuadrat et al., 2018; Kim et al., 2012; Tianero et al., 2012; Woodhouse et al., 2013).

9.7  Water quality management Water is a natural resource used for humans for several purposes such as drinking, irrigation and recreation. Surveillance of aquatic ecosystems is mandatory and in these cyanobacteria genomic research carries great impact. The evaluation on the presence of genes associated with cyanobacterial toxicity is extremely relevant in terms of water quality. Their presence in water systems can represent an increased risk to humans and wildlife constituting and an important vector of contamination to these resulting in illness and loss. In history some outbreaks of cyanotoxins have been reported either in a dialysis center in Brazil associated with the contamination of water with microcystins, in a reservoir in Australia that was contaminated with cylindrospermopsins and that affected human lives or through the death of dogs in a river in France after a bloom event contaminated with neurotoxins (Bourke et al., 1983; Gugger et al., 2005; Jochimsen et al., 1998). The fact that cyanobacteria are important producers of harmful toxins in water systems carries great negative impact not only on public health but also on the impairment of water quality through the release of odor and scums in surface waters. The application of all the genomic methods described in this book chapter can aid in the early detection and identification of both cyanotoxins and toxic cyanobacteria in aquatic ecosystems providing a prompt tools in its detection. This allows agencies and governments

156  PART | III  Algal genomics

to implement safety measures in the control and prevention of cyanobacteria outbreaks and consequently of cyanotoxins production and release to water systems. Apart of the scientific perspective of genomic studies in these microorganisms much remains in the application of these methods either solely or in a polyphasic approach in the evaluation of the cyanotoxicity in water quality management. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a, b, c, d, e, f, g, h, i, j, k, l, m, n, o).

References Al-Tebrineh, J., Pearson, L.A., Yasar, S.A., Neilan, B.A., 2012. A multiplex qPCR targeting hepato- and neurotoxigenic cyanobacteria of global significance. Harmful Algae 15, 19–25. Arnison, P.G., Bibb, M.J., Bierbaum, G., Bowers, A.A., Bugni, T.S., Bulaj, G., et al., 2013. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30 (1), 108–160. Bachmann, B.O., Ravel, J., 2009. Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data. Method. Enzymol. 458, 181–217. Bahl, J., Lau, M.C.Y., Smith, G.J., Vijaykrishna, D., Cary, S.C., Lacap, D.C., et al., 2011. Ancient origins determine global biogeography of hot and cold desert cyanobacteria. Nat. Commun. 2, 163. Balskus, E.P., Case, R.J., Walsh, C.T., 2011. The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities. FEMS Microbiol. Ecol. 77 (2), 322–332. Berry, M.A., Davis, T.W., Cory, R.M., Duhaime, M.B., Johengen, T.H., Kling, G.W., et al., 2017. Cyanobacterial harmful algal blooms are a biological disturbance to Western Lake Erie bacterial communities. Environ. Microbiol. 19 (3), 1149–1162. Blin, K., Medema, M.H., Kazempour, D., Fischbach, M.A., Breitling, R., Takano, E., et al., 2013. antiSMASH 2.0—a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 41, W204–W212. Bourke, A.T.C., Hawes, R.B., Neilson, A., Stallman, N.D., 1983. An outbreak of hepato-enteritis (the Palm Island mystery disease) possibly caused by algal intoxication. Toxicon 21 (3), 45–48. Brient, L., Ben Gamra, N., Periot, M., Roumagnac, M., Zeller, P., Bormans, M., et al., 2017. Rapid characterization of microcystin-producing cyanobacteria in freshwater lakes by TSA-FISH (tyramid signal amplification-fluorescent in situ hybridization). Front. Environ. Sci. 5, 43. Brito, A., Gaifem, J., Ramos, V., Glukhov, E., Dorrestein, P.C., Gerwick, W.H., et al., 2015. Bioprospecting Portuguese Atlantic coast cyanobacteria for bioactive secondary metabolites reveals untapped chemodiversity. Algal Res. 9, 218–226. Bukowska, A., Bielczynska, A., Karnkowska, A., Chrost, R.J., Jasser, I., 2014. Molecular (PCR-DGGE) versus morphological approach: analysis of taxonomic composition of potentially toxic cyanobacteria in freshwater lakes. Aquat. Biosyst. 10, 2. Burch, M.D., 2008. Effective doses, guidelines & regulations. Adv. Exp. Med. Biol. 619, 831–853. Burja, A.M., Dhamwichukorn, S., Wright, P.C., 2003. Cyanobacterial postgenomic research and systems biology. Trends Biotechnol. 21 (11), 504–511. Cadel-Six, S., Dauga, C., Castets, A.M., Rippka, R., Bouchier, C., de Marsac, N.T., et al., 2008. Halogenase genes in nonribosomal peptide synthetase gene clusters of Microcystis (cyanobacteria): sporadic distribution and evolution. Mol. Biol. Evol. 25 (9), 2031–2041. Churro, C., Pereira, P., Vasconcelos, V., Valerio, E., 2012. Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii. Arch. Microbiol. 194 (9), 749–757. Cimermancic, P., Medema, M.H., Claesen, J., Kurita, K., Brown, L.C.W., Mavrommatis, K., et al., 2014. Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters. Cell 158 (2), 412–421. Cuadrat, R.R.C., Ionescu, D., Davila, A.M.R., Grossart, H.P., 2018. Recovering genomics clusters of secondary metabolites from lakes using genomeresolved metagenomics. Front. Microbiol. 9, 251. Dittmann, E., Gugger, M., Sivonen, K., Fewer, D.P., 2015. Natural product biosynthetic diversity and comparative genomics of the cyanobacteria. Trends Microbiol. 23 (10), 642–652. Dittmann, E., Neilan, B.A., Borner, T., 2001. Molecular biology of peptide and polyketide biosynthesis in cyanobacteria. Appl. Microbiol. Biot. 57 (4), 467–473. Fergusson, K.M., Saint, C.P., 2003. Multiplex PCR assay for Cylindrospermopsis raciborskii and cylindrospermopsin-producing cyanobacteria. Environ. Toxicol. 18 (2), 120–125. Fewer, D.P., Jokela, J., Paukku, E., Osterholm, J., Wahlsten, M., Permi, P., et al., 2013. New structural variants of aeruginosin produced by the toxic bloom forming cyanobacterium Nodularia spumigena. PLoS One 8 (9), e73618. Fewer, D.P., Koykka, M., Halinen, K., Jokela, J., Lyra, C., Sivonen, K., 2009. Culture-independent evidence for the persistent presence and genetic diversity of microcystin-producing Anabaena (Cyanobacteria) in the Gulf of Finland. Environ. Microbiol. 11 (4), 855–866. Gan, N.Q., Huang, Q., Zheng, L.L., Song, L.R., 2010. Quantitative assessment of toxic and nontoxic Microcystis colonies in natural environments using fluorescence in situ hybridization and flow cytometry. Sci. China Life Sci. 53 (8), 973–980. Gugger, M., Lenoir, S., Berger, C., Ledreux, A., Druart, J.C., Humbert, J.F., et al., 2005. First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis. Toxicon 45 (7), 919–928. Hisbergues, M., Christiansen, G., Rouhiainen, L., Sivonen, K., Borner, T., 2003. PCR- based identification of microcystin-producing genotypes of different cyanobacterial genera. Arch. Microbiol. 180 (6), 402–410.



Genomics perspectives on cyanobacteria research Chapter | 9  157

Ishida, K., Welker, M., Christiansen, G., Cadel-Six, S., Bouchier, C., Dittmann, E., et al., 2009. Plasticity and evolution of aeruginosin biosynthesis in cyanobacteria. Appl. Environ. Microbiol. 75 (7), 2017–2026. Jasser, I., Krolicka, A., Jakubiec, K., Chrost, R.J., 2013. Seasonal and spatial diversity of picocyanobacteria community in the Great Mazurian lakes derived from DGGE analyses of 16S rDNA and cpcBA-IGS markers. J. Microbiol. Biotechnol. 23 (6), 739–749. Jochimsen, E.M., Carmichael, W.W., An, J.S., Cardo, D.M., Cookson, S.T., Holmes, C.E., et al., 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N. Engl. J. Med. 338 (13), 873–878. John, N., Koehler, A.V., Ansell, B.R.E., Baker, L., Crosbie, N.D., Jex, A.R., 2018. An improved method for PCR-based detection and routine monitoring of geosmin-producing cyanobacterial blooms. Water Res. 136, 34–40. Jungblut, A.D., Hawes, I., Mountfort, D., Hitzfeld, B., Dietrich, D.R., Burns, B.P., et al., 2005. Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo Ice Shelf, Antarctica. Environ. Microbiol. 7 (4), 519–529. Kellmann, R., Mills, T., Neilan, B.A., 2006. Functional modeling and phylogenetic distribution of putative cylindrospermopsin biosynthesis enzymes. J. Mol. Evol. 62 (3), 267–280. Kim, E.J., Lee, J.H., Choi, H., Pereira, A.R., Ban, Y.H., Yoo, Y.J., et al., 2012. Heterologous production of 4-O-demethylbarbamide, a marine cyanobacterial natural product. Org. Lett. 14 (23), 5824–5827. Kleigrewe, K., Almaliti, J., Tian, I.Y., Kinnel, R.B., Korobeynikov, A., Monroe, E.A., et al., 2015. Combining mass spectrometric metabolic profiling with genomic analysis: a powerful approach for discovering natural products from cyanobacteria. J. Nat. Prod. 78 (7), 1671–1682. Kleigrewe, K., Gerwick, L., Sherman, D.H., Gerwick, W.H., 2016. Unique marine derived cyanobacterial biosynthetic genes for chemical diversity. Nat. Prod. Rep. 33 (2), 348–364. Kleinteich, J., Hildebrand, F., Wood, S.A., Cires, S., Agha, R., Quesada, A., et al., 2014. Diversity of toxin and non-toxin containing cyanobacterial mats of meltwater ponds on the Antarctic Peninsula: a pyrosequencing approach. Antarct. Sci. 26 (5), 521–532. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Kristiansen, J., 1996. Dispersal of freshwater algae—a review. Hydrobiologia 336 (1–3), 151–157 (Chapter 16). Leao, T., Castelao, G., Korobeynikov, A., Monroe, E.A., Podell, S., Glukhov, E., et al., 2017. Comparative genomics uncovers the prolific and distinctive metabolic potential of the cyanobacterial genus Moorea. Proc. Natl. Acad. Sci. U. S. A. 114 (12), 3198–3203. Li, H., Singh, A.K., McIntyre, L.M., Sherman, L.A., 2004. Differential gene expression in response to hydrogen peroxide and the putative PerR Regulon of Synechocystis sp. strain PCC 6803. J. Bacteriol. 186 (11), 3331–3345. Lopes, V.R., Ramos, V., Martins, A., Sousa, M., Welker, M., Antunes, A., et al., 2012. Phylogenetic, chemical and morphological diversity of cyanobacteria from Portuguese temperate estuaries. Mar. Environ. Res. 73, 7–16. Martins, A., Moreira, C., Vale, M., Freitas, M., Regueiras, A., Antunes, A., et al., 2011. Seasonal dynamics of Microcystis spp. and their toxigenicity as assessed by qPCR in a temperate reservoir. Mar. Drugs 9 (10), 1715–1730.

158  PART | III  Algal genomics

Martins, J., Leao, P.N., Ramos, V., Vasconcelos, V., 2013. N-terminal protease gene phylogeny reveals the potential for novel cyanobactin diversity in cyanobacteria. Mar. Drugs 11 (12), 4902–4916. Martins, J., Saker, M.L., Moreira, C., Welker, M., Fastner, J., Vasconcelos, V.M., 2009. Peptide diversity in strains of the cyanobacterium Microcystis aeruginosa isolated from Portuguese water supplies. Appl. Microbiol. Biot. 82 (5), 951–961. Martins, J., Vasconcelos, V., 2015. Cyanobactins from cyanobacteria: current genetic and chemical state of knowledge. Mar. Drugs 13 (11), 6910–6946. Medema, M.H., Blin, K., Cimermancic, P., de Jager, V., Zakrzewski, P., Fischbach, M.A., et al., 2011. antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 39 (s2), W339–W346. Medlin, L.K., 2018. CYANO RT-Microarray: a novel tool to detect gene expression in cyanobacteria. J. Environ. Microbiol. 1 (1), 17–27. Metcalf, J.S., Reilly, M., Young, F.M., Codd, G.A., 2009. Localization of the microcystin synthetase genes in colonies of the cyanobacterium Microcystis using fluorescence in situ hybridization. J. Phycol. 45 (6), 1400–1404. Mihali, T.K., Kellmann, R., Muenchoff, J., Barrow, K.D., Neilan, B.A., 2008. Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis. Appl. Environ. Microbiol. 74 (3), 716–722. Mikalsen, B., Boison, G., Skulberg, O.M., Fastner, J., Davies, W., Gabrielsen, T.M., et al., 2003. Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. J. Bacteriol. 185 (9), 2774–2785. Moffitt, M.C., Neilan, B.A., 2001. On the presence of peptide synthetase and polyketide synthase genes in the cyanobacterial genus Nodularia. FEMS Microbiol. Lett. 196 (2), 207–214. Moreira, C., Azevedo, J., Antunes, A., Vasconcelos, V., 2013a. Cylindrospermopsin: occurrence, methods of detection and toxicology. J. Appl. Microbiol. 114 (3), 605–620. Moreira, C., Martins, A., Azevedo, J., Freitas, M., Regueiras, A., Vale, M., et al., 2011. Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential. Appl. Microbiol. Biot. 92 (1), 189–197. Moreira, C., Spillane, C., Fathalli, A., Vasconcelos, V., Antunes, A., 2014. African origin and Europe-mediated global dispersal of the cyanobacterium Microcystis aeruginosa. Curr. Microbiol. 69 (5), 628–633. Moreira, C., Vasconcelos, V., Antunes, A., 2013b. Phylogeny and biogeography of cyanobacteria and their produced toxins. Mar. Drugs 11 (11), 4350–4369. Moss, N.A., Leao, T., Glukhov, E., Gerwick, L., Gerwick, W.H., 2018. Collection, culturing, and genome analyses of tropical marine filamentous benthic cyanobacteria. Method. Enzymol. 604, 3–43. Murakami, M., Sun, Q., Ishida, K., Matsuda, H., Okino, T., Yamaguchi, K., 1997. Microviridins, elastase inhibitors from the cyanobacterium Nostoc minutum (NIES-26). Phytochemistry 45 (6), 1197–1202. Neilan, B.A., Dittmann, E., Rouhiainen, L., Bass, R.A., Schaub, V., Sivonen, K., et al., 1999. Nonribosomal peptide synthesis and toxigenicity of cyanobacteria. J. Bacteriol. 181 (13), 4089–4097. Neilan, B.A., Jacobs, D., del Dot, T., Blackall, L.L., Hawkins, P.R., Cox, P.T., et al., 1997. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. Int. J. Syst. Bacteriol. 47 (3), 693–697. Neilan, B.A., Jacobs, D., Goodman, A., 1995. Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus. Appl. Environ. Microbiol. 61 (11), 3875–3883. Nollau, P., Wagener, C., 1997. Methods for detection of point mutations: performance and quality assessment. IFCC Scientific Division, Committee on Molecular Biology Techniques. Clin. Chem. 43 (7), 1114–1128. Ouahid, Y., Perez-Silva, G., del Campo, F.F., 2005. Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions. Environ. Toxicol. 20 (3), 235–242. Palinska, K.A., Deventer, B., Hariri, K., Lotocka, M., 2011. A taxonomic study on Phormidium-group (cyanobacteria) based on morphology, pigments, RAPD molecular markers and RFLP analysis of the 16S rRNA gene fragment. Fottea 11 (1), 41–55. Ramos, V.M.C., Castelo-Branco, R., Leao, P.N., Martins, J., Carvalhal-Gomes, S., da Silva, F.S., et al., 2017. Cyanobacterial diversity in microbial mats from the hypersaline lagoon system of Araruama, Brazil: an in-depth polyphasic study. Front. Microbiol. 8, 1233. Rantala, A., Rizzi, E., Castiglioni, B., de Bellis, G., Sivonen, K., 2008. Identification of hepatotoxin-producing cyanobacteria by DNA-chip. Environ. Microbiol. 10 (3), 653–664. Rantala-Ylinen, A., Kana, S., Wang, H., Rouhiainen, L., Wahlsten, M., Rizzi, E., et al., 2011a. Anatoxin-a synthetase gene cluster of the cyanobacterium Anabaena sp. strain 37 and molecular methods to detect potential producers. Appl. Environ. Microbiol. 77 (20), 7271–7278. Rantala-Ylinen, A., Sipari, H., Sivonen, K., 2011b. Molecular methods: chip assay and quantitative real-time PCR: in detecting hepatotoxic cyanobacteria. Methods Mol. Biol. 739, 73–86. Rantala-Ylinen, A., Sivonen, K., Wilmotte, A., Matthijs, H.C.P., Schuurmans, J.M., 2017. DNA (diagnostic) and cDNA microarray. In: Kurmayer, R., Sivonen, K., Wilmotte, A., Salmaso, N. (Eds.), Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria. John Wiley & Sons Ltd, Chichester, pp. 241–261. Rudi, K., Skulberg, O.M., Skulberg, R., Jakobsen, K.S., 2000. Application of sequence-specific labeled 16s rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization. Appl. Environ. Microbiol. 66 (9), 4004–4011. Saker, M., Moreira, C., Martins, J., Neilan, B., Vasconcelos, V.M., 2009. DNA profiling of complex bacterial populations: toxic cyanobacterial blooms. Appl. Microbiol. Biot. 85 (2), 237–252. Savela, H., Spoof, L., Perala, N., Preede, M., Lamminmaki, U., Nybom, S., et al., 2015. Detection of cyanobacterial sxt genes and paralytic shellfish toxins in freshwater lakes and brackish waters on Aland Islands, Finland. Harmful Algae 47, 1–10. Schembri, M.A., Neilan, B.A., Saint, C.P., 2001. Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii. Environ. Toxicol. 16 (5), 413–421.



Genomics perspectives on cyanobacteria research Chapter | 9  159

Shokraei, R., Fahimi, H., Blanco, S., Nowruzi, B., 2019. Genomic fingerprinting using highly repetitive sequences to differentiate close cyanobacterial strains. Microb. Bioact. 2 (1), 068-07x. Tanabe, Y., Kasai, F., Watanabe, M.M., 2007. Multilocus sequence typing (MLST) reveals high genetic diversity and clonal population structure of the toxic cyanobacterium Microcystis aeruginosa. Microbiology 153 (11), 3695–3703. Tianero, M.D.B., Donia, M.S., Young, T.S., Schultz, P.G., Schmidt, E.W., 2012. Ribosomal route to small-molecule diversity. J. Am. Chem. Soc. 134 (1), 418–425. Tidgewell, K., Clark, B.R., Gerwick, W.H., 2010. The natural products chemistry of cyanobacteria. In: Liu, H.W.B., Mander, L. (Eds.), Comprehensive Natural Products II: Chemistry and Biology. Elsevier, Amsterdam, pp. 141–188. Tillett, D., Parker, D.L., Neilan, B.A., 2001. Detection of toxigenicity by a probe for the Microcystin Synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies. Appl. Environ. Microbiol. 67 (6), 2810–2818. Valerio, E., Chambel, L., Paulino, S., Faria, N., Pereira, P., Tenreiro, R., 2009. Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs. Microbiology 155 (2), 642–656. van Apeldoorn, M.E., van Egmond, H.P., Speijers, G.J.A., Bakker, G.J.I., 2007. Toxins of cyanobacteria. Mol. Nutr. Food Res. 51 (1), 7–60. van Gremberghe, I., Leliaert, F., Mergeay, J., Vanormelingen, P., van der Gucht, K., Debeer, A.E., et al., 2011. Lack of phylogeographic structure in the freshwater cyanobacterium Microcystis aeruginosa suggests global dispersal. PLoS One 6 (5), e19561. Vestola, J., Shishido, T.K., Jokela, J., Fewer, D.P., Aitio, O., Permi, P., et al., 2014. Hassallidins, antifungal glycolipopeptides, are widespread among cyanobacteria and are the end-product of a nonribosomal pathway. Proc. Natl. Acad. Sci. U. S. A. 111 (18), E1909–E1917. Watson, S.B., Ridal, J., Boyer, G.L., 2008. Taste and odour and cyanobacterial toxins: impairment, prediction, and management in the Great Lakes. Can. J. Fish. Aquat. Sci. 65 (8), 1779–1796. Welker, M., von Dohren, H., 2006. Cyanobacterial peptides—nature’s own combinatorial biosynthesis. FEMS Microbiol. Rev. 30 (4), 530–563. Wilmotte, A., Laughinghouse, I.V.H.D., Capelli, C., Rippka, R., Salmaso, N., 2017. Taxonomic identification of cyanobacteria by a polyphasic approach. In: Kurmayer, R., Sivonen, K., Wilmotte, A., Salmaso, N. (Eds.), Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria. John Wiley & Sons Ltd, Chichester, pp. 79–134. Wilson, K.M., Schembri, M.A., Baker, P.D., Saint, C.P., 2000. Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR. Appl. Environ. Microbiol. 66 (1), 332–338. Woodhouse, J.N., Fan, L., Brown, M.V., Thomas, T., Neilan, B.A., 2013. Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges. ISME J. 7 (9), 1842–1851. Ye, W.J., Liu, X.L., Tan, J., Li, D.T., Yang, H., 2009. Diversity and dynamics of microcystin—producing cyanobacteria in China’s third largest lake, Lake Taihu. Harmful Algae 8 (5), 637–644. Yen, H.K., Lin, T.F., Tseng, I.C., 2012. Detection and quantification of major toxigenic Microcystis genotypes in Moo-Tan reservoir and associated water treatment plant. J. Environ. Monitor JEM 14 (2), 687–696. Zeller, P., Mejean, A., Biegala, I., Contremoulins, V., Ploux, O., 2016. Fluorescence in situ hybridization of Microcystis strains producing microcystin using specific mRNA probes. Lett. Appl. Microbiol. 63 (5), 376–383. Zervou, S.K., Christophoridis, C., Kaloudis, T., Triantis, T.M., Hiskia, A., 2017. New SPE-LC-MS/MS method for simultaneous determination of multiclass cyanobacterial and algal toxins. J. Hazard. Mater. 323 (A), 56–66. Ziemert, N., Ishida, K., Weiz, A., Hertweck, C., Dittmann, E., 2010. Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Appl. Environ. Microbiol. 76 (11), 3568–3574. Ziemert, N., Podell, S., Penn, K., Badger, J.H., Allen, E., Jensen, P.R., 2012. The natural product domain seeker NaPDoS: a phylogeny based bioinformatic tool to classify secondary metabolite gene diversity. PLoS One 7 (3), e34064.

Chapter 10

Using new techniques to study old favorites: A case study of Euglena Ellis O’Neill School of Chemistry, University of Nottingham, Nottingham, United Kingdom

10.1  Introduction to Euglena Euglenids are a group of fast-growing algae, able to achieve a very high cell density, even in challenging environments. They show characteristics of both plants and animals, whilst, in evolutionary terms, are only distantly related to other algae and are most closely related to the well-studied protozoan parasites Trypanosoma and Leishmania. The taxonomy of Euglenid algae has largely been based on morphology, but more recent molecular techniques are requiring some re-evaluation of their relationships (Bicudo and Menezes, 2016). Euglena are able to grow using both photosynthesis and by absorbing nutrients from the surroundings. There are many different species of Euglenid algae, displaying a great diversity of shapes, number of chloroplasts, pigmentation and behavior (Triemer and Ciugulea, 2010), with the species Euglena gracilis being the most heavily studied. The chloroplast of Euglena is derived from a eukaryotic green alga which is surrounded by three membranes (Martin et al., 1992) and in some species is no longer photosynthetic (Hadariova et al., 2017). This endosymbiont was incorporated into the free-living ancestral protist, bringing many genes involved in the function and maintenance of the chloroplast. Euglena are known for their production of a wide range of vitamins and essential amino acids, as well as polyunsaturated fatty acids and β-glucans (Schwartzbach and Shigeoka, 2017), which has led to them being exploited as a food supplement (Zeng et al., 2016). They can also make large amounts of wax esters under anaerobic conditions which can be used for biofuel production, currently being explored for commercial use (Inui et al., 2017). The unique metabolism of Euglena has led to them being used in the Eu:CROPIS biological life support systems for growing plants in different levels of gravity on Mars and the Moon (Hauslage et al., 2018). These wide range of features have led to the investigation of Euglena as a biotechnological platform for the production of proteins and high value metabolites (Krajcovic et al., 2015).

10.1.1  Historical research Euglena have been studied for many years due to their ease of culture and combination of plant and animal like characteristics (Fig. 10.1). Euglena gracilis in particular has been of great interest in the development of biochemistry and cellular biology (Buetow, 1968a, b, c, 1989). A wide range of Euglenid algae have been studied from a huge diversity of ecological niches including fresh water, snow pack and acid mine drainage and including photosynthetic, eukaryovorous and purely heterotrophic species (Zakrys et al., 2017). Studies in Euglena were fundamental in defining different sub cellular compartments and organelles. As complete loss of the plastid is not lethal in Euglena, unlike in other chloroplast-containing organisms, they have been heavily studied for the metabolic role of the chloroplast. The easy with which large amounts can be grown and the metabolic flexibility has led to the use of Euglena to investigate photosynthesis and metabolism (Schwartzbach and Shigeoka 2017). They have also been used extensively in ecotoxicology testing and for waste water treatment. Euglena are therefore of great interest due to their evolutionary history, unique cell biology, complex metabolism and potential for biotechnological exploitation. However, in the modern molecular biology era it has become apparent that Euglena are not related to animals and plants at all and instead belongs to the Excavates, one of the earliest branching Eukaryote lineages. This led to a decrease in the interest in Euglena, as research has tended to focus more on model organisms. The development of new techniques for the study of model organisms has led to a resurgence in research in more varied species. The application of these modern research techniques not only provides a more thorough understanding of these fascinating algae, but also inform their industrial exploitation. In this chapter I will present new results from the use of various omics techniques in Euglena, what this can teach us about their fundamental biology and how this can be exploited industrially (Fig. 10.2). Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00010-3 © 2020 Elsevier Inc. All rights reserved.

161

162  PART | III  Algal genomics

FIG.  10.1  Timeline of Euglena research. Different strands of research in Euglena have been available at different times, with some breakthroughs required to allow the use of new technologies.

FIG. 10.2  Deployment of omics techniques in Euglena. The plastid, mitochondrial and nuclear genomes of Euglena gracilis have been separately sequenced. The glycans present in the paramylon granules, cell surface and flagella have been separated and studied. The proteome and metabolome of the whole cell have been studied under different growth conditions. (Image credit: Sahutchai Inwongwan.)

10.2 Sequencing Euglena Euglena have complex genomes, composed of nuclear, plastid and mitochondrial sequences, which have arisen from a series of endosymbiotic events during its evolution. The nuclear genome is derived from: the ancestral protozoa; the mitochondria, related to alphaproteobacteria; a cryptic red algae endosymbiont which has since been lost; the primary photosynthetic host, related to a green alga; and the primary photosynthetic endosymbiont, related to cyanobacteria, obtained from the green alga. Aside from the endosymbiotic gene transfer, there is also evidence for horizontal gene transfer of individual genes, or even entire pathways, from diverse organisms such as bacteria and fungi.

10.2.1  Transcriptome sequencing In order to avoid the complexities of sequencing the nuclear genome, transcriptome sequencing has been used to understand the metabolic capacity and cell biology of Euglena. This technique sequences the mRNA of the study organism, greatly simplified compared to the genome sequence, with introns removed and epigenetic markers not present. However, it only sequences genes derived from the nucleus and containing a polyadenylated tail, and only identifies sequences that are actively transcribed at the time of sampling. Surprisingly, for a well-studied single celled organism, the transcriptome of Euglena gracilis was found to contain over 30,000 unique protein-encoding genes with an average length of 425 amino acids (O’Neill et al., 2015a), around double the

Using new techniques to study old favorites: A case study of Euglena Chapter | 10  163



number of genes in the model green alga Chlamydomonas reinhardtii (Blaby et al., 2014). The large number of genes in E. gracilis support their metabolic flexibility, highlighting metabolic pathways for lipids, amino acids, and vitamins, with an overabundance of enzyme for the synthesis and degradation of carbohydrates. This transcriptome also highlighted the capacity for the biosynthesis of polyketides and non-ribosomal peptides and the redox-active thiols nor-trypanothione and ovothiol. Comparative transcriptomics of Euglena grown under aerobic and anaerobic conditions has been used to understand wax ester fermentation (Yoshida et al. 2016). It was found that the transcript abundance did not change substantially under different growth conditions and that Euglena controls protein expression post-transcriptionally. Transcriptomics has been used to study other species related to E. gracilis to understand their unique capabilities. The transcriptome of Eutreptiella, a marine bloom forming species, has been sequenced, but with an average transcript length of 318 bp and less than 3% of sequences able to be assigned an enzyme code, this is of limited use (Kuo et al. 2013). Sequencing the acid mine drainage indicator species Euglena mutabilis gave 12,000 contigs with an average length of 700 bp and was used to study its ability to transport and metabolize arsenic, as well as other adaptations to their extreme habitat (Halter et al. 2015). The new transcriptomes of Euglena have allowed great new insights into the cell biology and metabolism of these algae. The invariance of transcript abundance under different growth conditions indicates that few genes are likely present in the nuclear genome that are not transcribed, although genome sequencing is needed to confirm this.

10.2.2  Genome sequencing DNA sequencing has become routine as prices have come down and capacity has increased. However, sequencing the nuclear genome of Euglena has proved problematic due to its size, ploidy and repetitive nature (Linton et al., 2010). The presence of the epigenetic modification Base J, a unique feature of Euglenozoa, may also disrupt accurate DNA sequencing (Hazelbaker and Buratowski 2012).

10.2.2.1  Plastid genome The plastid genome of Euglena gracilis was one of the first plastid genomes to be sequenced. The gene content is similar to, though the overall organization quite different from, that of green algae and land plants (Hallick et al., 1993). The Euglena plastid genome does include a unique region containing a variable number of short, tandem repeats, some extremely large and complex introns and a unique class of very small introns. Many more chloroplast genomes have been sequenced across the Euglenid algae and display a large amount of variability regarding genome organization across the family, although gene content was highly conserved within the family (Bennett and Triemer 2015).

10.2.2.2  Mitochondrial genome The mitochondrial genome of Euglena gracilis has only recently been sequenced and found to be very small, with just seven protein coding genes (Dobakova et al., 2015). The DNA sequences were small and varied in size from 5 to 8 kb, and, although the gene content could be characterized, it is not possible to give a definitive genome structure. The transcripts do not seem to undergo any of the extremely complex posttranscriptional modifications used by diplonemids and kinetoplastids, close relatives of Euglena.

10.2.2.3  Nuclear genome The sequencing of the genome of Euglena has been complicated by its size and complexity, and the available data only allow initial analysis of genome structure (Ebenezer et al., 2019). The E. gracilis nuclear genome is estimated to be around 500 Mb in size (see Table 10.1). By comparing the genome to transcript sequences, most identified genes appear to be

TABLE 10.1  Features of the genomes of Euglena gracilis. Genome

Size

Number of genes

Reference

Nuclear

~500 Mbp

~30,000

Ebenezer et al. (2019)

Chloroplast

143,170 bp

97

Hallick et al. (1993)

Mitochondria

Total unknown; 5–8 kb fragments

7

Dobakova et al. (2015)

164  PART | III  Algal genomics

cis-spliced, with many conventional introns, and some intermediate and non-conventional splice sites (Guminska et al., 2018). There is evidence for alternate splicing and some introns are very large compared to the exon sequences between them (Ebenezer et al., 2019). There is also evidence for gene clustering, with many of the relatively short contigs containing more than one coding sequence. There are genes from both red and green algae in the E. gracilis nuclear genome, indicating multiple endosymbiotic events, with earlier symbionts being completely lost from the modern host (Ebenezer et al., 2019). The extant chloroplast is clearly derived from the endosymbiosis of a eukaryotic green alga (Martin et al., 1992), but there is clear evidence for a red algae endosymbiont which has since been lost, after transfer of some genes to the Euglena nuclear genome (Maruyama et al., 2011). This has given rise to a complicated euglena genome, with contributions from the ancestral mitochondria, chloroplast, green algae and red algae to the host genome.

10.3  Euglena proteomics It has long been known that in Euglena there is very little correlation between changes in transcript abundance and protein level, except for proteins encoded by the chloroplast genome (Ebenezer et al., 2019). This implies that protein expression is controlled at the post-translational stage. In order to characterize the abundance of proteins in Euglena, rather than measuring the transcript levels, it is necessary to measure the actual proteins, using proteomics. This is only feasible once the E. gracilis genome and transcriptome sequences became available. Various label-free proteomic studies have been carried out in Euglena, particularly looking at the production of metabolites under different growth conditions. For example, in studying the synthesis of ascorbate, α-tocopherol and amino acids, Hasan et al. identified several enzyme isoforms that were influenced by the metabolic growth condition, with many isoforms only detected under individual growth conditions (Hasan et al., 2017). Proteomic analysis of different strains of Euglena has allowed the study of the different growth rates and metabolites produced. For instance, although it was known that the Euglena gracilis var. saccharophila variant strain is able to convert glucose into paramylon at a higher rate than the Z strain, proteomic analysis showed that this is likely down to a higher expression of PFK-1, which catalyzes the first reaction that commits glucose to the glycolytic pathway (Sun et al., 2018). Most enzymes in the gluconeogenesis pathway were also more abundant in the saccharophila strain, whilst those involved in paramylon metabolism were mostly less abundant. Due to the complex evolution of Euglena and the triple membraned chloroplast, it is not possible to predict targeting of proteins from the nucleus to the plastid (Inwongwan et al., 2019). However, by separating various organelles and analysing their protein content, the distribution of proteins can be analyzed. 1345 proteins were detected from purified plastids of E. gracilis, of which 48 were encoded by the plastid genome (Vanclova et al., 2019). 774 of the identified proteins could be assigned functional annotations, while 571 had no known function. Intriguingly, an iron-sulphur cluster assembly pathway closely related to that found in Chlamydia was discovered in the chloroplast, alongside plant related enzymes, suggesting a duplication of pathways for the biosynthesis of these cofactors. This proteomic data has been used to reconstruct the distribution of enzymes within the chloroplast and to build a model of the subcellular location of various metabolic pathways (Inwongwan et al., 2019). Proteomics is much more reliable for determining which pathways are up or down regulated in Euglena than transcriptomics. However, good predicted protein sequences are needed to enable reliable proteomics. Now that these are available, there are many opportunities to monitor the response of Euglena to environmental or biotic stimuli.

10.4  Metabolites in Euglena Euglena is well known as a prolific producer of many central metabolites, such as essential amino acids and polyunsaturated fatty acids as well as more specialized metabolites, such as β-glucans and vitamins (Schwartzbach and Shigeoka, 2017). Whilst the wide range of metabolites produced by Euglena have been well characterized, the rise of high-throughput metabolomics has facilitated much greater understanding of their biosynthesis and informs optimization of production.

10.4.1  Central metabolites The metabolites in Euglena vary markedly depending on how the cells are grown, which can help to understand the metabolic fluxes and biosynthesis of these compounds. By understanding these fluxes and metabolite productions, growth conditions can be varied to optimize yield of the desired products. High throughput metabolomics techniques can be routinely applied to identify distribution of known metabolites in Euglena.



Using new techniques to study old favorites: A case study of Euglena Chapter | 10  165

For example, the profile of amino acids is altered substantially under different growth conditions, such as an increase in tyrosine under photosynthetic growth, with a decrease in phenylalanine, compared to heterotrophic growth (Hasan et al., 2017). A decrease in phenylalanine in all conditions was noted in stationary phase and it was also proposed that the balance between valine and isoleucine may be involved in controlling growth phase. Higher levels of the antioxidants ascorbate, α-tocopherol and histidine were also found in cells undergoing photosynthetic growth, consistent with their roles in protecting against photosynthesis related oxidative stress (Hasan et al., 2017). E. gracilis contains a range of poly unsaturated fatty acids, including omega-3 and omega-6 fatty acids (Wang et al., 2018). Most of these are found in the chloroplast membranes and thus the levels in of the cells is affected by the chloroplast activity, dependent on light levels. E. gracilis produces different profiles of fatty acids in responses to different culture conditions and this can be used to manipulate production of desired products (Zeng et al., 2016). These techniques can also be used to study environmental samples. For example, profiling E. mutabilis cells from an acid mine drainage identified 57 metabolites from Euglena cells recovered in situ, with an additional 21 from in vitro cultures (Halter et al., 2012). Only three metabolites were found in the Euglena cells in situ but not in in vitro, indicating that E. mutabilis synthesizes the majority of metabolites found from the cells in situ (Halter et al., 2012).

10.4.2  Specialized metabolites Euglena produce many specialized metabolites that have a wide range of uses. Aside from discovery of these molecules, monitoring the production under different growth conditions allows production to be optimized. These techniques can be limited in the compounds identified, with specific applications sometimes needing altered protocols and some metabolites are very difficult to monitor. However, having been developed in model organisms, these techniques can be applied to Euglena. A unique characteristic of Euglena is its ability to produce large amounts of wax esters under hypoxia using facultative anaerobic metabolism (Muller et al., 2012). Wax esters contain a wide range of lipids, with the most abundant being C14:0 fatty acid-C14:0 fatty alcohol ester (Furuhashi et al., 2015). These wax esters are being evaluated as biofuels, and so monitoring yields and product profiles is important for strain improvement. Only one natural products, has been isolated and characterized from a Euglena (E. sanguinea), the ichthyotoxic alkaloid euglenophycin (Zimba et al., 2010). Inspection of the E. gracilis transcriptomes reveals there are transcripts apparent for the complex multi-domain secondary metabolite synthases needed to make such compounds, as is evident for an increasing array of algae now that genome/transcriptome sequence data is becoming available (O’Neill et al., 2016). There is also evidence for uptake of iron by Euglena, which is proposed to be mediated by a small molecule which may be related to bacterial or fungal siderophores (O’Neill et al., 2015b). Whilst it is clear that there is potential for synthesis of complex natural products in Euglena gracilis, the exact products made are not known. High throughput metabolite profiling may be useful in identifying the compounds and optimizing the growth conditions for production (Crusemann et al., 2017). High throughput metabolomics techniques can be used to routinely monitor production of specific compounds under different conditions and at different growth stages. Applying this to Euglena, not only informs on the biology, but also can help to improve yields of desired products.

10.5  Euglena glycomics Euglena contain a protein-based cell wall, rather than a carbohydrate based one as is common for most organisms, and produce a linear β-glucan storage polysaccharide, paramylon. This led to the proposal that they would have a simple glycan profile and thus relatively few enzymes for the biosynthesis and degradation of carbohydrates. Instead the transcriptome of Euglena gracilis contains a wide range of these enzymes, similar to the number in multicellular animals, and much more than other single celled algae (O’Neill et al., 2017). Predicting the specificity of carbohydrate active enzymes is notoriously difficult and so it is not possible to predict the carbohydrates Euglena can make, but new techniques are beginning to be used to study the Euglena glycome.

10.5.1 Paramylon Paramylon, the storage polysaccharide in Euglena, is composed of β-1,3-linked glucose, rather than the α-1,4-linked glucan found as starch and glycogen in plants, animals and bacteria. Paramylon is found as membrane bound crystalline granules in the cytosol, and the shape is specific for individual species (Monfils et al., 2011). Paramylon is synthesized by transfer of glucose from UDP-glucose by paramylon synthase (Baumer et al., 2001). Two components from the E. gracilis transcriptomes

166  PART | III  Algal genomics

were identified as members of GT48 with significant similarity to β-1,3-Glucan synthase from fungi, with multiple transmembrane domains (Yoshida et al., 2016). Using RNAi, one of these was found to be necessary for the synthesis of paramylon, whilst the other was dispensable (Tanaka et al., 2017). Paramylon is degraded by hydrolysis by both endo- and exo-β-1,3-glucanases, which have been identified in E. gracilis (Muchut et al., 2018). The glucans can also be degraded by phosphorylases, which release glucose-1-P, maintaining the energy of the bond (O’Neill and Field, 2015). β-1,3-Glucan phosphorylases have been identified in Euglena and classified into a new Glycoside Hydrolase family, GH149 (Kuhaudomlarp et al., 2018), with a domain structure similar to bacterial GH94 disaccharide phosphorylases (Kuhaudomlarp et al., 2019).

10.5.2  Protein glycosylation Protein glycosylation is key to the correct folding of certain proteins and is involved in recognition and signalling. N-glycans are attached to proteins and modified in the Golgi, and can be extremely specific to certain cell types (Marth, 1999). Protein N-glycan profiling of E. gracilis shows that it produces mainly high mannose type glycans with a small proportion containing a modification which is consistent with the mass of aminoethylphosphonate (O’Neill et  al., 2017). Aminoethylphosphate-bearing mannose residues are a core component of human GPI anchor structures. There is no evidence for complex N-glycans or for the presence of typical O-linked glycans on Euglena proteins. Because E. gracilis apparently does not produce classic complex-type N-glycans, it offers a platform for the heterologous expression and manufacture of therapeutic proteins that will exclusively carry oligomannose-type N-glycans. Glycosylphosphatidylinositol (GPI) membrane anchors are involved in anchoring proteins to the cell surface. E. gracilis has all of the genes necessary for the biosynthesis of core GPI anchors, including the key transamidase for attaching the protein (O’Neill et al., 2015a, b), though the structure of the GPI anchor is not known. There are three members of GT41 family of intracellular protein glycosyltransferases in the E. gracilis transcriptome (O’Neill et al., 2015a, b). Animals have one well characterized enzyme in this family, which transfers N-acetylglucosamine to serine and threonine residues in the cytosol (Olszewski et al., 2010). Plants have two enzymes, one of which transfers N-acetylglucosamine to proteins, and one which is specific for fucose (Zentella et al., 2017). The carbohydrate specificity of the three members of this family present in Euglena is not clear. N-Acetylglucosamine-1-phosphate transferase activity has been detected in membrane preparations of E. gracilis cells (Ivanova et al., 2017), likely involved in modifying proteins to target them to different subcellular compartments. Overall it is clear Euglena has the capacity to glycosylate proteins and the N-glycans appear relatively simple whilst other glycan modifications have not been well studied. The identity of glycosylated proteins and the role these modifications play remain to be investigated in Euglena.

10.5.3  Surface glycans The kinetoplastids, pathogens related to Euglena, have well defined and complex surface glycans to evade the immune system of their hosts, unnecessary for free living Euglena. Euglena is not reported to have a carbohydrate-based cell wall, but it has also been reported that cells can become encased in a carbohydrate coating (Smith, 1951). The structure of the extracellular polysaccharide is not well described, but initial studies indicate it is likely complex. Analysis of the sugar nucleotide pool in E. gracilis showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose (O’Neill et al., 2017). Glucose, galactose, mannose, fucose, xylose, and rhamnose have all been detected in cell surface extracts by paper chromatography (Barras and Stone, 1965) and a complex xylose-containing material has been found associated with the flagella (Bouck et al., 1978). Lectin- and antibody-based profiling of whole Euglena cells and extracted carbohydrates revealed a complex sugar based surface, which may be related to plant galactans and xylans (O’Neill et al., 2017). These results suggest that there are structurally complex polymers related to xylan, mannan, arabinan and arabinogalactan, though these structures have not been fully elucidated. Glycomic profiling has been developed for use in the analysis of mammalian and plant cells, identifying variations of known structures. Applying these techniques to Euglena has revealed some details of the structures, but, as they are novel modifications to the glycans, further work is needed.

10.6  Biotechnology exploiting Euglena Due to its metabolic flexibility and robust growth, Euglena are currently being produced industrially as a nutritional supplement, for waste water treatment and for the production of biofuels. They can be used for production of a range of



Using new techniques to study old favorites: A case study of Euglena Chapter | 10  167

­compounds for use in cosmeceuticals and nutraceuticals, such as α-tocopherol, wax esters and polyunsaturated fatty acids. Paramylon, the storage polysaccharide in Euglena, stimulates the immune system and has interesting material properties. The biomass can be used directly as a nutritional supplement in aquaculture and in animal feed. Euglena can also be used in ecotoxicological risk assessment, heavy metal bioremediation, and for treatment of wastewater and contaminated water (Krajcovic et al., 2015). Manipulating the growth conditions has the potential to alter the yields and distribution of the various compounds of interest. Deploying the omics techniques outlined in this chapter allows selection of the optimum production conditions. A range of genetic manipulation techniques have been explored in Euglena. Plastid transformation has been achieved in Euglena gracilis, utilizing biolistics (Doetsch et al., 2001). Euglena contains components of the RNA-silencing machinery (O’Neill et al., 2015b) and successful knockdown experiments have been performed (Ntefidou et al., 2003; Ishikawa et al., 2008). Despite a large and high ploidy genome, mutagenesis techniques have shown some success in manipulating Euglena. A 40% increase in lipid content was achieved using Fe-ion irradiation and FACS-based isolation of top 0.5% lipidrich E. gracilis cells (Yamada et al., 2016). Nuclear transformation of Euglena has been attempted a number of times with varying success. Agrobacterium mediated transformation has been attempted to express resistance genes from viral promoters, but without expression of any other gene (Nakazawa et al., 2014). Biolistic transformation of a linear DNA fragment encoding a chloroplast localized protein has shown an increase in expression of the protein (Shigeoka et al., 2014). Whilst the transgene was shown to be present in extracted DNA, there was no evidence that this was not from the chloroplast. Additionally, the 35S and Nos promoters used in this study are derived from a virus and a bacterium respectively and are not specific to nuclear transcription, and thus this may just be a further example of plastid transformation. Successful expression of GFP was achieved using Agrobacterium mediated transformation of a construct which contained 600 bp regions homologous to the Euglena genome either side, to stimulate homologous recombination and insertion into the genome (Khatiwada et al., 2019). Euglena are currently used in a range of biotechnological applications, including for production of biofuels and food supplements. New uses, including in space exploration and sewage treatment, are being explored. Genetic manipulation techniques are being developed to expand the uses of Euglena in biotechnology. These techniques are not routine in Euglena, but there is great potential for the heterologous production of proteins or small molecules, as has been achieved in plants (O’Neill and Kelly, 2017).

10.7 Conclusions The development of modern high throughput technologies, with decreasing cost and improved usability, has renewed interest in the study of non-model organisms. Research in Euglena has helped to understand the biology of this unusual alga, with some important differences to those well-studied models. These results will help to inform further research into these algae, in particular for improvements for biotechnological uses. The great advances in the understanding of Euglena, achieved by the application of the wide range of omics technologies described in this chapter, provides inspiration for the study of many more diverse organisms. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a, b, c, d, e, f, g, h, i, j, k, l, m, n, o).

References Barras, D.R., Stone, B.A., 1965. Chemical composition of pellicle of Euglena gracilis var bacillaris. Biochem. J. 97 (2), 14–15. Baumer, D., Preisfeld, A., Ruppel, H.G., 2001. Isolation and characterization of paramylon synthase from Euglena gracilis (Euglenophyceae). J. Phycol. 37 (1), 38–46. Bennett, M.S., Triemer, R.E., 2015. Chloroplast genome evolution in the Euglenaceae. J. Eukaryot. Microbiol. 62 (6), 773–785. Bicudo, C.E.d.M., Menezes, M., 2016. Phylogeny and classification of Euglenophyceae: a brief review. Front. Ecol. Evol. 2016, 00017. Blaby, I.K., Blaby-Haas, C.E., Tourasse, N., Hom, E.F.Y., Lopez, D., Aksoy, M., et al., 2014. The Chlamydomonas genome project: a decade on. Trends Plant Sci. 19 (10), 672–680. Bouck, G.B., Rogalski, A., Valaitis, A., 1978. Surface organization and composition of Euglena. 2. Flagellar mastigonemes. J. Cell Biol. 77 (3), 805–826. Buetow, D.E. (Ed.), 1968a. The Biology of Euglena. Vol. 1. General Biology and Ultrastructure. Academic Press, New York, NY. Buetow, D.E. (Ed.), 1968b. The Biology of Euglena. Vol. 3. Physiology. Academic Press, New York, NY. Buetow, D.E. (Ed.), 1968c. The Biology of Euglena. Vol. 2. Biochemistry. Academic Press, New York, NY. Buetow, D.E. (Ed.), 1989. The Biology of Euglena. Vol. 4. Subcellular Biochemistry and Molecular Biology. Academic Press, New York, NY. Crusemann, M., O’Neill, E.C., Larson, C.B., Melnik, A.V., Floros, D.J., da Silva, R.R., et al., 2017. Prioritizing natural product diversity in a collection of 146 bacterial strains based on growth and extraction protocols. J. Nat. Prod. 80 (3), 588–597.

168  PART | III  Algal genomics

Dobakova, E., Flegontov, P., Skalicky, T., Lukes, J., 2015. Unexpectedly streamlined mitochondrial genome of the euglenozoan Euglena gracilis. Genome Biol. Evol. 7 (12), 3358–3367. Doetsch, N.A., Favreau, M.R., Kuscuoglu, N., Thompson, M.D., Hallick, R.B., 2001. Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic psbK operon. Curr. Genet. 39 (1), 49–60. Ebenezer, T.E., Zoltner, M., Burrell, A., Nenarokova, A., Vanclova, A.M.G.N., Prasad, B., et al., 2019. Transcriptome, proteome and draft genome of Euglena gracilis. BMC Biol. 17, 11. Furuhashi, T., Ogawa, T., Nakai, R., Nakazawa, M., Okazawa, A., Padermschoke, A., et al., 2015. Wax ester and lipophilic compound profiling of Euglena gracilis by gas chromatography-mass spectrometry: toward understanding of wax ester fermentation under hypoxia. Metabolomics 11 (1), 175–183. Guminska, N., Płecha, M., Zakrys, B., Milanowski, R., 2018. Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis. PLOS Gen. 14 (10), 1007761. Hadariova, L., Vesteg, M., Bircak, E., Schwartzbach, S.D., Krajcovic, J., 2017. An intact plastid genome is essential for the survival of colorless Euglena longa but not Euglena gracilis. Curr. Genet. 63 (2), 331–341. Hallick, R.B., Hong, L., Drager, R.G., Favreau, M.R., Monfort, A., Orsat, B., et al., 1993. Complete sequence of Euglena gracilis chloroplast DNA. Nucleic Acids Res. 21 (15), 3537–3544. Halter, D., Andres, J., Plewniak, F., Poulain, J., da Silva, C., Arsene-Ploetze, F., et al., 2015. Arsenic hypertolerance in the protist Euglena mutabilis is mediated by specific transporters and functional integrity maintenance mechanisms. Environ. Microbiol. 17 (6), 1941–1949. Halter, D., Goulhen-Chollet, F., Gallien, S., Casiot, C., Hamelin, J., Gilard, F., et al., 2012. In situ proteo-metabolomics reveals metabolite secretion by the acid mine drainage bio-indicator, Euglena mutabilis. ISME J. 6 (7), 1391. Hasan, M.T., Sun, A., Mirzaei, M., Te’o, J., Hobba, G., Sunna, A., et al., 2017. A comprehensive assessment of the biosynthetic pathways of ascorbate, α-tocopherol and free amino acids in Euglena gracilis var. saccharophila. Algal Res. 27, 140–151. Hauslage, J., Strauch, S.M., Essmann, O., Haag, F.W.M., Richter, P., Kruger, J., et al., 2018. Eu:CROPIS—“Euglena gracilis: combined regenerative organic-food production in space”—a space experiment testing biological life support systems under Lunar and Martian gravity. Microgravity Sci. Technol. 30 (6), 933–942. Hazelbaker, D.Z., Buratowski, S., 2012. Transcription: Base J blocks the way. Curr. Biol. 22 (22), R960–R962. Inui, H., Ishikawa, T., Tamoi, M., 2017. Wax ester fermentation and its application for biofuel production. Adv. Exp. Med. Biol. 979, 269–283. Inwongwan, S., Ratcliffe, R.G., Kruger, N.J., O’Neill, E.C., 2019. Euglena central metabolic pathways and their subcellular locations. Metabolites (under review). Ishikawa, T., Nishikawa, H., Gao, Y., Sawa, Y., Shibata, H., Yabuta, Y., et al., 2008. The pathway via d-galacturonate/l-galactonate is significant for ascorbate biosynthesis in Euglena gracilis. J. Biol. Chem. 283 (45), 31133–31141. Ivanova, I.M., Nepogodiev, S.A., Saalbach, G., O’Neill, E.C., Urbaniak, M.D., Ferguson, M.A.J., et  al., 2017. Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes. Carbohyd. Res. 438, 26–38. Khatiwada, B., Kautto, L., Sunna, A., Sun, A., Nevalainen, H., 2019. Nuclear transformation of the versatile microalga Euglena gracilis. Algal Res. 37, 178–185. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.



Using new techniques to study old favorites: A case study of Euglena Chapter | 10  169

Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Krajcovic, J., Matej, V., Schwartzbach, S.D., 2015. Euglenoid flagellates: a multifaceted biotechnology platform. J. Biotechnol. 202, 135–145. Kuhaudomlarp, S., Patron, N.J., Henrissat, B., Rejzek, M., Saalbach, G., Field, R.A., 2018. Identification of Euglena gracilis β-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149. J. Biol. Chem. 293 (8), 2865–2876. Kuhaudomlarp, S., Walpole, S., Stevenson, C.E.M., Nepogodiev, S.A., Lawson, D.M., Angulo, J., et al., 2019. Unravelling the specificity of laminaribiose phosphorylase from Paenibacillus sp. YM-1 towards donor substrates glucose/mannose-1-phosphate by using X-ray crystallography and saturation transfer difference NMR spectroscopy. Chembiochem 20 (2), 181–192. Kuo, R.C., Zhang, H., Zhuang, Y.Y., Hannick, L., Lin, S.J., 2013. Transcriptomic study reveals widespread spliced leader trans-splicing, short 5′-UTRs and potential complex carbon fixation mechanisms in the euglenoid alga Eutreptiella sp. Plos One 8 (4), 60826. Linton, E.W., Karnkowska-Ishikawa, A., Kim, J.I., Shin, W.G., Bennett, M.S., Kwiatowski, J., et al., 2010. Reconstructing Euglenoid evolutionary relationships using three genes: nuclear SSU and LSU, and chloroplast SSU rDNA sequences and the description of Euglenaria gen. nov (Euglenophyta). Protist 161 (4), 603–619. Marth, J.D., 1999. N-glycans. In: Varki, A., Cummings, R.D., Esko, J.D., Freeze, H., Hart, G., Marth, J. (Eds.), Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Martin, W., Somerville, C.C., Loiseaux-de Goer, S., 1992. Molecular phylogenies of plastid origins and algal evolution. J. Mol. Evol. 35 (5), 385–404. Maruyama, S., Suzaki, T., Weber, A.P.M., Archibald, J.M., Nozaki, H., 2011. Eukaryote-to-eukaryote gene transfer gives rise to genome mosaicism in Euglenids. BMC Evol. Biol. 11, 105. Monfils, A.K., Triemer, R.E., Bellairs, E.F., 2011. Characterization of paramylon morphological diversity in photosynthetic euglenoids (Euglenales, Euglenophyta). Phycologia 50 (2), 156–169. Muchut, R.J., Calloni, R.D., Herrera, F.E., Garay, S.A., Arias, D.G., Iglesias, A.A., et al., 2018. Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase. Biochimie 154, 176–186. Muller, M., Mentel, M., van Hellemond, J.J., Henze, K., Woehle, C., Gould, S.B., et al., 2012. Biochemistry and evolution of anaerobic energy metabolism in eukaryotes. Microbiol. Mol. Biol. R. 76 (2), 444–495. Nakazawa, M., Haruguchi, D. Ueda, M., Miyatake, K., 2014. Transformed Euglena and process for producing same. US, US20150368655A1. Ntefidou, M., Iseki, M., Watanabe, M., Lebert, M., Hader, D.P., 2003. Photoactivated adenylyl cyclase controls phototaxis in the flagellate Euglena gracilis. Plant Physiol. 133 (4), 1517–1521. O’Neill, E., Kuhaudomlarp, S., Rejzek, M., Fangel, J., Alagesan, K., Kolarich, D., et al., 2017. Exploring the glycans of Euglena gracilis. Biology 6 (4), 45. O’Neill, E.C., Field, R.A., 2015. Enzymatic synthesis using glycoside phosphorylases. Carbohyd. Res. 403, 23–37. O’Neill, E.C., Kelly, S., 2017. Engineering biosynthesis of high-value compounds in photosynthetic organisms. Crit. Rev. Biotechnol. 37 (6), 779–802. O’Neill, E.C., Saalbach, G., Field, R.A., 2016. Gene discovery for synthetic biology: exploring the novel natural product biosynthetic capacity of eukaryotic microalgae. Method. Enzymol. 576, 99–120. O’Neill, E.C., Trick, M., Henrissat, B., Field, R.A., 2015a. Euglena in time: evolution, control of central metabolic processes and multi-domain proteins in carbohydrate and natural product biochemistry. Perspect. Sci. 6, 84–93. O’Neill, E.C., Trick, M., Hill, L., Rejzek, L., Dusi, R.G., Hamilton, C.J., et al., 2015b. The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry. Mol. Biosyst. 11 (10), 2808–2820. Olszewski, N.E., West, C.M., Sassi, S.O., Hartweck, L.M., 2010. O-GlcNAc protein modification in plants: evolution and function. BBA-Gen. Subj. 1800 (2), 49–56. Schwartzbach, S., Shigeoka, S. (Eds.), 2017. Euglena: Biochemistry, Cell and Molecular Biology. Springer, Cham. Shigeoka, S., Tamoi, M., Suzuki, K., Yoshida, E., 2014. Method for introducing gene to Euglena, and transformant therefrom. US, US20160010070A1. Smith, G.M. (Ed.), 1951. Manual of Phycology, An Introduction to the Algae and Their Biology. Chronica Botanica Co., Waltham, MA. Sun, A., Hasan, M.T., Hobba, G., Nevalainen, H., Te’o, J., 2018. Comparative assessment of the Euglena gracilis var. saccharophila variant strain as a producer of the β-1,3-glucan paramylon under varying light conditions. J. Phycol. 54 (4), 529–538. Tanaka, Y., Ogawa, T., Maruta, T., Yoshida, Y., Arakawa, K., Ishikawa, T., 2017. Glucan synthase-like 2 is indispensable for paramylon synthesis in Euglena gracilis. FEBS Lett. 591 (10), 1360–1370. Triemer, R.E., Ciugulea, I., 2010. A Color Atlas of Photosynthetic Euglenoids. Michigan State University Press, Michigan, MI. Vanclova, A.M.G.N., Zoltner, M., Kelly, S., Soukal, P., Zahonova, K., Fussy, Z., et al., 2019. Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history. bioRxiv 2019, 573709. Wang, Y.M., Seppanen-Laakso, T., Rischer, H., Wiebe, M.G., 2018. Euglena gracilis growth and cell composition under different temperature, light and trophic conditions. PLoS One 13 (4), 0195329. Yamada, K., Suzuki, H., Takeuchi, T., Kazama, Y., Mitra, S., Abe, T., et  al., 2016. Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting. Sci. Rep. U. K. 6, 26327. Yoshida, Y., Tomiyama, T., Maruta, T., Tomita, M., Ishikawa, T., Arakawa, K., 2016. De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions. BMC Genomics 17 (1), 182. Zakrys, B., Milanowsk, R., Karnkowska, A., 2017. Evolutionary origin of Euglena. In: Schwartzbach, S., Shigeoka, S. (Eds.), Euglena: Biochemistry, Cell and Molecular Biology. Springer, Cham, pp. 3–17.

170  PART | III  Algal genomics

Zeng, M., Hao, W.L., Zou, Y.D., Shi, M.L., Jiang, Y.G., Xiao, P., et al., 2016. Fatty acid and metabolomic profiling approaches differentiate heterotrophic and mixotrophic culture conditions in a microalgal food supplement ‘Euglena. BMC Biotechnol. 16 (1), 49. Zentella, R., Sui, N., Barnhill, B., Hsieh, W.P., Hu, J.J., Shabanowitz, J., et al., 2017. The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA. Nat. Chem. Biol. 13 (5), 479–485. Zimba, P.V., Moeller, P.D., Beauchesne, K., Lane, H.E., Triemer, R.E., 2010. Identification of euglenophycin—a toxin found in certain Euglenoids. Toxicon 55 (1), 100–104.

Chapter 11

Exploring ‘omics’ approaches: Towards understanding the essence of stress phenomena in diatoms and haptophytes Deepi Dekaa,b, Shashanka Sonowala,b, Channakeshavaiah Chikkaputtaiahb,c, Natarajan Velmurugana,b a

Biological Sciences Division, CSIR-North East Institute of Science and Technology, Branch Laboratory, Itanagar, India, bAcademy of Scientific and Innovative Research (AcSIR), CSIR-NEIST, Jorhat, India, cBiological Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, India

11.1 Introduction Diatoms and haptophytes are a dominant calcifying group of coccolithophores in the phytoplankton communities (Holligan, 1992). Among the group of phytoplankton, diatoms and haptophytes play a major role in the biological carbon fixation in the carbon cycles, and responsible for more than 20% of global carbon fixation (Armbrust et al., 2004; Bowler et al., 2008; Araie et al., 2011). The silicified and calciferous cell walls of diatom and haptophytes, respectively, have been explored for studies related to global climate changes. Diatoms and haptophytes are well-known for frequent formation of massive blooms in the photic zone of the ocean. Diatoms Thalassiosira nordenskioeldii, Detonula confervacea, Chaetoceros diadema, Skeletonema costatum; haptophytes Phaeocystis and Emiliania huxleyi have been reported for formation of massive blooms, and play a potential role in climate change, marine food-web structures, and the deposition of large amount of silica and calcite in the marine sediments (Danbara and Shiraiwa, 1999; Fukuda et al., 2011; Schoemann et al., 2005; Tiselius and Kuylenstierna, 1996). Diatoms and haptophytes have been reported to accumulate a variety of biologically and commercially important carbon compounds, including triacylglycerides (TAG) which can be converted to biodiesel and jet fuels. A broad variety of long- and very long-chain poly unsaturated fatty acids (PUFA) from diatoms and haptophytes have been reported. Diatoms producing long- and very long-chain PUFA can be potentially used in aquaculture industries as direct feedstock materials. Therefore, diatoms and haptophytes have gained substantial support as second generation feedstock materials. Diatoms and haptophytes have unique ability to accumulate excessive amount of high-content intracellular lipids (about 40–60% of dry cell weight) with commercial importance (Velmurugan and Deka, 2018; Zhang and Hu, 2014). Especially, haptophytes have been extensively studied for their unique characteristics to produce a very long chain fatty acids, alkenone, and C36n-alkenoic acid (Volkman et al., 1981). Detailed fatty acids profiling revealed the predominant presence of even-numbered fatty acids in diatoms Nitzschia frustrula, N. closterium, N. incerta, Navicula pelliculosa, Phaeodactylum tricornutum, and Synedra fragilaroides; and presence of C14 to C22 fatty acids in haptophytes E. huxleyi, Hymenomonas carterae, Isochrysis galbana, and Crystallolithus hyalinus (Volkman et  al., 1981; Liang et  al., 2000). Apart from their role in climate regulation, biogeochemical cycles, and fatty acids accumulations, diatoms and haptophytes have also been explored for synthesis of several key bioinspired materials such as polysaccharides (Le Costaouec et al., 2017; Kayano et al., 2011). The cell membrane materials, amorphous silica in diatoms, coccoliths in haptophytes, and specific protein compounds frustulins, pleuralins, and silaffins have been well characterized from diatoms and haptophytes, and all these materials received immense interests in the fields of biotechnology and material sciences (Le Costaouec et  al., 2017; Kayano et al., 2011). Comprehensive cellular and functional analysis of diatoms and haptophytes in regard with their role in climate regulations, biogeochemical cycles, fatty acids and polysaccharides biosynthesis is necessary and of great interest to the world. So far, conventional methodologies related to cellular processing and functional studies, such as gene characterizations through markers, and enzyme assays have been used to study the diatoms and haptophytes. However, conventional methods Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00011-5 © 2020 Elsevier Inc. All rights reserved.

171

172  PART | III  Algal genomics

provide only little information about the molecular mechanisms related to biological responses in diatoms and haptophytes. In addition, conventional methodologies are labor intensive, time consuming and require high microbial biomass. ‘Omics’ methodologies coupled with various bioinformatics tools open a wide door to access a global picture on how the different molecular mechanisms operate inside the cell from the simplest genomics leading to the production of proteins and metabolites through the analysis of the genes, transcription and translation factors involved in the metabolic pathways. High-throughput ‘omics’ technologies are providing us a comprehensive molecular data for investigations on genomes (genome sequencing), transcriptomes (RNA sequencing), proteomes (protein profiling and analysis), as well metabolomes (metabolites profiling and analysis) (Blankenburg et al., 2009). Recent developments in high-throughput sequencing (HTS) methodologies create new capabilities for prediction of both quantitative and qualitative measurement of cellular processing at molecular level (Ashworth, 2017). When we combine innovative stress manipulation techniques with different ‘omics’ approaches, we can obtain new insights into various biosynthetic pathways. These new insights allow us to generate stable and adaptable new microbial strains to different environmental conditions and can potentially accumulate high-level bioproducts through manipulations at genome level. From the industrial standpoint, generation of highly stable phenotypes with high-level synthesis of bioproducts can significantly reduce the production cost (Velmurugan and Deka, 2018). Therefore, ‘omics’ methodologies provide a clear advantage to study and improve the diatom and haptophyta strains. In view of the above, the first part of this chapter briefly highlights recent ‘omics’ applications and developments in diatoms and haptophytes. Role of ‘omics’ approaches in stress response and reconstruction of biosynthetic pathways for the improvement of diatom and haptophyta industrial strains are discussed in the second part.

11.2  Revolution of ‘omics’ approaches in diatoms and haptophytes Different ‘omics’ platforms are providing us a comprehensive cellular and molecular data starting from genome (genomics) to transcriptional and post-transcriptional (transcriptomics) to protein (proteomics) to primary and secondary metabolites (metabolomics), and finally to lipids (lipidomics) levels (Fig. 11.1). Recent developments in ‘omics’ methodologies are revolutionizing the diatoms and haptophytes as true multipurpose feedstocks and climate regulation precursors. Major ‘omics’ components used to study and understand diatom and haptophyta biotechnology along with related data mining tools are discussed below individually.

11.2.1 Genomics With the development of advanced automatic sequencing technology, genomics has become a tremendous tool in unlocking the biosynthetic and metabolic potential of an organism’s complete genomic constituents (Table 11.1). It facilitates the assemblage of DNA sequences and the analysis of the gene structure and expression. The advent of the first ‘Sanger DNA Sequencing’ has revolutionized science over the past three decades. It was followed by the current ‘Next Generation Sequencing’ (NGS), which sharp fall in cost has revolutionized the acquisition of genomic data in the field of taxonomy, phylogeny and evolutionary history of diatoms and haptophytes (Kim et al., 2014). It provides a blueprint for engineering and optimizing of target bioactive product by mining the vast genomic data. So far, whole genome sequences of six different diatoms, T. pseudonana (Armbrust et al., 2004), P. tricornutum (Bowler et al., 2008), T. oceanica (Lommer et al., 2012), Cyclotella cryptica, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries (unpublished but available at http://genome. jgi.doe.gov.bacillariophyta/bacillariophyta.info.html; http://www.marinemicroeukaryotes.org; Sabatino et  al., 2015) are available in the public domains. The first whole genome sequence in diatom was published in 2004 for the model centric diatom T. pseudonana using a whole-genome shotgun approach. This approach yields 14-fold sequence coverage with genome size of 34 Mbp, 24 chromosomes, and 11,390 genes (Armbrust et al., 2004). This genomics study also identified the novel genes responsible for silicic acid transport, formation of silica-based cell walls, high-affinity iron uptake, and biosynthetic enzymes for different types of PUFAs, along with a complete urea cycle (Armbrust et al., 2004). This was followed by the whole genome sequencing of the pennate diatom P. tricornutum in 2008 (Bowler et al., 2008). The size of the pennate diatom P. tricornutum was approximately 27.4 Mbp, and this reported size was smaller than centric diatom T. pseudonana genome. P. tricornutum genome approximately encodes 10,000 genes, and among them 1328 genes were diatom-specific genes. This pennate diatom genome was compared with centric diatom genome, and found that both genomes showed dramatically different structures and ~ 40% of genes have not been shared by these representatives of the two lineages. Recently a consortium of laboratories sequenced the cold-adapted diatom F. cyclindrus (originally isolated from the Southern Ocean) using two different sequencing approaches, ‘Sanger Sequencing’ and ‘PacBio Sequencing’ methods

Exploring ‘omics’ approaches Chapter | 11  173



Genomics

Transcriptomics

Metabolomics and lipidomics

H

O H H H N O

H

Haptophytes H

O

Diatoms

N

H

O

Proteomics

(A)

2004

(B)

2008

2009

Genome Transcriptome Genome sequencing of T. sequencing of haploid and of diploid cells pseudonana P. tricornutum of E. huxleyi (Ambrust (Bowler (Van Dassow et al., 2004) et al., 2008) et al., 2009)

2013

Genome sequencing E. huxleyi (Read et al., 2013)

2013

2015

2015

Oil-body associated proteome of Fistulifera sp. (Nojima et al., 2016)

Genome and transcriptome of F. solaris (Tanaka et al., 2015)

Plastid proteome of T. pseudonana and P. tricornutum (Gruber et al., 2015)

FIG. 11.1  (A) Different ‘omics’ researches in diatoms and haptophytes. Schematic representation of different ‘omics’ platforms starting from genome (genomics) to transcriptional and post-transcriptional (transcriptomics) to protein (proteomics) to primary and secondary metabolites and finally to lipids (metabolomics and lipidomics) levels. A combination of above mentioned ‘omics’ approaches are providing us a comprehensive cellular and molecular data of diatoms and haptophytes. (B) A timeline depicting landmark studies and key events in the history of ‘omics’ developments in diatoms and haptophytes.

(Mock et al., 2017). The study revealed the nuclear genome size of F. cyclindrus was about 61.1 Mbp which was far higher than pennate and centric diatom genomes. The study compared the F. cylindrus genome with those of T. pseudonana and P. tricornutum, both live in different marine environment than F. cylindrus. High numbers (121 proteins) of zinc-binding proteins were appeared in F. cylindrus which was higher than 7 and 12 numbers of zinc-binding proteins appeared in T. pseudonana and P. tricornutum, respectively. This high divergence of zinc-binding proteins in F. cylindrus may be due to the presence of relatively high concentration of zinc in Southern Ocean surface waters. De novo genomic assembly and a comprehensive transcriptomics and proteomics analysis uncovered metabolic flexibility in T. oceanica CCMP1005 (Lommer et al., 2012). These genomics results had shown high gene diversification in diatoms. Substantial fractions of diatom genes were not able to assign functions when compared with their similarity to

TABLE 11.1  List of key ‘omics’ findings in diatoms and haptophytes. Organisms

Omics approaches used

Applications

References

T. pseudonana

Whole genome shotgun approach

Determination of the whole genome with special emphasis on ecology, evolution and metabolism

Armbrust et al., 2004

P. tricornutum

Whole genome shotgun approach

Determination of the whole genome sequence

Bowler et al., 2008

E. huxleyi

Illumina Sanger sequencing method

Determination of whole genome sequencing

Read et al., 2013

Tisochrysis lutea

PacBio RSII sequencer

Development of pipeline named PiRATE for annonation of potentially autonomous transposable elements (TEs)

Berthelier et al., 2018

T. pseudonana, P. tricornutum

Transcriptome-wide microarray expression data available in public domains

Pan-transcriptomic analysis for identification of coordinated and orthologous functional modules

Ashworth et al., 2015

P. arenysensis, P. delicatissima, P. multistriata

Illumina Hi-Seq2000

Identification of comparable gene sets and the presence of nitric oxide synthase genes among selective diatoms

Di Dato et al., 2015

F. solaris

Illumina sequencing

Identification of the major metabolic pathways and characterize their transcriptional regulators, responsible for simultaneous growth and lipid accumulation

Tanaka et al., 2015

E. huxleyi

EST sequencing

Analysis of gene expression of transcripts promoting coccolithogenesis using EST approach

Wahlund et al., 2004

E. huxleyi

EST sequencing

Identification of genes specific to diploid calcification or haploid motility

Van Dassow et al., 2009

Fistulifera sp.

1D gel separation and LC-MS identification

Investigation of oil body-associated proteins

Nojima et al., 2013

T. pseudonana, P. tricornutum

Genome-wide prediction of nucleus-encoded plastid proteins

Identification of plastid proteome

Gruber et al., 2015

T. pseudonana

1D gel separation

Investigation of role of proteins in biomineralization of SiO2 (silica)

Kotzsch et al., 2017

N. laevis, N. incerta, N. pelliculosa, C. fusiformis

Gas chromatography-mass spectrometry

Screening for heterotrophic EPA production

Tan and Johns, 1996

E. huxleyi

Gas chromatography-mass spectrometry

Development of GC-MS based metabolic profiling

Obata et al., 2013

E. huxleyi

Gas chromatography-mass spectrometry

Metabolic profiling in haploid and diploid phases

Mausz and Georg, 2015

N. closterium f. minutissima

Ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (UPLC-ESI-Q-TOF-MS)

Investigation of the lipid metabolic changes during growth to identify and select the potential biomarkers for biological functions

Su et al., 2013

Pleurochrysis haptonemofera

One dimensional gel separation and ELISA

Investigation of the localization and associative strength of acid polysaccharides in coccoliths

Hirokawa et al., 2013

Genomics

Transcriptomics

Proteomics

Metabolomics

Lipidomics



Exploring ‘omics’ approaches Chapter | 11  175

genes in other organisms (Armbrust et al., 2004). However, diatom genomes also shared hundreds of common genes with bacteria, and exhibited rapid divergence with yeast and metazoans genomes (Bowler et al., 2008). This high gene diversification in diatoms may be attributed for their origins, evolution, and distinctive features. So far, in the haptophytes, whole genome sequencing was reported only for the model coccolithophore, E. huxleyi with the genome size of 142 Mbp, using ‘Sanger Sequencing’ method. The relative composition of the genome comprises of 21.9% protein-coding regions, 14.1% introns, 19.7% non-repetitive DNA, 6.5% exclusive tandem repeats and lowcomplexity regions, 30.9% unclassified repeats, 2.9% rDNA-related and 4.1% transposable elements (Read et al., 2013). This pan genome sequencing of E. huxleyi showed high abundance (~ 64%) of repetitive elements than in diatoms (~ 15%). The lone genome sequencing in haptophytes showed high-level gene diversity based on their similarity to genes in other organisms. Therefore, this lone genome sequencing may not be a typical representative of other strains in haptophytes. Very surprisingly, pan genome sequencing of E. huxleyi has not identified gene encoding the DmdA protein (a key catalyst for the initial demethlyation of dimethylsulphoniopropionate DMSP) as E. huxleyi is well-known to produce DMSP (a prominent natural source of atmospheric sulphur, DMS). However, genes responsible in later stage of DMSP degradation were identified in E. huxleyi genome. Soon after genome characterization of E. huxleyi, Alcolombri et al. (2015) identified and characterized Alma1, a DMSP lyase gene from the E. huxleyi which was responsible for the synthesis of DMS from DMSP. Another key gene DSYB, responsible for DMSP synthesis was also reported from the haptophyta Prymnesium parvum (Curson et al., 2018). DSYB encodes for methylthiohydroxybutryate methyltransferase enzyme which was localized in the chloroplasts and mitochondria of the P. parvum. Evolutionary analysis suggests that eukaryotic DSYB originated in bacteria, and was passed to eukaryotes in their early stages of evolution (Curson et al., 2018). The intracellular DMSP levels in P. tricornutum and T. pseudonana decreased during vitamin B12 limitation conditions while increased during nitrogen deprivation (Lee et al., 2017; Kageyama et al., 2018). It is reported that DMSP could potentially protect the cells from sudden changes in the amount of salt in the seawater (salinity) or from other damage, such as oxidative stress - a build-up of harmful chemicals inside cells (Raina et al., 2017). E. huxleyi genome sequencing partially characterized two functionally redundant pathways for the synthesis of omega-3 polyunsaturated, eicosapentaenoic (EPA) and docosahexanoic (DHA) fatty acids. In addition to this ‘lone genome sequencing’ in haptophytes, recently, a new pipeline named PiRATE, ‘Pipeline to Retrieve and Annotate Transposable Elements’, https://doi.org/10.17882/51795) was designed to identify potentially autonomous transposable elements (TEs), known to be drivers of genome evolution, in de novo genome assembly of Tisochrysis lutea (Berthelier et al., 2018). PiRATE established that the genomic data in T. lutea comprised of 15.89% Class I and 4.95% Class II TEs. The annotation data was compared with transcriptomics and proteomics data for identification of 17 expressed TE families, of which ‘TIR/Mariner’ and a ‘TIR/hAT’ family were capable of transposing or triggering the transposition of potentially related MITE elements.

11.2.2 Transcriptomics ‘High throughput sequencing’ of gene encoding mRNA paves the way for transcriptomic profiling in different microbes. Comparative transcriptomic analysis enables deeper understanding for identification of individual transcriptional units, differential gene expression, the expression of unique transcripts restricted to cell conditions or cell types, the transcript diversity at each transcriptional locus (i.e., splice isoforms, alternative transcription start sites, and polyadenylation sites), and the examination of the protein factors that control the transcriptional cassettes of a cell and their mechanisms of action of the sequenced as well as un-sequenced genomes (Hemaiswarya et al., 2013). Microarray and illumina RNASeq were the two NGS methodologies mostly used for transcriptomics analysis. The transcriptomics data were often validated by ‘quantitative reverse-transcriptase PCR’ (qRT-PCR). This is followed by the in-depth analysis of the generated data using the global databases like NCBI and JGI. In late of 1990s, the transcriptomics analysis of centric diatom T. weissflogii identified few new genes during the sexual reproduction (Armbrust, 1999). The identified genes, SIG1, SIG2, and SIG3, belong to a novel gene family, and encode three different polypeptides. Further characteristics of these SIG polypeptides were designated as targets to determine the sexual reproduction in diatoms. Prior to genome sequencing of cold-adapted diatom F. cylindrus, a transcriptome library was constructed using expressed sequence tags (EST) (Mock et al., 2005). Twenty seven percentage of genes in ESTs library showed similarity with centric diatom T. pseudonana (Mock et al., 2005). Most of the predicted genes were responsible for cellular growth and development. The cold adaptation requires significantly high level expression of genes and proteins in microbes. However, the key genes and proteins responsible for extreme cold adaptation in diatoms were still unclear. The Thomas Mock laboratory at University of East Anglia, UK developed the diatom EST database specific for diatom digital transcriptomics (http://www. biologie.ens.fr/diatomics/EST3). This database consists of 130,000 and 77,000 ESTs of P. tricornutum and T. pseudonana,

176  PART | III  Algal genomics

respectively (Maheswari et al., 2009). As majority of diatoms genes assigned a putative functions due to the lack of genetic information available in the public domains, this EST database facilitates to explore diatom genes with respect to their origin, ecological relevance, environmental adaptations, and biological functions. Further another diatom portal was constructed in 2015 (http://networks.systemsbiology.net/diatom-portal/) for the exploration of all publicly available transcriptome-based expression data of different diatoms (Ashworth et  al., 2015). This portal provides an opportunity for an integrated analysis of all publicly available transcriptome data of diatoms T. pseudonana and P. tricornutum exposed under different environmental and stress conditions. This approach for the integrated analysis of independent transcriptome experiments in diatoms revealed the coordination in transcriptional response in diatoms when exposed to different environmental and stress conditions. The analysis exposed (i) involvement of heat shock transcription factors during nitrate stress, moderate and elevated CO2 levels, and low temperature and elevated pH levels and (ii) role of multiple distinctly regulated groups of genes during silicic acid depletion. ‘Expressed Sequence Tags’ (ESTs) and clusters of E. huxleyi have studied extensively for the genome properties (Wahlund et al., 2004). Transcriptome analysis of the haploid (N) and diploid (2N) cells of E. huxleyi identified key genes involved in diploid-specific biomineralization, haploid-specific motility, and transcriptional controls (Van Dassow et al., 2009). A study on comparative transcriptome analysis of four bloom-forming haptophytes, P. parvum, Chrysochromulina brevifilum, C. ericina, and Phaeocystis antarctica revealed a set of core genes that is responsible for the essential metabolic and cellular pathways in the cell (Koid et al., 2014). Transcriptomics analysis of the C. tobin in line with circardian photoperiod exhibited distinct expression patterns of the genes responsible for fatty acid synthesis, modification and catabolism (Hovde et al., 2015). Further, the study provides insights into the evolutionary history, ecology and economic importance of haptophytes (Hovde et al., 2015).

11.2.3 Proteomics Transcriptomics profiling of mRNA shows only transient expression of genes, but the depth understanding of many key genes could be achieved at post-transcriptional level using proteomics studies which is the ultimate biomolecule in the central dogma (Arora et al., 2018). Proteomic technologies are based on peptide generation, automated multidimensional separation, mass spectrometry that detects numerous different types of peptides, post-translational modifications, protein structures, protein-protein interactions at high resolution resulting in high throughput analysis at femtomole and attomole levels (Ashworth, 2017; Jamers et  al., 2009). Proteomics was studied in diatoms from the late 90s in different species including, S. costatum, Clindrotheca fusiformis, Nitzschia angularis, P. tricornutum, T. pseudonana, and Fistulifera sp. A study by Chan et al. (2011) reported the evolutionary origin of membrane transporters (MTs) in T. pseudonana and P. tricornutum. Diatom MTs shares similarities with the mesophilic red algae Porphyridium cruentum, Calliarthron tuberculosum and the stramenopile Ectocarpus siliculosus (Chan et al., 2011). Two separate protein-level analyses in diatoms revealed the potential involvement of key protein precursors for lipid body formation and SiO2 biogenesis in the diatom membrane (Nojima et  al., 2013; Kotzsch et  al., 2017). A study by Nojima et al., (2013) extracted and identified oil body associated proteins from the oleaginous diatom Fistulifera sp. JPCC DA0580. In this study, 15 proteins have been successfully isolated as oil body-associated protein compounds. Majority of the identified proteins belonged to chloroplast and few proteins were transmembrane-associated and transporter proteins. Two transmembrane proteins were found to specifically localize to the lipid bodies in JPCC DA0580. The study also predicts a potassium channel protein in endoplasmic reticulum (ER), thus, suggests that lipid bodies may originate from ER in JPCC DA0580. In a separate study by Kotzsch et al. (2017) predicts Sin1, a SDV highly conserved transmembrane protein, plays a major role in the biogenesis of silica membrane in diatom. Reports on proteomics in haptophytes are scarce. A proteome-based study by Hirokawa et al. (2013) identified acid polysaccharides (PS-2 and PS-3) which are playing an important role in the crystal nucleation and crystal growth in Pleurochrysis haptonemofera (Hirokawa et al., 2013).

11.2.4  Metabolomics and lipidomics Metabolomics involves the qualitative and quantitative measurement of all low molecular weight organic molecules or metabolites, and combined with statistical analysis which enables us to comprehend the relationship between metabolites, organism physiology and the environment (Llewellyn et al., 2015). Diatoms and haptophytes produce a diverse kind of metabolites, therefore, single analytical method is not good enough for complete metabolite profiling in diatoms and haptophytes. Complete metabolite profiling in diatoms and haptophytes improves our understanding on function of individual genes and of metabolic process.



Exploring ‘omics’ approaches Chapter | 11  177

To extract complete intracellular metabolites from diatoms and haptophytes, researchers have used different solvents and mixtures of solvents. In addition to standard metabolite analyzer GC-MS (Gas Chromatography Mass Spectrophotometry), FTIR (Fourier Transform Infrared Spectroscopy), RP UHPLC-FT-ICR-MS (Reverse Phase Ultra High Performance Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, FIA-MS/MS (Flow Injection), and IC-MS/MS (Ion Chromatography Tandem Mass Spectroscopy), were also used for metabolic profiling in diatoms (Jungandreas et al., 2014; Llewellyn et al., 2015). A comparative metabolomics analysis in S. marinoi revealed that intracellular metabolites profiling in S. marinoi differ during different growth phases, exponential, stationary and declining phases (Vidoudez and Pohnert, 2012). It has been observed that metabolites related to sugar and amino acid biosynthesis were the highest in exponential phase while catabolism related metabolites, terpens, and putrescine were the highest in declining phase. The investigation also revealed that diatom metabolism is a highly dynamic process. In line with this finding, a very recent study by Remmers et al. (2018) related to metabolome in P. tricornutum found highest accumulation of several sugar compounds, intermediate metabolites of TCA cycle and alternation in lipid composition, under nitrogen limitation conditions. Metabolites specially related to sulfur metabolism, acrylic acid and dimethyl sulfoniopropionate (DMSP) also found to be highly accumulated in P. tricornutum. The studies also recommend further studies to understand the fluid balance regulations during nitrogen limitation conditions. Metabolomics analyses in diatoms were used to expand our chemodiversity knowledge of diatoms for the production of natural antifouling compounds. Chemodiversity of diatoms were highlighted using NMR and LC-MS based metabolomics. Metabolomics analysis in line with chemodiversity provides alternative route to find novel eco-friendly antifoulants from diatoms and haptophytes. A first comprehensive metabolome analysis of haptophytes E. huxleyi under nitrogen and phosphorus limitation conditions provides more insights related to intracellular metabolic processes (Wordenweber, 2017). The comprehensive analysis was carried out using different analytical instruments such as GC-MS, LC-MS/MS, FTIR, UV-VIS-HPLC, GC-FID, and ‘Charged Aerosol Detector’, CAD-HPLC for the complete analysis of entire set of different metabolites, pigments, and cellular content. The investigation in haptophytes suggests metabolic processes related to cellular growth and development were primarily arrested during phosphate limitation, and decrease in majority of central metabolites was observed during nitrogen limitation condition. The nitrogen and phosphate limitations have caused different cellular and metabolic responses in haptophytes E. huxleyi. Lipidomics is a branch of metabolomics to study all lipids and their functions within the cell. Lipids plays a crucial role in the structural composition, storing energy, photosynthesis, secretion, signal transduction, vesicle trafficking, cytoskeleton recognition and environmental responses in diatoms and haptophytes (Velmurugan and Deka, 2018). Lipids analyses have passed a long journey starting from traditional ‘Thin Layer Chromatography’, TLC, method, to GC, and to GC-MS, to lately applied in ‘Electrospray Ionization Mass Spectrometry’, ESI-MS, (Su et al., 2013). The traditional lipid profiling methods are time consuming and require high quantity of lipids whereas the ESI-MS can rapidly detect large number of different types of lipids within a short period. Although traditional methods are time consuming, they are widely and frequently used for lipid analysis in diatoms and haptophytes. A study by Bromke et al. (2013) reported the lipid profiling of 13 diatoms using lipidomics platform comprises of UPLC separation with a high resolution/high mass accuracy mass spectrometer. The study measured and annotated a total of 142 lipid species from T. pseudonana CCMP 1335, T. pseudonana CCMP 1007, T. weissflogii CCMP 1587, T. weissflogii CCMP 1336, Chaetoceros simplex CCMP 200, Amphitetrasante diluviana ECT 3627, Biddulphia biddulphiana ECT3902, Cerataulina daemon AP 8, Eunotogramma sp. AP 8 Eunoto, Hemiaulus sinensis 24I10-1A Hemi, Leptocylindrus danicus ECT 3929 araphid 3, Rhizosolenia setigera 25VI12-2A Rhizo, and Thalassionema frauenfeldii ECT 3929 Thal XL. Among 142 lipid species, 32 lipids were found in all 13 tested cultures. The lipidomic profile of 13 diatoms belongs to six classes of glycerolipids. The study also extended to check the metabolomics profiling of these 13 diatoms under nitrogen limitation conditions, and found alternation in metabolite profiling between nitrogen starved and nitrogen repleted conditions. However, surprisingly, there were no major effects found on the basis of species-specific metabolomics profile. Lipidomics was also used to understand the ecology and evolution of haptophytes during marine viruses infection (Hunter et al., 2015). A comparative lipidomic profiling of marine viral infected and uninfected, diploid and haploid cells of E. huxleyi revealed that glycosphingolipids play a major role during viral infection in E. huxleyi, and also found traceable quantity of glycosphingolipids in uninfected diploid cells. This lipidomics profiling of E. huxleyi highlighted the potential novel lipids-based biomarkers for viral infection and susceptibility in E. huxleyi. Diatom specific lipid-based biomarkers were developed using UPLC-Q-TOF-MS methods in N. clostridium (Su et al., 2013). HPLC and HPLC coupled with MS/MS or QTOF were used to measure organic acids and lipid accumulations in P. tricornutum and T. pseudonana (Mus et al., 2013; Mock et al., 2008). GC along with non-polar capillary column and FID were also used to measure total petroleum hydrocarbons upon exposure to P. tricornutum (Hook and Osborn, 2012).

178  PART | III  Algal genomics

11.3  Role of ‘omics’ approaches in understanding stress response in diatoms Gene regulation in most of the cellular and molecular functions in diatoms can be studied using different ‘omics’ approaches (Fig. 11.2). The advancements in ‘omics’-based technologies have boosted the research in diatoms to globally understand gene regulation under different stress conditions (Fig. 11.2). Advanced ‘omics’ approaches can be a promising tool for ecological monitoring including, environmental quality assessment, systems ecotoxicology in environmental samples (Carvalho et al., 2011). Various ‘omics’ analyses have been carried out in different diatoms under different nutrient stress conditions including nitrogen, phosphorus, iron, silicic acid, cadmium, silicon and aluminum toxicity (Fig. 11.2 and Table 11.2). Nitrogen and phosphorus are major macronutrients for the normal metabolic activities in diatoms. The deprivation of nitrogen source in diatoms facilitates the understanding of molecular mechanisms related to lipid/TAG accumulation, nitrogen metabolism, TCA, PPP, carbon storage, phosphorylation pathways, genetic expression of transcripts leading to the production of value-added metabolites and adaptation mechanisms (Table 11.2). Majority of ‘omics’ studies in diatoms under different stress conditions have been carried out in P. tricornutum and T. pseudonana (Fig. 11.2 and Table 11.2). Most of the analysis shows species-specific responses in diatoms to nutrient stress conditions. For example, T. pseudonana exhibits highly-sensitive or less adaptation to iron starvation conditions due to absence of the ferritin and plastocyanin genes while T. oceanica and P. tricornutum show better adaptation as these species have alternative electron carriers (Lommer et al., 2012). A comprehensive survey in ‘omics’ analysis in diatoms after nutrient starvation indicated that the metabolic responses in diatom after nutrient starvation were different that found in other photosynthetic eukaryotes (Matthijs et al., 2017). Studies related to enhanced lipid accumulation in diatoms after nutrient starvation showed highly differential regulation pattern in genes involved in the TCA and the urea cycles (Villanova et al., 2017; Ge et al., 2014; Yang et al., 2014; Longworth et al., 2016; Osada et al., 2017; Bromke et al., 2013; McCarthy et al., 2017; Matthijs et al., 2017). A study of Matthijs et al. (2016) reported molecular regulation at early stage (first 20 h) of nitrogen starvation in P. tricornutum. The study found the potential involvement of RING-GAPF-Gln (RGQ) proteins of transcription factors in nitrogen stress response in P. tricornutum. Following it, another study at metabolite and transcriptome level by Matthijs et al. (2017) revealed that transcription factor bZIP14 plays main role in regulating central carbon metabolism in TCA cycle during nitrogen starvation in P. tricornutum. Mixotrophic metabolism in P. tricornutum was studied by a combined effect of transcriptomics and metabolomics (Villanova et  al., 2017). High-level accumulation of intracellular lipids in P. tricornutum was induced by glycerol, and found that glycerol showed no inhibition on the photosynthetic potential of the strain. This study also delivers potential metabolic pathways for the strain improvement through metabolic engineering. A proteome level analysis in P. tricornutum under nitrogen starvation conditions found that carbon flux, carbon and energy metabolisms were redirected towards lipid accumulation in P. tricornutum while proteins involved in amino acid metabolism and light-harvesting functions were down-regulated (Yang et al., 2014). At the same time, the deprivation of other macronutrients such as phosphate, triggers up-regulation of genes in putative carbon pathways in P. tricornutum for photosynthetic carbon accumulation (Valenzuela et al., 2012). Silica is a very important ingredient in the formation of siliceous membranes in diatoms. Biological manipulation of silicon in diatom T. pseudonana was studied by genome-wide transcriptome analysis (Mock et al., 2008). The analysis revealed that iron and silicon stress in T. pseudonana triggers up-regulation of a common set of 84 genes. This finding suggests that iron and silicon shares same biological pathways in T. pseudonana or iron acts as alternative cofactor for silicon processes in the absence of silicon. A comprehensive genomic, transcriptomic and proteomic analysis in T. pseudonana found important metabolic flexibility including the preservation of iron-rich mitochondrial proteins and an overall decrease in photosynthetic electron transfer complexes, during iron-limitation conditions (Lommer et al., 2012). The molecular mechanisms responsible for diatoms adaptation in various silicon levels were studied by genome-wide transcriptome analyses in P. tricornutum, and found the differential expression in genes involved in glutamine-nitrogen pathways, genes encoding putative extracellular matrix components, and genes involved in iron regulations (Sapriel et al., 2009). Besides, different light sources play a major role in cellular and molecular responses of diatoms. A couple of recent reports by Jungandreas et al. (2014) and Valle et al. (2014) studied the biological system responses to different light sources in P. tricornutum. The ‘omics’ studies in diatoms under different light (blue, white, and red) conditions revealed several interesting biological phenomena (i) the cellular pigment concentrations in diatoms depend on light intensity and quality, (ii) high quality light was essential for the efficient processing of photosynthesis-associated nuclear genes, (iii) blue light efficiently triggers genes responsible for photoprotection and photosystem II (PS II) repair, (iv) diatoms were not adapted to acclimate higher intensities of red light due to lack of red light controlling mechanisms, and (v) prolonged exposure to blue and red light, separately, shown similar cellular and molecular functions in P. tricornutum, however, shift between blue to red and red to blue potentially influences carbon partitioning in diatom.

Exploring ‘omics’ approaches Chapter | 11  179



FIG. 11.2  Schematic representation of responses of selective diatoms and haptophytes under different stress conditions. Various stress conditions, light (

), temperature (

), and nutrient (

), or combinations have been used to obtain different biologically and commercially important compounds

hyper-accumulating diatoms and haptophytes (see Table 11.2). In this stress-based strain improvement strategy, diatoms and haptophytes mutagenized under different stress conditions first, and the stress conditions caused significant alterations in various metabolic pathways in diatoms and haptophytes for enhanced production of different biologically and commercially important compounds. Alterations in metabolic pathways can be understood well by various ‘omics’ approaches and this will lead us for creation of metabolically engineered strains (see Table 11.2).

Stress conditions applied

Method used for detection/ analysis

Metabolic pathways and biological compounds targeted

References

T. oceanica

Iron depletion

Pyrosequencing

Photosynthesis

Lommer et al., 2012

C. cryptica

Silicon depletion

Illumina sequencing

TAG biosynthesis

Traller et al., 2016

T. pseudonana

Nitrogen, silicon, iron, temperature depletion

Tiling arrays

Silicon metabolism

Mock et al., 2008

P. tricornutum

Silicic acid stress

Microarray

Silicon metabolism

Sapriel et al., 2009

P. tricornutum

Nutrient stresses (nitrogen, CO2, silicate, iron), abiotic stresses (low temperature, low salinity)

EST analysis

Stress metabolism

Maheswari et al., 2010

P. tricornutum

Cadmium stress

Microarray

Cadmium uptake mechanism

Brembu et al., 2011

T. pseudonana

Benzo[a]pyrene stress

Microarray

Lipid metabolism, silicon metabolism, stress response

Carvalho et al., 2011

T. pseudonana

Phosphorus depletion

Illumina sequencing

Phosphorus metabolism

Dyhrman et al., 2012

T. pseudonana

Iron depletion

Microarray

Death-related genes in stress acclimation

Thamatrakoln et al., 2012

P. tricornutum

Phosphate, nitrate, carbon depletion

Illumina sequencing

Lipid accumulation

Valenzuela et al., 2012

T. pseudonana

Photoperiod stress

Microarray

Diel cycling

Ashworth et al., 2013

P. tricornutum

Photoperiod stress

Microarray

Carbohydrate, lipid metabolism

Chauton et al., 2013

Nitzschia sp.

Salinity stress

Illumina sequencing

TAG accumulation

Cheng et al., 2013

P. tricornutum

Light depletion

Microarray

Photosynthetic mechanism

Nymark et al., 2013

P. tricornutum

Iron depletion

Microarray

Photosynthesis, mitochondrial electron transport and nitrate assimilation

Allen et al., 2014

Tetraselmis, subcordiformis

Oxygen depletion

Illumina sequencing

Hydrogen production

Cao et al., 2014

P. tricornutum

Blue, green, and red light stress

Microarray

Photosynthetic mechanism

Valle et al., 2014

T. pseudonana, T. rotula, S. costatum

Nitrogen and phosphorus depletion

Illumina sequencing

Nitrogen, phosphorus metabolism

Alexander et al., 2015

Organisms studied Genomics

Transcriptomics

180  PART | III  Algal genomics

TABLE 11.2  A brief overview of the different ‘omics’ approaches in diatoms and haptophytes under different stress conditions.



T. pseudonana

Nitrogen depletion

Microarray

Carbon concentrating mechanisms

Hennon et al., 2015

P. tricornutum

Nitrogen depletion

Illumina sequencing

Nitrogen metabolism

Matthijs et al., 2016

T. pseudonana

Silicon depletion

Microarray

Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, photosynthetic parameters, lipid accumulation

Smith et al., 2016

P. tricornutum

Nitrate depletion

Illumina sequencing

Nitrate assimilation

McCarthy et al., 2017

P. tricornutum

Nitrogen depletion

Microarray

TCA

Matthijs et al., 2017

F. solaris

Nutrient depletion

Illumina sequencing

Lipid metabolism (PPP)

Osada et al., 2017

P. tricornutum

Nitrogen depletion

Microarray

Carbon storage and lipid metabolism

Villanova et al., 2017

T. pseudonana

Iron depletion

SDS-PAGE, Western blotting detection

Metacaspases, programmed cell death (PCD)

Bidle and Bender, 2008

P. tricornutum

Blue light (BL), white light (WL), red light (RL), low light (LL), medium light (ML)

SDS-PAGE, LC-ESI-MS/MS

Light acclimation and photoprotection

Costa et al., 2013

T. pseudonana

Phosphorus repletion, depletion

HPLC, LC-MS

Phosphorus metabolism

Dyhrman et al., 2012

T. oceanica

Iron depletion

SDS-PAGE, LC-MS/MS

Photosynthesis

Lommer et al., 2012

T. pseudonana

Iron depletion

LC-MS, LTQ-OT

Nitrate and nitrite transporters, Photosystem II and photosystem I complexes

Nunn et al., 2013

P. tricornutum

High light exposure, nitrogen depletion, iron depletion

SDS-PAGE, LC-MS/MS, LC-ESIMS, Western blotting

Phosphorylation

Chen et al., 2014

P. tricornutum

Nitrate stress

SDS-PAGE, immunoblotting, HPLC, LC-MS/MS

TAG accumulation

Ge et al., 2014

P. tricornutum

Oxidative stress

UPLC, triple-quadrupole mass spectrometetry

Redox-sensitive signaling network (redoxome)

Rosenwasser et al., 2014

P. tricornutum

Nitrogen depletion

2-D gel electrophoresis

Lipid accumulation, fatty acid biosynthesis

Yang et al., 2014

P. tricornutum

Aluminum stress

LC-MS/MS

Aluminum toxicity mechanism

Xie et al., 2015

T. pseudonana

High light stress

(SCX) HPLC, Nano RP HPLC MS

Photoprotective mechanism

Dong et al., 2016

P. tricornutum

Nitrogen depletion

HPLC, LC-MS/MS

Lipid accumulation

Longworth et al., 2016

P. tricornutum

Nitrate depletion

Immunoblotting

Nitrate assimilation

McCarthy et al., 2017

F. solaris

Nutrient depletion

SDS-PAGE, Western blotting

Lipid metabolism (PPP)

Osada et al., 2017

Proteomics

Exploring ‘omics’ approaches Chapter | 11  181

(Continued)

Metabolomics N. laevis

Photoautotrophic, mixotrophic and heterotrophic

HPLC, GC-FID

Glucose, EPA

Wen and Chen, 2000

P. tricornutum

Cadmium stress

HPLC

Xanthophyll cycle

Bertrand et al., 2001

P. tricornutum

Cadmium stress

HPLC

Photosynthetic pigment

Brembu et al., 2011

P. tricornutum

Blue light (BL), white light (WL), red light (RL), low light (LL), medium light (ML)

HPLC

Light acclimation and photoprotection

Costa et al., 2013

T. pseudonana

Nitrogen depletion, iron depletion, sea salt repletion, carbonate repletion

GC-MS, GC-TOF MS,

Lipid metabolism, TCA

Bromke et al., 2013

P. tricornutum

Nitrogen depletion, alkaline pH stress, bicarbonate, organic acid repletion

Spectrophotometry, fluorometry, HPLC

Chlorophyll, carotenoids, nitrate, phosphate, protein, carbohydrate, DIC, lipids, organic acid

Mus et al., 2013

P. tricornutum

Light depletion

HPLC

Photosynthetic pigment

Nymark et al., 2013

P. delicatissima, S. marinoi

Iron depletion

UPLC

Domoic acid

Prince et al., 2013

P. tricornutum

Iron depletion

GC-MS

Photosynthesis,mitochondrial electron transport and nitrate assimilation

Allen et al., 2014

P. tricornutum

Nitrate depletion

HPLC-MS, NMR

TAG accumulation

Ge et al., 2014

P. tricornutum

Blue-red light stress

HPLC, FTIR

Photoacclimation metabolism

Jungandreas et al., 2014

P. tricornutum

Blue, green, and red light stress

HPLC

Photosynthetic pigment

Valle et al., 2014

P. tricornutum

Light intensity stress

HPLC

Photoprotection

Lavaud et al., 2002

P. tricornutum

Photo-oxidative stress

UPLC-UV-MS, LC-MS

Cartenoid accumulation

Yi et al., 2015

T. pseudonana

High light stress

Phyto-PAM phytoplankton analyzer, HPLC, GC-MS

Photoprotective mechanism

Dong et al., 2016

P. tricornutum

Nitrate depletion

FTIR, spectrophotometry, GCFID

Nitrate assimilation

McCarthy et al., 2017

P. tricornutum

Nitrogen depletion

GC-MS

TCA

Matthijs et al., 2017

P. tricornutum

Nitrogen depletion

Spectroscopy, colorimetry, HPLC, GC-MC/FID

Carbon storage and lipid metabolism

Villanova et al., 2017

182  PART | III  Algal genomics

TABLE 11.2  A brief overview of the different ‘omics’ approaches in diatoms and haptophytes under different stress conditions—cont’d



Lipidomics C. cryptica

Nitrogen, silica depletion, temperature stress

GC

Lipid, fatty acid biosynthesis

Sriharan et al., 1991

P. tricornutum

Nitrogen phosphate, sodium chloride, growth factors, CO2, initial temperature, pH, precursors stress

GC

ω-3 fatty acids

Yongmanitchai and Ward, 1991

N. closterium (C. fusiformis)

Light stress

TLC, GLC

Lipids, fatty acids biosynthesis

Orcutt and Patterson, 1974

P. tricornutum

Low temperature stress

GC

PUFA, EPA

Jiang and Gao, 2004

P. tricornutum, C. muelleri

UV-radiation stress

GC

Fatty acids biosynthesis

Liang et al., 2006

P. tricornutum

Nitrogen depletion

GC-MS

Lipid accumulation, fatty acid biosynthesis

Yang et al., 2014

T. weissflogii, C. cryptica

Nitrogen, silicon depletion

GC

Lipid metabolism

d’Ippolito et al., 2015

F. solaris

Nutrient depletion

UPLC-TOF-MS

Lipid biosynthesis

Liang et al., 2015

Nitrogen depletion

Illumina sequencing

Lipid metabolism

Carrier et al., 2018

E. huxleyi

Selenium depletion

Northern blotting

Selenoprotein

Obata and Shiraiwa, 2005

E. huxleyi

Nitrogen, phosphorus depletion

ABI DNA sequencing

Nitrogen, phosphorus metabolism

Dyhrman et al., 2006

E. huxleyi

Phosphate depletion

Microarray

Biomineralization

Quinn et al., 2006

P. tricornutum

Oil exposure stress (WAF, dispersant, CEWAF)

Microarray

Oil toxicity metabolism

Hook and Osborn, 2012

Silicon depletion

Microarray

Silicon metabolism (silicified cell wall synthesis, recovery from silicon Starvation and co-regulation with silicon transporter)

Shrestha et al., 2012

E. huxleyi

Sulphate depletion

Illumina sequencing

Sulfate deficiency metabolism

Bochenek et al., 2013

T. lutea

Nitrogen depletion

Illumina sequencing

Lipid metabolism and life cycle

Carrier et al., 2014

P. parvum

Salinity stress

Illumina sequencing

Osmolyte production, salinity stress, ion transport and acetate metabolism, polyketide synthase (PKS)

Talarski et al., 2016

I. zhangjiangensis

Nitrogen depletion

Illumina sequencing

Regulatory mechanism of oxidative species (O2−, H2O2, ClO−, NO), correlations among oxidative species and salicylic acid, jasmonic acid, folic acid

Wu et al., 2016

Haptophytes Genomics T. lutea Transcriptomics

Exploring ‘omics’ approaches Chapter | 11  183

(Continued)

Proteomics E. huxleyi

Selenium depletion

IEF/SDS-PAGE, HPLC

Selenoprotein

Obata and Shiraiwa, 2005

E. huxleyi

Bicarbonate depletion

SDS-PAGE, Western blotting

Carbonic anhydrases (γ-EhCA2 and δ-EhCA1)

Soto et al., 2006

E. huxleyi

High and low light intensity stress

LC-MS/MS

PSI and PSII, Lhcfs, L1818 proteins

McKew et al., 2013

I. galbana

Nitrogen depletion

2-DE, MALDI-TOF/TOF-MS

Lipid metabolism

Song et al., 2013

T. lutea

Nitrogen depletion

LC-MS/MS

Fatty acids, carbon homeostasis and carbohydrate catabolism

Garnier et al., 2014

Isochrysis sp.

Light intensity stress

Analytical methods, GC-MS

Protein, carbohydrate, lipid, chlorophyll a, fatty acids

Renaud et al., 1991

I. galbana

High light intensity stress, nitrogen depletion

GC-MS

Carbohydrates, lipids

Sukenik and Wahnon, 1991

I. galbana

Nitrogen depletion

GC-MS

Protein, neutral lipids (phospholipids, galactolipids) and total fatty acids (PUFA, EPA, DHA), carbohydrates, chlorophyll-a,c, carotenes, carotenoids biosynthesis

Fidalgo et al., 1998

E. huxleyi

Sulphate depletion

GC, HPLC

Sulfate metabolism

Bochenek et al., 2013

I. zhangjiangensis

Nitrogen depletion

UV-Vis spectrophotometer, carbon analyzer (TOC-L), WaterPAM fluorometer, cary eclipse fluorospectrophotometer, UPLC

Regulatory mechanism of oxidative species (O2−, H2O2, ClO−, NO), correlations among oxidative species and salicylic acid, jasmonic acid, folic acid

Wu et al., 2016

E. huxleyi

Nitrogen depletion

GC-MS, GC-FID, LC-MS/MS, UV-Vis HPLC, CAD-HPLC

Pigment, lipid, FAME, TAG, alkene, alkenone, amino acid, small organic acid, osmolyte, polyamine

Wordenweber, 2017

T. lutea

Nitrogen depletion

HPTLC

Lipid metabolism

Carrier et al., 2018

I. galbana

Photoperiod stress (24:0, 12:12, 16:8)

GC-FID

DHA profiling

Tzovenis et al., 1997

P. lutheri

Light intensity stress (low, intermediate)

TLC, GC

Lipids, fatty acids

Guedes et al., 2010

P. lutheri

High light with 14C and 1-14C radioisotope stress

TLC, HPLC

LC-PUFA biosynthesis

Guiheneuf et al., 2011

I. galbana

Intermittent light color stress

GC

Lipids and fatty acids profiling

Yoshioka et al., 2012

Metabolomics

Lipidomics

184  PART | III  Algal genomics

TABLE 11.2  A brief overview of the different ‘omics’ approaches in diatoms and haptophytes under different stress conditions—cont’d



Exploring ‘omics’ approaches Chapter | 11  185

An iTRAQ-based proteomic analysis shown that high intensity light exposure to T. pseudonana resulted differential expression in large numbers (143) of proteins (Dong et al., 2016). Photoprotective pigments in chlorophyll, diadinoxanthin and diatoxanthin along with other photoprotective proteins were up-regulated after exposure to high light intensity in T. pseudonana. In addition, a change in fatty acid composition (high level of C14:0 and C16:0 and reduced levels of C20:5ω) was observed after exposure to high light intensity in T. pseudonana. Altogether, global ‘omics’ analysis of diatoms under different stress conditions revealed key messages that (i) diatoms exhibited species-specific response towards different stress conditions as substantial fractions of genes of diatoms originally evolved from other microbes and (ii) adaptation of diatoms in various marine and freshwater environmental conditions may partially arise by global regulations from their gene expression level to genomic organization in clusters and to functional distribution of function-specific proteins.

11.4  Role of ‘omics’ approaches in understanding stress response in haptophytes The model haptophyta E. huxleyi has already been targeted for the various ‘omics’ studies upon exposure to different stresses, to understand several metabolic pathways related to biomineralization and biosyntheses of selenoprotein, DMSP, cysteine, glutathione, carbohydrate, lipids, proteins and pigments (Fig. 11.2 and Table 11.2). Nitrogen is an essential macronutrient for normal growth and metabolic activities of haptophytes. Its abundance and deficiency may cause significant changes in the expressions of genes, proteins and metabolites in the haptophytes, E. huxleyi, I. galbana and P. parvum (Fig. 11.2 and Table 11.2). Nitrogen starvation in I. galbana leads to the dramatic changes in metabolic profiling under different growth phases, and molecular findings indicate the opportunity to increase PUFA accumulation in haptophytes under nitrogen stress conditions (Fidalgo et al., 1998; Garnier et al., 2014). The nitrogen starvation in haptophytes leads to the identification of differentially expressed genes with unknown functions, and extends our knowledge about metabolic level responses to different life-cycle stages of haptophytes, E. huxleyi (Dyhrman et al., 2006; Wordenweber, 2017). A study by Carrier et  al. (2018) reported the phenotypic characteristics of few high-lipid accumulating strains of T. lutea along with their draft genomes, under nitrogen starvation conditions. The study isolated high-level lipid accumulating phenotypes using ‘Fluorescence Activated Cell Sorter’, FACS. The combined studies of FACS-based selection of high-lipid producing phenotypes with their genome changes were providing us a better platform for the development of improved strains (Carrier et al., 2018). The genome level study in T. lutea under nitrogen stress conditions revealed that transposable elements may play a major role for the determination of intra-strain diversity within T. lutea (Carrier et al., 2018). Apart from nitrogen starvation, limitations in other macronutrients, especially, urea, phosphorus, bicarbonate, phosphate, and sulphate have exhibited myriad effects in haptophytes at transcriptome, proteome and metabolome levels (Table 11.2). Selenium plays a major role for the formation of the calcified exoskeleton called coccolithophore in E. huxleyi. Selenium deficiency in E. huxleyi causes drastic changes at proteome and transcriptome levels. 75Se and 32P radio labeling identified major selenium-containing proteins in E. huxleyi that originally promote growth in haptophytes (Obata and Shiraiwa, 2005). A 27-kDa selenoprotein, EhSEP2, characterized in E. huxleyi, contains a highly conserved thioredoxin domain and shares homology with protein disulphide isomerase (PDI) localized in the endoplasmic reticulum. An in-depth transcriptomics analysis in selenium deficiency in E. huxleyi suggests that EhSEP2 transcript level increased with the increase in selenium availability while the reverse consequences show the non-translation of selenium mRNA transcripts (Obata and Shiraiwa, 2005). Further, transcriptomic profiling of P. parvum under salinity stress identified a group of differentially expressed genes associated with various cellular functions including transmembrane, vesicular transport and acetate metabolism (Talarski et al., 2016). Moreover, the transcriptome study also revealed that the ichthyotoxicity creates the contributory effects in PUFA production in P. parvum. Apart from above discussed, abiotic stress factors such as light intensities, colors, and temperatures caused significant changes in haptophytes, especially in E. huxleyi, I. galbana, and P. parvum, at proteomics, metabolomics and lipid levels (Durmaz et al., 2007; Guedes et al., 2010; Guiheneuf et al., 2011; McKew et al., 2013; Renaud et al., 1991; Tzovenis et al., 1997; Yoshioka et al., 2012).

11.5  Potential application of ‘omics’ tools in reconstruction of biosynthetic pathways for the production of commercially important compounds in diatoms and haptophytes Extensive in-depth knowledge in ‘whole genome sequencing’ analysis and expression of transcripts, proteomes and metabolites together is laying the groundwork for implementation in the reconstruction of biosynthetic pathways for the highly controlled regulations of commercially important compounds. Developments in ‘omics’ in combination with synthetic

186  PART | III  Algal genomics

biology and microbial bioengineering have made it possible to reconstruct genome-scale metabolic networks of model organisms, or organisms of interest for biotechnological potential. This would be possible after successful modifications in the expression levels of target genes, transcription factors and proteins leading to the highly controlled regulations of target metabolites. The reconstruction of metabolic pathways involves five major stages, including, draft reconstruction, refinement of reconstruction, conversion of reconstruction into computable format, network evaluation and data assembly and dissemination (Thiele and Palsson, 2010). This could be a promising strategy to obtain an improved strains of diatoms and haptophytes. Diatoms possess unique combination of genes due to its endosymbiotic origin (Levering et al., 2016). The ‘whole genome sequencing’ of the two model diatoms, T. pseudonana and P. tricornutum provide insights for the reconstruction of biosynthetic pathways. Diatoms accumulate TAGs as storage lipids, but little is known about the molecular mechanisms of lipid metabolism. A report on P. tricornutum by Barka et al. (2015) reveals that Tgl1 encoding TAG lipases enzyme mediates the first initial step of TAG breakdown from the storage lipids. The identification of the TAG degrading function of Tgl1 in P. tricornutum was achieved using antisense RNA approach. The mutant cells synthesized a reduced level (~ 20%) of Tgl1mRNA transcripts than wild type, and the reduction in Tgl1 transcription level increased TAGs storage in P. tricornutum. ‘DiatomCyc’ (http://www.diatomcyc.org), a detailed metabolic pathway and genome database specific for P. tricornutum, was created using the genome-wide approach. ‘DiatomCyc’ offers a range of software tools for the visualization and analysis of metabolic networks and ‘omics’ data specific to diatoms. It was anticipated that ‘DiatomCyc’ can be a potential key in gaining further understanding of diatom metabolism and, ultimately, will feed metabolic engineering strategies for the industrial valorization of diatoms (Fabris et al., 2012). ‘Flux balance analysis’, FBA, is a widely used approach for studying metabolic networks, based on genomic information or biochemical data, that calculates the fluxes of metabolites through this network using linear programming (Kim et al., 2016). Flux balance analysis of P. tricornutum predicts some of unusual features of diatom metabolism, such as the presence of lower glycolysis pathways in the mitochondria, and differences between P. tricornutum and other photosynthetic organisms (Kim et al., 2016). This may be beneficial in higher predictive capacity of the fluxes in this model diatom, thereby, metabolic engineering of biosynthetic pathways using different knockout and knock-in approaches. Genome-scale model in P. tricornutum reveals glutamine-ornithine shunt that could be potentially used to transfer reducing equivalents generated by photosynthesis to the mitochondria, and reflecting the known biochemical composition in defined culture conditions (Levering et al., 2016). This strategy further enables metabolic engineering for strain improvement for different biotechnological applications (Levering et al., 2016). A team from University of California at San Diego developed an integrated regulatory and metabolic model with the help of various transcriptome sequencing expression libraries are available for different environmental responses (Levering et al., 2017). The model shows highly connected modules within carbon and nitrogen metabolism, and the predicting the response to rising CO2 levels (Levering et al., 2017). The coccolithophores-haptophytes are well-known for the biosynthesis of various commercially important compounds, especially long chain PUFAs (Velmurugan and Deka, 2018). Biosynthesis pathways involving in the production of ‘long chain PUFAs’, LC-PUFAs, especially EPA and DHA, were also investigated in Pavlova lutheri in response to light limitation (Guiheneuf et al., 2011). This study shows two different metabolic pathways, (i) chloroplastic light-dependent and (ii) non-chloroplastic light-independent, were responsible for LC-PUFAs production in P. lutheri. cDNA microarray analysis of E. huxleyi exhibits the changes at transcription level that are associated with ­biomineralization-related pathways in promoting the formation of coccolithophore (Quinn et al., 2006). The sulfur limitations in E. huxleyi resulted in the up-regulation of four carbohydrate metabolism pathways (ascorbate, aldarate, butanoate, and pyruvate metabolism, and citrate cycle) and five pathways related to lipid metabolism (fatty acid, bile acid, sphingoglycolipid, prostaglandin and leukotriene, and glycerolipids) (Bochenek et al., 2013). This study revealed that sulfur and carbon were redirected from DMSP instead of lowering the photosynthetic activity (Bochenek et al., 2013). A ‘fluorescent activated cell sorter’, FACS, based methodology yields high lipid accumulating (~ 520% more TAGs than wild type) strain of I. galbana from a population of I. galbana (Carrier et al., 2018). The transcriptome analysis among selective high lipid accumulation strains of I. galbana reveals major pathways involved in lipid biosynthesis and this facilitates further engineering for the improvement of I. galbana strains for the increased accumulation of lipids (Carrier et al., 2018). Parallel investigations in I. galbana at proteomics level under nitrogen limitation conditions identified proteins involved in various metabolic pathways including lipid, carbohydrate, amino acid, energy, pigment metabolisms, glycolysis, TCA cycle, glyoxylate cycle, sulfur assimilation and citrate transport systems (Garnier et al., 2014; Song et al., 2013). Most of the metabolic engineering studies target fatty acids biosynthesis pathways in diatoms and haptophytes, for the production of value-added metabolites especially PUFA (Velmurugan and Deka, 2018).



Exploring ‘omics’ approaches Chapter | 11  187

Hence, the reconstruction of the novel pathways (that were originally outcomes of ‘omics’ approaches) could be achievable using any of the following six approaches: 1. High-level expression of the starting material. 2. High-level expression of genes involved in fatty acids biosynthesis. 3. Controllable expression of genes involved in TAG assembly. 4. Modification of fatty acids composition. 5. Knock-down of genes in catabolism. 6. Knock-down of genes in competitive pathways (Hess et al., 2017).

11.6  Conclusions and future prospects In this chapter, we have briefed about the current scenario of utilization of different ‘omics’ methodologies in diatoms and haptophytes, and further expended the role of ‘omics’ approaches to understand the molecular responses in diatoms and haptophytes during different stress conditions. To date, various ‘omics’-based information of diatoms and haptophytes is available in public database domains. ‘Omics’ data-sets provide valuable information to understand the ecology, evolution, cellular and functional biology, and industrial importance of these globally important organisms. Significantly, the ‘high-throughput technique’, FACS,-based isolation of high-lipid accumulating haptophyta phenotypes with their genome changes has initiated a platform for the development of improved strains. Many considerable challenges still remain that must be addressed well. An important challenge is that, despite the importance of diatoms and haptophytes, very few species have been sequenced, and the sequencing results clearly indicate the fact that substantial fraction of genes have not been shared by sequenced species and genomes shown dramatically different structures. Thus, sequencing of more ecologically important species of diatoms and haptophytes is necessary for better understanding of their origins, evolution and distinctive features. It is also necessary to conduct comparative studies under different stress conditions to find the global regulations of diatoms and haptophytes genes in regard with ecology and industrial important, so that, creation of improved strains can be achievable in future. Highly specific single-cell level selection from a population, and regeneration and metabolic engineering in diatoms and haptophytes, are still most challenging task, thus, development in this regard is of great interest (Sonowal et al., 2019). More efficient combined study of stress biology and multi-omics approaches in diatoms and haptophytes can potentially be used to study the biologically important pathways and produce improved strains for various biotechnological applications. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a, b, c, d, e, f, g, h, i, j, k, l, m, n, o).

Acknowledgments The authors wish to thank the Director, CSIR-NEIST, Assam Jorhat, for his kind encouragement in carrying out this work. We express our sincere thanks to the Editor for extending invitation to write this chapter, and Ms. A. Vinnarasi, (KCET, India) for her assistance for arranging figures in this chapter. This work was supported by research fund of DST-INSPIRE Faculty program (IFA 15-LSPA31) funded by the Department of Science and Technology (DST), Government of India.

References Alcolombri, U., Ben-Dor, S., Feldmesser, E., Levin, Y., Tawfik, D.S., Vardi, A., 2015. Identification of the algal dimethylsulfide-releasing enzyme: a missing link in the marine sulfur cycle. Science 348 (6242), 1466–1469. Alexander, H., Bethany, D.J., Jenkins, B.D., Rynearson, T.A., Dhyrman, S.T., 2015. Metatranscriptome analyses indicate resource partitioning between diatoms in the field. Proc. Natl. Acad. Sci. U. S. A. 112 (17), E2182–E2192. Allen, A.E., LaRoche, J., Maheswari, U., Lommer, M., Schauer, N., Lopez, P.J., et al., 2014. Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc. Natl. Acad. Sci. U. S. A. 105 (30), 10438–10448. Araie, H., Sakamoto, K., Suzuki, I., Shiraiwa, Y., 2011. Characterization of the selenite uptake mechanism in the coccolithophore Emiliania huxleyi (Haptophyta). Plant Cell Physiol. 52 (7), 1204–1210. Armbrust, E.V., 1999. Identification of a new gene family expressed during the onset of sexual reproduction in the centric diatom Thalassiosira weissflogii. Appl. Environ. Microb. 65 (7), 3121–3128. Armbrust, E.V., Berges, J.A., Bowler, C., Green, B.R., Martinez, D., Putnam, N.H., et al., 2004. The genome of the diatom Thalassiosira Pseudonana: ecology, evolution, and metabolism. Science 306 (5693), 79–86. Arora, N., Pienkos, P.T., Pruthi, V., Poluri, K.M., Guarnieri, M.T., 2018. Leveraging algal omics to reveal potential targets for augmenting TAG accumulation. Biotechnol. Adv. 36 (4), 1274–1292. Ashworth, J., 2017. Marine microalgae: systems biology from ‘omics’. In: Kumar, M., Ralph, P. (Eds.), Systems Biology of Marine Ecosystems. Springer, Cham, pp. 207–221.

188  PART | III  Algal genomics

Ashworth, J., Coesel, S., Lee, A., Ambrust, E.V., Orellana, M.V., Baliga, N.S., 2013. Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana. Proc. Natl. Acad. Sci. USA 110 (18), 7518–7523. Ashworth, J., Turkarslan, S., Harris, M., Orellana, M.V., Baliga, N.S., 2015. Pan-transcriptomic analysis identifies coordinated and orthologous functional modules in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Mar. Genom. 26, 21–28. Barka, F., Angstenberger, M., Ahrendt, T., Lorenzen, W., Bode, H.B., Buchel, C., 2015. Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum. Biochim. Biophys. Acta 1861 (3), 239–248. Berthelier, J., Casse, N., Daccord, N., Jamilloux, V., Saint-Jean, B., Carrier, G., 2018. A transposable element annotation pipeline and expression analysis reveal potentially active elements in the microalga Tisochrysis lutea. BMC Genomics 19 (1), 378. Bertrand, M., Schoefs, B., Siffel, P., Rohacek, K., Molnar, I., 2001. Cadmium inhibits epoxidation of diatoxanthin to diadinoxanthin in the xanthophyll cycle of the marine diatom Phaeodactylum tricornutum. FEBS Lett. 508 (1), 153–156. Bidle, K.D., Bender, S.J., 2008. Iron starvation and culture age activate metacaspases and programmed cell death in the marine diatom Thalassiosira pseudonana. Eukaryot. Cell 7 (2), 223–236. Blankenburg, M., Haberland, L., Elvers, H.D., Tannert, C., Jandrig, B., 2009. High-throughput omics technologies: potential tools for the investigation of influences of EMF on biological systems. Curr. Genomics 10 (2), 86–92. Bochenek, M., Etherington, G.J., Koprivova, A., Mugford, S.T., Bell, T.G., Malin, G., et al., 2013. Transcriptome analysis of the sulfate deficiency response in the marine microalga Emiliania huxleyi. New Phytol. 199 (3), 650–662. Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., et al., 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456 (7219), 239–244. Brembu, T., Jorstad, M., Winge, P., Valle, K.C., Bones, A.M., 2011. Genome-wide profiling of responses to cadmium in the diatom Phaeodactylum tricornutum. Environ. Sci. Technol. 45 (18), 7640–7647. Bromke, M.A., Giavalisco, P., Willmitzer, L., Hesse, H., 2013. Metabolic analysis of adaptation to short-term changes in culture conditions of the marine diatom Thalassiosira pseudonana. PLoS One 8 (6), e67340. Cao, X.P., Wu, X.D., Ji, C.F., Yao, C.H., Chen, Z.A., Li, G.H., et al., 2014. Comparative transcriptional study on the hydrogen evolution of marine microalga Tetraselmis subcordiformis. Int. J. Hydrogen Energ. 39 (32), 18235–18246. Carrier, G., Matthieu, G., Le Cunff, L., Bougaran, G., Probert, I., de Vargas, C., et al., 2014. Comparative transcriptome of wild type and selected strains of the comparative transcriptome of wild type and selected strains of the microalgae Tisochrysis lutea provides insights into the genetic basis, lipid metabolism and the life cycle. PLoS One 9 (1), e86889. Carrier, G., Baroukh, C., Rouxel, C., Duboscq-Bidot, L., Schreiber, N., Bougaran, G., 2018. Draft genomes and phenotypic characterization of Tisochrysis lutea strains. Toward the production of domesticated strains with high added value. Algal Res. 29 (7), 1–11. Carvalho, R.N., Bopp, S.K., Lettieri, T., 2011. Transcriptomics responses in marine diatom Thalassiosira pseudonana exposed to the polycyclic aromatic hydrocarbon benzo[a]pyrene. PLoS One 6 (11), e26985. Chan, C.X., Reyes-Prieto, A., Bhattacharya, D., 2011. Red and green algal origin of diatom membrane transporters: insights into environmental adaptation and cell evolution. PLoS One 6 (12), e29138. Chauton, M.S., Winge, P., Brembu, T., Vadstein, O., Bones, A.M., 2013. Gene Regulation of carbon fixation, storage, and utilization in the diatom Phaeodactylum tricornutum acclimated to light/dark cycles. Plant. Physiol. 161 (12), 1034–1048. Chen, Z., Yang, M.K., Li, C.Y., Wang, Y., Zhang, J., Wang, D.B., et al., 2014. Phosphoproteomic analysis provides novel insights into stress responses in Phaeodactylum tricornutum, a model diatom. J. Proteome Res. 13 (5), 2511–2523. Cheng, R.L., Feng, J., Zhang, B.X., Huang, Y., Cheng, J., Zhang, C.X., 2013. Transcriptome and gene expression analysis of an oleaginous diatom under different salinity conditions. Bioenerg. Res. 7 (1), 192–205. Costa, B.S., Jungandreas, A., Jakob, T., Weisheit, W., Mittag, M., Wilhelm, C., 2013. Blue light is essential for high light acclimation and photoprotection in the diatom Phaeodactylum tricornutum. J. Exp. Bot. 64 (2), 483–493. Curson, A.R.J., Williams, B.T., Pinchbeck, B.J., Sims, L.P., Martinez, A.B., Rivera, P.P.L., et al., 2018. DSYB catalyses the key step of dimethylsulfoniopropionate biosynthesis in many phytoplankton. Nat. Microbiol. 3 (4), 430–439. d’Ippolito, G., Sardo, A., Paris, D., Vella, F.M., Adelfi, M.G., Botte, P., et al., 2015. Potential of lipid metabolism in marine diatoms for biofuel production. Biotechnol. Biofuels 8, 28. Danbara, A., Shiraiwa, Y., 1999. The requirement of selenium for the growth of marine coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp. (Prymnesiophyceae). Plant Cell Physiol. 40 (7), 762–766. Di Dato, V., Musacchia, F., Petrosino, G., Patil, S., Montresor, M., Sanges, R., et al., 2015. Transcriptome sequencing of three Pseudo-nitzschia species reveals comparable gene sets and the presence of nitric oxide synthase genes in diatoms. Sci. Rep. U. K. 5, 12329. Dong, H.P., Dong, Y.L., Cui, L., Balamurugan, S., Gao, J., Lu, S.H., et al., 2016. High light stress triggers distinct proteomic responses in the marine diatom Thalassiosira pseudonana. BMC Genomics 17 (1), 994. Durmaz, Y., Monteiro, M., Bandarra, N., Gokpinar, S., Isik, O., 2007. The effect of low temperature on fatty acid composition and tocopherols of the red microalga Porphyridium cruentum. J. Appl. Phycol. 19 (3), 223–227. Dyhrman, S.T., Haley, S.T., Birkeland, S.R., Wurch, L.L., Cipriano, M.J., McArthur, A.G., 2006. Long serial analysis of gene expression for gene discovery and transcriptome profiling in the widespread marine coccolithophore Emiliania huxleyi. Appl. Environ. Microb. 72 (1), 252–260. Dyhrman, S.T., Jenkins, B.D., Rynearson, T.A., Saito, M.A., Mercier, M.L., Alexander, H., et al., 2012. The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse phosphorus stress response. PLoS One 7 (3), e33768. Fabris, M., Matthijs, M., Rombauts, S., Vyverman, W., Goossens, A., Baart, G.J.E., 2012. The metabolic blueprint of Phaeodactylum tricornutum reveals a eukaryotic Entner-Doudoroff glycolytic pathway. Plant J. 70 (6), 1004–1014.



Exploring ‘omics’ approaches Chapter | 11  189

Fidalgo, J.P., Cid, A., Torres, E., Sukenik, A., Herrero, C., 1998. Effects of nitrogen source and growth phase on proximate biochemical composition, lipid classes and fatty acid profile of the marine microalga Isochrysis galbana. Aquaculture 166 (1–2), 105–116. Fukuda, S.Y., Suzuki, I., Hama, T., Shiraiwa, Y., 2011. Compensatory response of the unicellular-calcifying alga Emiliania huxleyi (Coccolithophoridales, Haptophyta) to ocean acidification. J. Oceanogr. 67 (1), 17–25. Garnier, M., Carrier, G., Rogniaux, H., Nicolau, E., Bougaran, G., Saint-Jean, B., et al., 2014. Comparative proteomics reveals proteins impacted by nitrogen deprivation in wild-type and high lipid-accumulating mutant strains of Tisochrysis lutea. J. Proteomics 105 (10), 107–120. Ge, F., Huang, W.C., Chen, Z., Zhang, C.Y., Xiong, Q., Bowler, C., et al., 2014. Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum. Plant Cell 26 (4), 1681–1697. Gruber, A., Rocap, G., Kroth, P.G., Ambrust, E.V., Mock, T., 2015. Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage. Plant. J 81 (3), 519–528. Guedes, A.C., Meireles, L.A., Amaro, H.M., Malcata, F.X., 2010. Changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity. J. Am. Oil Chem. Soc. 87 (7), 791–801. Guiheneuf, F., Ulmann, L., Tremblin, G., Mimouni, V., 2011. Light-dependent utilization of two radiolabelled carbon sources, sodium bicarbonate and sodium acetate, and relationships with long chain polyunsaturated fatty acid synthesis in the microalga Pavlova lutheri (Haptophyta). Eur. J. Phycol. 46 (2), 143–152. Hemaiswarya, S., Raja, R., Ravikumar, R., Kumar, A.Y., de Carvalho, I.S., 2013. Microalgal omics and their applications. In: Barh, D., Zambare, V., Azevedo, V. (Eds.), Omics: Applications in Biomedical, Agricultural, and Environmental Sciences. CRC Press, Boca Raton, pp. 439–450. Hennon, G.M.M., Ashworth, J., Groussman, R.D., Berthiaume, C., Morales, R.L., Baliga, N., et al., 2015. Diatom acclimation to elevated CO2 via cAMP signalling and coordinated gene expression. Nat. Clim. Change 5 (8), 751–765. Hess, S.K., Lepetit, B., Kroth, P.G., Mecking, S., 2017. Production of chemicals from microalgae lipids—status and perspectives. Eur. J. Lipid Sci. Tech. 120 (1), 1700152. Hirokawa, Y., Matsuzuka, S., Itayama, S., Uchida, T., Fujiwara, S., Ozaki, N., et al., 2013. Localization and associative strength of acid polysaccharides in coccoliths of Pleurochrysis haptonemofera (Haptophyta) predicted from their extractability from partially decalcified coccoliths. Open J. Mar. Sci. 3 (1), 48–54. Holligan, P.M., 1992. Do marine phytoplankton influence global climate? In: Falkowski, P.G., Woodhead, A.D. (Eds.), Primary Productivity and Biogeochemical Cycles in the Sea. Plenum Press, Brookhaven, pp. 487–501. Hook, S.E., Osborn, H.L., 2012. Comparison of toxicity and transcriptomic profiles in a diatom exposed to oil, dispersants, dispersed oil. Aquat. Toxicol. 124–125, 139–151. Hovde, B.T., Deodato, C.R., Hunsperger, H.M., Ryken, S.A., Yost, W., Jha, R.K., et  al., 2015. Genome sequence and transcriptome analyses of Chrysochromulina tobin: metabolic tools for enhanced algal fitness in the prominent order prymnesiales (Haptophyceae). PLoS Genet. 11 (9), e1005469. Hunter, J.E., Frada, M.J., Fredricks, H.F., Vardi, A., van Mooy, A.S., 2015. Targeted and untargeted lipidomics of Emiliania huxleyi viral infection and life cycle phases highlights molecular biomarkers of infection, susceptibility, and ploidy. Front. Mar. Sci. 2, 00081. Jamers, A., Blust, R., de Coen, W., 2009. Omics in algae: paving the way for a systems biological understanding of algal stress phenomena? Aquat. Toxicol. 92 (3), 114–121. Jiang, H.M., Gao, K.S., 2004. Effects of lowering temperature during culture on the production of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum (bacillariophyceae). J. Phycol. 40 (4), 651–654. Jungandreas, A., Costa, B.S., Jakob, T., von Bergen, M., Baumann, S., Wilhelm, C., 2014. The acclimation of Phaeodactylum tricornutum to blue and red light does the acclimation of Phaeodactylum tricornutum to blue and red light does not influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern. PLoS One 9 (8), e99727. Kageyama, H., Tanaka, Y., Shibata, A., Waditee-Sirisattha, R., 2018. Dimethylsulfoniopropionate biosynthesis in a diatom Thalassiosira pseudonana: identification of a gene encoding MTHB-methyltransferase. Arch. Biochem. Biophys. 645, 100–106. Kayano, K., Saruwatari, K., Kogure, T., Shiraiwa, Y., 2011. Effect of coccolith polysaccharides isolated from the coccolithophorid, Emiliania huxleyi, on calcite crystal formation in in vitro CaCO3 crystallization. Mar. Biotechnol. 13 (1), 83–92. Kim, K.M., Park, J.H., Bhattacharya, D., Yoon, H.S., 2014. Applications of next-generation sequencing to unravelling the evolutionary history of algae. Int. J. Syst. Evol. Micr. 64 (2), 333–345. Kim, J., Fabris, M., Baart, G., Kim, M.K., Goossens, A., Vyverman, W., et  al., 2016. Flux balance analysis of primary metabolism in the diatom Phaeodactylum tricornutum. Plant J. 85 (1), 161–176. Koid, A.E., Liu, Z.F., Terrado, R., Jones, A.C., Caron, D.A., Heidelberg, K.B., 2014. Comparative transcriptome analysis of four prymnesiophyte algae. PLoS One 9 (6), e97801. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

190  PART | III  Algal genomics

Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Kotzsch, A., Groger, P., Pawolski, D., Bomans, P.H.H., Sommerdijk, N.A.J.M., Schlierf, M., et al., 2017. Silicanin-1 is a conserved diatom membrane protein involved in silica biomineralization. BMC Biol. 15, 65. Lavaud, J., Rousseau, B., Gorkom, H.J.V., Etienne, A.L., 2002. Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum. Plant. Physiol. 129 (3), 1398–1406. Le Costaouec, T., Unamunzaga, C., Mantecon, L., Helbert, W., 2017. New structural insights into the cell-wall polysaccharide of the diatom Phaeodactylum tricornutum. Algal Res. 26, 172–179. Lee, P.A., Bearden, D.W., Casu, F., Pound, H., Janech, M.G., 2017. Short-term impact of vitamin B12 and nitrate deprivation on intracellular DMSP levels and DMSP production in marine diatoms. In: Association for the Sciences of Limnology and Oceanography Aquatic Sciences Meeting, Honolulu, Hawaii. Levering, J., Broddrick, J., Dupont, C.L., Peers, G., Beeri, K., Mayers, J., et al., 2016. Genome-scale model reveals metabolic basis of biomass partitioning in a model diatom. PLoS One 11 (5), e0155038. Levering, J., Dupont, C.L., Allen, A.E., Palsson, B.O., Zengler, K., 2017. Integrated regulatory and metabolic networks of the marine diatom Phaeodactylum tricornutum predict the response to rising CO2 levels. mSystems 2 (1), e00142-16. Liang, Y., Mai, K.S., Sun, S.C., 2000. Total lipid and fatty acid composition of eight strains of marine diatoms. Chin. J. Oceanol. Limn. 18 (4), 345–349. Liang, Y., Beardall, J., Heraud, P., 2006. Effect of UV Radiation on growth, chlorophyll fluorescence and fatty acid composition of Phaeodactylum tricornutum and Chaetoceros muelleri (Bacillariophyceae). Phycologia 45 (6), 605–615. Liang, Y., Osada, K., Sunaga, Y., Yoshino, T., Bowler, C., Tanaka, T., 2015. Dynamic oil body generation in the marine oleaginous diatom Fistulifera solaris in response to nutrient limitation as revealed by morphological and lipidomic analysis. Algal Res. 12, 359–367. Llewellyn, C.A., Sommer, U., Dupont, C.L., Allen, A.E., Viant, M.R., 2015. Using community metabolomics as a new approach to discriminate marine microbial particulate organic matter in the western English Channel. Prog. Oceanogr. 137 (B), 421–433. Lommer, M., Specht, M., Roy, A.S., Kraemer, L., Andreson, R., Gutowska, M.A., et al., 2012. Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation. Genome Biol. 13 (7), R66. Longworth, J., Wu, D., Huete-ortega, M., Wright, P.C., Vaidyanathan, S., 2016. Proteome response of Phaeodactylum tricornutum, during lipid accumulation induced by nitrogen depletion. Algal Res. 18 (6), 213–224. Maheswari, U., Mock, T., Armbrust, E.V., Bowler, C., 2009. Update of the diatom EST database: a new tool for digital transcriptomics. Nucl. Acids Res. 37, D1001–D1005. Maheswari, U., Jabbari, K., Petit, J.-L., Porcel, B.M., Allen, A.E., Cadoret, J.-P., Martino, A.D., Heijde, M., Kaas, R., Roche, J.L., Lopez, P.J., MartinJézéquel, V., Meichenin, A., Mock, T., Parker, M.S., Vardi, A., Armbrust, E.V., Weissenbach, J., Katinka, M., Bowler, C., 2010. Digital expression profiling of novel diatom transcripts provides insight into their biological functions. Genome Biol. 11, R85. Matthijs, M., Fabris, M., Broos, S., Vyverman, W., Goossens, A., 2016. Profiling of the early nitrogen stress response in the diatom Phaeodactylum tricornutum reveals a novel family of ring-domain transcription factors. Plant Physiol. 170 (1), 489–498. Matthijs, M., Fabris, M., Obata, T., Foubert, I., Franco-Zorrilla, J.M., Solano, R., et al., 2017. The transcription factor bZIP 14 regulates the TCA cycle in the diatom Phaeodactylum tricornutum. EMBO J. 36 (11), 1559–1576. Mausz, M.A., Georg, P., 2015. Phenotypic diversity of diploid and haploid Emiliania huxleyi cells and of cells in different growth phases revealed by comparative. J. Plant. Phys. 172, 137–148. McCarthy, J.K., Smith, S.R., McCrow, J.P., Tan, M., Zheng, H., Beeri, K., et al., 2017. Nitrate reductase knockout uncouples nitrate transport from nitrate assimilation and drives repartitioning of carbon flux in a model pennate diatom. Plant Cell. 29 (8), 2047–2070.



Exploring ‘omics’ approaches Chapter | 11  191

McKew, B.A., Lefebvre, S.C., Achterberg, E.P., Metodieva, G., Raines, C.A., Metodiev, M.V., et al., 2013. Plasticity in the proteome of Emiliania huxleyi CCMP 1516 to extremes of light is highly targeted. New Phytol. 200 (1), 61–73. Mock, T., Krell, A., Glockner, G., Kolukisaoglu, U., Valentin, K., 2005. Analysis of expressed sequence tags (ESTs) from the polar diatom Fragilariopsis cylindrus. J. Phycol. 42 (1), 78–85. Mock, T., Samanta, M.P., Iverson, V., Berthiaume, C., Robison, M., Holtermann, K., et al., 2008. Whole-genome expression profiling of the marine diatom Thalassiosira pseudonana identifies genes involved in silicon bioprocesses. Proc. Natl. Acad. Sci. U. S. A. 105 (5), 1579–1584. Mock, T., Otillar, R.P., Strauss, J., McMullan, M., Paajanen, P., Schmutz, J., et al., 2017. Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus. Nature 541 (7638), 78–85. Mus, F., Toussaint, J.P., Cooksey, A.E., Fields, M.W., Gerlach, R., Peyton, B.M., et al., 2013. Physiological and molecular analysis of carbon source supplementation and pH stress-induced lipid accumulation in the marine diatom Phaeodactylum tricornutum. Appl. Microbiol. Biot. 97 (8), 3625–3642. Nojima, D., Yoshino, T., Maeda, Y., Tanaka, M., Nemoto, M., Tanaka, T., 2013. Proteomics analysis of oil body-associated proteins in the oleaginous diatom. J. Proteome Res. 12 (11), 5293–5301. Nunn, B.L., Faux, F.A., Hippman, A.A., Maldonado, M.T., Harvey, H.R., Goodlet, L.R., et al., 2013. Diatom proteomics reveals unique acclimation strategies to mitigate Fe limitation. PLoS One 8 (10), e75653. Nymark, M., Valle, K.C., Hancke, K., Winge, P., Andresen, K., Johnsen, G., et al., 2013. Molecular and photosynthetic responses to prolonged darkness and subsequent acclimation to re-illumination in the diatom Phaeodactylum tricornutum. PLoS One 8 (3), e58722. Obata, T., Shiraiwa, Y., 2005. A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi. J. Biol. Chem. 280 (18), 18462–18468. Obata, T., Schoenefeld, S., Krahnert, I., Bergmann, S., Scheffel, A., Fernie, A.R., 2013. Gas-chromatography mass-spectrometry (GC-MS) based metabolite profiling reveals mannitol as a major storage carbohydrate in the coccolithophorid alga Emiliania huxleyi. Metabolites 3 (1), 168–184. Orcutt, D.M., Patterson, G.W., 1974. Effect of light intensity upon lipid composition Nitzschia closterium (Cylindrotheca fusiformis). Lipids 9 (12), 1000–1003. Osada, K., Maeda, Y., Yoshino, T., Nojima, D., Bowler, C., Tanaka, T., 2017. Enhanced NADPH production in the pentose phosphate pathway accelerates lipid accumulation in the oleaginous diatom Fistulifera solaris. Algal Res. 23, 126–134. Prince, E.K., Irmer, F., Pohnert, G., 2013. Domoic acid improves the competitive ability of Pseudo-Nitzschia delicatissima against the diatom Skeletonema marinoi. Mar. Drugs 11 (7), 2398–2412. Quinn, P., Bowers, R.M., Zhang, X.Y., Wahlund, T.M., Fanelli, M.A., Olszova, D., et al., 2006. cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta). Appl. Environ. Microb. 72 (8), 5512–5526. Raina, J.B., Clode, P.L., Cheong, S., Bougoure, J., Kilburn, M.R., Reeder, A., et al., 2017. Subcellular tracking reveals the location of dimethylsulfoniopropionate in microalgae and visualises its uptake by marine bacteria. ELife 6, e23008. Read, B.A., Kegel, J., Klute, M.J., Kuo, A., Lefebvre, S.C., Maumus, F., et al., 2013. Pan genome of the phytoplankton Emiliania underpins its global distribution. Nature 499 (7457), 209–213. Remmers, I.M., D'Adamo, S., Martens, D.E., de Vos, R.C.H., Mumm, R., America, A.H.P., Cordewener, J.H.G., Bakker, L.V., Peters, S.A., Wijffels, R.H., Lamers, P.P., 2018. Orchestration of transcriptome, proteome and metabolome in the diatom Phaeodactylum tricornutum during nitrogen limitation. Algal Res. 35, 33–49. Renaud, S.M., Parry, D.L., Thinh, L.V., Kuo, C., Padovan, A., Sammy, N., 1991. Effect of light intensity on the proximate biochemical and fatty acid composition of Isochrysis sp. and Nannochloropsis oculata for use in tropical aquaculture. J. Appl. Phycol. 3 (1), 43–53. Rosenwasser, S., van Creveld, S.G., Schatz, D., Malitsky, S., Tzfadi, O., Aharoni, A., et al., 2014. Mapping the diatom redox-sensitive proteome provides insight into response to nitrogen stress in the marine environment. Proc. Natl. Acad. Sci. U. S. A. 111 (7), 2740–2745. Sabatino, V., Russo, M.T., Patil, S., d’Ippolito, G., Fontana, A., Ferrante, M.I., 2015. Establishment of genetic transformation in the sexually reproducing diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and inheritance of the transgene. Mar. Biotechnol. 17 (4), 452–462. Sapriel, G., Quinet, M., Heijde, M., Jourdren, L., Tanty, V., Luo, G.Z., et  al., 2009. Genome-wide transcriptome analyses of silicon metabolism in Phaeodactylum tricornutum reveal the multilevel regulation of silicic acid transporters. PLoS One 4 (10), e7458. Schoemann, V., Becquevort, S., Stefels, J., Rousseau, V., Lancelot, C., 2005. Phaeocystis blooms in the global ocean and their controlling mechanisms: a review. J. Sea. Res. 53 (1–2), 43–66. Shrestha, R.P., Tesson, B., Norden-Krichmar, T., Federowicz, S., Hildebrand, M., Allen, A.E., et al., 2012. Whole transcriptome analysis of the silicon response. BMC Genomics 13, 499. Smith, S.R., Gle, C., Abbriano, R.M., Traller, J.C., Davis, A., Trentacoste, E., Vernet, M., et al., 2016. Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana. New Phytol. 210 (3), 890–904. Song, P.P., Li, L., Liu, J.G., 2013. Proteomic analysis in nitrogen-deprived Isochrysis galbana during lipid accumulation. PLoS One 8 (12), e82188. Sonowal, S., Chikkaputtaiah, C., Velmurugan, N., 2019. Role of flow cytometry for the improvement of bioprocessing of oleaginous microorganisms. J. Chem. Technol. Biot. 94 (6), 1712–1726. Soto, A.R., Zheng, H., Shoemaker, D., Rodriguez, J., Read, B.A., Wahlund, T.M., 2006. Identification and preliminary characterization of two cDNAs encoding unique carbonic anhydrases from the marine alga Emiliania huxleyi. Appl. Environ. Microb. 72 (8), 5500–5511. Sriharan, S., Bagga, D., Nawaz, M., 1991. The effects of nutrients and temperature on biomass, growth, lipid production, and fatty acid composition of Cyclotella cryptica Reimann, Lewin, and Guillard. Biotechnol. Appl. Bioc. 28-29 (1), 317–326. Su, X.L., Xu, J.L., Yan, X.J., Zhao, P., Chen, J.J., Zhou, C.X., et al., 2013. Lipidomic changes during different growth stages of Nitzschia closterium f. minutissima. Metabolomics 9 (2), 300–310. Sukenik, A., Wahnon, R., 1991. Biochemical quality of marine unicellular algae with special emphasis on lipid composition. I. Isochrysis galbana. Aquaculture 97 (1), 61–72.

192  PART | III  Algal genomics

Talarski, A., Manning, S.R., la Clarie, J.W., 2016. Transcriptome analysis of the euryhaline alga, Prymnesium parvum (Prymnesiophyceae): effects of salinity on differential gene expression. Phycologica 55 (1), 33–44. Tan, C.K., Johns, M.R., 1996. Screening of diatoms for heterotrophic eicosapentaenoic acid production. J. Appl. Phycol. 8 (1), 59–64. Tanaka, T., Maeda, Y., Veluchamy, A., Tanaka, M., Abida, H., Marechal, E., et al., 2015. Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome. Plant Cell. 27 (1), 162–176. Thamatrakoln, K., Korenovska, O., Niheu, A.K., Bidle, K.D., 2012. Whole-genome expression analysis reveals a role for death-related genes in stress acclimation of the diatom Thalassiosira pseudonana. Environ. Microbiol. 14 (1), 67–81. Thiele, I., Palsson, B.O., 2010. A protocol for generating a high-quality genome-scale metabolic reconstruction. Nat. Protoc. 5 (1), 93–121. Tiselius, P., Kuylenstierna, M., 1996. Growth and decline of a diatom spring bloom: phytoplankton species composition, formation of marine snow and the role of heterotrophic dinoflagellates. J. Plankton Res. 18 (2), 133–155. Traller, J.C., Cokus, S.J., Lopez, D.A., Gaidarenko, O., Smith, S.R., McCrow, J.P., et al., 2016. Biotechnology for biofuels genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnol. Biofuels 9, 258. Tzovenis, I., de Pauw, N., Sorgeloos, P., 1997. Effect of different light regimes on the docosahexaenoic acid (DHA) content of Isochrysis aff. galbana (clone T-ISO). Aquacult. Int. 5 (6), 489–507. Valenzuela, J., Mazurie, A., Carlson, R.P., Gerlach, R., Cooksey, K.E., Peyton, B.M., et al., 2012. Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum. Biotechnol. Biofuels 5, 40. Valle, K.C., Nymark, M., Aamot, I., Hancke, K., Winge, P., Andrsen, K., et al., 2014. System responses to equal doses of photosynthetically usable radiation of blue green, and red light in the marine diatom Phaeodactylum tricornutum. PLoS One 9, 114211. Van Dassow, P., Ogata, H., Probert, I., Wincker, P., da Silva, C., Audic, S., et al., 2009. Transcriptome analysis of functional differentiation between haploid and diploid cells of Emiliania huxleyi, a globally significant photosynthetic calcifying cell. Genome Biol. 10 (10), R114. Velmurugan, N., Deka, D., 2018. Transformation techniques for metabolic engineering of diatoms and haptophytes: current state and prospects. Appl. Microbiol. Biot. 102 (10), 4255–4267. Vidoudez, C., Pohnert, G., 2012. Comparative metabolomics of the diatom Skeletonema marinoi in different growth phases. Metabolomics 8 (4), 654–669. Villanova, V., Fortunato, A.E., Singh, D., Bo, D.D., Conte, M., Obata, T., et  al., 2017. Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum. Philos. T. R. Soc. B 372 (1728), 20160404. Volkman, J.K., Smith, D.J., Eglinton, G., Forsberg, T.E.V., 1981. Sterol and fatty acid composition of four marine haptophycean algae. J. Mar. Biol. Assoc. UK 61 (2), 509–527. Wahlund, T.M., Hadaegh, A.R., Clark, R., Nguyen, B., Fanelli, M., Read, B.A., 2004. Analysis of expressed sequence tags from calcifying cells of marine coccolithophorid (Emiliania huxleyi). Mar. Biotechnol. 6 (3), 278–290. Wen, Z.Y., Chen, F., 2000. Production potential of eicosapentaenoic acid by the diatom Nitzschia laevis. Biotechnol. Lett. 22 (9), 727–733. Wordenweber, R., 2017. Comprehensive Metabolome Analysis of the Marine Microalga Emiliania huxleyi Regarding Calcification Status, Growth Phase and Nutrient-Starvation Response. PhD, University of Bielefeld, Bielefeld. Wu, S.A., Meng, Y.Y., Cao, X.P., Xue, S., 2016. Regulatory mechanisms of oxidative species and phytohormones in marine microalgae Isochrysis zhangjiangensis under nitrogen deficiency. Algal. Res. 17, 321–329. Xie, J., Bai, X.C., Lavoie, M., Lu, H.P., Fan, X.J., Pan, X.L., et al., 2015. Analysis of the proteome of the marine diatom Phaeodactylum tricornutum exposed to aluminum providing insights into aluminum toxicity mechanisms. Environ. Sci. Technol. 49 (18), 11182–11190. Yang, Z.K., Ma, Y.H., Zheng, J.W., Yang, W.D., Liu, J.S., Li, H.Y., 2014. Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum. J. Appl. Phycol. 26 (1), 73–82. Yi, Z.Q., Xu, M.N., Magnusdottir, M., Zhang, Y.T., Brynjolfsson, S., Fu, W.Q., 2015. Photo-oxidative stress-driven mutagenesis and adaptive evolution on the marine diatom Phaeodactylum tricornutum for enhanced carotenoid accumulation. Mar. Drugs 13 (10), 6138–6151. Yongmanitchai, W., Ward, O.P., 1991. Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions. Appl. Environ. Microb. 57 (2), 419–425. Yoshioka, M., Yago, T., Yoshie-Stark, Y., Arakawa, H., Morinaga, T., 2012. Effect of high frequency of intermittent light on the growth and fatty acid profile of Isochrysis galbana. Aquaculture 338–341 (5), 111–117. Zhang, C.Y., Hu, H.H., 2014. High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation. Mar. Genomics 16 (4), 63–66.

Chapter 12

The scientometric analysis of the research on the algal photosystems and photosynthesis Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

12.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 papers published since 1980. The algal photosystems and photosynthesis have been among the most-prolific research fronts over time as evidenced with over 11,500 papers published during the same study period, corresponding to the public concerns about the development of sustainable and environment-friendly bioproducts. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field, the stakeholders should have timely access to the information on the relevant research (Konur, 2000, 2002a, b, c, 2004, 2006a, b, 2007a, b, 2012a). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal toxicology and the underlying research areas as in other fields of the algal research (Konur, 2011, 2015a, b, c, d, e, f, g, h, i, j, k, 2016a, 2017a, 2019a, 2020a, b, c, d, e, f, g, h, i, j, k, l, m, n), bioenergy and biofuels (Konur, 2012b, c, d, e, f, g, h, i, j, k, 2018a, b, c), nanobiomaterials (Konur, 2016b, c, d, e, f, g, 2017b, c, d, e, f, 2019b), biomedicine (Konur, 2016h, 2018d), and social sciences (Konur, 2012o, p, q, r, s, t). Although there have been over 600 literature reviews on the algal photosystems and photosynthesis, there has been no published scientometric studies in the journal literature. However, there has been a book chapter covering algal photosynthesis in part (Konur, 2015e). Furthermore, there has been only one paper on the scientometric studies in photosynthesis at large (Yu et al., 2012). Therefore, this paper presents the first-ever scientometric study of the research in algal photosystems and photosynthesis covering the whole range of research fronts as well as whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a low funding rate.

12.2  Materials and methodology The search for the scientometric analysis of the literature on the algal photosystems and photosynthesis was carried out in January 2019 using 4 databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI) and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in Appendix. These keyword sets have been devised in three major parts: the keywords related to photosystems and photosynthesis and keywords related to the algae as well as the cross-subject keywords related to algal photosynthesis.

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00012-7 © 2020 Elsevier Inc. All rights reserved.

195

196  PART | IV  Algal photosystems and photosynthesis

There have been two distinct keyword sets for the first part: the set of core journal titles related to photosystems and photosynthesis and keywords related to the photosystems and photosynthesis. On the other hand, the second part consists of the keywords related to the algae and phytoplankton in general, dinoflagellates and coccolithophores, microalgae, macroalgae, diatoms, cyanobacteria, and journal titles related to the algae. The papers found through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subject to the quality control exercise to ensure that these references have been primarily related to the algal photosystems and photosynthesis. This refined list of papers have formed the sample for the scientometric and content overview of the literature on the algal photosystems and photosynthesis. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. Additionally, the search has been carried out for the papers with at least 100 citations has been collected and the relevant scientometric data has been recovered. These papers have been termed as ‘influential papers’. Furthermore, the data on the scientometric analysis and content overview of 20 most-cited papers have also been provided focusing on the determination of the key research fronts for these citation classics, complementing sections on the keywords and research fronts. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

12.3 Results 12.3.1  Documents and indexes The search has resulted in 12,823 papers where there have been 10,781 articles, 1,048 meeting abstracts, 620 reviews, 211 notes, 67 corrections, 55 editorial materials, 24 letters, and 9 corrections and additions. In the first instance, the papers excluding meeting abstracts and corrections have been selected resulting in 11,691 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 11,519 papers in English. The other major languages have been Russian, French, German, and Chinese. This set of 11,519 papers has formed the sample for the scientometric analysis of the literature on the algal photosystems and photosynthesis. The articles have formed 92.2% of the final sample whilst reviews, notes, editorial matters, and letters have formed 5.3%, 1.8%, 0.5%, and 0.2% of this sample, respectively. Additionally, 3.6% of these papers have been ‘proceedings papers’ and there have been no ‘retracted papers’. On the other hand, 99.9% of these papers have been indexed by the SCI-E whilst only 9 papers have been indexed by the SSCI and A&HCI focusing on the societal and humanitarian aspects of algal photosystems and photosynthesis. Additionally, 0.2% of the papers have been indexed by the ESCI.

12.3.2 Keywords The most-prolific keywords used in algal photosystems and photosynthesis have been determined based on the influential papers to determine the hot topics and the primary research fronts in the algal photosystems and photosynthesis There have been a number of most-prolific keywords for the first set of keywords for the photosystems and photosynthesis: ‘photosynth*, photosystem*, * cytochrom*’. The other prolific key words have been ‘“light harvest*”, “electron transport”, “carbonic anhydrase*”, polypeptide*, ferredoxin*, antenna*, “reaction center*”, nadp*’. On the other hand, there have been two journals related to photosynthesis: ‘“Photosynthesis Research”, Photosynthetica*’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, macroalga*, phytoplankton, rhodophyt*, seaweed*, bacillariophycea*, diatom, diatoms, and cyanobacter*’. The other prolific keywords for the algae have been ‘coccolith*, dinophycea*, Alexandrium, chlorophycea*, chlorophyt*, “green alga*”, microalga*, “micro-alga*”, Chlamydomonas, *Chlorella, Dunaliella, Euglena, Scenedesmus, “brown alga*”, phaeophycea*, kelp*, phaeophyt*, “red alga*”, Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, Ulva, bacillariophyt*, “blue green alga*”, “blue-green alga*”, *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, Synechocystis’. On the other hand, the most-prolific journals related solely to the algal research have been ‘Algal Research*’, ‘European Journal of Phycology’, ‘Harmful Algae’, ‘Journal of Applied Phycology’, ‘Journal of Phycology’, ‘Phycologia’.

The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  197



12.3.3 Authors There have been 18,671 authors contributing to the research on the algal photosystems and photosynthesis in total. The information on the most-prolific and influential 20 authors is provided in Table 12.1: Authors’ names, gender, institutions, countries, primary research fronts, number of papers produced by these authors, the number of influential papers with at least 100 citations received (I-100), and the percentage of the number of influential papers with relative to the number of all the papers published (I-100%).

TABLE 12.1  The most-prolific and influential authors in algal photosystems and photosynthesis. Author

Gender

Institution

Country

Research fronts

I-0

I-100

I-100%

1

Murray R Badger

M

Australian Natl. Univ.

Australia

CO2 concentrating mechanisms

71

23

32.4

2

G Dean Price

M

Australian Natl. Univ.

Australia

CO2 concentrating mechanisms

63

19

30.2

3

Paul G Falkowski

M

Brookhaven Natl. Lab.

US

Photosynthesis

40

19

47.5

4

Bruce A Diner

M

Dupont

US

Photosystems

59

17

28.8

5

Norio Murata

M

Natl. Inst. Basic Biol.

Japan

Photosystems

59

15

25.4

6

Arthur R Grossman

M

Carnegie Inst. Sci.

US

Photosynthesis

60

13

21.7

7

Petra Fromme

F

Arizona St. Univ.

US

Photosystems

47

13

27.7

8

Richard J Geider

M

Univ. Essex

Untd. King.

Photosynthesis

38

13

34.2

9

Jean-David Rochaix

M

Univ. Geneva

Switzerland

Photosystems

54

12

22.2

10

Suleyman I Allakhverdiev

M

Russ. Acad. Sci.

Russia

Photosystems

42

12

28.6

11

John A Raven

M

Univ. Dundee

Untd. King.

CO2 concentrating mechanisms

50

11

22.0

12

Richard J Debus

M

Univ. Calif. Riverside

US

Photosystems

48

11

22.9

13

Peter J Nixon

M

Imperial Coll. Lond.

Untd. King.

Photosystems

69

9

13.0

14

Eberhard Schlodder

M

Tech. Univ. Berlin

Germany

Photosystems

44

9

20.5

15

Yoshitaka Nishiyama

M

Saitama Univ.

Japan

Photosystems

43

9

20.9

16

Horst Tobias Witt

M

Tech. Univ. Berlin

Germany

Photosystems

35

9

25.7

17

James Barber

M

Imperial Coll. Lond.

Untd. King.

Photosystems

77

8

10.4

18

Anastasios Melis

M

Univ. Calif. Berkeley

US

Photosynthesis

67

8

11.9

19

Francois-Andre Wollman

M

Inst. Biol. Phys. Chem.

France

Photosynthesis

47

8

17.0

20

John J Cullen

M

Dalhouise Univ.

Canada

Photosynthesis

44

8

18.2

Average

52.8

12.3

24.1

Total

1057

246

Total %

7.0

37.8

M: Male. F: Female. I-0: No. papers, the number of papers for at least 13 papers. I-100: The number of influential papers with at least 100 citations for at least 2 papers, I-100%: The percentage of the number of influential papers with relative to the number of all the papers published.

198  PART | IV  Algal photosystems and photosynthesis

The data on these authors shows that the most-prolific author with the highest citation impact has been ‘Murray R Badger’ of the Australian National University, working primarily on the CO2 concentrating mechanisms, with 71 papers. His citation impact is highest with 23 influential papers. The other most-prolific authors with the high citation impact have been ‘G Dean Price’, ‘Paul G Falkowski’, ‘Bruce A Diner’, and ‘Norio Murata’ with 15 or more influential papers each. It is notable that only one of these authors are female: ‘Petra Fromme’. The US and the United Kingdom have been the most-prolific countries for these authors with six and four authors, respectively, whilst Australia, Germany, and Japan followed these top countries with two authors each. On the other hand, Europe has had eight authors as a whole. Similarly, the most-prolific institutions have been ‘Australian National University’, ‘Imperial College London’, and ‘Technical University of Berlin’ with two authors each. The most-prolific research fronts have been the ‘algal photosystems’, ‘algal photosynthesis’, and ‘algal CO2 concentrating mechanisms’ with 11, 6, and 3 authors, respectively. The number of papers published by these authors have ranged from 35 to 77. These most-prolific authors have contributed to nearly 7.0% and 37.8% of all the papers and influential papers, respectively. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the influential papers with relative to the number of all the papers published for each author in the last column (I-100%). These data shows that ‘Paul G Falkowski’ has been the top influential author with 47.5% ratio. The other most-influential authors have been ‘Richard J Geider’, ‘Murray G Badger’, ‘G Dean Price’, ‘Bruce A Diner’, and ‘Suleyman I Allakhverdiev’ with over 28% ratio each.

12.3.4 Countries Nearly 99.8% of the papers have had country information in their abstract pages and 100 countries and territories have contributed to these papers overall. Table 12.2 provides the information about the most-prolific and influential 20 countries. These 20 most-prolific countries have produced 175.6% and 197.7% of all the papers and influential papers, respectively, as a whole. The most-prolific and influential country has been the US producing 27.1% and 45.4% of all the papers and influential papers, respectively.

TABLE 12.2  The most-prolific and influential countries in algal photosystems and photosynthesis. Country

I-0

I-0%

I-100

I-100%

Europe

6640

57.6

381

58.6

1

US

3124

27.1

295

45.4

2

Germany

1792

15.6

110

16.9

3

France

876

7.6

78

12.0

4

Japan

1477

12.8

75

11.5

5

Untd. King.

864

7.5

69

10.6

6

Australia

507

4.4

52

8.0

7

Canada

650

5.6

48

7.4

8

Sweden

347

3.0

24

3.7

9

Israel

344

3.0

23

3.5

11

Russia

522

4.5

20

3.1

10

Netherlands

385

3.3

20

3.1

12

Switzerland

169

1.5

15

2.3

13

Spain

598

5.2

13

2.0

14

Italy

295

2.6

11

1.7

15

Denmark

140

1.2

11

1.7

The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  199



TABLE 12.2  The most-prolific and influential countries in algal photosystems and photosynthesis—cont’d Country

I-0

I-0%

I-100

I-100%

16

Czech Rep.

228

2.0

10

1.5

17

Finland

120

1.0

9

1.4

18

Hungary

158

1.4

8

1.2

19

China

852

7.4

7

1.1

20

New Zealand

113

1.0

6

0.9

Total

20,201

175.6

1285

197.7

I-0: The number of all the papers. I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations. I-100%: The percentage of the number of influential papers.

Additionally, Germany, France, Japan, and the United Kingdom have emerged as the other most-prolific and influential countries following the US producing 15.6%, 7.6%, 12.8%, and 7.5% of all the papers, respectively. These counties have also produced 16.9%, 12.0%, 11.5%, and 10.6% of the influential papers, respectively. Australia and Canada have been the other prolific and influential countries. On the other hand, China has produced 7.4% and 1.1% of all the papers and influential papers, respectively. The European countries have been dominant in the top-20 country list as they have produced 57.6% and 58.6% of all the papers and influential papers, respectively, as a whole, surpassing significantly the US, Japan, and Australia.

12.3.5 Institutions Over 99.8% of the papers have had their institutions listed in their abstract pages. For these papers, 2870 institutions have contributed to the research on the algal photosystems and photosynthesis in total. The information about the 20 mostprolific and influential institutions is given in Table 12.3. The most-prolific and influential institution has been the ‘Centre National de la Recherche Scientifique’ (CNRS) publishing 7.7% and 5.3% of the influential and all papers, respectively. ‘University of California Berkeley’,

TABLE 12.3  The most-prolific and influential institutions in algal photosystems and photosynthesis. Institutions

Country

I-0

I-0%

I-00

I-100% papers

Europe

2479

16.4

234

36.0

US

659

4.4

100

15.4

1

Ctr. Natl. Rec. Sci.-CNRS

France

615

5.3

50

7.7

2

Univ. Calif. Berkeley

US

222

1.9

31

4.8

3

Commis. Ener. Atom. Ener. Alt.-CEA

France

311

2.7

30

4.6

4

Australian Natl. Univ.

Australia

144

1.3

26

4.0

5

Tech. Univ. Berlin

Germany

165

1.4

25

3.8

6

Brookhaven Natl. Lab.

US

62

0.5

20

3.1

7

Univ. Paris Saclay

France

297

2.6

19

2.9

8

Helmholtz Assoc.

Germany

175

1.5

18

2.8

9

Dupont

US

67

0.6

18

2.8

Sorbonne Univ.

France

176

1.5

17

2.6

Max Planck Soc.

Germany

191

1.7

16

2.5

11

Continued

200  PART | IV  Algal photosystems and photosynthesis

TABLE 12.3  The most-prolific and influential institutions in algal photosystems and photosynthesis—cont’d Institutions

Country

I-0

I-0%

I-00

I-100% papers

12

Imperial Coll. Lond.

Untd. King.

157

1.4

16

2.5

13

Natl. Inst. Basic Biol.

Japan

139

1.2

16

2.5

14

Carnegie Inst. Sci.

US

115

1.0

16

2.5

15

Russian Acad. Sci.

Russia

294

2.6

15

2.3

16

Arizona State Univ.

US

193

1.7

15

2.3

17

Free Univ. Berlin

Germany

141

1.2

15

2.3

18

Alfred Wegener Res. Inst.

Germany

124

1.1

15

2.3

19

RIKEN

Japan

131

1.1

14

2.2

20

Umea Univ.

Sweden

127

1.1

13

2.0

3846

33.4

405

62.3

Total

I-0: The number of all the papers. I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations. I-100%: The percentage of the number of influential papers.

‘French Alternative Energies and Atomic Energy Commission’ (CEA), ‘Australian National University’, ‘Technical University of Berlin’, and ‘Brookhaven National Laboratory’ have been the other influential institutions as they have produced 4.8%, 4.6%, 4.0%, 3.8%, and 3.1% of the influential papers, respectively. The most-prolific countries for these institutions have been the US, Germany, and France with six, five, and four institutions, respectively. On the other hand, Japan has had 2 institutions and Europe has had 11 institutions as a whole where these European institutions produced 16.4% and 30.7% of the all the papers and influential papers, respectively. The contribution of these institutions has ranged from 0.5% to 5.3% for all the papers and from 2.0% to 7.7% for the influential papers. Overall, these 20 institutions have contributed to 33.4% and 62.3% of these papers, respectively.

12.3.6  Research funding bodies Only 33.4% of these papers have had declared any research funding in their abstract pages and overall, 4873 funding bodies have funded these papers. The most-prolific funding bodies have been the ‘National Natural Science Foundation of China’, ‘National Institute of General Medical Sciences’, and ‘National Science Foundation’ of the US funding 2.8%, 2.5%, and 1.6% of the papers, respectively. The other prolific funding bodies have been the ‘German Research Foundation’, ‘Biotechnology and Biological Sciences Research Council’, ‘Japan Society for the Promotion of Science’, ‘Russian Foundation for Basic Research’, ‘Natural Environment Research Council’, and ‘Australian Research Council’ with at least 0.5% of the papers each.

12.3.7  Publication years Fig. 12.1 shows the number of papers on the algal photosystems and photosynthesis, published between 1980 and 2018 as of January 2019. The data in this figure shows that the number of papers has risen from 178 papers in 1980 to 435 papers in 2017. The most prolific decades have been the 1990s, 2000s, and 2010s with 27.5%, 26.2%, and 30.1% of the papers, respectively. Additionally, 16.1% of the papers have been published in the 1980s. Thus, the figure shows that there has been a steadily increasing trend between 1980 and January 2019.

12.3.8  Source titles Overall, these papers have been published in 970 journals. Table 12.4 provides the information on the 20 most-prolific and influential journals. These 20 journals have published 42.7% and 62.1% of all the papers and influential papers, ­respectively, in total.

The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  201



500 450

Number of papers

400 350 300 250 200 150 100 50 0

Publication years

FIG. 12.1  The number of publications in the algal photosystems and photosynthesis between 1980 and 2018.

TABLE 12.4  The most-prolific and influential journals in algal photosystems and photosynthesis. Journals

Abbr.

Subject

I-0

I-0 %

I-100

I-100%

1

Plant Physiology

Plant Physiol.

Plant Sci.

501

4.3

47

7.2

2

Proceedings of the National Academy of Sciences of the United States of America

P. Natl. Acad. Sci. USA

Mult. Sci.

170

1.5

46

7.1

3

Biochemistry

Biochemistry-US

Bioch. Mol. Biol.

422

3.7

42

6.5

4

Photosynthesis Research

Photosynth. Res.

Plant Sci.

842

7.3

36

5.5

5

Biochimica et Biophysica Acta Bioenergetics

BBA-Bioenergetics

Bioch. Mol. Biol.; Biophys.

432

3.8

32

4.9

6

Journal of Phycology

J. Phycol.

Plant Sci.; Mar. Fresh. Biol.

383

3.3

31

4.8

7

Nature

Nature

Mult. Sci.

36

0.3

26

4.0

8

Journal of Biological Chemistry

J. Biol. Chem.

Bioch. Mol. Biol.

347

3.0

25

3.8

9

Limnology and Oceanography

Limnol. Oceanogr.

Limnol.; Oceanogr.

125

1.1

25

3.8

10

Marine Ecology Progress Series

Mar. Ecol. Prog. Ser.

Ecol.; Mar. Fresh. Biol.; Oceanogr.

183

1.6

18

2.8

11

EMBO Journal

EMBO J

Bioch. Mol. Biol.; Cell Biol.

40

0.3

16

2.5

12

Marine Biology

Mar. Biol.

Mar. Fresh. Biol.

162

1.4

14

2.2

13

Plant Cell

Plant Cell

Bioch. Mol. Biol.; Plant Sci.; Cell Biol.

82

0.7

13

2.0

14

Plant and Cell Physiology

Plant Cell Physiol.

Plant Sci.; Cell Biol.

258

2.2

11

1.7

15

Science

Science

Mult. Sci.

22

0.2

11

1.7

16

Biochimica et Biophysica Acta

Biochim. Biophys. Acta

Bioch. Mol. Biol.; Biophys.

432

3.8

10

1.5

17

Planta

Planta

Plant Sci.

155

1.3

10

1.5 Continued

202  PART | IV  Algal photosystems and photosynthesis

TABLE 12.4  The most-prolific and influential journals in algal photosystems and photosynthesis—cont’d Journals

Abbr.

Subject

I-0

I-0 %

I-100

I-100%

18

FEBS Letters

FEBS Lett.

Bioch. Mol. Biol.; Biophys. Cell Biol.

229

2.0

8

1.2

19

Annual Review of Plant Physiology and Plant Molecular Biology

Annu. Rev. Plant Phys.

Bioch. Mol. Biol.; Plant Sci.

9

0.1

8

1.2

20

Biophysical Journal

Biophys. J.

Biophys.

84

0.7

7

1.1

4914

42.7

436

62.1

Total

I-0: The number of all the papers. I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations. I-100%: The percentage of the number of influential papers.

The most-prolific and influential journals have been ‘Plant Physiology’ and ‘Proceedings of the National Academy of Sciences of the United States of America’ publishing 7.2% and 7.1% of the influential papers, respectively and 4.3% and 1.3% of all the papers, respectively. ‘Biochemistry’, ‘Photosynthesis Research’, ‘Biochimica et Biophysica Acta Bioenergetics’, ‘Journal of Phycology’, and ‘Nature’ have followed these top journals as the other most prolific and influential journals with at least 4.0% of the influential papers each. The most-prolific subject category for these journals has been ‘Biochemistry and Molecular Biology’ with eight journals, followed by ‘Plant Sciences’ with six journals. The other prolific subjects have been ‘Biophysics’, ‘Cell Biology’, and ‘Oceanography’ with five, four, and four journals respectively. Additionally, ‘Multidisciplinary Sciences’ and ‘Marine and Freshwater Biology’ have covered with two journals each. It is notable that ‘Journal of Phycology’ has been the only journal related to the algae in this top-20 journal list. Similarly, ‘Photosynthesis Research’ has been the only journal related to the photosynthesis directly.

12.3.9  Subject categories The information about the 10 most-prolific and influential subject categories are given in Table 12.5. As expected, the mostprolific subject categories have been ‘Plant Sciences’ and ‘Biochemistry & Molecular Biology’ indexing 35.0% and 29.0% of all the papers and 32.0% and 21.8% of the influential papers, respectively. The other prolific and influential subjects have been ‘Marine Freshwater Biology’, ‘Multidisciplinary Sciences’, ‘Biophysics’, ‘Cell Biology’, and ‘Oceanography’ with at least 9.7% of the influential papers each. Thus, the first-two categories have been the key pillars of the research in algal photosystems and photosynthesis, indexing together 63.8% of the influential papers.

12.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers with at least 100 citations, 5.6% of the research sample of 11,519 papers, Table 12.6. The records in this dataset has been refined from 650 papers to 602 papers to focus on the core papers for the field of algal photosystems and photosynthesis. The data shows that the field of ‘cyanobacterial photosynthesis’ and ‘cyanobacterial photosystems’ have been the most prolific research fronts with 142 and 109 papers, forming 23.6% and 18.1% of these influential papers, respectively. The other key research fronts have been ‘algal photosynthesis’, ‘microalgal photosynthesis’, ‘photosynthesis in dinoflagellates and coccolithophores’, ‘cyanobacterial CO2 concentrating mechanisms’, and ‘photosynthesis in diatoms’ with 14.5%, 13.5%, 5.0%, 4.3%, and 4.2% of the influential papers, respectively. The most-studied types of algae have been ‘cyanobacteria’, ‘microalgae’ and ‘algae or phytoplankton in general’ with 48.1%, 21.5%, and 18.6% of the influential papers, respectively. Additionally, the papers on the ‘dinoflagellates and coccolithophores’, ‘diatoms’, and ‘macroalgae’ formed 5.9%, 5.9% and 4.5% of these papers, respectively. On the other hand, the most-studied individual research front has been ‘photosynthesis’ with 67.5% of these papers. Additionally, the papers related to ‘photosystems’ and ‘CO2 concentrating mechanisms’ formed 24.8% and 12.5% of these papers, respectively.

The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  203



TABLE 12.5  The most-prolific and influential subject categories in algal photosystems and photosynthesis. Subject categories

I-0 no. papers

I-0% papers

I-100 no. papers

I-100% papers

1

Plant Sciences

4031

35.0

208

32.0

2

Biochemistry Molecular Biology

3338

29.0

207

31.8

3

Marine Freshwater Biology

1970

17.1

87

13.4

4

Multidisciplinary Sciences

477

4.1

85

13.1

5

Biophysics

1596

13.9

71

10.9

6

Cell Biology

794

6.9

66

10.2

7

Oceanography

606

5.3

63

9.7

8

Ecology

537

4.7

35

5.4

9

Microbiology

660

5.7

33

5.1

10

Limnology

223

1.9

26

4.0

Total

14,232

123.7

881

135.5

I-0: The number of all the papers. I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations. I-100%: The percentage of the number of influential papers.

TABLE 12.6  The most-prolific research fronts in algal photosystems and photosynthesis. Research fronts

Algae

Microalgae

Cyanobacteria

Diatoms

Dinoflagellates

Macroalgae

Total

1

Photosystems

6

24

109

2

2

0

143 (24.8%)

2

Photosynthesis

87

81

142

25

30

23

388 (67.4%)

3

CO2 concentration mechanisms

14

19

26

7

2

3

71 (12.3%)

Total

107 (18.6%)

124 (21.5%)

277 (48.1%)

34 (5.9%)

34 (5.9%)

26 (4.5%)

576 (100%)

Numbers: The number of influential papers for each research front and type of algae: %: The percentage of influential papers for each research front and type of algae. The number in bold: the most-prolific research fronts with at least 15 influential papers. Dinoflagellates: it includes coccolithophores.

12.3.11  Citation classics This section provides the information on both the scientometric analysis and brief content overview of the most-cited 20 papers in algal photosystems and photosynthesis. The information on these papers is given in Table 12.7: authors’ names, publication years, document type, number of authors per paper, lead authors’ names, gender, and paper numbers (lead authors were determined based on the number of papers produced in this field with at least 13 papers and 5 influential papers), journals, subject areas, research fronts, types of algae, academic focus of the papers, number of citations received, and the number of citations per year.

12.3.11.1  Scientometric overview of the citation classics These papers have been published between 1980 and 2015. The most-prolific decades have been the 1990s and 2000s with six and nine papers, respectively. Additionally, there have been three and two papers published in the 1980s and 2010s, respectively. The reviews have been over-represented in these classical papers as there have been 15 articles and 5 reviews. The number of the authors of these papers has ranged from 1 to 11 whilst the mean number of authors has been 4.3. The most-prolific lead author has been ‘Wolfram Saenger’ with four citation classics. The other prolific lead authors have been ‘Jan Kern’ and ‘Athina Zouni’ with three citation classics each and ‘Petra Fromme’, ‘Ingrid Witt’, and ‘Jian-Ren Shen’ with two papers each. In total, 17 lead authors contributed to these citation classics. Three of these top authors are female.

TABLE 12.7  The citation classics in algal photosystems and photosynthesis. Authors

Year

Doc.

N Auths.

Lead authors

Journal

Subject area

Res. fronts

Algae

Topic

Cits.

Av. cits

1

Ferreira et al.

2004

A

5

J Barber-8

Science

Mult. Sci.

Photosystems

Cyanobacteria— Thermosynechococcus

PSII structure

2406

172

2

Jordan et al.

2001

A

6

P Fromme-13; HT Witt-9; W Saenger-8

Nature

Mult. Sci.

Photosystems

Cyanobacteria— Synechococcus

PSI structure

1659

98

3

Zouni et al.

2001

A

7

P Fromme-13; HT Witt-9; W Saenger-8; A Zouni-5; J Kern-5

Nature

Mult. Sci.

Photosystems

Cyanobacteria— Synechococcus

PSII structure

1568

92

4

Loll et al.

2005

A

5

W Saenger-8; A Zouni-5; J Kern-5

Nature

Mult. Sci.

Photosystems

Cyanobacteria

PSII structure

1420

109

5

Behrenfeld and Falkowski

1997

A

2

PG Falkowski-19

Limnol. Oceanogr.

Limnol.; Oceanogr.

Photosynthesis

Phytoplankton

Photosynthetic rates

1370

65

6

Platt et al.

1980

A

3

T Platt-8

J. Mar. Res.

Oceanogr.

Photosynthesis

Phytoplankton

Photosynthesis photoinhibition

1329

35

7

Niyogi

1999

R

1

KK Niyogi-5

Annu. Rev. Plant Phys.

Bioch. Mol. Biol.; Plant Sci.

Photosynthesis

Microalgae

Photoprotective mechanisms

1295

68

8

Collini et al.

2010

A

6

Nature

Mult. Sci.

Photosynthesis

Microalgae

Light-harvesting

960

120

9

Kamiya and Shen

2003

A

2

JR Shen-6

P. Natl. Acad. Sci. USA

Mult. Sci.

Photosystems

Cyanobacteria— Thermosynechococcus

PSII Structure

894

60

10

Van Grondelle et al.

1994

R

4

R van Grondelle-5; JP Dekker-5

BBABioenergetics

Bioch. Mol. Biol.; Biophys.

Photosystems

Cyanobacteria, microalgae

Energy transfer

887

37

11

Guskov et al.

2009

A

6

W Saenger-8; A Zouni-5; J Kern-5

Nat. Struct. Mol. Biol.

Bioch. Mol. Biol.; Biophys.; Cell Biol.

Photosystems

Cyanobacteria— Thermosynechococcus

PSII structure

812

90

12

Williams

1988

A

1

Method. Enzymol.

Bioch. Res. Meth.; Bioch. Mol. Biol.

Photosystems

Cyanobacteria— Synechocystis

PSII structure

765

26

13

Giordano et al.

2005

R

3

Annu. Rev. Plant Phys.

Bioch. Mol. Biol.; Plant Sci.

CO2 concentrating mechanisms-CCMs

Algae

CCM modulation

702

54

J Beardall-6; JA Raven-11

14

Melis et al.

2000

A

5

15

Summons et al.

1999

A

16

Kurisu et al.

2003

17

Badger and Price

18

A Melis-8

Plant Physiol.

Plant Sci.

Photosynthesis

Microalgae— Chlamydomonas

Biohydrogen production

619

34

4

Nature

Mult. Sci.

Photosynthesis

Cyanobacteria

Biomarkers for oxygenic photosynthesis

569

30

A

4

Science

Mult. Sci.

Photosynthesis

Cyanobacteria— Mastigocladus

Cytochrome b6f complex

477

32

1994

R

2

Annu. Rev. Plant Phys.

Bioch. Mol. Biol.; Plant Sci.

CO2 concentrating mechanisms-CCMs

Microalgae, cyanobacteria, macroalgae

Role of carbonic anhydrases

476

20

Eilers and Peeters

1988

A

2

Ecol. Model.

Ecol.

Photosynthesis

Phytoplankton

Role of light intensity

472

16

19

Suga et al.

2015

A

11

JR Shen-6

Nature

Mult. Sci.

Photosystems

Cyanobacteria— Thermosynechococcus

PSII structure

468

156

20

Kaplan and Reinhold

1999

R

2

A Kaplan-7

Annu. Rev. Plant Phys.

Bioch. Mol. Biol.; Plant Sci.

CO2 concentrating mechanisms-CCMs

Cyanobacteria

Overview

460

24

MR Badger-24; GD Price-19

Total

81

19608

1338

Average

4

980

67

Doc.: Document. A: Article. R: Review. Gender: gender of lead authors- female authors in italic. N paper: for the authors with at least 13 papers with 0 citations and with at least 2 influential papers—number after the author names. Subject: Web of Science subjects. Topic: primary topic of the papers. Algae: type of algae studied. Res. fronts: primary research fronts studied. Cits.: number of citations received in total. Av. Cits.: number of citations per year.

206  PART | IV  Algal photosystems and photosynthesis

There has been a significant gender deficit among the lead authors of these classical papers as only 3 out of 17 lead authors listed in Table 12.7 are female. In total, these citation classics have been published by only 11 journals. The most-prolific journals have been ‘Nature’, ‘Annual Review of Plant Physiology’, and ‘Science’ with 6, 4, and 2 papers, respectively. In total, these papers have been indexed by nine subject categories. The most-prolific category has been ‘Multidisciplinary Sciences’ with nine papers indexing two most prolific journals, closely followed by ‘Biochemistry and Molecular Biology’ with seven papers. The fields of ‘Biophysics’ and ‘Oceanography’ have followed these top subjects with two papers each. In total, there have been three research fronts. The most-prolific research fronts have been ‘algal photosystems’ and ‘algal photosynthesis’ with nine and eight papers, respectively. The other research front of ‘CO2 concentrating mechanisms’ has followed these top research fronts with three papers. There have been five types of algae covered by these classical papers. The most prolific type of algae has been ‘cyanobacteria’ with 13 papers. The other prolific types of algae have been ‘microalgae’ and ‘phytoplankton’ with four and three papers, respectively. Additionally, there have been one paper each related to ‘algae in general’ and ‘macroalgae’. There have been no papers related to ‘diatoms’ and ‘dinoflagellates and coccolithophores’. On the other hand, the most-studied types of algae on the individual basis have been ‘Synechococcus’ and ‘Thermosynechococcus’ with two papers each. The other types of algae have been ‘Synechocystis’, ‘Mastigocladus’, and ‘Chlamydomonas’ with one paper each. The most-studied topic has been the ‘structures of photosystem II’ (PSII) with seven papers. These papers have received between 460 and 2406 citations each, totaling in 19,608 citations with a mean value of 1029 citations. On the other hand, the number of citations per year has ranged from 16 to 172 with a mean value of 70 citations per year. The paper by Ferreira et al. (2004) on the PSII structure of Thermosynechococcus with 2406 total citations and 172 citations per year has been the most-cited paper.

12.3.11.2  Brief overview of the content of the citation classics There have been three major classes of papers: ‘algal photosystems’, ‘algal photosynthesis’, and ‘CO2 concentrating mechanisms’ with 9, 8, and 3 papers, respectively. Photosystems Algae/phytoplankton van Grondelle et al. (1994) discuss the energy transfer and trapping in photosynthesis focusing on Photosystem I (PSI) and Photosystem II (PSII) in a paper with 887 citations. After outlining the excitation energy transfer, they focus on energy transfer in plants, microalgae, and cyanobacteria. In doing so they focus more on PSI and PSII in detail. Cyanobacteria Ferreira et al. (2004) study the structure of PSII from Thermosynechococcus elongatus at 3.5 Å resolution to reveal its molecular structure in a seminal paper with 2406 citations. Based on their findings, they propose a structure of the oxygenevolving center (OEC) containing a cubane-like Mn3CaO4 cluster and they discuss the implications for a possible oxygenevolving mechanism in cyanobacteria. Jordan et al. (2001) study the crystal structure of PSI from Synechococcus elongatus at 2.5 Å resolution in a seminal paper with 1659 citations. Based on their findings, they argue that the structural information on the proteins and cofactors and their interactions provides a basis for understanding the high efficiency of PSI in both light capturing and electron transfer. Zouni et al. (2001) study the crystal structure of photosystem PSII from Synechococcus elongatus at 3.8 Å resolution in a seminal paper with 1568 citations. The structure shows how protein subunits and cofactors are spatially organized. They also provide the information on the position, size and shape of the manganese cluster. Loll et al. (2005) study the crystal structure of photosystem PSII at 3.0 Å resolution in a seminal paper with 1420 citations. They show locations of and interactions between 20 protein subunits and 77 cofactors per monomer and provide insights into electron and energy transfer and photoprotection mechanisms in the reaction center and antenna subunits. They propose a lipophilic pathway for the diffusion of secondary plastoquinone and provide information about the Mn4Ca cluster. Kamiya and Shen (2003) study the crystal structure of PSII from Thermosynechococcus vulcanus at 3.7 Å resolution to gain insights into the mechanism of PSII reactions in a paper with 894 citations. They show the arrangement of chlorophylls and cofactors which provide important clues to the secondary electron transfer pathways around the reaction center. Furthermore, they determine possible ligands for the Mn-cluster.



The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  207

Guskov et al. (2009) study the structure of PSII from Thermosynechococcus elongatus at 2.9 Å resolution focusing on the role of quinones, lipids, channels, and chloride in a paper with 812 citations. Their results suggest mechanisms for plastoquinol-plastoquinone exchange, and calculate other possible water or dioxygen and proton channels. They argue for a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. They also argue for a role for chloride position in proton-transfer reactions. Williams (1988) develops a new genetic method in Synechocystis 6803 for the molecular analysis of electron transport in the PSII reaction center in a paper with 765 citations. He describes a procedure for deleting PSII genes from Synechocystis 6803 to create a PSII mutant, replacement of the deleted genes to restore photosynthetic function, and some of the properties of the genetic transformation system in this cyanobacterium. Suga et al. (2015) study the ‘radiation-damage-free’ structure of photosystem II from Thermosynechococcus vulcanus at 1.95 Å resolution in a paper with 468 citations. They find that compared with the structure from X-ray diffraction (XRD), the oxygen evolving complex (OEC) in the X-ray free electron laser structure has Mn–Mn distances that are shorter by 0.1–0.2 Å. One of the oxo-bridged oxygens has significantly longer distances to Mn than do the other oxo-oxygen atoms. Their findings provide a structural basis for the mechanism of oxygen evolution, and they foresee that this structure will provide a blueprint for the design of artificial catalysts for water oxidation. Photosynthesis Algae/phytoplankton Platt et al. (1980) study the photoinhibition of photosynthesis in natural assemblages of marine phytoplankton in a paper with 1329 citations. They propose a new empirical equation to describe the photosynthesis by phytoplankton as a single, continuous function of available light. They show the versatility of the equation by data collected on natural phytoplankton assemblages. Behrenfeld and Falkowski (1997) study the photosynthetic rates based on chlorophyll concentration in phytoplankton in a paper with 1370 citations. They seek to understand the critical variables required for accurate assessment of daily depth-integrated phytoplankton carbon fixation from measurements of sea surface pigment concentrations. They develop a light-dependent, depth-resolved model for carbon fixation. They argue for focusing model development on temporal and spatial variability. Eilers and Peeters (1988) develop a dynamic model for the relationship between light intensity and the rate of photosynthesis in phytoplankton in a paper with 472 citations. The model describes the photosynthetic processes and those connected with photoinhibition and recovery from photoinhibition. They propose to present production curves in a dimensionless form on logarithmic scales. They argue that the main advantage of the model is its foundation on physiological mechanisms. Finally, they present a procedure to integrate the phytoplankton production of a water column analytically. Microalgae Niyogi (1999) outlines several photoprotective mechanisms against oxidative damage to the photosynthetic apparatus, operating within chloroplasts of plants and microalgae in a paper with 1295 citations. He argues that the use of genetic and molecular biological approaches is providing new insights into photoprotection, especially with respect to thermal dissipation of excess absorbed light energy, alternative electron transport pathways, chloroplast antioxidant systems, and repair of photosystem II. Collini et al. (2010) study coherently wired light-harvesting in photosynthetic marine microalgae at ambient temperature in a paper with 960 citations. They carry out two-dimensional photon echo spectroscopy measurements on two evolutionarily related light-harvesting proteins. Their results provide compelling evidence for quantum-coherent sharing of electronic excitation across the 5-nm-wide proteins under biologically relevant conditions. Based on their results, they argue that distant molecules within the photosynthetic proteins are ‘wired’ together by quantum coherence for more efficient light-harvesting in these algae. Melis et al. (2000) study the 2-stage photobiological hydrogen gas production through the reversible hydrogenase pathway in Chlamydomonas reinhardtii in a paper with 619 citations. They find that the mechanism of biohydrogen production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b6f and PSI complexes. They suggest that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. Cyanobacteria Summons et al. (1999) study the 2-methylhopanoids as biomarkers for photosynthesis in cyanobacteria in a paper with 569 citations. They show that 2-methylbacteriohopanepolyols occur in a high proportion of cultured cyanobacteria and

208  PART | IV  Algal photosystems and photosynthesis

c­yanobacterial mats. Their 2-methylhopane hydrocarbon derivatives are abundant in organic-rich sediments as old as 2500 Myr. They argue that these biomarkers may help constrain the age of the oldest cyanobacteria and the advent of oxygenic photosynthesis. They assert that these biomarkers could also be used to quantify the ecological importance of cyanobacteria through geological time. Kurisu et al. (2003) study the crystal structure of the cytochrome b6f complex from Mastigocladus laminosus at 3.01 Å resolution in a paper with 466 citations. Their results reveal a large quinone exchange cavity. The core of the b6f complex is similar to the analogous respiratory cytochrome bc1 complex. However, the domain arrangement outside the core and the complement of prosthetic groups are different. Similarly, the motion of the Rieske iron-sulfur protein extrinsic domain is also different in the b6f complex. Carbon concentration mechanisms Algae/phytoplankton Giordano et al. (2005) discuss the CO2 concentrating mechanisms (CCMs) in algae focusing on the mechanisms, environmental modulation, and evolution in a review paper with 702 citations. They note that modulating the CCMs may be crucial in the energetic and nutritional budgets of a cell, and a multitude of environmental factors can exert regulatory effects on the expression of the CCM components. They discuss the diversity of CCMs, their evolutionary origins, and the role of the environment in CCM modulation. Badger and Price (1994) discuss CO2 concentrating mechanisms in algae focusing on the role of carbonic anhydrases in photosynthesis in a review paper with 476 citations. They focus on cyanobacteria, microalgae, macroalgae, and other plants. Cyanobacteria Kaplan and Reinhold (1999) discuss CO2 concentrating mechanisms in cynanobacteria in a review paper with 460 citations. They note that the presence of membrane mechanisms for inorganic carbon (Ci) transport are central to the concentrating mechanism. They show the presence of multiple Ci transporting systems in cyanobacteria. They argue that massive Ci fluxes associated with the CO2-concentrating mechanism have wide-reaching ecological and geochemical implications.

12.4 Discussion As there have been over 11,500 core papers related to the algal photosystems and photosynthesis, comprising more than 7% of the papers on the algae as a whole, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts. The primary mode of scientific communication has been articles whilst reviews have formed 5.3% of the sample. The primary index has been SCI-E indexing more than 99.9% of the papers whilst only nine papers have been indexed by the SSCI and A&HCI focusing on the societal and humanitarian aspects of algal photosystems and photosynthesis. These findings suggest that there is substantial room for the research in these aspects such as policy-related studies as well as scientometric studies in this field. The most-prolific keywords related to the algal photosystems and photosynthesis have been determined through the detailed examination of the 602 influential papers with at least 100 citations. A detailed keyword set has been devised for the search (given in Appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the photosystems and photosynthesis have been ‘photosynth*, photosystem*, * cytochrom*’. The other prolific key words have been ‘“light harvest*”, “electron transport”, “carbonic anhydrase*”, polypeptide*, ferredoxin*, antenna*, “reaction center*”, nadp*’. On the other hand, there have been two journals related to photosynthesis: ‘“Photosynthesis Research”, Photosynthetica*’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, macroalga*, phytoplankton, rhodophyt*, seaweed*, bacillariophycea*, diatom, diatoms, cyanobacter*’. These keywords have formed the primary research fronts for the algal photosystems and photosynthesis. The findings show that although over 18,600 authors have contributed to the research, 20 most-prolific and influential authors have shaped the literature on the algal photosystems and photosynthesis publishing 7.0% and 37.8% of all the papers and the influential papers, respectively (Table 12.1). The success of these authors, their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988). The data provides the evidence for the presence of the significant gender deficit among both the lead authors (Table 12.1) and the lead authors of the citation classics (Table 12.7) (Bordons et al., 2003).



The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  209

The data on the papers by the most-prolific authors highlight the primary research fronts as the ‘algal photosystems’, ‘algal photosynthesis’, and ‘algal CO2 concentrating mechanisms’. The data in Table  12.1 provides information on the most-prolific and influential authors, institutions, countries, journals, topics, and their citation impact in terms of the I-100 and I-100% by these authors. It has been found during the search process that the author names with two or more forenames or surnames have been spelt differently by the databases with significant implications for the recovery of their papers. For example, there have been 41 and 17 papers for ‘Dela Rosa MA’, and ‘DelaRosa MA’, respectively. As another example, there have been 16 and 3 papers for ‘Falkowski PG’ and ‘Falkowski P’, respectively. Similar difficulties have also been observed for the common names such as ‘Wang Y’ or ‘Li Y’ for the recovery of their papers and the related analysis. The data shows that although 100 countries and territories have contributed to the research in algal photosystems and photosynthesis, most-prolific 20 countries contributed to 175.6% and 197.7% of all the papers and the influential papers, respectively (Table 12.2). The major producers of the research have been the US, Japan, and Europe as these countries have had the ‘first-mover advantage’ over the other countries. It is notable that the citation impact of China has been small in relation to these top producers as China has produced 7.4% and 1.1% of all the papers and influential papers, respectively. (Guan and Ma, 2007). As in the case of countries, although over 2800 institutions have contributed to the research in algal photosystems and photosynthesis, the 20 most-prolific institutions mainly from the US and Europe, having the first-mover advantages, have published more than 33.4% of all the papers and 62.3% of the influential papers, respectively (Table 12.3). As only 33.4% of the papers have declared a research funding, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). It is notable that there has been significant research funding opportunities in China in relation to the US and Europe. Thus, there is ample room to extend the research funding opportunities to support the research and development in algal photosystems and photosynthesis. The steady rise in the number of the publications in this field in the 2000s and 2010s (as of January 2019) provides the strong evidence for the increasing public importance of the algal photosystems and photosynthesis in recent years (Fig. 12.1). The annual number of publications have risen to nearly 450 papers and it is expected that the number of papers would continue to rise in the next decade provided that the opportunities for research funding would increase significantly, corresponding to the increasing importance of the algal photosystems and photosynthesis to the global society at large. Although nearly 1000 journals have contributed to the research in algal photosystems and photosynthesis, the 20 mostprolific journals, having the first-mover advantages, have published over 23% and 55% of all the papers and influential papers, respectively (Table 12.4). This finding has been most relevant for ‘Proceedings of the National Academy of Sciences of the United States of America’ publishing 1.5% and 7.1% of all the papers and influential papers, respectively. The data on the Web of Science subject categories suggests that the first two subjects, covering over 50% of the influential papers have formed the scientific basis of the research in this field: ‘Biochemistry & Molecular Biology’ and ‘Plant Sciences’. The other prolific and influential subjects have been ‘Marine Freshwater Biology’, ‘Multidisciplinary Sciences’, ‘Biophysics’, ‘Cell Biology’, and ‘Oceanography’ (Table 12.5). As the journals related to algae and photosynthesis have published only a small part of both all the papers and influential papers, the broad search strategy, covering all subject categories and journals, developed for this study, has been justified. The data on the research fronts have confirmed that the major research fronts have been ‘cyanobacterial photosynthesis’ and ‘cyanobacterial photosystems’. The other key research fronts have been ‘algal photosynthesis’, ‘microalgal photosynthesis’, ‘photosynthesis in dinoflagellates and coccolithophores’, ‘cyanobacterial CO2 concentrating mechanisms’, and ‘photosynthesis in diatoms’. The most-studied the types of algae has been ‘cyanobacteria’, ‘microalgae’ and ‘algae or phytoplankton in general’. On the other hand, the most-studied individual research fronts have been ‘algal photosynthesis’, ‘algal photosystems’, and ‘CO2 concentrating mechanisms’. The extensive data on the 20 citation classics largely confirm the findings of the earlier sections based on the 11,519 papers (Table 12.7). There has been significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely over-represented in the citation classic sample as there has been 5 reviews. Similarly, the most-prolific research fronts have been ‘algal photosystems’, ‘algal photosynthesis’, and ‘CO2 concentrating mechanisms’. The most prolific type of algae have been ‘cyanobacteria’, ‘microalgae’ and ‘phytoplankton’. The most-studied types of algae on the individual basis have been ‘Synechococcus’ and ‘Thermosynechococcus’ with two papers each. The most-studied topics have been ‘structures of photosystem II’ (PSII) with seven papers.

210  PART | IV  Algal photosystems and photosynthesis

It appears that the structure-processing-property relationships form the basis of the research in algal photosystems and photosynthesis as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

12.5 Conclusion This analytical study of the research in algal photosystems and photosynthesis at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the development of the research and practice in this field. Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as bioenergy and biofuels, complementing 614 literature reviews. The data has shown that the annual number of papers in this field has risen to nearly 450 papers whilst there have been over 11,500 papers over the study period from 1980 to 2018. It is further expected that the size of the research output would continue to increase in the incoming years and decades, corresponding to the increasing public importance of the algal photosystems and photosynthesis to the global society at large. The provision of the adequate research funding is essential for the further development of the research and practice in this field as only 33.4% of the papers have declared a research funding. The key research fronts have been ‘cyanobacterial photosynthesis’ and ‘cyanobacterial photosystems’. The other key research fronts have been ‘algal photosynthesis’, ‘microalgal photosynthesis’, ‘photosynthesis in dinoflagellates and coccolithophores’, ‘cyanobacterial CO2 concentrating mechanisms’, and ‘photosynthesis in diatoms’. The most-studied types of algae has been ‘cyanobacteria’, ‘microalgae’ and ‘algae or phytoplankton in general’. On the other hand, the most-studied individual research fronts have been ‘algal photosynthesis’, ‘algal photosystems’, and ‘CO2 concentrating mechanisms’. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. It has been found that the detailed keyword set provided in the appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts.

Appendix: The keyword sets A.1  Photosystems and photosynthesis related keywords TI=(‘light harvest*’ or photosynth* or photorecep* or xanthophyll* or quench* or ‘electron transport’ or photosystem* or ‘carbonic anhydrase*’ or photoprotect* or photoinhibit* or ‘electron flow*’ or *cytochrom* or *plastocyanin* or polypeptide* or ferredoxin* or antenna* or ‘reaction center*’ or photodamage* or photoacclim* or photolyas* or photochrom* or photoreduction* or ‘light regulation*’ or nadp* or ‘carbon metabolism*’ or photota* or thioredoxin* or ‘oxygen evol*’ or hco3 or bicarbonate or peroxiredoxin* or carboxysome* or photoadapt* or ‘concentrating mechanism*’ or ndh or ‘light acclimation’ or ‘ps-ii’ or phytochrom* or ‘carbon acquisition’) OR SO=(‘Photosynthesis Research’ or photosynthetica*).

A.2  Algae related keywords A.2.1  Algal general TI=(alga or algae or algal or phycolog* or chlorarachn* or Bigelowiella or periphyton* or *phytoplankton) OR SO=(Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘European Journal of Phycology’ or Fottea* or ‘Harmful Algae’ or ‘International Journal on Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Research’).

A.2.2  Dinoflagellates & coccolithophores TI=(chrysophycea* or chlorococcales or chrysophyt* or *coccolith* or dinocyst* or dinoflagell* or dinophyceae or dinophyt* or haptophyt* or peridiniales or prymnesiophycea* or raphidophycea* or raphidophyt* or zooxanthella* or Akashiwo



The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  211

or Alexandrium or Amphidinium or Aureococcus or *Ceratium or *Chattonella or Cochlodinium or Crypthecodinium or Dinophysis or Emiliania or Gambierdiscus or *Gonyaulax or *Gymnodinium or Gyrodinium or Hematodinium or Heterocapsa or Heterosigma or Isochrysis or Karenia* or Karlodinium or Mallomonas or Noctiluca or Ochromonas or Ostreopsis or Oxyrrhis or Peridinium or Pfiesteria or Phaeocystis or Prorocentrum or Prymnesium or Scrippsiella or Symbiodinium or Vaucheria).

A.2.3 Microalgae TI=(chlorophycea* or chlorophyt* or cryptomonad* or cryptophycea* or cryptophyt* or euglen* or eustigmatophycea* or ‘green alga*’ or microalga* or ‘micro-alga*’ or ‘micro alga*’ or prasinophycea* or streptophyt* or trebouxiophycea* or volvocales or Acetabularia or Ankistrodesmus or Asteromonas or Aurantiochytrium or Botryococcus or Chaetoceros or Chlamydomonas or *Chlorella or *Chlorococcum or Coccomyxa or Cyanidioschyzon or Cyanidium or Desmodesmus or Dunaliella or Euglena or Galdieria or Haematococcus or Micrasterias or Micromonas or Monoraphidium or Nannochloropsis or Neochloris or Ostreococcus or Pediastrum or Phormidium or Platymonas or Polytomella or Porphyridium or Prototheca or Pseudokirchneriella or Pyramimonas or Scenedesmus or Schizochytrium or Selenastrum or Tetraselmis or Trebouxia or Volvox).

A.2.4 Macroalgae TI=(‘brown alga*’ or ‘macro-alga*’ or ‘macro alga*’ or ‘red alga*’ or agarophyt* or characea* or charophyt* or cladophorales or cryptonemiales or dictyotales or florideophycea* or fucale* or gelidiales or gigartinale* or gracilariales or kelp* or laminariale* or macroalga* or phaeophycea* or phaeophyt* or rhodophycea* or rhodophyt* or seaweed* or ulvale* or ulvophycea* or zygnematophycea* or ‘Chara Vulgaris’ or Alaria or Ascophyllum or Asparagopsis or Bangia* or Bostrychia or Bryopsis or Catenella or Caulerpa or Ceramium or Chaetomorpha or Chondrus or Cladophora or Codium or Corallina or Cystoseira or Ecklonia or Ectocarpus or ‘Eisenia bicyclis’ or Enteromorpha or Eucheuma or Fucus or Gelidium or Gigartina* or Gracilaria or Gracilariopsis or Grateloupia or Halimeda or Halocynthia or Hizikia or Hypnea or Kappaphycus or Laminaria or Laurencia* or Lessonia or Lomentaria or Macrocystis or Monostroma or Mougeotia or Nitella or Nitellopsis or Oedogonium or Padina or Palmaria or Pelvetia or Plocamium or Polysiphonia or Pyropia or Saccharina or Sargassum or Scytosiphon or Solieria or Spirogyra or Turbinaria or Ulva or Undaria).

A.2.5 Diatoms (TI=( bacillariophycea* or bacillariophyt* or diatom or diatoms or Asterionella or Chaetoceros or Coscinodiscus or Cyclotella or Cylindrotheca or Didymosphenia or Navicula or *Nitzschia or Phaeodactylum or Skeletonema or Stephanodiscus or Thalassiosira) OR SO=(‘Diatom Research’)) NOT (TI=(diatomic* or atom* or *molecule*or amphorae or diatomyid* or dissociation or ‘rare gas*’) OR SO=(‘Journal of Chemical Physics’)).

A.2.6 Cyanobacteria TI=(‘blue green alga*’ or ‘blue-green alga*’ or cryptophycin* or cyanelle or *cyanobacter* or cyanophage* or cyanophycin* or cyanophyt* or cyanophycea* or glaucophyt*or nostocales or oscillatoriales or prochlorophyt* or Acaryochloris or *Anabaena or Anacystis or Aphanizomenon or Aphanothece or Arthrospira or Calothrix or Cyanophora or Cyanothece or Cylindrospermopsis or Glaucocystis or *Lyngbya* or Mastigocladus or Microcoleus or Microcystis or Moorea or Nodularia or Nostoc or Oscillatoria or Planktothrix or Plectonema or Prochlorococcus or Prochloron or Prochlorothrix or Scytonema or Spirulina or *Synechococcus or Synechocystis or Tolypothrix or Trichodesmium).

A.2.7 Journals SO=(Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘Cryptogamie Algologie’ or ‘Diatom Research’ or ‘European Journal of Phycology’ or Fottea* or ‘Harmful Algae’ or ‘International Journal on Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Bulletin’ or ‘Phycological Research’).

A.3  Cross-subject keywords (TI=(photosystem* or photosynth*) and TS=(*synech*)) or TI=(phycobilisome* or cyanobacteriochrome* or diadinoxanthin*).

212  PART | IV  Algal photosystems and photosynthesis

References Abramo, G., D’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Badger, M.R., Price, G.D., 1994. The role of carbonic anhydrase in photosynthesis. Annu. Rev. Plant Phys. 45, 369–392. Behrenfeld, M.J., Falkowski, P.G., 1997. Photosynthetic rates derived from satellite-based chlorophyll concentration. Limnol. Oceanogr. 42 (1), 1–20. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sust. Energ. Rev. 14 (2), 557–577. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Collini, E., Wong, C.Y., Wilk, K.E., Curmi, P.M.G., Brumer, P., Scholes, G.D., 2010. Coherently wired light-harvesting in photosynthetic marine algae at ambient temperature. Nature 463 (7281), 644–647. Eilers, P.H.C., Peeters, J.C.H., 1988. A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton. Ecol. Model. 42 (3-4), 199–215. Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., Iwata, S., 2004. Architecture of the photosynthetic oxygen-evolving center. Science 303 (5665), 1831–1838. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Giordano, M., Beardall, J., Raven, J.A., 2005. CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Annu. Rev. Plant Biol. 56, 99–131. Guan, J., Ma, N., 2007. China's emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., Saenger, W., 2009. Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride. Nat. Struct. Mol. Biol. 16 (3), 334–342. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems - a review. Biotechnol. Adv. 29 (2), 189–198. Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W., Krauss, N., 2001. Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature 411 (6840), 909–917. Kamiya, N., Shen, J.R., 2003. Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-A resolution. P. Natl. Acad. Sci. U. S. A. 100 (1), 98–103. Kaplan, A., Reinhold, L., 1999. CO2 concentrating mechanisms in photosynthetic microorganisms. Annu. Rev. Plant Phys. 50, 539–570. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books Ltd, Stoke-on-Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energ. 88 (10), 3532–3540. Konur, O., 2012a. The policies and practices for the academic assessment of blind students in higher education and professions. Ener. Educ. Sci. Technol. B 4 (si1), 240–244. Konur, O., 2012b. Prof. Dr. Ayhan Demirbas’ scientometric biography. Ener. Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012c. The evaluation of the biogas research: a scientometric approach. Ener. Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012d. The evaluation of the bio-oil research: a scientometric approach. Ener. Educ. Sci. Technol. A 30 (si1), 379–392. Konur, O., 2012e. The evaluation of the biorefinery research: a scientometric approach. Ener. Educ. Sci. Technol. A 30 (si1), 347–358. Konur, O., 2012f. The evaluation of the research on the biodiesel: a scientometric approach. Ener. Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012g. The evaluation of the research on the bioethanol: a scientometric approach. Ener. Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012h. The evaluation of the research on the biofuels: a scientometric approach. Ener. Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012i. The evaluation of the research on the biohydrogen: a scientometric approach. Ener. Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012j. The evaluation of the research on the microbial fuel cells: a scientometric approach. Ener. Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012k. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenerg. 47, 504–515. Konur, O., 2012o. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Ener. Educ. Sci. Technol. B 4 (4), 1893–1908.



The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  213

Konur, O., 2012p. The evaluation of the research on the arts and humanities in Turkey: a scientometric approach. Ener. Educ. Sci. Technol. B 4 (3), 1603–1618. Konur, O., 2012q. The evaluation of the educational research: a scientometric approach. Ener. Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012r. The scientometric evaluation of the research on the deaf students in higher education. Ener. Educ. Sci. Technol. B 4 (3), 1573–1588. Konur, O., 2012s. The scientometric evaluation of the research on the students with ADHD in higher education. Ener. Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2012t. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Ener. Educ. Sci. Technol. B 4 (si1), 250–257. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63.

214  PART | IV  Algal photosystems and photosynthesis

Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019b. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Kurisu, G., Zhang, H.M., Smith, J.L., Cramer, W.A., 2003. Structure of the cytochrome b6f complex of oxygenic photosynthesis: tuning the cavity. Science 302 (5647), 1009–1014. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Poly. Sci. 37 (1), 106–126. Lieberman, M.B., Montgomery, D.B., 1988. First-mover advantages. Strateg. Manage. J. 9 (S1), 41–58. Loll, B., Kern, J., Saenger, W., Zouni, A., Biesiadka, J., 2005. Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II. Nature 438 (7070), 1040–1044. Melis, A., Zhang, L.P., Forestier, M., Ghirardi, M.L., Seibert, M., 2000. Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol. 122 (1), 127–135. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Edit. 48 (14), 2474–2499. Niyogi, K.K., 1999. Photoprotection revisited: genetic and molecular approaches. Annu. Rev. Plant Phys. 50, 333–359. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Platt, T., Gallegos, C.L., Harrison, W.G., 1980. Photoinhibition of photosynthesis in natural assemblages of marine-phytoplankton. J. Mar. Res. 38 (4), 687–701. 1305. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes-towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Suga, M., Akita, F., Hirata, K., Ueno, G., Murakami, H., Nakajima, Y., et al., 2015. Native structure of photosystem II at 1.95 A resolution viewed by femtosecond X-ray pulses. Nature 517 (7532), 99–103. Summons, R.E., Jahnke, L.L., Hope, J.M., Logan, G.A., 1999. 2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis. Nature 400 (6744), 554–557. van Grondelle, R., Dekker, J.P., Gillbro, T., Sundstrom, V., 1994. Energy transfer and trapping in photosynthesis. BBA-Bioenergetics 1187 (1), 1–65. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biot. 79 (5), 707–718. Williams, J.G.K., 1988. Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Method. Enzymol. 167, 766–778.



The scientometric analysis of the research on the algal photosystems and photosynthesis Chapter | 12  215

Yu, J.J., Wang, M.H., Xu, M., Ho, Y.S., 2012. A bibliometric analysis of research papers published on photosynthesis: 1992–2009. Photosynthetica 50 (1), 5–14. Zouni, A., Witt, H.T., Kern, J., Fromme, P., Krauss, N., Saenger, W., et al., 2001. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 A resolution. Nature 409 (6821), 739–743.

Further reading Konur, O., 2012l. The evaluation of the global energy and fuels research: a scientometric approach. Ener. Educ. Sci. Technol. A 30 (1), 613–628. Konur, O., 2012m. 100 citation classics in energy and fuels. Ener. Educ. Sci. Technol. A 30 (si1), 319–332. Konur, O., 2012n. What have we learned from the citation classics in energy and fuels: a mixed study. Ener. Educ. Sci. Technol. A 30 (si1), 255–268. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489.

Chapter 13

Photosynthesis in diatoms Reimund Goss, Christian Wilhelm, Torsten Jakob University of Leipzig, Leipzig, Germany

13.1 Introduction Diatoms are eukaryotic microalgae which have a different phylogenetic origin than green algae, the green lineage mosses, ferns and higher plants. The diatoms developed from a secondary endocytobiosis of a eukaryotic host and a eukaryotic red alga. During evolution, most of the genes from the red algal nucleus had been transferred to the host by lateral gene transfer. In this gene transfer endocytobiotic bacteria delivered the molecular machinery for lateral gene transfer with the result that the genome of diatoms contains genes of two eukaryotic nuclei and two prokaryotic genomes, one of the mitochondrium and the second one of the chloroplast. Total genome sequencing of different diatom species revealed that the nuclear genome is merged from plant and animal genomes, together with a large amount of bacterial genes from different phylogenetic origin (Armbrust, 2009; Archibald, 2015). The result of this second endocytobiological event becomes obvious by some peculiarities of the chloroplast in diatoms. One consequence of the second endocytobiosis is the fact that the chloroplast is surrounded by four membranes instead of two in the green lineage. Since the chloroplast envelope is an extremely selective membrane, on the one hand controlling the mass transfer from photosynthates out of the chloroplast and on the other hand supporting the biosynthetic machinery with metabolites which cannot be produced by the chloroplast itself, the presence of four membranes leads to a different compartmentalization of biosynthetic routes. For example, the green plastid possesses his own machinery to produce nucleotides and, therefore, nucleotide dependent regulations in the cytosol and in the plastid are controlled independently (Linka and Weber, 2010). Therefore, the envelope should be more or less impermeable for nucleotides. By contrast, the chloroplast in diatoms is not completely autonomous with respect to nucleotide biosynthesis and therefore, the envelope must contain nucleotide transport systems (Ast et al., 2009). Taking into account this complex genetic background, it can be expected that phylogenetically conserved components of the photosynthetic apparatus like the photosystems, the electron transport components and the enzymatic apparatus of CO2 fixation are quite similar to those in the green lineage, whereas biosynthesis, assembling and functional regulation of plastidic proteins are substantially different. Therefore, the flux regulation of metabolic pathways distributing the primary products of photosynthesis into the macromolecular pools of carbohydrates, proteins, lipids or secondary products is clearly distinguished from the green plant lineage. Recent comparative analyses of diatom genomes showed that the lower half of glycolytic enzymes had been retained in the mitochondria, whereas the upper half of glycolytic enzymes have been transferred into the cytosol (Smith et al., 2012). One of the most prominent examples of this difference is the intracellular localization of the storage product of photosynthesis. In diatoms the major storage product of diatom photosynthesis is Chrysolaminarin, which is a polymer of glucose polymerized by a 1,3-ß bond between the glucose units. In contrast to starch in the green lineage, Chrysolaminarin is not stored in the chloroplast but outside and therefore, the transitory accumulation of the storage product in the chloroplast is not observed in diatoms (Hildebrand et al., 2017). Further genetic and metabolic peculiarities in diatoms have been reviewed by Kroth et al. (2008) and Depauw et al. (2012). The most prominent unique feature of the photosynthesis apparatus in diatoms is the pigmentation of the light-­harvesting apparatus and the thylakoid macrodomain organization. Diatoms are characterized by the brown color which originates from a high content of fucoxanthin being bound to “light-harvesting proteins” (LHC) in an equal or even higher ratio than chlorophyll (Chl) a. Therefore, these proteins are called “fucoxanthin-chlorophyll proteins” (FCPs, for more details see Section 13.3 below). In green chloroplasts the most prominent LHC protein is bound to PSII which is specifically localized in the grana membranes. These PSII-LHCII supercomplexes are responsible for the membrane stacking and therefore, these

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00013-9 © 2020 Elsevier Inc. All rights reserved.

217

218  PART | IV  Algal photosystems and photosynthesis

complexes are the functional basis for the light-dependent grana re-organization in green chloroplasts (Albanese et al., 2017 and references therein). However, the thylakoids in diatoms are running in bands of three membranes (Tanaka et al., 2015) and this structure is more or less independent on the light intensity (for review see Wilhelm et al., 2014). Recently it was shown that these three membranes possess a different biochemical composition and can be understood as a membrane domain organization to optimize photosynthesis (Flori et al., 2017). Therefore, this triple structure can only be altered by the application nonnatural far red illumination during growth (Bina et al., 2016). It is the highly adaptive and efficient photosynthetic apparatus of the diatoms which has been claimed to be the reason for this extremely successful phylogenetic group: It contains about 100,000 species, which during evolution, have emerged in a relatively short time. Diatoms inhabit extremely different ecological niches from marine to freshwater, to benthic environments and aerophilic biofilms in extreme cold habitats. The following paragraphs will outline the biochemical and physiological features which allowed the diatoms such a successful phylogenetic/ecological history.

13.2  Diatom lipids 13.2.1  Lipid composition of thylakoid membranes Diatom cells contain a large variety of lipids, the number depending on the growth conditions. Lipids can be stored as “triacylglycerols” (TAGs) in cytosolic lipid droplets, “diacylglycerols” represent the main building blocks of the cellular biomembranes. Here, the thylakoid lipids are usually predominant with a proportion of about 30% of the total carbon. In general, diatom thylakoid membranes contain the same lipid classes as thylakoid membranes of higher plants and green algae (Goss et  al., 2009; Lepetit et  al., 2012). These comprise the neutral galactolipids “monogalactosyldiacylglycerol” (MGDG) and “digalactosyldiacylglycerol” (DGDG), the anionic sulfolipid “sulphoquinovosyldiacylglycerol” (SQDG) and the negatively charged phospholipid “phosphatidylglycerol” (PG). In diatom thylakoid membranes a second phospholipid, namely “phosphatidylcholine” (PC), can be present in significant concentration. This is different to higher plant thylakoids where minor amounts of PC are seen as a contamination with lipids of the chloroplast envelope membranes (Joyard et al., 1998). Besides these well-studied lipid classes more exotic lipids like the betaine lipids “diacylglycerylcarboxyhydroxymethylcholine” (DGCC), “diacylgyceryl-N-trimethylhomoserine” (DGTS) and “diacylglycerylhydroxymethyl-N,N,N-trimethylβ-alanine” (DGTA) can be found in trace amounts in diatom thylakoid membranes (Vieler et al., 2007; Canavate et al., 2016). Although the major lipid classes are comparable between diatoms and higher plants and green algae, the contribution of the lipid classes to the total thylakoid membrane lipid is significantly different. In higher plants and green algae, the neutral galactolipids MGDG and DGDG represent the main membrane lipids (Siegenthaler and Murata, 1998). MGDG amounts to around 40–50% of the total lipid, while DGDG contributes around 20 to 30%. The high amount of neutral lipids leads to a ratio of neutral to negatively charged lipids of around 4–5. In diatom thylakoids the anionic lipids SQDG and PG are present in much higher concentrations and 40–50% of the total membrane lipid is represented by these two lipids (Goss et al., 2009; Lepetit et al., 2012). The concomitant decrease of the concentration of MGDG and DGDG results in a ratio of neutral to negatively charged lipids of around 1–2. This results in a diatom thylakoid membrane, which contains a significantly higher negative charge compared to the thylakoid membrane of higher plants and green algae. The relevance of the membrane charge for the thylakoid arrangement is described in brief below (Section 13.2.4).

13.2.2  Fatty acid composition of thylakoid membrane lipids The “fatty acid” (FA) composition of the main thylakoid lipid classes reflects the specific features of the diatom FA biosynthesis (for a recent review see Zulu et al., 2018). Diatoms are characterized by the synthesis of very long chain “polyunsaturated fatty acids” (PUFAs) with chain lengths up to 28 C atoms (Guschina and Harwood, 2006). Analysis of the FAs of the main lipid classes (Yongmanitchai and Ward, 1993; Dodson et al., 2013, 2014) shows that MGDG is enriched in the main long chain PUFA of diatoms, “eicosapentaenoic acid” (EPA, 20:5). EPA is bound at the sn-1 position of the glycerol backbone, other FAs located at the sn-1 position are 20:3 and 20:4. These are, however, found in lower concentration. The sn-2 position of MGDG is normally occupied by a C16 FA. 16:3 seems to be the most abundant FA at this position but 16:1, 16:2 or 16:4 are also found. In addition, the long chain PUFA “docosahexaenoic acid” (DHA, 22:6) and C18 FAs are found in minor amounts in MGDG. The second galactolipid DGDG exhibits a comparable FA composition as MGDG (Yongmanitchai and Ward, 1993; Dodson et al., 2013, 2014).



Photosynthesis in diatoms Chapter | 13  219

The main part of DGDG is enriched in EPA, which occupies the sn-1 position and 16:3 is usually found at the sn-2 position. In contrast to the galactolipids, SQDG contains a higher quantity of shorter FAs and the main SQDG fraction is characterized by the presence of 16:1 FAs at both the sn-1 and sn-2 positions (Yongmanitchai and Ward, 1993). Recent results have indicated that the FA composition of MGDG and DGDG is more diverse in diatoms and that differences exist between centric and pennate diatoms (Dodson et al., 2013). While the FA composition of MGDG and DGDG described above seems typical for centric diatoms (Skeletonema marinoi, Thalassiosira weissflogii) and the pennate Phaeodactylum tricornutum, other pennate diatoms primarily contain C18/C16 and C18/C18 forms of MGDG and DGDG. The main forms of MGDG and DGDG found in the pennate diatoms Haslea ostreria and Nitzschia perminuta are 18:3 at the sn-1 position and 16:3 at the sn-2 position. MGDG and DGDG of these two pennate diatoms are thus comparable to MGDG and DGDG from higher plants and green algae, which use the prokaryotic pathway of galactolipid synthesis and contain 18:3 and 16:3 at the sn-1 and sn-2 positions, respectively (Eichenberger, 1982; Cho and Thompson, 1987). The occurrence of higher amounts of C18 FAs in the pennate diatoms is interesting because so far there is no evidence for a synthesis of the C18 acyl carrier protein in diatoms, which may be responsible for the low amounts of C18 FAs usually observed in diatom cells (Zulu et al., 2018).

13.2.3  Changes of lipid and fatty acid composition by abiotic stress or nutrient deprivation In general, favorable growth conditions lead to the synthesis of membrane lipids. However, when diatoms are exposed to abiotic stress, i.e., light and temperature stress, or supply of nutrients falls short, a degradation of membrane lipids and an accumulation of storage lipids can be observed (Hu et al., 2008). On the level of thylakoid lipid classes an increase of the negatively charged lipids SQDG and PG, accompanied by a concomitant decrease of the galactolipids MGDG and DGDG, has been observed during cultivation of diatoms with high light intensities (Lepetit et al., 2012). With respect to the impact of the light climate on FA composition, data from the literature are somewhat contradictory and both increases of EPA during high light cultivation (Guiheneuf et al., 2008) or complete darkness (Bai et al., 2016) have been reported. The effect of temperature on the FA composition of diatoms is unambiguous and it can be generalized that under higher growth temperatures diatoms show an increased synthesis of shorter chain FAs with a high degree of saturation, while at lower temperatures the production of long chain PUFA is favored (Jiang and Gao, 2004). This also holds true for the thylakoid membrane galactolipids and it was recently demonstrated that MGDG and DGDG isolated from the pennate diatoms H. ostrearia and P. tricornutum grown at moderate temperatures contained EPA at the sn-1 position and C16 FAs at the sn-2 position (Dodson et al., 2014). Growth at high temperatures leads to a complete loss of EPA and an exchange by C18 FAs in the galactolipids. In addition, at high temperatures the maximum number of double bonds in the FA residues of MGDG and DGDG is three. Increases in the concentration of long chain PUFAs can be seen as a way to maintain the membrane in a fluid state at low temperatures. At high temperatures the shift to shorter FAs with a high degree of saturation must be seen as a mechanism to avoid the loss of membrane structural integrity. Nitrogen starvation leads to lipid remodeling in diatoms and membrane galactolipids and phospholipids are degraded and the free FAs are directly used for the synthesis of TAGs (Abida et al., 2015). Phosphate and sulfate limitation have an impact on the thylakoid lipid class composition. Phosphate limitation induces an increased synthesis of SQDG while, on the other hand, sulfate deprivation leads to increased concentrations of PG (Sato et al., 2000). This indicates that the amount of negatively charged thylakoid membrane lipids has to be kept constant. Phosphate limitation, although not as severe as nitrate limitation, also leads to lipid remodeling and degradation of membrane lipids in favor of storage lipids (Abida et al., 2015). With respect to FA composition, an increase of shorter chain FAs can be observed accompanied by reduced amounts of long chain PUFAs like EPA, possibly in order to minimize energy consumption during FA ­biosynthesis (Yang et al., 2014).

13.2.4  Function of thylakoid membrane lipids The thylakoid membrane lipids fulfill important functions on different levels of organization. They build up the thylakoid membrane bilayer incorporating the photosystems and the electron transport components, which consist of membrane and peripheral membrane proteins. Thylakoid lipids serve as structural elements of the photosynthetic pigment protein complexes (Kern and Guskov, 2011). MGDG is enriched at the monomer-monomer interface of PSII core complexes and is thought to play a role in the dimerization of PSII. At the donor site of PSII DGDG may play a role in the stabilization of the oxygen evolving complex and at the acceptor site a cluster of lipids forms a transfer pathway which targets QB molecules to their binding site. The phospholipid PG is most likely required for the trimerization of the LHCII of higher plants and seems to be important for the oligomerization of PSI (Wada and Murata, 2007). Although these data have been gathered for

220  PART | IV  Algal photosystems and photosynthesis

cyanobacteria and higher plants, the structural similarities between the pigment protein complexes suggest that membrane lipids perform comparable tasks in the thylakoids of diatoms. Few studies exist where the influence of thylakoid membrane lipids on pigment protein complexes of diatoms has been investigated. Recently, it was shown that MGDG stabilizes FCP complexes of diatoms in a comparable manner to the LHCII of higher plants (Schaller-Laudel et al., 2017). The negatively charged lipids SQDG and PG, however, which exert a strong disaggregating effect on the LHCII, are not able to reduce the aggregate size of FCPs. MGDG has been shown to preferentially associate with the LHCs of both higher plants (Schaller et al., 2010) and diatoms (Lepetit et al., 2010) where, during high light illumination, it forms a special nonbilayer lipid phase. The nonbilayer phases can be seen as special membrane domains in the vicinity of the diatom LHCs (see Section 13.3) where, after binding of the xanthophyll deepoxidases, the xanthophyll cycles, i.e., the conversions of violaxanthin to zeaxanthin in higher plants and diadionoxanthin to diatoxanthin in diatoms, take place (Goss and Lepetit, 2015; Garab et al., 2016). The xanthophyll cycle itself plays an important role in photoprotection via NPQ (see Sections 13.3.3 and 13.4.1). With respect to the arrangement of thylakoid membranes within the chloroplast, it has been proposed that the high concentration of the negatively charged lipid SQDG in diatom thylakoids leads to electrostatic repulsion between adjacent membranes (Lepetit et al., 2012). Thus, a dense packing of thylakoid membranes, as it is typical for higher plant grana stacks, is not possible and the diatom thylakoids adopt the more regular arrangement of the typical stacks of three membranes.

13.3  Diatom light-harvesting complexes The light-harvesting system of diatoms consists of the so-called “fucoxanthin-chlorophyll-protein” (FCP) complexes (for a recent review see Buchel, 2015). The FCPs of both pennate and centric diatoms bind Chl a and Chl c. Besides the xanthophyll “fucoxanthin” (Fx), which represents the main light-harvesting pigment of diatoms, the xanthophyll cycle pigments “diadinoxanthin” (DD) and “diatoxanthin” (Dt) are additionally present. Different pools of Fx exist within the light-harvesting system and one of these pools shows light absorption up to 550 nm, thus permitting the use of green light for photosynthesis (Szabo et al., 2010), which cannot be captured by the chlorophylls and the other xanthophylls.

13.3.1  FCP proteins The diatom FCP proteins belong to the large family of light-harvesting proteins and are thus related to the LHC proteins of higher plants and green algae (Koziol et al., 2007). The genes for the FCP proteins are encoded in the nucleus and, thus, the proteins have to be imported into the chloroplast. The FCP proteins can be divided into three groups: (1) The Lhcf proteins, which constitute the main, peripheral FCP complexes. These serve as light-harvesting antenna of both PSI and PSII (Lepetit et al., 2010; Grouneva et al., 2011; Gundermann et al., 2013; Nagao et al., 2013). (2) The Lhcr proteins, which are found in association with PSI and are thus thought to represent a PSI-specific antenna (Veith et al., 2009; Lepetit et al., 2010; Grouneva et al., 2011; Ikeda et al., 2013). (3) The Lhcx proteins, which are related to the Lhcsr proteins of green algae and mosses (Beer et al., 2006; Lepetit et al., 2010; Grouneva et al., 2011; Nagao et al., 2013). Lhcx proteins play an important role in photoprotection via NPQ (Bailleul et al., 2010). Lhcf and Lhcr proteins are encoded by a high number of genes. In the pennate diatom P. triconutum 17 Lhcf and 14 Lhcr genes have been found so far (Bowler et al., 2008), in the centric T. pseudonana 11 Lhcf and 14 Lhcr genes seem to be present (Armbrust et al., 2004). The number of genes encoding for the Lhcx proteins is significantly lower and so far 4 and 6 Lhcx genes have been annotated for P. tricornutum and T. pseudonana, respectively (Armbrust et al., 2004; Bowler et al., 2008). Expression of the Lhcx genes depends on the light climate during culture growth, however, in the pennate diatom P. tricornutum the Lhcx1 protein is constitutively present (Taddei et al., 2018). Longer periods of illumination with high light intensities can increase the concentration of Lhcx1, but especially the Lhcx3 protein is accumulated under these light conditions (Taddei et al., 2016, 2018). A second Lhcx, namely Lhcx2, is also expressed during prolonged periods of high light illumination, albeit at lower concentration than Lhcx3. The centric diatom T. pseudonana also shows changes in the Lhcx composition in dependence of the light conditions. With respect to the Lhcx1 protein results from the literature are somewhat contradictory. While a study based on immunodetection indicated a constitutive expression (Zhu and Green, 2010), as it is observed for the pennate diatoms, another study detected Lhcx1 only under conditions of high light exposition (Grouneva et al., 2011). Short high light periods lead to the expression of Lhcx6 in the centric diatoms (Zhu and Green, 2010), while Lhcx6_1 seems to be constitutively present (Grouneva et al., 2011).



Photosynthesis in diatoms Chapter | 13  221

Another Lhcx protein that is found during prolonged high light illumination in the centric diatoms is Lhcx4 (Grouneva et al., 2011). Interestingly, expression of Lhcx proteins is not only sensitive to high light illumination but has also been observed in diatoms under nutrient stress. Taddei et al. (2016) observed a strong induction of Lhcx2 in iron-limited cells, while nitrogen limitation triggered the induction of Lhcx3 and Lhcx4 mRNAs and proteins. In addition, a small increase of Lhcx1 and Lhcx2 on the protein level was detected under nitrogen starvation.

13.3.2  Native structure of FCPs Until today, no crystal structures of FCP complexes exist and knowledge about the native FCP structure stems from biochemical investigations. These investigations have demonstrated that differences exist between pennate and centric diatoms. In pennate diatoms like P. tricornutum FCP trimers seem to represent the basic unit of the antenna complexes (Lepetit et al., 2007; Grouneva et al., 2011; Gundermann et al., 2013). However, under mild solubilization conditions FCP oligomers can be observed, suggesting that the FCP trimers form higher order structures in the native membrane of pennate diatoms (Lepetit et al., 2007). The FCP trimers of pennate diatoms consist of several Lhcf proteins in different composition (Gundermann et al., 2013). Under low light conditions Lhcf5 seems to be the most abundant protein in the FCP trimers, whereas under high light cultivation a significant amount of trimers composed of Lhcf4 can be observed. The precise localization of the Lhcx proteins in the antenna system of the pennates is still unknown, as well as the possible interaction between Lhcx proteins and specific FCP trimers. The antenna system of the centric diatoms seems to be more complex and two different FCP complexes, termed FCPa and FCPb have been found (Buchel, 2003). Like in the pennate diatoms FCP trimers are the basic unit of the FCPa complexes, the FCPb, however, seems to be composed of FCP nonamers (Beer et al., 2006; Grouneva et al., 2011; Nagao et al., 2013). While earlier results have already shown that FCPa and FCPb contain specific Lhcf proteins and that Lhcx proteins are associated with the trimeric FCPs (Beer et al., 2006; Grouneva et al., 2011), a very recent study has elucidated the FCPa and FCPb composition of Cyclotella meneghiniana in great detail (Gundermann et al., 2019). According to these new data, four different trimeric subtypes of the FCPa exist, while the FCPb is represented by two different nonameric subtypes. The different subtypes can be differentiated by their Lhcf composition. Lhcf1 is the main subunit of the abundant FCPa3 and FCPa4 complexes, Lhcf4 and Lhcf6 dominate the protein composition of the FCPa3 complex. With respect to the FCPb nonameric complexes, Lhcf3 and Fcp5 are the prominent subunits of the two FCPb subtypes. In general, the protein composition of the FCPa and FCPb complexes of C. meneghiniana is comparable to that of another centric diatom, namely T. pseudonana, which has been analyzed in an earlier study (Grouneva et al., 2011). Gundermann et al. (2019) also present evidence for the association of different Lhcx proteins with specific FCP subunits and they observe that Lhcx1 is a component of the major FCPa4. Under high light cultivation the amount of Lhcx1 associated with FCPa4 increases and Lhcx1 can additionally be found in the FCPa1 and FCPb2. Another Lhcx protein, namely Lhcx6_1, can be found in association with FCPa1, FCPa2 and FCPb2 and only in the latter high illumination leads to an increased amount of associated Lhcx protein. In the native membrane the FCP complexes can undergo structural changes. Aggregation of FCPs at low pH-values has been shown for both pennate (Schaller et al., 2014) and centric diatoms (Gundermann and Buchel, 2008). FCP complexes of the pennate P. tricornutum aggregate at low pH-values, Mg2+-ions increase the aggregation of the antenna complexes (Schaller et al., 2014). In comparison to the LHCII of higher plants, FCP aggregation is significantly less pronounced and most likely restricted to the plane of the membrane, i.e., macroaggregation across several thylakoid membranes, as it has been documented for LHCII in grana membranes (Garab and Mustardy, 1999), does not occur. Recent results have shown that the presence of the de-epoxidized xanthophyll cycle pigment Dt strongly increases the aggregation of FCP complexes of P. tricornutum (Schaller-Laudel et al., 2015). A comparable result was obtained for the centric diatom C. meneghiniana where the trimeric FCPa complexes, but not the nonameric FCPb complexes, show a pHand Dt-dependent aggregation (Gundermann and Buchel, 2008).

13.3.3  Function of FCPs The LHCs of diatoms exert two important, yet fundamentally different, functions. Besides the efficient harvesting of light energy, the FCP complexes play an important role in photoprotection via nonphotochemical quenching (NPQ see also Section 13.4.1). NPQ is driven by the build-up of the proton gradient, the conversion of DD to Dt in the xanthophyll cycle and the presence of Lhcx proteins. During the induction of NPQ a structural change, i.e., aggregation, of the FCP complexes is taking place. Recent models predict the presence of two quenching centers, which are responsible for the conversion of excessive excitation energy into heat (Lavaud and Goss, 2014; Goss and Lepetit, 2015; Taddei et al., 2018). Q1 consists of

222  PART | IV  Algal photosystems and photosynthesis

FCP complexes, which have detached from the PSII core complex and have aggregated. Aggregation of FCP complexes in Q1 becomes visible as strong quenching of the FCP fluorescence maximum at around 680 nm. In addition, it shows a spectral fingerprint and can be detected both in vivo and in vitro as long-wavelength fluorescence emission at around 705 nm (Miloslavina et al., 2009; Schaller-Laudel et al., 2015). Aggregation of the FCP complexes in Q1 seems to be independent on the presence of Dt. NPQ at Q2, however, requires the presence of the de-epoxidized xanthophyll cycle pigments. Q2 seems to consist of FCP complexes, which stay attached to the PSII core complex and thus form a dissipative center in the vicinity of the PSII reaction center. With respect to the Lhcx proteins as regulatory element in the generation of NPQ, it has been shown that Lhcx1 is essential for NPQ in P. ­tricornutum and C. meneghiniana (Bailleul et al., 2010). It has been proposed that Lhcx1-dependent NPQ is located in Q2 in the vicinity of the PSII reaction center. Lhcx3 is supposed to be part of Q1, the quenching site consisting of detached, aggregated FCP complexes. Recent results about the protein composition of the FCPa and FCPb complexes of C. meneghiniana (Gundermann et al., 2019) suggest that the role of Lhcx proteins is different in centric diatoms. Here it is suggested that Lhcx1 is responsible for the quenching in Q1 under prolonged high light illumination, whereas Lhcx 6_1 seems to induce NPQ in Q2 at the PSII core.

13.4  Regulation of photosynthetic electron transport 13.4.1  Nonphotochemical quenching The process of “nonphotochemical quenching” (NPQ) describes the thermal dissipation of excessively absorbed radiation at the level of excited singlet states of chlorophyll whereby the emission of variable chlorophyll fluorescence is decreased (quenched; Li et al., 2009). Quenching of singlet Chl a is performed by carotenoids, alternatively the establishment of a Chl a-Zeaxanthin radical pair has been proposed (Ruban, 2016). Particularly in diatoms, the rapidly inducible NPQ is considered as an important regulatory mechanism of photosynthesis (Ruban et al., 2007). NPQ consists of three components which are defined by their kinetics of relaxation: (1) the “inhibitory quenching” (qI) is the slowest relaxing (hours) component and is mainly related to inactivation or even damage of the reaction center of PS II, (2) the slower relaxing (tens of minutes) state-transition dependent quenching qT which is related to the translocation of part of the PS II antenna to PS I, and (3) the fast relaxing (seconds to minutes) energy-dependent component qE. Whereas qT appears to be absent in diatoms (Owens, 1986), qI components can be observed but the precise attribution of fluorescence quenching to photoinhibitory events is not an easy task. First, photoinhibition of photosynthesis rates is hardly observed in diatoms, most probably due to the presence of very effective energy/electron-dissipating mechanisms (i.e., alternative electron pathways; see Section 13.4.2). Second, XC pigments are likely involved in sustained quenching events in the antenna and may interfere with quenching effects due to photodamage of the PSII reaction center (Lavaud and Goss, 2014). Thus, the most important NPQ component in diatoms is qE. For the regulation of photosynthetic electron transport, the capacity and mechanistic aspects of NPQ are important. In previous publications, quite large species- and even strain-specific differences in the NPQ capacity of diatoms have been shown (Lepetit et al., 2013; Lavaud and Lepetit, 2013). Under normal light conditions the induction of qE is dependent on the presence of Dt, however, at higher Dt concentrations in the thylakoid membrane the correlation between the amount of Dt and the resulting quenching capacity gets lost (e.g., Schumann et al., 2007; Lavaud and Lepetit, 2013). It was shown that large amounts of Dt can be dissolved in the lipid phase of the thylakoid membrane instead of being bound to antennae proteins. In this case, Dt molecules do not participate in qE but are considered to function as antioxidants to prevent lipid peroxidation (Lepetit et al., 2010). Besides the involvement of Dt in the NPQ process, it has been demonstrated that the presence of specific Lhcx proteins is essential (Bailleul et al., 2010). Whereas, Lhcx1 shows a constitutively high expression level irrespective of the light conditions, Lhcx2 and Lhcx3 expression levels are clearly dependent on the intensity of irradiance (Zhu and Green, 2010; Taddei et al., 2016). It was demonstrated for P. tricornutum that Lhcx1 plays an important role in short-term photoprotection of the photosynthetic apparatus, i.e., during a fast exposition of low light adapted diatom cells to high light intensities (Taddei et al., 2018). In addition, the maximum NPQ capacity was shown to be connected to an increase of the Lhcx1 protein (Costa et al., 2013). Further, under conditions where Lhcx2 was expressed, NPQ was linearly correlated with the amount of Lhcx2 protein. The correlation between NPQ capacity and newly synthesized Lhcx3 protein was less obvious (Lepetit et al., 2017).



Photosynthesis in diatoms Chapter | 13  223

It was further concluded that this Lhcx-induced regulation of NPQ in diatoms is of strong advantage specifically under fluctuating light conditions (i.e., due to the drift of phytoplankton within the water body) in comparison to more stable light conditions at the surface of aquatic habitats. In this respect, it is interesting that the expression of Lhcx2 and Lhcx3 is differently regulated in response to fluctuating and nonfluctuating light conditions, respectively (Lepetit et al., 2017). The different effects of the Lhcx proteins on NPQ could be also due to a different distribution of the proteins within the FCPs and the photosystems and therefore, different Lhcx proteins may play different roles in the two different NPQ quenching sites predicted by the recent NPQ models (Taddei et al., 2018; see below). This holds true also for the Lhxc6 protein in T. pseudonana. Zhu and Green (2010) showed that Lhcx6 increased under 6 h of high light treatment. However, this increase correlated with an increase in qI. Since there was no photodamage under the respective experimental conditions, it was concluded that Lhcx6 binds Dt and thus, plays a role in the long-term photoprotection by maintaining a high NPQ capacity under pro-longed high light stress. In addition to the inevitable presence of Lhcx proteins, the NPQ capacity depends on the presence of larger amounts of Dt at the Q2 quenching site and on the formation and functional disconnection of oligomeric FCP complexes at the Q1 site. This ensures an effective contribution of Dt to NPQ and an amplification of this Dt-dependent quenching process (Lavaud and Lepetit, 2013). Whereas Q2, located in the vicinity of the PSII reaction center, is dominating under light stress and could be important particularly in the short-term acclimation, the Lhcx3-mediated NPQ in the FCP quenching site Q1 was shown to be beneficial upon high light exposure for a few days (Miloslavina et al., 2009). In the regulation of photosynthetic electron transport, the dark relaxation of NPQ is an important parameter particularly under fluctuating light conditions. A slow NPQ relaxation after high light illumination could limit the quantum efficiency of photosynthesis in a subsequent light period with a gradual increase of irradiance. In contrast, a slow NPQ relaxation would be advantageous in habitats with slow and moderate light changes. Indeed, the fast NPQ characteristics of a specific strain of P. tricornutum and the slower NPQ kinetics of Skeletonema costatum are in accordance with their original habitats of estuarine waters and a Mediterranean semienclosed bay, respectively (Lavaud and Lepetit, 2013). With the importance of Lhcx proteins for NPQ the question of their gene regulation arises. Since NPQ is a photo-­ protective mechanism it is reasonable to assume that an overexcitation of the electron transport chain should be a trigger of gene expression. Generally, the redox state of the “plastoquinone pool” (PQ pool) serves (1) as a sensor of the balance between light absorption and the actual demand of photosynthetic energy, and (2), with the resulting redox signaling, it regulates both the chloroplast and nuclear gene expression (e.g., Pfannschmidt, 2003). Although it was shown that the redox state of the PQ pool is important for photosynthetic processes, i.e., the induction of cyclic electron transport at PS II (Feikema et al., 2006; see also Section 13.4.2), there was no evidence for an involvement of the PQ pool in redox signaling in diatoms for a long time. Lepetit et al. (2013) provided first indications for a retrograde signaling mechanism in diatoms and the regulation of gene expression of Lhcx1 and Lhcx2 by the redox state of PQ. However, it was also concluded that the formation of reactive oxygen species could influence the expression of specific Lhcx genes (i.e., Lhcx3) and would thus interfere with the redox signaling from the PQ pool (Lepetit et al., 2017). Further evidence for chloroplast-derived signals in the regulation of gene expression in diatoms came from experiments conducted by Taddei et al. (2016). It was shown that the expression of Lhcx4 was downregulated after a dark to light transition. In the presence of the PS II inhibitor DCMU, which keeps the PQ pool in an oxidized state, this repression was not observed anymore. It could be assumed that the gene regulation described above is important under high light illumination. However, for phytoplankton cells the light quality is an extremely important source of information, too. The underwater light field shows tremendous depth-related changes, with blue light usually being the waveband with the deepest penetration within the water body. Diatoms possess a large number of different photoreceptor proteins, with the blue light-sensing Cryptochromes and Aureochromes being the most important [for a detailed review on photoreceptors in diatoms see Depauw et al. (2012) and Kroth et al. (2017)]. Consequently, the proteins CPF1 and CRYP, both belonging to the ­cryptochrome-photolyase family, modulate the light-dependent expression of Lhcx1, Lhcx2, and Lhcx3 (Coesel et al., 2009; Juhas et al., 2014). In addition, the study of Costa et al. (2013) provided first evidence that the Aureochrome 1a is of pivotal importance in the photoacclimation process of P. tricornutum. More recently, the study of Mann et al. (2017) delivered final evidence for the photoprotective role of Aureochrome 1a by showing that aureochrome knockout mutants “AUREO1a” of P. tricornutum were strongly impaired in the acclimation to blue light. In addition, these mutants were characterized by a decreased NPQ capacity. In this respect, it is of further importance that the promoter region of Lhcx1 of P. tricornutum possesses two potential binding sites for Aureochrome 1a (Costa et al., 2013). It should be noted that particularly nutrient limitation (i.e., iron and nitrogen deficiency) strongly affects the expression of Lhcx proteins. However, this acclimation reaction appears to be different from high light-acclimation and thus reveals the complexity of NPQ regulation in diatoms (discussed in detail in Taddei et al., 2016).

224  PART | IV  Algal photosystems and photosynthesis

13.4.2  Alternative electron transport The photosynthetic electron transport does not only produce reducing equivalents (i.e., NADPH) but also generates a proton motive force across the thylakoid membrane. This proton motive force is composed of the proton gradient and the membrane potential across the thylakoid membrane, both of which drive the ATP synthesis via the ATP synthetase (Armbruster et al., 2017). It is generally accepted that the “linear electron flow” (LEF) generates an ATP/NADPH ratio that is insufficient to meet the energy demand of CO2 assimilation in the Calvin cycle (Allen, 2002). Therefore, alternative electron sinks/pathways (alternative electron flow, AEF) are necessary to adjust the ATP/NADPH ratio but also to dissipate excessively absorbed light energy that exceeds the capacity of its usage in cellular metabolism. AEF generally includes “cyclic electron flow” (CEF) around Photosystem I (PS I; Shikanai, 2007), the water-to-water cycle (Mehler reaction; Asada, 1999), and the photorespiration (Ort and Baker, 2002). Under nutrient-replete conditions, the assimilation of nitrate including the reduction of nitrite to ammonium is a significant sink for photosynthetic electrons supplied via Ferredoxin (Raven et al., 1992). The cyclic electron transport around PSII appears to be an AEF that is characteristic for diatoms (Lavaud et al., 2002). For a long time, the quantitative contribution of AEF to the regulation of the ATP/NADPH ratio in diatoms remained largely unknown. However, it was shown that the extent of AEF in relation to LEF is rather low in diatoms compared to other phytoplankton groups (i.e., Chlorophyta; Wagner et al., 2006). It was assumed that the large capacity of NPQ in diatoms (see Section 13.4.1) limits the necessity for AEF in the dissipation of excessively absorbed light energy. Nevertheless, this would not explain by which mechanism diatoms adjust the ATP/NADPH ratio. Tremendous progress in resolving this question was made during recent years. In a very elegant study of Bailleul et al. (2015) a combination of different measuring techniques was used to unravel the mechanisms (including their quantitative contribution), which regulate of the ATP/NADPH ratio under illumination in P. tricornutum. Interestingly, clear evidence was found for a very low CEF activity that did not contribute to the regulation of ATP/NADPH fluxes. Instead, a light stimulated oxygen consumption, fed by electrons generated at PS II, was observed. This electron flow was estimated as 10% of photosynthetic electron flow. Whereas earlier studies referred such an oxygen consumption preferentially to the activity of the Mehler reaction (Claquin et al., 2004; Waring et al., 2010), Bailleul et al. (2015) could clearly prove a connection to mitochondrial activity. An intense metabolic cross-talk between chloroplast and mitochondria in diatoms was suggested already by Prihoda et al. (2012). The idea of a close interaction between chloroplast and mitochondrium is supported by a very close spatial relationship of the two organelles and by the fact that the mitochondrial “alternative oxidase” (AOX) is highly active under conditions of excessive supply of photosynthetic energy in relation to metabolic energy demand, e.g., under iron limitation (Allen et al., 2008). Bailleul et al. (2015) provided further evidence by the investigation of AOX knockdown cell lines of P. tricornutum that revealed a strong regulatory influence of AOX activity on the photosynthetic capacity. From the analysis of ATP, NADPH, and NADP+ levels in WT and knock down cells it was concluded that mitochondrial AOX contributes to the ATP/NADPH ratio, not by an additional ATP production but by an increased NADPH consumption. Thus, AOX can be seen as an important sink for excess reductants (Prihoda et al., 2012) and the energetic coupling between plastid and mitochondrium allows sharing/balancing of ATP/NADPH flows in response to specific demands. The correct adjustment of the ATP/NADPH ratio is not only important under excess light conditions but also under light limitation. Diatoms are known for their high photosynthetic efficiency particularly under fluctuating light conditions (Wagner et al., 2006). Fisher and Halsey (2016) showed that his extraordinary light use efficiency to produce biomass is due to the switch between ATP generating pathways. Accordingly, at very low light intensities the relative contribution of mitochondrial ATP production is reduced to conserve carbon stores. To compensate for this reduction, light-dependent oxygen consumption in the chloroplast (Mehler reaction, CEF) is increased. Thereby, the proton motive force in the chloroplast is enhanced to generate additional ATP. Another process with significant potential to regulate the ATP/NADPH ratio is nitrate assimilation and the associated usage of photosynthetically generated NADPH for the reduction of nitrite to ammonium in the chloroplast. This reduction reaction requires 6 electrons per nitrite molecule (Glibert et al., 2016) and in nutrient-replete cells this process can consume at least 20% of photosynthetic electrons (with the assumption of a C/N ratio of the biomass of >5). However, in diatoms, nitrate uptake and reduction can take place even when the nutritional demand for nitrogen is saturated (Lomas and Glibert, 1999), which separates diatoms from other phytoplankton groups (Glibert et al., 2016). This seems to be of particular importance for marine diatoms under fluctuating light conditions in combination with low temperatures and nitrate-enriched waters and is thought to represent a tremendous sink for excessive photosynthetic energy. Temperature, light, and the nitrogen source are also important triggers for another energy-dissipating mechanism: photorespiration. In the photorespiratory process, the enzyme “Ribulose-bisphosphate Carboxylase/Oxygenase” (RubisCO) uses O2 rather than CO2 as substrate resulting in the formation of 2-phosphoglycolate which can be excreted as glycolate



Photosynthesis in diatoms Chapter | 13  225

into the surrounding medium or metabolized in the so-called C2 cycle. There have been early proofs of photorespiratory activity and its metabolic pathways in diatoms (Winkler and Stabenau, 1995). The study of Parker and Armbrust (2005) was an important step to unravel the regulation of photorespiratory pathways in response to environmental conditions. Accordingly, the highest glycolate excretion was observed under the combination of high light, high temperatures and nitrate as nitrogen source. Obviously, this strategy allows for maintaining a high activity of the Calvin cycle, whereby the ATP/NADPH ratio is adjusted by the reduction of nitrate and excessively assimilated carbon is dissipated by the release of glycolate. In contrast, the lowest glycolate excretion was observed under high light, low temperatures and ammonium as nitrogen source. However, the highest metabolic activity of the C2 cycle was detected under this condition. It could be concluded that the downregulation of the Calvin cycle by low temperatures forces the cells to dissipate excess light energy via the high energy requirement of glycolate recycling, thereby compensating for the absence of energy dissipation by nitrate reduction and minimizing the loss of organic carbon (Davis et al., 2017). The cyclic electron flow within PS II re-donates electrons to the reaction center, thereby avoiding electron release from water splitting at the oxygen evolving complex. This way, the PS II cycle opens a valve to dissipate excessively absorbed light energy while simultaneously preventing an overreduction of QA, the primary acceptor of PS II (Feikema et al., 2006). Thus, this AET pathway does not regulate the ATP/NADPH ratio but is used under conditions where a fast and effective energy dissipation is required to prevent photoinhibitory damage of PS II reaction centers (Lavaud, 2007). Typically, high PS II cycle activity is observed under high irradiance in diatoms (Lavaud et al., 2002). This activity is remarkably increased under nitrogen limited conditions in comparison to nutrient replete cells of P. tricornutum (Wagner et al., 2016). It should be noted that under these conditions of N limitation, NPQ, as an alternative energy-dissipating mechanism, is not enhanced. Thus, it can be concluded that the energy-dissipating capacity of cyclic electron transport at PS II is sufficient to replace the amount of energy that is usually dissipated by nitrate reduction.

13.5  Dark reactions in photosynthesis In diatoms, the process of carbon fixation is very efficient and reflects the evolutionary adaptation to environments with low availability of dissolved CO2, e.g., in sea water. The peculiarities in CO2 assimilation in diatoms compared to the green lineage refer to two major differences: (a) the redox regulation of the key enzymes and (b) the carbon concentrating mechanisms. As in green algae and higher plants, the Calvin Cycle is the operating unit for CO2 fixation. In the green lineage the Calvin Cycle is activated only in the light by the light-driven thioredoxin system which becomes reduced by the photosynthetic electron transport. Although diatoms possess also a plastidic thioredoxin system, some key enzymes of the Calvin cycle are not regulated by this redox mechanism. For example, the “glyceraldehyde-phosphate dehydrogenase” (GAPDH) is strictly redox-regulated in green chloroplasts but not in diatoms. In addition, the “phospho-ribulokinase” (PRK)-GAPDH-CP12 complex, typically present in most green plastids, has not been identified in diatoms so far. In the regeneration phase of the Calvin cycle sedoheptulose-1,7-bisphosphatase and the phosphoribulokinase are regulatory steps under strict redox control in green chloroplast, however, in diatoms these enzymes operate independent of their redox state (Jensen et al., 2017). Another difference in the enzymatic regulation is found for the RubisCO activase. In green algae and higher plants this enzyme is nuclear encoded but is replaced in diatoms by an analogous chloroplast-encoded CbbX, “Calvin-Benson-Bassham protein X.” The second important difference with respect to the regulation of CO2 assimilation in diatoms refers to the carbon concentrating mechanisms. Reinfelder et al. (2000) identified in a diatom for the first time the C4 pathway as a potential CO2 fixation mechanism. However, the crucial step in C4 photosynthesis, the decarboxylation of malate, which is necessary to deliver CO2 to the RubisCO, was not shown so far and is still a matter of debate. Therefore, it was postulated that the C4 mechanism in diatoms serves as a carbon concentrating mechanism in parallel to inorganic carbon transporters. Recently, based on genome analyses, it was shown that in the diatom P. tricornutum all the enzymes required to operate a C4 pathway are genetically present, however, silencing of the “pyruvate orthophosphate dikinase” (PPDK) in a genetically transformed cell line did not reduce photosynthetic carbon fixation (Kroth et al., 2008). A comparison of the localization of the C4 specific enzymes between diatoms and higher plants showed a significantly different distribution, and indicated that at least the model diatom P. tricornutum does not use C4 decarboxylation in the plastid to provide the RubisCO with inorganic carbon (Ewe et al., 2018). Also the transport of inorganic carbon from outside the cell into the plastid shows some important deviations compared to green cells. Diatoms can take up dissolved inorganic carbon either by the use of a special plasma-membrane localized carrier (SLC4), which belongs to the family of HCO_3 transporters, or by passive diffusion of CO2 formed by an external “carbonic anhydrase” (CA). The resulting bicarbonate taken up into the cytoplasm can be used either by PEP or can be actively

226  PART | IV  Algal photosystems and photosynthesis

transported into the plastid by SLC4-type transporters located in the chloroplast membrane system. Here it is re-converted to CO2 close to the RubisCO preventing either CO2 loss or photorespiration (for review see Matsuda et al., 2017). In summary, the peculiarities reported show that diatoms are extremely well adapted to restrictions in CO2 availability and that our knowledge about the diversity of molecular strategies to cope with these limitations is still incomplete. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c,d, e,f,g,h,i,j,k,l,m,n,o).

References Abida, H., Dolch, L.J., Mei, C., Villanova, V., Conte, M., Block, M.A., et al., 2015. Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum. Plant Physiol. 167 (1), 118–136. Albanese, P., Melero, R., Engel, B.D., Grinzato, A., Berto, P., Manfredi, M., et al., 2017. Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap. Sci. Rep. 7, 10067. Allen, J.F., 2002. Photosynthesis of ATP—electrons, proton pumps, rotors, and poise. Cell 110 (3), 273–276. Allen, A.E., LaRoche, J., Maheswari, U., Lommer, M., Schauer, N., Lopez, P.J., et al., 2008. Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc. Natl. Acad. Sci. U. S. A. 105 (30), 10438–10443. Archibald, J.M., 2015. Evolution: gene transfer in complex cells. Nature 524 (7566), 423–424. Armbrust, E.V., 2009. The life of diatoms in the world’s oceans. Nature 459 (7244), 185–192. Armbrust, E.V., Berges, J.A., Bowler, C., Green, B.R., Martinez, D., Putnam, N.H., et al., 2004. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306 (5693), 79–86. Armbruster, U., Galvis, V.C., Kunz, H.H., Strand, D.D., 2017. The regulation of the chloroplast proton motive force plays a key role for photosynthesis in fluctuating light. Curr. Opin. Plant Biol. 37, 56–62. Asada, K., 1999. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. 50, 601–639. Ast, M., Gruber, A., Schmitz-Esser, S., Neuhaus, H.E., Kroth, P.G., Horn, M., et al., 2009. Diatom plastids depend on nucleotide import from the cytosol. Proc. Natl. Acad. Sci. USA 106 (9), 3621–3626. Bai, X.C., Song, H., Lavoie, M., Zhu, K., Su, Y.Y., Ye, H.Q., et al., 2016. Proteomic analyses bring new insights into the effect of a dark stress on lipid biosynthesis in Phaeodactylum tricornutum. Sci. Rep. UK 6, 25494. Bailleul, B., Berne, N., Murik, O., Petroutsos, D., Prihoda, J., Tanaka, A., et al., 2015. Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms. Nature 524 (7565), 366–369. Bailleul, B., Rogato, A., de Martino, A., Coesel, S., Cardol, P., Bowler, C., et al., 2010. An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light. Proc. Natl. Acad. Sci. U. S. A. 107 (42), 18214–18219. Beer, A., Gundermann, K., Beckmann, J., Buchel, C., 2006. Subunit composition and pigmentation of fucoxanthin-chlorophyll proteins in diatoms: evidence for a subunit involved in diadinoxanthin and diatoxanthin binding. Biochemistry-US 45 (43), 13046–13053. Bina, D., Herbstova, M., Gardian, Z., Vacha, F., Litvin, R., 2016. Novel structural aspect of the diatom thylakoid membrane: lateral segregation of photosystem I under red-enhanced illumination. Sci. Rep. 6, 25583. Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., et al., 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456 (7219), 239–244. Buchel, C., 2003. Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states. Biochemistry-US 42 (44), 13027–13034. Buchel, C., 2015. Evolution and function of light harvesting proteins. J. Plant Physiol. 172, 62–75. Canavate, J.P., Armada, I., Rios, J.L., Hachero-Cruzado, I., 2016. Exploring occurrence and molecular diversity of betaine lipids across taxonomy of marine microalgae. Phytochemistry 124, 68–78. Cho, S.H., Thompson, G.A., 1987. On the metabolic relationship between monogalactosyldiacylglycerol and digalactosyldiacylglycerol molecular species in Dunaliella salina. J. Biol. Chem. 262 (16), 7586–7593. Claquin, P., Kromkamp, J.C., Martin-Jezequel, V., 2004. Relationship between photosynthetic metabolism and cell cycle in a synchronized culture of the marine alga Cylindrotheca fusiformis (Bacillariophyceae). Eur. J. Phycol. 39 (1), 33–41. Coesel, S., Mangogna, M., Ishikawa, T., Heijde, M., Rogato, A., Finazzi, G., et al., 2009. Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity. EMBO Rep. 10 (6), 655–661. Costa, B.S., Jungandreas, A., Jakob, T., Weisheit, W., Mittag, M., Wilhelm, C., 2013. Blue light is essential for high light acclimation and photoprotection in the diatom Phaeodactylum tricornutum. J. Exp. Bot. 64 (2), 483–493. Costa, B.S., Sachse, M., Jungandreas, A., Bartulos, C.R., Gruber, A., Jakob, T., et al., 2013. Aureochrome 1a is involved in the photoacclimation of the diatom Phaeodactylum tricornutum. PLoS One 8 (9), e74451. Davis, A., Abbriano, R., Smith, S.R., Hildebrand, M., 2017. Clarification of photorespiratory processes and the role of malic enzyme in diatoms. Protist 168 (1), 134–153. Depauw, F.A., Rogato, A., d’Alcala, M.R., Falciatore, A., 2012. Exploring the molecular basis of responses to light in marine diatoms. J. Exp. Bot. 63 (4), 1575–1591.



Photosynthesis in diatoms Chapter | 13  227

Dodson, V.J., Dahmen, J.L., Mouget, J.L., Leblond, J.D., 2013. Mono- and digalactosyldiacylglycerol composition of the marennine-producing diatom, Haslea ostrearia: comparison to a selection of pennate and centric diatoms. Phycol. Res. 61 (3), 199–207. Dodson, V.J., Mouget, J.L., Dahmen, J.L., Leblond, J.D., 2014. The long and short of it: temperature-dependent modifications of fatty acid chain length and unsaturation in the galactolipid profiles of the diatoms Haslea ostrearia and Phaeodactylum tricornutum. Hydrobiologia 727 (1), 95–107. Eichenberger, W., 1982. Distribution of diacylglyceryl-O-4′-(N,N,N-trimethyl)-homoserine in different algae. Plant Sci. Lett. 24 (1), 91–95. Ewe, D., Tachibana, M., Kikutani, S., Gruber, A., Bartulos, C.R., Konert, G., et al., 2018. The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum. Photosynth. Res. 137 (2), 263–280. Feikema, W.O., Marosvolgyi, M.A., Lavaud, J., van Gorkom, H.J., 2006. Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum. Biochim. Biophys. Acta 1757 (7), 829–834. Fisher, N.L., Halsey, K.H., 2016. Mechanisms that increase the growth efficiency of diatoms in low light. Photosynth. Res. 129 (2), 183–197. Flori, S., Jouneau, P.H., Bailleul, B., Gallet, B., Estrozi, L.F., Moriscot, C., et al., 2017. Plastid thylakoid architecture optimizes photosynthesis in diatoms. Nat. Commun. 8, 15885. Garab, G., Mustardy, L., 1999. Role of LHCII-containing macrodomains in the structure, function and dynamics of grana. Aust. J. Plant Physiol. 26 (7), 649–658. Garab, G., Ughy, B., Goss, R., 2016. Role of MGDG and non-bilayer lipid phases in the structure and dynamics of chloroplast thylakoid membranes. In: Nakamura, Y., Li-Beisson, Y. (Eds.), Lipids in Plant and Algae Development. Springer, Cham, pp. 127–157. Glibert, P.M., Wilkerson, F.P., Dugdale, R.C., Raven, J.A., Dupont, C.L., Leavitt, P.R., et al., 2016. Pluses and minuses of ammonium and nitrate uptake and assimilation by phytoplankton and implications for productivity and community composition, with emphasis on nitrogen-enriched conditions. Limnol. Oceanogr. 61 (1), 165–197. Goss, R., Lepetit, B., 2015. Biodiversity of NPQ. J. Plant Physiol. 172, 13–32. Goss, R., Nerlich, J., Lepetit, B., Schaller, S., Vieler, A., Wilhelm, C., 2009. The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane. J. Plant Physiol. 166 (17), 1839–1854. Grouneva, I., Rokka, A., Aro, E.M., 2011. The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery. J. Proteome Res. 10 (12), 5338–5353. Guiheneuf, F., Mimouni, V., Ulmann, L., Tremblin, G., 2008. Environmental factors affecting growth and omega 3 fatty acid composition in Skeletonema costatum. The influences of irradiance and carbon source. Diatom Res. 23 (1), 93–103. Gundermann, K., Buchel, C., 2008. The fluorescence yield of the trimeric fucoxanthin-chlorophyll-protein FCPa in the diatom Cyclotella meneghiniana is dependent on the amount of bound diatoxanthin. Photosynth. Res. 95 (2-3), 229–235. Gundermann, K., Schmidt, M., Weisheit, W., Mittag, M., Buchel, C., 2013. Identification of several sub-populations in the pool of light harvesting proteins in the pennate diatom Phaeodactylum tricornutum. Biochim. Biophys. Acta 1827 (3), 303–310. Gundermann, K., Wagner, V., Mittag, M., Buchel, C., 2019. Fucoxanthin-chlorophyll protein complexes of the centric diatom Cyclotella meneghiniana differ in Lhcx1 and Lhcx6_1 content. Plant Physiol. https://doi.org/10.1104/pp.18.01363. Guschina, I.A., Harwood, J.L., 2006. Lipids and lipid metabolism in eukaryotic algae. Prog. Lipid Res. 45 (2), 160–186. Hildebrand, M., Manandhar-Shrestha, K., Abbriano, R., 2017. Effects of chrysolaminarin synthase knockdown in the diatom Thalassiosira pseudonana: implications of reduced carbohydrate storage relative to green algae. Algal Res. 23, 66–77. Hu, C.W., Li, M., Li, J.L., Zhu, Q., Liu, Z.L., 2008. Variation of lipid and fatty acid compositions of the marine microalga Pavlova viridis (Prymnesiophyceae) under laboratory and outdoor culture conditions. World J. Microbiol. Biotechnol. 24 (7), 1209–1214. Ikeda, Y., Yamagishi, A., Komura, M., Suzuki, T., Dohmae, N., Shibata, Y., et al., 2013. Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms. Biochim. Biophys. Acta 1827 (4), 529–539. Jensen, E., Clement, R., Maberly, S.C., Gontero, B., 2017. Regulation of the Calvin–Benson–Bassham cycle in the enigmatic diatoms: biochemical and evolutionary variations on an original theme. Philos. Trans. Roy. Soc. B 372 (1728), 20160401. Jiang, H.M., Gao, K.S., 2004. Effects of lowering temperature during culture on the production of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum (Bacillariophyeae). J. Phycol. 40 (4), 651–654. Joyard, J., Marechal, E., Miege, C., Block, M.A., Dorne, A.J., Douce, R., 1998. Structure, distribution and biosynthesis of glycerolipids from higher plant chloroplasts. In: Siegenthaler, P.A., Murata, N. (Eds.), Lipids in Photosynthesis: Structure, Function and Genetics. Springer, Dordrecht, pp. 21–52. Juhas, M., von Zadow, A., Spexard, M., Schmidt, M., Kottke, T., Buchel, C., 2014. A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light-harvesting protein levels. FEBS J. 281 (9), 2299–2311. Kern, J., Guskov, A., 2011. Lipids in photosystem II: multifunctional cofactors. J. Photochem. Photobiol. B 104 (1-2), 19–34. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

228  PART | IV  Algal photosystems and photosynthesis

Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Koziol, A.G., Borza, T., Ishida, K.I., Keeling, P., Lee, R.W., Durnford, D.G., 2007. Tracing the evolution of the light-harvesting antennae in chlorophyll a/b-containing organisms. Plant Physiol. 143 (4), 1802–1816. Kroth, P.G., Chiovitti, A., Gruber, A., Martin-Jezequel, V., Mock, T., Parker, M.S., et  al., 2008. A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis. PLoS One 3 (1), e1426. Kroth, P.G., Wilhelm, C., Kottke, T., 2017. An update on aureochromes: phylogeny – mechanism – function. J. Plant Physiol. 217, 20–26. Lavaud, J., 2007. Fast regulation of photosynthesis in diatoms: mechanisms, evolution and ecophysiology. Funct. Plant Sci. Biotechnol. 1, 267–287. Lavaud, J., Goss, R., 2014. The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae. In: Demmig-Adams, B., Adams, W.W., Garab, G., Govindjee (Eds.), Non-Photochemical Quenching and Thermal Energy Dissipation in Plants, Algae and Cyanobacteria. Springer, Dordrecht, pp. 421–443. Lavaud, J., Lepetit, B., 2013. An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms. Biochim. Biophys. Acta 1827 (3), 294–302. Lavaud, J., van Gorkom, H., Etienne, A.L., 2002. Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms. Photosynth. Res. 74 (1), 51–59. Lepetit, B., Gelin, G., Lepetit, M., Sturm, S., Vugrinec, S., Rogato, A., et al., 2017. The diatom Phaeodactylum tricornutum adjusts nonphotochemical fluorescence quenching capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis. New Phytol. 214 (1), 205–218. Lepetit, B., Goss, R., Jakob, T., Wilhelm, C., 2012. Molecular dynamics of the diatom thylakoid membrane under different light conditions. Photosynth. Res. 111 (1-2), 245–257. Lepetit, B., Sturm, S., Rogato, A., Gruber, A., Sachse, M., Falciatore, A., et al., 2013. High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool. Plant Physiol. 161 (2), 853–865. Lepetit, B., Volke, D., Gilbert, M., Wilhelm, C., Goss, R., 2010. Evidence for the existence of one antenna-associated, lipid-dissolved, and two proteinbound pools of diadinoxanthin cycle pigments in diatoms. Plant Physiol. 154 (4), 1905–1920. Lepetit, B., Volke, D., Szabo, M., Hoffmann, R., Garab, G., Wilhelm, C., et al., 2007. Spectroscopic and molecular characterization of the oligomeric antenna of the diatom Phaeodactylum tricornutum. Biochemistry-US 46 (34), 9813–9822. Li, Z.R., Wakao, S., Fischer, B.B., Niyogi, K.K., 2009. Sensing and responding to excess light. Annu. Rev. Plant Biol. 60, 239–260. Linka, N., Weber, A.P., 2010. Intracellular metabolite transporters in plants. Mol. Plant 3 (1), 21–53. Lomas, M.W., Glibert, P.M., 1999. Temperature regulation of nitrate uptake: a novel hypothesis about nitrate uptake and reduction in cool-water diatoms. Limnol. Oceanogr. 44 (3), 556–572. Mann, M., Serif, M., Jakob, T., Kroth, P.G., Wilhelm, C., 2017. PtAUREO1a and PtAUREO1b knockout mutants of the diatom Phaeodactylum tricornutum are blocked in photoacclimation to blue light. J. Plant Physiol. 217, 44–48. Matsuda, Y., Hopkinson, B.M., Nakajima, K., Dupont, C.L., Tsuji, Y., 2017. Mechanisms of carbon dioxide acquisition and CO2 sensing in marine diatoms: a gateway to carbon metabolism. Philos. Trans. Roy. Soc. B 372 (1728), 20160403. Miloslavina, Y., Grouneva, I., Lambrev, P.H., Lepetit, B., Goss, R., Wilhelm, C., et al., 2009. Ultra-fast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms. Biochim. Biophys. Acta 1787 (10), 1189–1197. Nagao, R., Takahashi, S., Suzuki, T., Dohmae, N., Nakazato, K., Tomo, T., 2013. Comparison of oligomeric states and polypeptide compositions of fucoxanthin chlorophyll a/c-binding protein complexes among various diatom species. Photosynth. Res. 117 (1-3), 281–288. Ort, D.R., Baker, N.R., 2002. A photoprotective role for O2 as an alternative electron sink in photosynthesis? Curr. Opin. Plant Biol. 5 (3), 193–198. Owens, T.G., 1986. Light-harvesting function in the diatom Phaeodactylum tricornutum: II. Distribution of excitation energy between the photosystems. Plant Physiol. 80 (3), 739–746. Parker, M.S., Armbrust, E.V., 2005. Synergistic effects of light, temperature, and nitrogen source on transcription of genes for carbon and nitrogen metabolism in the centric diatom Thalassiosira pseudonana (Bacillariophyceae). J. Phycol. 41 (6), 1142–1153.



Photosynthesis in diatoms Chapter | 13  229

Pfannschmidt, T., 2003. Chloroplast redox signals: how photosynthesis controls its own genes. Trends Plant Sci. 8 (1), 33–41. Prihoda, J., Tanaka, A., de Paula, W.B.M., Allen, J.F., Tirichine, L., Bowler, C., 2012. Chloroplast-mitochondria cross-talk in diatoms. J. Exp. Bot. 63 (4), 1543–1557. Raven, J.A., Wollenweber, B., Handley, L.L., 1992. A comparison of ammonium and nitrate as nitrogen sources for photolithotrophs. New Phytol. 121 (1), 19–32. Reinfelder, J.R., Kraepiel, A.M.L., Morel, F.M.M., 2000. Unicellular C-4 photosynthesis in a marine diatom. Nature 407 (6807), 996–999. Ruban, A.V., 2016. Nonphotochemical chlorophyll fluorescence quenching: mechanism and effectiveness in protecting plants from photodamage. Plant Physiol. 170 (4), 1903–1916. Ruban, A.V., Berera, R., Ilioaia, C., van Stokkum, I.H.M., Kennis, J.T.M., Pascal, A.A., et al., 2007. Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450 (7169), 575–578. Sato, N., Hagio, M., Wada, H., Tsuzuki, M., 2000. Environmental effects on acidic lipids of thylakoid membranes. Biochem. Soc. Trans. 28 (6), 912–914. Schaller, S., Latowski, D., Jemiola-Rzeminska, M., Wilhelm, C., Strzałka, K., Goss, R., 2010. The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII). Biochim. Biophys. Acta 1797 (3), 414–424. Schaller, S., Richter, K., Wilhelm, C., Goss, R., 2014. Influence of pH, Mg2+, and lipid composition on the aggregation state of the diatom FCP in comparison to the LHCII of vascular plants. Photosynth. Res. 119 (3), 305–317. Schaller-Laudel, S., Latowski, D., Jemioła-Rzeminska, M., Strzałka, K., Daum, S., Bacia, K., et al., 2017. Influence of thylakoid membrane lipids on the structure of aggregated light-harvesting complexes of the diatom Thalassiosira pseudonana and the green alga Mantoniella squamata. Physiol. Plant. 160 (3), 339–358. Schaller-Laudel, S., Volke, D., Redlich, M., Kansy, M., Hoffmann, R., Wilhelm, C., et  al., 2015. The diadinoxanthin diatoxanthin cycle induces structural rearrangements of the isolated FCP antenna complexes of the pennate diatom Phaeodactylum tricornutum. Plant Physiol. Biochem. 96, 364–376. Schumann, A., Goss, R., Jakob, T., Wilhelm, C., 2007. Investigation of the quenching efficiency of diatoxanthin in cells of Phaeodactylum tricornutum (Bacillariophyceae) with different pool sizes of xanthophyll cycle pigments. Phycologia 46 (1), 113–117. Shikanai, T., 2007. Cyclic electron transport around photosystem I: genetic approaches. Annu. Rev. Plant Biol. 58, 199–217. Siegenthaler, P.A., Murata, N. (Eds.), 1998. Lipids in Photosynthesis: Structure, Function, and Genetics. Springer, Dordrecht and Boston, MA. Smith, S.R., Abbriano, R.M., Hildebrand, M., 2012. Comparative analysis of diatom genomes reveals substantial differences in the organization of carbon partitioning pathways. Algal Res. 1 (1), 2–16. Szabo, M., Premvardhan, L., Lepetit, B., Goss, R., Wilhelm, C., Garab, G., 2010. Functional heterogeneity of the fucoxanthins and fucoxanthin-­ chlorophyll proteins in diatom cells revealed by their electrochromic response and fluorescence and linear dichroism spectra. Chem. Phys. 373 (1-2), 110–114. Taddei, L., Chukhutsina, V.U., Lepetit, B., Stella, G.R., Bassi, R., van Amerongen, H., et al., 2018. Dynamic changes between two LHCX-related energy quenching sites control diatom photoacclimation. Plant Physiol. 177 (3), 953–965. Taddei, L., Stella, G.R., Rogato, A., Bailleul, B., Fortunato, A.E., Annunziata, R., et al., 2016. Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum. J. Exp. Bot. 67, 3939–3951. Tanaka, A., de Martino, A., Amato, A., Montsant, A., Mathieu, B., Rostaing, P., et al., 2015. Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum. Protist 166 (5), 506–521. Veith, T., Brauns, J., Weisheit, W., Mittag, M., Buchel, C., 2009. Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana. Biochim. Biophys. Acta 1787 (7), 905–912. Vieler, A., Wilhelm, C., Goss, R., Suss, R., Schiller, J., 2007. The lipid composition of two different algae (Chlamydomonas reinhardtii, Chlorophyceae and Cyclotella meneghiniana, Bacillariophyceae) investigated by MALDI-TOF MS and TLC. Chem. Phys. Lipids 150 (2), 143–155. Wada, H., Murata, N., 2007. The essential role of phosphatidylglycerol in photosynthesis. Photosynth. Res. 92 (2), 205–215. Wagner, H., Jakob, T., Lavaud, J., Wilhelm, C., 2016. Photosystem II cycle activity and alternative electron transport in the diatom Phaeodactylum tricornutum under dynamic light conditions and nitrogen limitation. Photosynth. Res. 128 (2), 151–161. Wagner, H., Jakob, T., Wilhelm, C., 2006. Balancing the energy flow from captured light to biomass under fluctuating light conditions. New Phytol. 169 (1), 95–108. Waring, J., Klenell, M., Bechthold, U., Underwood, G.J.C., Baker, N.R., 2010. Light-induced responses of oxygen photoreduction, reactive oxygen species production and scavenging in two diatom species. J. Phycol. 46 (6), 1206–1217. Wilhelm, C., Jungandreas, A., Jakob, T., Goss, R., 2014. Light acclimation in diatoms: from phenomenology to mechanisms. Mar. Genomics 16, 5–15. Winkler, U., Stabenau, H., 1995. Isolation and characterization of peroxisomes from diatoms. Planta 195 (3), 403–407. Yang, Z.K., Ma, Y.H., Zheng, J.W., Yang, W.D., Liu, J.S., Li, H.Y., 2014. Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum. J. Appl. Phycol. 26 (1), 73–82. Yongmanitchai, W., Ward, O.P., 1993. Positional distribution of fatty acids, and molecular species of polar lipids, in the diatom Phaeodactylum tricornutum. J. Gen. Microbiol. 139 (3), 465–472. Zhu, S.H., Green, B.R., 2010. Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress. Biochim. Biophys. Acta 1797 (8), 1449–1457. Zulu, N.N., Zienkiewicz, K., Vollheyde, K., Feussner, I., 2018. Current trends to comprehend lipid metabolism in diatoms. Prog. Lipid Res. 70, 1–16.

Chapter 14

The pioneering research on the cyanobacterial photosystems and photosynthesis Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

14.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 indexed-papers published since 1980 (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). The field of algal photosystems and photosynthesis (Konur, 2015d,e, 2020f) has been among the most-prolific research fronts over time as evidenced with over 11,500 papers, comprising nearly 8% of the algal research as a whole, published during the same study period, corresponding to the public concerns about the development of sustainable and environmentfriendly bioproducts and bioprocesses at large. The field of the cyanobacterial photosystems and photosynthesis has been a special case of the research on the algal photosystems and photosynthesis (Konur, 2020f). This book chapter covers the 33 pioneering research papers on the cyanobacterial photosystems and photosynthesis with at least 300 citations each providing the ample data for the primary stakeholders about the contents of these papers to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate. It also provides the strategic information on the research landscape for these pioneering papers.

14.2  Materials and methodology The search for the literature on the cyanobacterial photosystems and photosynthesis was carried out in April 2019 using 4 databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI) and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been developed by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in the Appendix. These keyword sets have been devised in 3 major parts: the keywords related to photosynthesis, keywords related to the cyanobacteria, and cross-subject keywords. The papers with at least 300 citations each were selected for this study. First, the strategic information on the research landscape for these papers were presented in summary to put these papers in the context (Konur, 2020a,b,c,d,e,f,g,h,i,j,k,l, m,n). Next, the concise information about each paper was presented under the 2 topical headings of cyanobacterial photosystems and cyanobacterial photosynthesis. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00014-0 © 2020 Elsevier Inc. All rights reserved.

231

232  PART | IV  Algal photosystems and photosynthesis

14.3 Results 14.3.1  The research landscape There have been 15 prolific authors with 2 or more pioneering papers researching the cyanobacterial photosystems and photosynthesis. Ten, 2, 2, and one of them have been from Germany, Australia, the US, and Japan, respectively. These are ‘Wolfram Saenger’ and ‘Olaf Klukas’ of Free University Berlin; ‘Petra Fromme’, ‘Horst Tobias Witt’, ‘Jan Kern’, and ‘Athina Zouni’ of Technical University Berlin; ‘Norbert Krauss’ of Humboldt University Berlin; ‘Wolfram Bode’, ‘Tilman Schirmer’, and ‘Robert Huber’ of Max Planck Institute from Germany. The others are ‘Murray R Badger’ and ‘G Dean Price’ of Australian National University from Australia; ‘Alexander N. Glazer’ of University of California Berkeley and ‘Aaron Kaplan’ of Carnegie Institution of Washington from the US; and ‘Jian-Ren Shen’ of RIKEN from Japan. In total, 104 authors have contributed to these papers. There have been 8 prolific institutions with 2 or more pioneering papers researching this field. Three, 2, 2, and one institutions have been from Germany, the US, Japan, and Australia, respectively. These are. ‘Free University of Berlin’, ‘Technical University of Berlin’, and ‘Max Planck Institute’ from Germany; ‘University of California Berkeley’ and ‘Carnegie Institution Washington’ from the US; ‘Japan Science Technology Agency’ and RIKEN from Japan; and ‘Australian National University’ from Australia. In total, 44 institutions have contributed to these papers. There have been 7 journals publishing 2 or more pioneering papers in this field. These are ‘Nature’, ‘Science’, ‘Annual Review of Plant Physiology and Plant Molecular Biology’, ‘Journal of Experimental Botany’, ‘Journal of Molecular Biology’, ‘Microbiology and Molecular Biology Reviews’, and ‘Proceedings of the National Academy of Sciences of the United States of America’. It is notable that the first 2 journals have published 9 papers. In total, 21 journals have published these papers. There have been 5 countries publishing 2 or more pioneering papers in this field. These are the US, Germany, Japan, Australia, and United Kingdom. The US and Germany have published 14 and 9 papers, respectively. In total, 13 countries have published these papers. There have been 14, 9, 8, and 3 papers published in the 2000s and 1990s, 1980s, and 2010s, respectively. The mostprolific years have been 1999 and 2003 with 3 papers each. There have been 8 prolific subject categories indexing 2 or more of these papers. These are ‘Biochemistry Molecular Biology’, ‘Multidisciplinary Sciences’, ‘Plant Sciences’, ‘Biophysics’, ‘Cell Biology’, ‘Microbiology’, ‘Biotechnology Applied Microbiology’, and ‘Marine Freshwater Biology’. It is notable that the first 4 subject categories have indexed 35 papers. In total, 11 categories have indexed these papers. The reviews have been overrepresented with 12 papers. Only 8 papers have declared any research funding in their abstract pages. There have been 20,545 citations to these papers, in total where the average number of citations per paper has been 623 and H-index has been 33.

14.3.2  The pioneering research on the cyanobacterial photosystems There have been 11 pioneering studies regarding cyanobacterial photosystems, PSI and PSII (Table 14.1). Ferreira et al. (2004) study the structure of photosystem II (PSII) of the Thermosynechococcus elongatus at 3.5 A resolution focusing on the structure of the oxygen-evolving center (OEC) in a paper with 2440 citations. They assigned most of the amino acid residues of this 650-kilodalton dimeric multisubunit complex and refined the structure to reveal its molecular architecture. They describe details of the binding sites for cofactors and propose a structure of the OEC. They find that the OEC contains a cubane-like Mn3CaO4 cluster linked to a fourth Mn by a mono-μ-oxo bridge. They then discuss the details of the surrounding coordination sphere of the metal cluster and the implications for a possible oxygen-evolving mechanism. Jordan et al. (2001) study the crystal structure of photosystem I (PSI) of the Synechococcus elongatus at 2.5 A resolution in a paper with 1678 citations. They provide a picture of this structure at atomic detail of 12 protein subunits and 127 cofactors comprising 96 chlorophylls, 2 phylloquinones, 3 Fe4S4 clusters, 22 carotenoids, 4 lipids, a putative Ca2+ ion and 201 water molecules. They note that the structural information on the proteins and cofactors and their interactions provides a basis for understanding how the high efficiency of photosystem I in light capturing and electron transfer is achieved. Zouni et al. (2001) study the crystal structure of PSII from Synechococcus elongatus at 3.8 A on the basis of crystals fully active in water oxidation in a paper with 1583 citations. The structure shows how protein subunits and cofactors are spatially organized. They assign the larger subunits and define the locations and orientations of the cofactors. They also provide information on the position, size and shape of the manganese cluster, which catalyzes water oxidation.

The pioneering research on the cyanobacterial photosystems and photosynthesis Chapter | 14  233



TABLE 14.1  The research on the cyanobacterial photosystems. Cyanobacteria

Key topics

Secondary topics

Cits.

References

1

Thermosynechococcus elongatus

PSII

Structure of the oxygenevolving center (OEC)

2440

Ferreira et al. (2004)

2

Synechococcus elongatus

PSI

Proteins and cofactors structure

1678

Jordan et al. (2001)

3

Synechococcus elongatus

PSII

Protein subunits and cofactors structure

1583

Zouni et al. (2001)

4

Cyanobacteria

PSII

Complete cofactor arrangement

1428

Loll et al. (2005)

5

Thermosynechococcus vulcanus

PSII

Mechanism of PSII reactions

899

Kamiya and Shen (2003)

6

Thermosynechococcus elongatus

PSII

Role of quinones, lipids, channels and chloride

819

Guskov et al. (2009)

7

Synechocystis-6803

PSII

Photosynthetic reaction center

772

Williams (1988)

8

Thermosynechococcus vulcanus

PSII

Mechanism of oxygen evolution

502

Suga et al. (2015)

9

Synechococcus sp.

PSI

PSI structure

334

Krauss et al. (1993)

10

Synechococcus elongatus

PSI

Photosynthetic reaction centre and core antenna system

322

Krauss et al. (1996)

11

Synechococcus sp.

PSI PSII

Nacl-induced inactivation of PSI and PSII

300

Allakhverdiev et al. (2000)

Loll et al. (2005) study the complete cofactor arrangement in the 3.0 A resolution structure of PSII to understand the processes that convert light to chemical energy in a paper with 1428 citations. They describe the most complete cyanobacterial PSII structure showing locations of and interactions between 20 protein subunits and 77 cofactors per monomer. They note that the assignment of 11 β-carotenes yields insights into electron and energy transfer and photoprotection mechanisms in the reaction center and antenna subunits. The high number of 14 integrally bound lipids reflects the structural and functional importance of these molecules for flexibility within and assembly of PSII. They propose a lipophilic pathway for the diffusion of secondary plastoquinone that transfers redox equivalents from PSII to the photosynthetic chain. The structure provides information about the Mn4Ca cluster. Kamiya and Shen (2003) study the crystal structure of PSII from Thermosynechococcus vulcanus at 3.7-A resolution focusing on the mechanism of PSII reactions in a paper with 899 citations. The present structure was built on the basis of the sequences of PSII large subunits D1, D2, CP47, and CP43; extrinsic 33- and 12-kDa proteins and cytochrome c550; and several low molecular mass subunits. This yielded much information concerning the molecular interactions within this large protein complex. They then show the arrangement of chlorophylls and cofactors, including two beta-carotenes recently identified in a region close to the reaction center. They further determine possible ligands for the Mn cluster. In particular, they show that the C terminus of D1 polypeptide was connected to the Mn cluster directly. Guskov et al. (2009) study the Thermosynechococcus elongatus PSII at 2.9-A resolution focusing on the role of quinones, lipids, channels and chloride in a paper with 819 citations. They assign all 20 protein subunits and model all 35 chlorophyll a molecules and 12 carotenoid molecules, 25 integral lipids and 1 chloride ion per monomer. The presence of a third plastoquinone QC and a second plastoquinone-transfer channel suggests mechanisms for plastoquinol-plastoquinone exchange, and they calculate other possible water or dioxygen and proton channels. Putative oxygen positions obtained from a Xenon derivative indicate a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. The chloride position suggests a role in proton-transfer reactions because it is bound through a putative water molecule to the Mn4Ca cluster at a distance of 6.5 A and it is close to two possible proton channels.

234  PART | IV  Algal photosystems and photosynthesis

Williams (1988) studies specific mutations in PSII photosynthetic reaction center by genetic engineering methods in Synechocystis-6803 in a paper with 772 citations. He develops a genetic technique for the molecular analysis of electron transport in the PSII reaction center. This methodology permits specific PSII genes to be deleted from the cyanobacterial genome. The deleted genes can then be replaced with copies modified by site-directed mutagenesis. In this way, specific amino acid changes can be engineered in the polypeptides that bind the pigments and electron carriers in the PSII protein complex. He notes that this experimental system depends on two important characteristics of Synechocystis 6803: a naturally occurring genetic transformation system and the ability to grow photo-heterotrophically on glucose. Suga et al. (2015) study the structure of PSII of Thermosynechococcus vulcanus at 1.95 A resolution viewed by femtosecond X-ray pulses to provide a structural basis for the mechanism of oxygen evolution in a paper with 502 citations. They report a ‘radiation-damage-free’ structure of PSII from T. vulcanus in the S1 state and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from X-ray diffraction, they find that the oxygen-evolving center (OEC) in the X-ray free electron laser structure has Mn-Mn distances that are shorter by 0.1–0.2 A. The valences of each manganese atom were tentatively assigned as Mn1D(iii), Mn2C(iv), Mn3B(iv) and Mn4A(iii), based on the average Mn-ligand distances and analysis of the Jahn-Teller axis on Mn(iii). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo‑oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion. Krauss et al. (1996) study the PSI structure of Synechococcus elongatus at 4 A focusing on the photosynthetic reaction center and core antenna system in a paper with 322 citations. The X-ray structure model of trimeric PSI of the S. elongatus reveals 31 transmembrane, 9 surface and 3 stromal α-helices per monomer, assigned to the 11 protein subunits: PsaA and PsaB are related by a pseudo two-fold axis normal to the membrane plane, along which the electron transfer pigments are arranged, 65 antenna chlorophyll a (Chi a) molecules separated by less than or equal to 16 A form an oval, clustered net, continuous with the electron transfer chain through the second and third Chi a pairs of the electron transfer system, This suggests a dual role for these Chi a both in excitation energy and electron transfer. The architecture of the protein core indicates quinone and iron‑sulfur type reaction centers to have a common ancestor. Krauss et al. (1993) study the PSI structure of Synechococcus sp. at 6A resolution in a paper with 334 citations. X-ray structure analysis shows that the monomer of trimeric photosystem I (PS I) of S. sp. consists of a catalytic domain and a smaller domain that connects the monomers. The 4Fe-4S clusters FX, FA and FB, 28 α-helices and 45 chlorophyll a molecules were located. The two large subunits of PS I are represented by 9 α-helices each; they are related by a local 2-fold rotation axis passing through FX. Electron densities close to this axis are interpreted as carriers of the electron transfer chain.

14.3.3  The pioneering research on the cyanobacterial photosynthesis There have been 22 pioneering studies regarding cyanobacterial photosynthesis (Table 14.2). Partensky et al. (1999) discuss Prochlorococcus in a review paper with 851 citations. Prochlorococcus is the smallest known photosynthetic organism with its tiny size (0.5 to 07 μm in diameter) and it is presumably the most abundant photosynthetic organism on Earth. Prochlorococcus typically divides once a day in the subsurface layer of oligotrophic areas, where it dominates the photosynthetic biomass. It also possesses a remarkable pigment complement which includes divinyl derivatives of chlorophyll a (Chl a) and Chl b, the so-called Chl a2 and Chl b2. Its tiny size is an advantage for its adaptation to nutrient-deprived environments. Furthermore, genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, are present at depth and in surface waters. This vertical species variation has allowed Prochlorococcus to adapt to the natural light gradient occurring in the upper layer of oceans. Giordano et al. (2005) discuss CO2 concentrating mechanisms (CCMs) in the proximity of RUBISCO in cyanobacteria focusing on the mechanisms, environmental modulation, and evolution in a review paper with 724 citations. They note that modulating the CCMs may be crucial in the energetic and nutritional budgets of a cell, and a multitude of environmental factors can exert regulatory effects on the expression of the CCM components. They detail the diversity of CCMs, their evolutionary origins, and the role of the environment in CCM modulation. Summons et al. (1999) study the 2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis in a paper with 574 citations. They show that 2-methylbacteriohopanepolyols occur in a high proportion of cultured cyanobacteria and cyanobacterial mats. Their 2-methylhopane hydrocarbon derivatives are abundant in organic-rich sediments as old as 2500 Myr. They note that these biomarkers may help constrain the age of the oldest cyanobacteria and the advent of oxygenic photosynthesis. They could also be used to quantify the ecological importance of cyanobacteria through geological time. Kurisu et al. (2003) study the structure of the cytochrome b6f complex of oxygenic photosynthesis in Mastigocladus laminosus in a paper with 488 citations. A 3.0 A crystal structure of the dimeric b6f complex reveals a large quinone

The pioneering research on the cyanobacterial photosystems and photosynthesis Chapter | 14  235



TABLE 14.2  The research on the cyanobacterial photosynthesis. Cyanobacteria

Key topics

Secondary topics

Cits.

References

1

Prochlorococcus

Photosynthetic organism

Structure and properties

851

Partensky et al. (1999)

2

Cyanobacteria

CO2 concentrating mechanisms

Mechanisms, environmental modulation, and evolution

724

Giordano et al. (2005)

3

Cyanobacteria

Oxygenic photosynthesis

2-Methylhopanoids as biomarkers

502

Summons et al. (1999)

4

Mastigocladus laminosus

Cytochrome b6f complex

Structure of the cytochrome b6f complex

488

Kurisu et al. (2003)

5

Cyanobacteria

CO2 concentrating mechanisms

Organization of the carboxysomes

467

Kaplan and Reinhold (1999)

6

Cyanobacteria

Photosynthesis and acclimation

Chlorophyll fluorescence analysis

464

Campbell et al. (1998)

7

Synechococcus elongatus

CO2 biomitigation

Isobutyraldehyde and isobutanol production

444

Atsumi et al. (2009)

8

Cyanobacteria

CO2 concentrating mechanisms

Molecular components, their diversity and evolution

436

Badger and Price (2003)

9

Cyanobacteria

Light harvesting

Light harvesting by phycobilisomes

432

Glazer (1985)

10

Cyanobacteria

Light energy transfer

Role of phycobilisomes in light energy transfer

428

Glazer (1984)

11

Cyanobacteria

Light energy transfer

Role of phycobilisomes in light energy transfer

397

Gantt (1981)

12

Cyanobacteria

Photoacclimation

Photoacclimation of photosynthesis irradiance response (PE) curves

396

MacIntyre et al. (2002)

13

Cyanobacteria

Light harvesting

Complementary chromatic adaptation

395

Grossman et al. (1993)

14

Cyanobacteria

Light sensory system

Signal transduction by phytochrome

392

Yeh et al. (1997)

15

Cyanobacteria

Light harvesting

Complementary chromatic adaptation

376

MacColl (1998)

16

Cyanobacteria

CO2 concentrating mechanisms

Functional components, Ci transporters, diversity, genetic regulation

333

Price et al. (2008)

17

Mastigocladus laminosus

Light harvesting

Structure of the C-phycocyanin

319

Schirmer et al. (1985)

18

Anabaena variabilis

Photosynthesis

Photosynthesis and the intracellular inorganic carbon pool

307

Kaplan et al. (1980)

19

Anabaena, aphanizomenon, microcystis, oscillatoria

Photosynthetic capacity

Photosynthetic capacity, respiration, and growth-rates

306

Robarts and Zohary (1987)

20

Synechocystis

CO2 biomitigation

Isoprene generation

303

Lindberg et al. (2010)

21

Prochlorococcus

Oxygenic photosynthesis

Transfer of photosynthesis genes

300

Lindell et al. (2004)

22

Mastigocladus laminosus, Agmenellum quadruplicatum

Light harvesting

Structure of the C-phycocyanin

300

Schirmer et al. (1987)

236  PART | IV  Algal photosystems and photosynthesis

e­ xchange cavity, stabilized by lipid, in which plastoquinone, a quinone-analog inhibitor, and a novel heme are bound. The core of the b6f complex is similar to the analogous respiratory cytochrome bc1 complex, but the domain arrangement outside the core and the complement of prosthetic groups are strikingly different. The motion of the Rieske iron‑sulfur protein extrinsic domain, essential for electron transfer, must also be different in the b6f complex. Kaplan and Reinhold (1999) discuss CO2 concentrating mechanisms in cyanobacteria in a review paper with 467 ­citations. They note that the organization of the carboxysomes in prokaryotes and of the pyrenoids in eukaryotes, and the presence of membrane mechanisms for inorganic carbon (Ci) transport, are central to the concentrating mechanism. The presence of multiple Ci transporting systems in cyanobacteria has been indicated. Certain genes involved in structural organization, Ci transport and the energization of the latter have been identified. Massive Ci fluxes associated with the CCM have wide-reaching ecological and geochemical implications. Campbell et al. (1998) discuss the chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation in a review paper with 464 citations. They consider how the special properties of cyanobacteria can be accommodated and used to extract biologically useful information from cyanobacterial in vivo chlorophyll fluorescence signals. They describe how the pattern of fluorescence yield versus light intensity can be used to predict the acclimated light level for a cyanobacterial population. The size of the change in fluorescence yield during dark-to-light transitions can provide information on respiration and the iron status of the cyanobacteria. Finally, fluorescence parameters can be used to estimate the electron transport rate at the acclimated growth light intensity. Atsumi et al. (2009) study the direct bioconversion of CO2 to isobutyraldehyde and isobutanol in a paper with 444 citations. They genetically engineer Synechococcus elongatus PCC7942 to produce isobutyraldehyde and isobutanol directly from CO2 and increase productivity by overexpression of ‘ribulose 1,5-bisphosphate carboxylase/oxygenase’ (RUBISCO). Isobutyraldehyde is a precursor for the synthesis of other chemicals, and isobutanol can be used as a gasoline substitute. The high vapor pressure of isobutyraldehyde allows in situ product recovery and reduces product toxicity. The engineered strain remained active for 8 d and produced isobutyraldehyde at a higher rate than those reported for ethanol, hydrogen or lipid production by cyanobacteria or algae. Badger and Price (2003) discuss CO2 concentrating mechanisms in cyanobacteria focusing on the molecular components, their diversity and evolution in a review paper with 436 citations. Cyanobacteria have evolved an extremely effective single-cell CO2 concentrating mechanism. Recent molecular, biochemical and physiological studies have significantly extended current knowledge about the genes and protein components of this system and how they operate to elevate CO2 around RUBISCO during photosynthesis. The CCM components include at least 4 modes of active inorganic carbon ­uptake, including 2 bicarbonate transporters and 2 CO2 uptake systems associated with the operation of specialized NDH-1 complexes. There have been some surprising findings. Firstly, cyanobacteria have evolved two types of carboxysomes, correlated with the form of RUBISCO present (Form 1A and 1B). Secondly, the two HCO3-and CO2 transport systems are distributed variably, with some cyanobacteria appearing to lack CO2 uptake systems entirely. Finally, there are multiple carbonic anhydrases (CA) in many cyanobacteria, but, surprisingly, several cyanobacterial genomes lack any identifiable CA genes. Glazer (1985) discusses light harvesting by phycobilisomes in a review paper with 432 citations. He details phycobilisomes, their components, structures of bilins, biliproteins, and structure-property relationships in phycobilisomes. Glazer (1984) discusses phycobilisomes with a focus on their role in light energy transfer in a review paper with 428 citations. He details structure, composition, and properties of biliproteins, morphology, composition, structure, and assembly of phycobilisomes, and role of phycobiliomes in energy transfer. Gantt (1981) discusses phycobilisomes in a review paper with 397 citations. He details composition, morphology, structure, and assembly of phycobilisomes, energy transfer and phycobiliprotein synthesis. MacIntyre et al. (2002) study the photoacclimation of photosynthesis irradiance response (PE) curves and photosynthetic pigments in cyanobacteria in a review paper with 396 citations. They note that the chl a-specific initial slope (αchl) declines as irradiance increases in the cyanobacteria where phycobiliproteins dominate light absorption because of plasticity in the phycobiliprotein:chl a ratio. By definition, light-saturated photosynthesis (Pm) is limited by a factor other than the rate of light absorption. Within species, Pm C is independent of growth irradiance. Among species, Pm C covaries with the resource-saturated growth rate. The chl a:C ratio is a key physiological variable because the appropriate currencies for normalizing light-limited and light-saturated photosynthetic rates are, respectively, chl a and carbon. Typically, chl a:C is reduced to about 40% of its maximum value at an irradiance that supports 50% of the species-specific maximum growth rate and light-harvesting accessory pigments show similar or greater declines. In the steady state, this downregulation of pigment content prevents cyanobacteria from maximizing photosynthetic rates throughout the light-limited region for growth. They argue that maximizing the rate of photosynthetic carbon assimilation is not the only criterion governing photoacclimation.



The pioneering research on the cyanobacterial photosystems and photosynthesis Chapter | 14  237

Grossman et al. (1993) discuss complementary chromatic adaptation in cyanobacteria in a review paper with 395 citations. They note that the phycobilisome is not an integral membrane complex but is attached to the surface of the photosynthetic membranes. It is composed of both the pigmented phycobiliproteins and the nonpigmented linker polypeptides; the former are important for absorbing light energy, while the latter are important for stability and assembly of the complex. The composition of the phycobilisome is very sensitive to a number of different environmental factors. Some of the filamentous cyanobacteria can alter the composition of the phycobilisome in response to the prevalent wavelengths of light in the environment. This process, ‘complementary chromatic adaptation’, allows these organisms to efficiently utilize available light energy to drive photosynthetic electron transport and CO2 fixation. Under conditions of macronutrient limitation, many cyanobacteria degrade their phycobilisomes in a rapid and orderly fashion. Since the phycobilisome is an abundant component of the cell, its degradation may provide a substantial amount of nitrogen to nitrogen-limited cells. Furthermore, degradation of the phycobilisome during nutrient-limited growth may prevent photodamage that would occur if the cells were to absorb light under conditions of metabolic arrest. Yeh et al. (1997) discuss a cyanobacterial phytochrome two-component light sensory system in a review paper with 393 citations. Phytochrome is an ancient molecule that evolved from a more compact light sensor in cyanobacteria. The cyanobacterial phytochrome Cph1 is a light-regulated histidine kinase that mediates red, far-red reversible phosphorylation of a small response regulator, Rcp1, response regulator for cyanobacterial phytochrome, encoded by the adjacent gene, thus implicates protein phosphorylation-dephosphorylation in the initial step of light signal transduction by phytochrome. MacColl (1998) discusses cyanobacterial phycobilisomes in a review paper with 376 citations. Cyanobacterial phycobilisomes harvest light and cause energy migration usually towards PSII reaction centers. Energy transfer from phycobilisomes directly to PSI may occur under certain light conditions. The phycobilisomes are highly organized complexes of various biliproteins and linker polypeptides. Phycobilisomes are composed of rods and a core. The biliproteins have their bilins (chromophores) arranged to produce rapid and directional energy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane. The modulation of the energy levels of the 4 chemically different bilins by a variety of influences produces more efficient light harvesting and energy migration. Acclimation of cyanobacterial phycobilisomes to growth light by ‘complementary chromatic adaptation’ is a complex process that changes the ratio of phycocyanin to phycoerythrin in rods of certain phycobilisomes to improve light harvesting in changing habitats. The Linkers govern the assembly of the biliproteins into phycobilisomes, and in certain cases they have improve the energy migration process. The Lcm polypeptide has several functions, including the linker function of determining the organization of the phycobilisome cores. Price et al. (2008) discuss cyanobacterial CO2 concentrating mechanism (CCM) focusing on the functional components, Ci transporters, diversity, and genetic regulation in a review paper with 333 citations. In cyanobacteria, RUBISCO is encapsulated in unique micro-compartments known as carboxysomes. Cyanobacteria can possess up to 5 distinct transport systems for Ci uptake. Through database analysis of some 33 complete genomic DNA sequences for cyanobacteria considerable diversity exists in the composition of transporters employed. In addition, 2 types of carboxysomes are known within the cyanobacteria that have apparently arisen by parallel evolution, and considerable progress has been made towards understanding the proteins responsible for carboxysome assembly and function. Progress has also been made towards identifying the primary signal for the induction of the subset of CCM genes known as CO2-responsive genes, and transcriptional regulators CcmR and CmpR regulate these genes. Schirmer et al. (1985) study the structure of the C-phycocyanin from Mastigocladus laminosus in a paper with 319 citations. They determine the structure of the biliprotein C-phycocyanin from M. laminosus at 3 A resolution by X-ray diffraction methods. They find that the protein consists of 3 identical (αβ)-units which are arranged around a 3-fold symmetry axis to form a disc of approximate dimensions 110 Å × 30 Å with a central channel of 35 Å in diameter. Both subunits, α and β, exhibit a similar structure and are related by a local 2-fold rotational axis. Each subunit is folded into 8 helices and irregular loops. Six helices are arranged to form a globular part, whereas 2 helices stick out and mediate extensive contact between the subunits. The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent Cα-atoms have a root-mean-square deviation of 2.9 Å. Kaplan et al. (1980) study the photosynthesis and the intracellular inorganic carbon pool in the Anabaena variabilis in response to external CO2 concentration in a paper with 307 citations. The photosynthetic affinity of A. variabilis to CO2 is greatly affected by the CO2 concentration in the medium during growth. Both high- and low-CO2-grown Anabaena accumulate Cinorg within the cell. However, the rate of accumulation and the steady-state internal Cinorg concentration reached is much higher in low as compared with high-CO2-grown cells. Anabaena cells actively accumulate Cinorg. The affinity of the transport mechanism for Cinorg is similar in both high- and low-CO2-grown cells. However, Vmax is 10-fold higher in the latter case. This higher Vmax for transport is the basis of the superior capability to accumulate Cinorg and the higher apparent photosynthetic affinity for external Cinorg in low-CO2-grown Anabaena.

238  PART | IV  Algal photosystems and photosynthesis

Robarts and Zohary (1987) study the temperature effects on photosynthetic capacity, respiration, and growth-rates of cyanobacteria in a paper with 306 citations. They determine the direct temperature effects on photosynthetic capacity (Pmax), specific respiration rate (Rest), and growth rate of Anabaena, Aphanizomenon, Microcystis, and Oscillatoria and assess the importance of direct temperature effects on cyanobacterial dominance in lakes. Pmax, Rest, and growth rate are temperature-dependent with optima usually at 25 °C or greater. The 4 genera varied in their response to low temperatures with Microcystis being most severely limited below about 15 °C. Oscillatoria tended to tolerate the widest range of temperatures. The direct temperature effects were secondary to indirect temperature effects (mixing) and nutrients in determining the dominance of bloom-forming cyanobacteria in lakes, direct temperature effects act synergistically with other factors in this process. Lindberg et  al. (2010) study the photosynthetic isoprene production in Synechocystis in a paper with 303 citations. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO2, without a prior requirement for the harvesting, dewatering and processing of the respective biomass. Lindell et al. (2004) study the transfer of photosynthesis genes to and from Prochlorococcus viruses in a paper with 300 citations. They report the presence of genes central to oxygenic photosynthesis in the genomes of 3 phages from 2 viral families (Myoviridae and Podoviridae) that infect Prochlorococcus. The genes that encode the PSII core reaction center protein D1 (psbA), and a high-light-inducible protein (HLIP) (hli) are present in all three genomes. Both myoviruses contain additional hli gene types, and one of them encodes the second PSII core reaction center protein D2 (psbD), whereas the other encodes the photosynthetic electron transport proteins plastocyanin (petE) and ferredoxin (petF). These uninterrupted, full-length genes are conserved in their amino acid sequence, suggesting that they encode functional proteins that may help maintain photosynthetic activity during infection. Phage D1, D2, and HLIP proteins cluster with those from Prochlorococcus, indicating that they are of cyanobacterial origin. The genes encoding these proteins were transferred from host to phage multiple times. Phage may be mediating the expansion of the hli gene family by transferring these genes back to their hosts after a period of evolution in the phage. Allakhverdiev et  al. (2000) study the ionic and osmotic effects of NaCl-induced inactivation of PSI and PSII in Synechococcus sp. in a paper with 300 citations. They report that osmotic effects and ionic effects are both involved in the NaCl-induced inactivation of the photosynthetic machinery in S. sp. PCC 7942. Incubation of the cyanobacterial cells in 0.5 M NaCl induced a rapid and reversible decline and subsequent slow and irreversible loss of the oxygen-evolving activity of PSII and the electron transport activity of PSI. An Na+-channel blocker protected both PSII and PSI against the slow, but not the rapid, inactivation. The presence of both a Na+-channel blocker and a water-channel blocker protected PSI and PSII against the short- and long-term effects of NaCl. Salt stress also decreased cytoplasmic volume and this effect was enhanced by the Na+-channel blocker. NaCl had both osmotic and ionic effects. The osmotic effect decreased the amount of water in the cytosol, rapidly increasing the intracellular concentration of salts. The ionic effect was caused by an influx of Na+ ions through potassium/Na+ channels that also increased concentrations of salts in the cytosol and irreversibly inactivated PSI and PSII. Schirmer et al. (1987) study the crystal structure of two C-phycocyanins (CPC) from Mastigocladus laminosus and Agmenellum quadruplicatum at 2.1 and 2.5 A resolution, respectively, focusing on the phycobilin-protein interaction in a paper with 300 citations. The 2C-PC structures are very similar, 213 Cα positions have a root-mean-square deviation of 0.49 Å. All 3 chromophores are completely defined and their tetrapyrroles exhibit very similar geometry. The structure of a C-PC chromophore resembles a cleaved porphyrin which has been twisted roughly 180 degrees around the C-5C-6 and C-14C-15 bonds. Accordingly, the configuration/conformation of the chromophores is Z-anti, Z-syn, Z-anti (with the exception of the ‘configuration’ of C-15 of chromophore B155, which is almost midway between Z and E). They conclude that hexameric aggregates are probably the basic functional units, and that interhexameric energy transfer takes place preferentially via the central B84 chromophores.

14.4 Discussion 14.4.1  The research landscape The section on the research landscape highlights the most-prolific authors, institutions, journals, countries, publication years, and subject categories putting the pioneering research in a context.



The pioneering research on the cyanobacterial photosystems and photosynthesis Chapter | 14  239

The authors and institutions from Germany, the US, Japan, and Australia have primarily contributed to the pioneering research in this field. ‘Nature’, ‘Science’, ‘Annual Review of Plant Physiology and Plant Molecular Biology’, and ‘Journal of Experimental Botany’ have been the primary research outlets. The US and Germany have published 14 and 9 papers, respectively. The data on the publication years show that this research field has progressively developed over the last four decades. It is notable that most of these papers have been indexed by the subject categories of ‘Biochemistry Molecular Biology’, ‘Multidisciplinary Sciences’, ‘Plant Sciences’, ‘Biophysics’, and ‘Cell Biology’ highlighting the biological science emphasis of the research. The citation impact of these papers has been relatively significant with 4216 20,545 citations in total, 623 citations per paper and H-index of 33. There have been 11 and 22 pioneering papers related to the cyanobacterial photosystems and photosynthesis, respectively.

14.4.2  The pioneering research on the cyanobacterial photosystems The research on the cyanobacterial photosystems, PS1 and PSII, has formed a significant part of the research in this field with 11 papers (Table 14.1). The research in this field has focused on the PSII with 9 papers. These papers have studied the structure of the oxygenevolving center (Ferreira et al., 2004), protein subunits and cofactors structure (Zouni et al., 2001), complete cofactor arrangement (Loll et al., 2005), mechanism of PSII reactions (Kamiya and Shen, 2003), role of quinones, lipids, channels and chloride (Guskov et al., 2009), photosynthetic reaction center (Williams, 1988), mechanism of oxygen evolution (Suga et al., 2015), and NaCl-induced inactivation of PSI and PSII (Allakhverdiev et al., 2000). Additionally, studies on the PSI have focused on the proteins and cofactors structure (Jordan et al., 2001), photosynthetic reaction center and core antenna system (Krauss et al., 1996), PSI structure (Krauss et al., 1993), and NaCl-induced inactivation of PSI and PSII (Allakhverdiev et al., 2000). These studies have focused on the photosystems of Synechococcus elongates (Jordan et al., 2001; Krauss et al., 1996; Zouni et al., 2001), Synechococcus sp. (Allakhverdiev et al., 2000; Krauss et al., 1993), Thermosynechococcus elongates (Ferreira et al., 2004; Guskov et al., 2009), and Thermosynechococcus vulcanus (Kamiya and Shen, 2003; Suga et al., 2015). Thus, the pioneering research on the cyanobacterial photosystems has provided ample evidence for the structure of these photosystems. The optimization of methodology for the determination of these structures emerges as a strategic point in these studies.

14.4.3  The pioneering research on the cyanobacterial photosynthesis The research on the cyanobacterial photosynthesis has formed a significant part of the research in this field with 22 papers (Table 14.2). There have been a number of research streams in this field: CO2 concentrating mechanisms (Badger and Price, 2003; Giordano et al., 2005; Kaplan and Reinhold, 1999; Price et al., 2008); light energy transfer (Gantt, 1981; Glazer, 1984); light harvesting (Glazer, 1985; Grossman et al., 1993; MacColl, 1998; Schirmer et al., 1985, 1987); oxygenic photosynthesis (Lindell et al., 2004; Summons et al., 1999); and CO2 biomitigation (Atsumi et al., 2009; Lindberg et al., 2010). The other topics have been cytochrome b6f complex (Kurisu et al., 2003), light sensory system (Yeh et al., 1997), photoacclimation (MacIntyre et al., 2002); photosynthesis (Kaplan et al., 1980); and photosynthesis and acclimation (Campbell et al., 1998). Although, most of these studies study cyanobacteria in general, some studies have focused on the specific cyanobacterial species: Anabaena (Kaplan et al., 1980; Robarts and Zohary, 1987); Mastigocladus (Kurisu et al., 2003; Schirmer et al., 1985, 1987); Prochlorococcus (Lindell et al., 2004; Partensky et al., 1999). The other studies have studied photosynthesis of Synechococcus (Atsumi et al., 2009) and Synechocystis (Lindberg et al., 2010). Thus, these pioneering studies have focused on the specific topics relevant to photosynthesis, forming the background for the applied studies in the production of bioenergy and biofuels as well as biomedical products among others (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a,b,c,d,e,f,g,h, 2017a,b,c,d,e,f, 2019, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n).

14.5 Conclusion This study of the pioneering research on the cyanobacterial photosystems and photosynthesis at the global scale covering the whole range of research fronts as well as all types of cyanobacteria has provided the ample data for the primary

240  PART | IV  Algal photosystems and photosynthesis

s­ takeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. The research in this field have progressed in two subfields: cyanobacterial photosystems and photosynthesis with 11 and 22 papers, respectively (Tables 14.1 and 14.2). These papers related to PSII have studied the structure of the oxygen-evolving center, protein subunits and cofactors structure, complete cofactor arrangement, mechanism of PSII reactions, role of quinones, lipids, channels and chloride, photosynthetic reaction center, mechanism of oxygen evolution, and NaCl-induced inactivation of PSI and PSII. Additionally, studies on the PSI have focused on the proteins and cofactors structure, photosynthetic reaction center and core antenna system, and PSI structure. There have been a number of research streams in the field of cyanobacterial photosynthesis: CO2 concentrating mechanisms, light energy transfer, light harvesting, oxygenic photosynthesis, and CO2 biomitigation. The other topics have been cytochrome b6f complex, light sensory system, photoacclimation, photosynthesis, photosynthesis and acclimation. Although some studied have focused on cyanobacteria general, there has also been a specific focus on cyanobacterial species: Synechococcus, Thermosynechococcus for photosystems and Anabaena, Mastigocladus, Prochlorococcus, Synechococcus, and Synechocystis for cyanobacterial photosynthesis. It has been found that the detailed keyword set provided in the appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. The citation impact of these pioneering studies has been significant, contributing to the wider research fields of energy and fuels, catalysts, PSII and PSI in general, phototransfer, proteins, ocean acidification, plant photosynthesis, plant physiology, and marine viruses among others. It is notable that the applications of nanotechnology in this field has not been significant. It is expected the applications of nanotechnology in this field would accelerate to further optimize the methodology for the determination of structures of photosystems and photosynthesis-related structures and processes (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e,f). It appears that the structure-processing-property relationships form the basis of the research in this field as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002). These pioneering studies with at least 300 citations each in this field have formed the basis for the rapid expansion of this research field with the promising results for the determination of the structures of photosystems and key research fronts in cyanobacterial photosynthesis. It is recommended that the complementary studies should be carried out in other related fields such as photosystems and photosynthesis of microalgae, diatoms, macroalgae, and dinoflagellates. It is further recommended that a full scientometric study of these fields should be carried out.

Appendix. The keyword sets A.1  Photosynthesis related keywords TI = (‘light harvest*’ or photosynth* or photorecep* or xanthophyll* or quench* or ‘electron transport’ or photosystem* or ‘carbonic anhydrase*’ or photoprotect* or photoinhibit* or ‘electron flow*’ or *cytochrom* or *plastocyanin* or polypeptide* or ferredoxin* or antenna* or ‘reaction center*’ or photodamage* or photoacclim* or photolyas* or photochrom* or photoreduction* or ‘light regulation*’ or nadp* or ‘carbon metabolism*’ or photota* or thioredoxin* or ‘oxygen evol*’ or hco3 or bicarbonate or peroxiredoxin* or carboxysome* or photoadapt* or ‘concentrating mechanism*’ or ndh or ‘light acclimation’ or ‘ps-ii’ or phytochrom* or ‘carbon acquisition’) OR SO = (‘Photosynthesis Research’ or photosynthetica*).

A.2  Cyanobacteria-related keywords TS = (‘blue green alga*’ or ‘blue-green alga*’ or cryptophycin* or cyanelle or *cyanobacter* or cyanophage* or cyanophycin* or cyanophyt* or cyanophycea* or glaucophyt*or nostocales or oscillatoriales or prochlorophyt* or Acaryochloris or *Anabaena or Anacystis or Aphanizomenon or Aphanothece or Arthrospira or Calothrix or Cyanophora or Cyanothece or Cylindrospermopsis or Glaucocystis or *Lyngbya* or Mastigocladus or Microcoleus or Microcystis or Moorea or Nodularia or Nostoc or Oscillatoria or Planktothrix or Plectonema or Prochlorococcus or Prochloron or Prochlorothrix or Scytonema or Spirulina or *Synechococcus or Synechocystis or Tolypothrix or Trichodesmium).



The pioneering research on the cyanobacterial photosystems and photosynthesis Chapter | 14  241

A.3  Cross-subject keywords TI = (phycobilisome* or cyanobacteriochrome*).

Acknowledgment The significant contributions of the authors of these pioneering studies in cyanobacterial photosystems and photosynthesis to the development of the research in in this field have been gratefully acknowledged.

References Allakhverdiev, S.I., Sakamoto, A., Nishiyama, Y., Inaba, M., Murata, N., 2000. Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp. Plant Physiol. 123 (3), 1047–1056. Atsumi, S., Higashide, W., Liao, J.C., 2009. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat. Biotechnol. 27 (12), 1177–1180. Badger, M.R., Price, G.D., 2003. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J. Exp. Bot. 54 (383), 609–622. Brennan, L., Owende, P., 2010. Biofuels from microalgae-a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14 (2), 557–577. Campbell, D., Hurry, V., Clarke, A.K., Gustafsson, P., Oquist, G., 1998. Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation. Microbiol. Mol. Biol. R. 62 (3), 667–683. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., Iwata, S., 2004. Architecture of the photosynthetic oxygen-evolving center. Science 303 (5665), 1831–1838. Gantt, E., 1981. Phycobilisomes. Annu. Rev. Plant Phys. 32, 327–347. Giordano, M., Beardall, J., Raven, J.A., 2005. CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Annu. Rev. Plant Biol. 56, 99–131. Glazer, A.N., 1984. Phycobilisome – a macromolecular complex optimized for light energy transfer. Biochim. Biophys. Acta 768 (1), 29–51. Glazer, A.N., 1985. Light harvesting by phycobilisomes. Annu. Rev. Biophys. Biol. 14, 47–77. Grossman, A.R., Schaefer, M.R., Chiang, G.G., Collier, J.L., 1993. The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbiol. Rev. 57 (3), 725–749. Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., Saenger, W., 2009. Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride. Nat. Struct. Mol. Biol. 16 (3), 334–342. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems – a review. Biotechnol. Adv. 29 (2), 189–198. Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W., Krauss, N., 2001. Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature 411 (6840), 909–917. Kamiya, N., Shen, J.R., 2003. Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-A resolution. Proc. Natl. Acad. Sci. USA 100 (1), 98–103. Kaplan, A., Badger, M.R., Berry, J.A., 1980. Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena-variabilis – response to external CO2 concentration. Planta 149 (3), 219–226. Kaplan, A., Reinhold, L., 1999. CO2 concentrating mechanisms in photosynthetic microorganisms. Annu. Rev. Plant Phys. 50, 539–570. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energ. 88 (10), 3532–3540. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244.

242  PART | IV  Algal photosystems and photosynthesis

Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2019. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam.



The pioneering research on the cyanobacterial photosystems and photosynthesis Chapter | 14  243

Konur, O., 2020m. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science. Technology and Medicine, Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Krauss, N., Hinrichs, W., Witt, I., Fromme, P., Pritzkow, W., Dauter, Z., et al., 1993. Three-dimensional structure of system-I of photosynthesis at 6 A resolution. Nature 361 (6410), 326–331. Krauss, N., Schubert, W.D., Klukas, O., Fromme, P., Witt, H.T., Saenger, W., 1996. Photosystem I at 4 A resolution represents the first structural model of a joint photosynthetic reaction centre and core antenna, system. Nat. Struct. Biol. 3 (11), 965–973. Kurisu, G., Zhang, H.M., Smith, J.L., Cramer, W.A., 2003. Structure of the cytochrome b6f complex of oxygenic photosynthesis: tuning the cavity. Science 302 (5647), 1009–1014. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Lindberg, P., Park, S., Melis, A., 2010. Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metab. Eng. 12 (1), 70–79. Lindell, D., Sullivan, M.B., Johnson, Z.I., Tolonen, A.C., Rohwer, F., Chisholm, S.W., 2004. Transfer of photosynthesis genes to and from Prochlorococcus viruses. Proc. Natl. Acad. Sci. USA 101 (30), 11013–11018. Loll, B., Kern, J., Saenger, W., Zouni, A., Biesiadka, J., 2005. Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II. Nature 438 (7070), 1040–1044. MacColl, R., 1998. Cyanobacterial phycobilisomes. J. Struct. Biol. 124 (2–3), 311–334. MacIntyre, H.L., Kana, T.M., Anning, T., Geider, R.J., 2002. Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria. J. Phycol. 38 (1), 17–38. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Partensky, F., Hess, W.R., Vaulot, D., 1999. Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol. Mol. Biol. R. 63 (1), 106–127. Price, G.D., Badger, M.R., Woodger, F.J., Long, B.M., 2008. Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. J. Exp. Bot. 59 (7), 1441–1461. Robarts, R.D., Zohary, T., 1987. Temperature effects on photosynthetic capacity, respiration, and growth-rates of bloom-forming cyanobacteria. N. Z. J. Mar. Fresh. 21 (3), 391–399. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes-towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Schirmer, T., Bode, W., Huber, R., 1987. Refined three-dimensional structures of two cyanobacterial C-phycocyanins at 2.1 and 2.5 Å resolution: a common principle of phycobilin-protein interaction. J. Mol. Biol. 196 (3), 677–695. Schirmer, T., Bode, W., Huber, R., Sidler, W., Zuber, H., 1985. X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures. J. Mol. Biol. 184 (2), 257–277. Suga, M., Akita, F., Hirata, K., Ueno, G., Murakami, H., Nakajima, Y., et al., 2015. Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature 517 (7532), 99–103. Summons, R.E., Jahnke, L.L., Hope, J.M., Logan, G.A., 1999. 2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis. Nature 400 (6744), 554–557. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Williams, J.G.K., 1988. Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Method. Enzymol. 167, 766–778. Yeh, K.C., Wu, S.H., Murphy, J.T., Lagarias, J.C., 1997. A cyanobacterial phytochrome two-component light sensory system. Science 277 (5331), 1505–1508. Zouni, A., Witt, H.T., Kern, J., Fromme, P., Krauss, N., Saenger, W., et al., 2001. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 A resolution. Nature 409 (6821), 739–743.

Chapter 15

Carbon and nitrogen metabolism in cyanobacteria: Basic traits, regulation and biotechnological application Marcelo Gomes Marçal Vieira Vaz, Allan Victor Martins Almeida, Naira Valle de Castro, Adriano Nunes-Nesi, Wagner L. Araújo Federal University of Viçosa, Viçosa, Brazil

15.1 Introduction Cyanobacteria, prokaryote microorganisms belonging to Bacteria domain, are one of the most ancient, versatile and ecologically successful group. They also show great morphological variation, ranging from unicellular to true-branching heterocytous filamentous forms (Castenholz et  al., 2001; Komarek et  al., 2014), as well as significant physiological and metabolic diversity (Schirrmeister et al., 2013; de Alvarenga et al., 2018; Genuario et al., 2019). The phylum Cyanobacteria is a phylogenetically coherent group characterized mainly by the ability of its members to perform oxygenic photosynthesis (Stanier and Cohen-Bazine, 1977; Giovannoni et al., 1988), despite of the occurrence of nonphotosynthetic strains, which have been characterized as obligate endosymbionts (Zehr et al., 2016). In addition, some cyanobacterial strains can use sulfide as electron donor during anoxygenic photosynthesis (Cohen et al., 1986) or even perform fermentation, under anoxic conditions or darkness (Stal and Moezelaar, 1997). It is important to mention that the recent description and characterization of two groups of nonphotosynthetic bacteria, Melainabacteria (di Rienzi et al., 2013; Soo et al., 2014) and Sericytochromatia (Soo et al., 2017), which have been considered as 'sibling phyla' to Cyanobacteria, added some confusion in the known and well-established cyanobacterial group. Soo et al. (2017) confined Melainabacteria and Sericytochromatia as classes into the phylum Cyanobacteria, in addition to the class Oxyphotobacteria, composed by oxygenic-photoautotrophic strains (true-cyanobacteria). However, the phylogenetic coherence of members of the phylum Cyanobacteria (Stanier et al., 1978; Woese, 1987; Giovannoni et al., 1988; Castenholz et al., 2001; Komarek et al., 2014) and photoautotrophy as the main mode of growth, despite of the usage of organic carbon source (Rippka et al., 1979; Nieves-Morion and Flores, 2018), must be recognized, as diacritical traits for delimitation of Cyanobacteria as a phylum harboring only the class Oxyphotobacteria (Herrero and Flores, 2019). Coupled to photoautotrophy, which relies on the fixation of the inorganic carbon (C) as CO2, via oxygenic photosynthesis (Zhang et al., 2018; Herrero and Flores, 2019), some cyanobacterial genera also perform the 'biological nitrogen fixation' (BNF) (Woese, 1987; Flores and Herrero, 2009; Esteves-Ferreira et al., 2017, 2018; Herrero and Flores, 2019). Accordingly, after CO2 fixation, BNF is the most important process in terms of ecosystem services, providing assimilable forms of nitrogen (N) for aquatic and terrestrial environments (Arp, 2000; Galloway, 2005). Thus, cyanobacteria are important players in the biogeochemical cycles of these elements (Knoll, 2008). N is also the most limiting nutrient in primary production (Zehr, 2011), since it restricts the fixation of atmospheric CO2 and is therefore a key element in the various trophic chains. In addition to BNF, cyanobacteria can assimilate inorganic N compounds, such as nitrite and nitrate, as well as simple organic compounds (Flores and Herrero, 2005). Notably, cyanobacterial genomes also encode for transporters of other N-containing organic compounds (such as amino acids), widening the range of N-substrates that can be up taken and assimilated (Hobbie and Hobbie, 2013; Herrero and Flores, 2019). Since studies dealing with organic compounds as C-sources are relatively scarce (Rippka et al., 1979; Nieves-Morion and Flores, 2018; Herrero and Flores, 2019), and considering atmospheric CO2 as the most abundant source of inorganic C, here our discussion regarding C-metabolism will be focused Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00015-2 © 2020 Elsevier Inc. All rights reserved.

245

246  PART | IV  Algal photosystems and photosynthesis

on CO2 as well as bicarbonate and carbonate concentration. Remarkably, the prevalence and metabolization of each of these C-inorganic compounds will depend on environmental physicochemical parameters, such as solubility of CO2 in water, which is temperature-dependent and pH values. Considering that a balanced C and N concentration/ratio is required for cyanobacterial growth (Zhang et al., 2018), many levels of regulation must be found in cells for controlling the uptake and assimilation of various C and N sources, according to their availability (Flores and Herrero, 2005; Burnap et al., 2015). Such regulation may occur ranging from the modulation of the activity of proteins related to nutrient assimilation to mechanisms controlling the expression of genes encoding for transcriptional factors, enzymes as well as structural proteins (Esteves-Ferreira et al., 2018; Zhang et al., 2018; Herrero and Flores, 2019). Accordingly, we will discuss how C and N are assimilated and metabolized as well as the signaling mechanisms used by cyanobacterial to sense C/N ratio, focusing on metabolic and physiological mechanisms underlying the responses to C and N sources, concentration and availability. Furthermore, we highlight how and to which extent the understanding of C/N balance contributes for optimum growth and production of specific compounds with biotechnological application in different cyanobacteria, mainly regarding biofuel production and cyanobacterial engineering.

15.2  Carbon (C) metabolism Although some cyanobacterial strains can use sugar or other organic carbon, CO2 is the primary C-source (Rippka et al., 1979; Zhang and Bryant, 2011; Burnap et al., 2015). In aquatic environments, the availability of CO2, that depends on pH and diffusion rates, among others environmental factors, may limit the growth (Wang et al., 2004). In addition, dissolved CO2 is found into different forms of inorganic carbon (Ci): CO2 + H2O ↔ H2CO3 ↔ HCO3− + H+ ↔ CO32− + 2 H+. The equilibrium of this reaction is pH-dependent, being the CO2 favored under acidic conditions, while bicarbonate (HCO3−) is by neutral and alkaline pH values, being both used by cyanobacteria (Zhang et al., 2018). Cyanobacteria evolved a complex 'Ci Concentrating Mechanism' (CCM), that acts to increase the intracellular concentration of CO2, overcoming the low CO2 affinity of the cyanobacterial enzyme 'ribulose 1,5-bisphosphate carboxylase/ oxygenase' (Rubisco) (Kaplan and Reinhold, 2002; Badger and Price, 2003; Wang et al., 2004). The CCM is expressed at constitutive levels under Ci replete conditions, while high expression rates are observed under Ci limitation (Wang et al., 2004; Zhang et al., 2018). Accordingly, Ci is actively acquired, either as HCO3− or as CO2, using a complex set of transporters and enzymes. HCO3− uptake further involves active membrane transporters, such as the Na+-dependent bicarbonate transporters, SbtA or BicA, as well as an ABC-type of HCO3− transporter, encoded by cmpABCD operon. These specific bicarbonate transporters are, respectively, driven by the Na+ gradient and ATP (Omata et al., 1999; Shibata et al., 2002; Wang et al., 2004). In its turn, CO2 uptake occurs via the enzymatic hydration of CO2 catalyzed by specialized modified forms of plastoquinone oxidoreductase 'NADPH dehydrogenase respiratory complexes' (NDH-1), located in thylakoid membrane (Kaplan et al., 2001; Wang et al., 2004; Zhang et al., 2018). In addition, CO2 continuously enters into cells by diffusion, mainly through water channels, following its conversion to HCO3− (Tchernov et al., 2001) (Fig. 15.1). Coupled to highly efficient uptake systems, CCM systems are integrated by carbonic anhydrases, as well as by microcompartments, called carboxysomes, which are polyhedral bodies constituted by a protein shell, that represents a diffusion barrier for CO2 exit and O2 entrance, allowing the transference of the Rubisco enzyme and the movement of HCO3−, and the substrates as well as products of Rubisco (Rae et al., 2013; Kerfeld and Erbilgin, 2015). The Ci sources absorbed by cyanobacterial cells through active mechanisms (Wang et al., 2004; Klahn et al., 2015) are assimilated into organic compounds by the action of Rubisco, located into the carboxysomes. This enzymatic complex performs a carboxylation reaction using CO2 and 'ribulose 1,5-biphosphate' (RuBP), yielding two molecules of '3-phosphoglycerate' (3-PGA), a molecule with three carbons. However, the cyanobacterial Rubisco has low affinity for CO2 (Km > 150 mM) and also catalyzes an oxygenase reaction between RuBP and O2, yielding one 2-phosphoglycolate (2-PG), a molecule of the two-carbon and only one 3-PGA (Allahverdiyeva et al., 2011; Hagemann et al., 2013; Moroney et al., 2013; Hagemann and Bauwe, 2016). That being said, the carbon fixation in cyanobacteria greatly relies on CCM (Price et al., 2013; Burnap et al., 2015; Turmo et al., 2017; Zhang et al., 2018), that enhances the CO2 concentration around the Rubisco, increasing the C assimilation efficiency (Herrero and Flores, 2019) (Fig. 15.1). Two classes of cyanobacteria have been distinguished depending on the type of carboxysome: α-cyanobacteria and β-cyanobacteria, which seems to have deep phylogenetic divergence in the cyanobacterial phylum. The β-carboxysomes contain form 1-B RubisCo and the CcmM and/or CcaA carbonic anhydrases. The genes encoding the large and small Rubisco subunits (rbcL and rbcS) are arranged in the rbc operon. β-Carboxysomes are widely distributed among the cyanobacterial lineages, being present in freshwater and marine coastal cyanobacteria (the β-cyanobacteria). Conversely, the αcarboxysomes contain form 1-A RubisCo and the CsoSCA carbonic anhydrase. The α-carboxysomes are found in a group of phylogenetically related oceanic cyanobacteria (α-cyanobacteria) (Rae et al., 2013; Kerfeld and Erbilgin, 2015; Kerfeld and Melnicki, 2016; Turmo et al., 2017).



Carbon and nitrogen metabolism in cyanobacteria Chapter | 15  247

FIG. 15.1  Schematic pathways of C and N transport, assimilation and metabolization in a hypothetical cyanobacterial cell. ABC-transporters for NO3− and NO2− (NrtABCD) and Urea (UrtABCDE); NH4+ translocator (Amt). The nitrate reduction system (1) consists of nitrate reductase (NarB) and nitrite reductase (NirA), which are ferredoxin (Fdred)-dependent. Urea is decarboxylated by UreABC, yielding one CO2 and two NH4+ molecules (2). HNO2 and NH3 can diffuse through the membrane. BNF (3), which is an energetically expensive metabolic reaction, is catalyzed by the nitrogenase enzymatic complex (NifHDK). Once N sources are converted to NH4+, it can be further incorporated into organic compounds by the glutamine synthetase (GS)glutamate synthase pathway, yielding glutamine in an ATP-dependent reaction. In sequence, the amino group of glutamine is transferred to 2-oxoglutarate (2-OG) by GOGAT in a reaction that requires reduced ferredoxin (4). 2-OG is a product of carbon assimilation, linking C and N metabolisms. NDH-13 (localized in the thylakoid) and NDH-14 (thylakoid (?) and plasma membrane) act on CO2 uptake producing bicarbonate and consuming NADPH (5), showing high and low affinity, respectively; SbtA and BicA are sodium-dependent HCO3− transporters, with high and low affinity, respectively (6); Cmp/ Bct1 is an ABC HCO3− transporter (7). HCO3− diffuses into the Carboxysome, being converted to CO2 by carbonic anhydrase (CA) (8). Then, CO2 is combined with ribulose 1,5-bis-phosphate by Rubisco, yielding 3-phospho-glycerate (3-PG) (9), which can be used to further produce 2-OG, through the TCA Cycle (10).

To date, five membrane Ci transporters have been described in cyanobacteria (Price, 2011; Zhang et al., 2018). CO2 can passively enter the membrane via aquaporins with subsequent energy-dependent conversion to HCO3− (Tchernov et al., 2001), or actively through two complexes: NDH-I3, located on the thylakoid membrane; and NDH-I4, placed on cell membrane (Ohkawa et al., 2000; Price, 2011). According to Price (2011), these complexes are not true transporters since the uptake process involves passive entry of CO2 into the cell, followed by its conversion to HCO3−. NDH-I3 and NDH-14 has low and high affinity for CO2, showing, respectively, K0.5 of 1–2 and 10–15 μM CO2 (Maeda et al., 2002; Price et al., 2002; Price, 2011). NDH-I4 is constitutively expressed, while NDH-I3 is expressed in response to Ci limitation (Klughammer et al., 1999; Ohkawa et al., 2000; Price et al., 2002) (Fig. 15.1). BCT1 is a high affinity HCO3− transporter, belonging to the ATP binding cassette family (Omata et al., 1999; Higgins, 2001). It is encoded by the operon cmpABCD, being expressed under carbon limitation and also under high-light conditions, which seems to enhance the operon transcription (Omata et al., 1999; McGinn et al., 2004). Genomic analysis indicates that BCT1 is only found in β-cyanobacteria, which are mainly restricted to freshwater or brackish strains (Badger et al., 2006) (Fig. 15.1). SbtA is a Na+-dependent transporter, detected as a cytoplasmic membrane protein, with high affinity for HCO3− (Shibata et al., 2002; Zhang et al., 2004). As shown for BCT1, SbtA abundance is dramatically increased under Ci limitation (Zhang et al., 2004; Price, 2011). BicA is another sodium-dependent but low affinity bicarbonate (~38 μM HCO3−) carrier (Price et al., 2004). Surprisingly, BicA belongs to a large family, the SulP/SLC26 family, reported as sulfate transporters. In addition, homologs of this transporter are present in most β- and α-cyanobacteria genomes so far examined (Price et al., 2004; Price and Howitt, 2010; Shelden et al., 2010). In some cyanobacteria, BicA may be induced (Synechococcus sp. PCC7002) or be constitutively expressed (Synechocystis sp. PCC6803) (Price et al., 2004; Wang et al., 2004; Price, 2011) (Fig. 15.1). In cyanobacteria, Ci uptake is tightly regulated, mainly at the transcriptional level. As consequence, CCM-related genes encoding components of Ci transporters have maximum expression under Ci-limiting conditions, while elevated CO2 levels lead to their repression (Wang et al., 2004; Klahn et al., 2015). For the model unicellular Synechocystis sp. PCC6803, three

248  PART | IV  Algal photosystems and photosynthesis

transcriptional regulators of Ci usage have been identified, for which homologous in other cyanobacterial strains have also been found. First, the transcriptional regulator cyAbrB2, which is a cyanobacterial homolog of the 'transition state regulator' found in Bacillus subtilis, regulates the transcription of genes associated with C and N metabolism (Ishii and Hihara, 2008; Lieman-Hurwitz et al., 2009; Kaneko et al., 2013). Second, the regulator of the ATP-dependent bicarbonate transport system (CmpR), activates the expression of the cmpABCD operon encoding the high-affinity bicarbonate transporter, BCT1, which is induced under low Ci conditions (Omata et al., 2001). Last, the transcriptional regulator NdhR (also CcmR, from 'CO2-concentrating mechanism regulator'), which is considered the most important regulator of Ci utilization, since it acts, primarily, as a repressor of the genes encoding components of Ci transporters (e.g., NDH-13 and the Na+-dependent bicarbonate transporter, SbtA) under high CO2 conditions (Figge et al., 2001; Wang et al., 2004; Woodger et al., 2007). The transcriptional regulators NdhR and CmpR are members of large family of LysR-type transcriptional regulators (LTTRs), which action is based on changing DNA-binding properties upon binding to small effector molecules. The DNA-binding activity of CmpR is stimulated through 2-PG and RuBP (Nishimura et al., 2008) while 2-oxoglutarate (2OG) and NADP+ have been stated as corepressor for NdhR (Daley et al., 2012; Zhang et al., 2018; Herrero and Flores, 2019).

15.3  Nitrogen (N) metabolism Considering cyanobacterial N metabolism, a variety of sources can be assimilated including inorganic forms as nitrate, nitrite and ammonium as well as organic compounds, such as urea, cyanate and some amino acids, up to atmospheric nitrogen (N2), via BNF (Esteves-Ferreira et al., 2017; Zhang et al., 2018; Herrero and Flores, 2019). However, ammonium, as the most reduced inorganic form of N, represents the preferred source for cyanobacteria. As consequence, when ammonium is provided together with other N sources (nitrate, nitrite or urea), cyanobacteria will uptake ammonium preferably (Herrero et al., 2001; Muro-Pastor and Florencio, 2003; Muro-Pastor et al., 2005). It is also important to mention that whatever the form of inorganic N taken up by the cells, it is always converted into ammonium, which plays a central role in assimilatory pathways, being directly incorporated into C skeletons to produce organic N compounds (Muro-Pastor et al., 2005). Thus, when ammonium is available in sufficient amounts, it determines the repression of genes encoding proteins for the assimilation of alternative N sources, which are, in turn, expressed on ammonium deprivation, a process known as global N control (Herrero et al., 2001; Forchhammer, 2004). Under alkaline conditions, ammonium can be found as ammonia (pKa [NH4+/NH3] = 9.3), which readily diffuse through biological membranes (Herrero and Flores, 2019). However, low ammonium concentrations are found in the majority of natural environments/conditions (Zehr, 2011) and specific ammonium translocators of the Amt family act for an efficient ammonium uptake (Montesinos et al., 1998; Wacker et al., 2014) (Fig. 15.1). Three amt genes have been described clustered together in Anabaena sp. PPC7120 (Khademi et al., 2004; Paz-Yepes et al., 2008). At transcription level, similar increased response to ammonium withdrawal are observed. However, they are expressed at different levels, being the amt1 homologous expressed at the highest levels (Flaherty et al., 2011), which is consistent with the recognized role of Amt1 as the major cyanobacterial ammonium transporter (Montesinos et al., 1998). In environmental conditions and also in commercial culture media (e.g. BG-11—Rippka et al., 1979), nitrate (NO3−) and nitrite (NO2−) are the most common sources of N for cyanobacteria (Flores and Herrero, 2005). Regarding some environmental conditions (pH values, redox potential), nitrite can be converted to its protonated form (nitrous acid—HNO2) and diffuse through the membrane (Sakamoto et al., 1999; Flores and Herrero, 2005). However, given that in aquatic environments NO3− and NO2− are found in relatively low concentrations, coupled to their negative residual charges, specific transporters are required (Zhang et al., 2018). Both NO3− and NO2− are actively transported by NrtABCD (ABC-type) transporters, which are found in freshwater cyanobacterial strains (Maeda et al., 2015). The genes encoding for NO3− assimilation structural proteins form an operon in which the genes are arranged in a conserved order (5′ to 3′): nirA (Fd-nitrite reductase), transporter genes (nrtABCD) and narB (Fd-nitrate reductase) (Flores and Herrero, 2005; Herrero and Flores, 2019). NirA and NarB act on NO3− and NO2− reduction, yielding ammonium (Esteves-Ferreira et al., 2018; Herrero and Flores, 2019) (Fig. 15.1). Other high-affinity NO3− and NO2− transporter, encoded by nrtP (Sakamoto et al., 1999), which is a 'Major Facilitator Superfamily' (MFS) NO3−/NO2− transporter (Aichi et al., 2006), has been identified in strains from freshwater and saline environments (Bird and Wyman, 2003; Maeda et al., 2015). Some cyanobacteria from marine/saline environments also present a NO2− transporter that belongs to the 'formate/NO2− transporter' (FNT) family (Maeda and Omata, 2009; Maeda et al., 2015). After NO3−/NO2− transport and concentration inside the cells, 'NO3− reductase' (NarB) catalyzes the reduction of NO3− producing NO2−, which is then used by 'NO2− reductase' (NirA), that catalyzes its reduction producing ammonium (Flores and Herrero, 2005). Cyanobacterial nitrate and nitrite reductases are metalloenzymes that



Carbon and nitrogen metabolism in cyanobacteria Chapter | 15  249

use reduced ferredoxin as electron donor, liking their assimilation to photosynthesis (Flores and Herrero, 2005), consuming up to 30% of the reducing equivalents produced by the photosynthetic light reactions (Sakamoto et al., 1999) (Fig. 15.1). BNF is an energetically expensive metabolic reaction catalyzed by the nitrogenase enzymatic complex, which is encoded by nifHDK operon (Herrero et al., 2001; Herrero and Flores, 2019). As an expensive metabolic process, BNF is only triggered when no source of combined N is available (Flores and Herrero, 2005). Additionally, the nitrogenase complex is inhibited by O2 (Stal, 2015). Some filamentous cyanobacteria form heterocytes, providing a micro-oxic intracellular environment for BNF (Flores and Herrero, 2009). In contrast, in nonheterocytous morphotypes, in which BNF and photosynthetic O2 evolution occur in the same cell, their activities are diurnally regulated, so that N2 fixation is restricted to the night (Schneegurt et al., 2000; Zehr, 2011). The heterocytes are specialized cell for the BNF, surrounded by a glycolipid envelope formed by laminated layer, acting as a barrier for gases, mainly O2 (Flores and Herrero, 2009). Accordingly, the activity of PSII is lost during heterocyte differentiation, contributing to the establishment of a lower O2 concentration environment. In addition, since Rubisco is absent in heterocyte, it does not fix CO2 photosynthetically, being dependent on the fixed C received from the adjacent vegetative cells (Flores and Herrero, 2009). In nonheterocytous cyanobacteria, photosynthesis and BNF are temporally separated and high nitrogenase activity is observed after the photosynthesis reached its peak, coupled with higher respiratory rates (Schneegurt et al., 2000; Stal, 2015) (Fig. 15.1). The genes nifD and nifK encode for nitrogenase, holding the Fe-S cluster known as the P-cluster and the Fe-Mo cofactor in which the nitrogen fixation reaction takes place. The nifH encodes for nitrogenase reductase, harboring an Fe-S cluster, and also binds to Mg2+-ATP, transferring electrons to nitrogenase (nifDK) (Rubio and Ludden, 2008), which were received from an electron carrier such as ferredoxin or flavodoxin (Esteves-Ferreira et al., 2017). It is worth to mention that alternative nitrogenases that use an Fe-V cofactor or an Fe-Fe cofactor are also known (Mus et al., 2018). Interestingly, most of heterocyte-forming cyanobacteria contain only the Fe-Mo nitrogenase, confined at heterocyte (Mus et al., 2018); however, in Anabaena variabilis ATCC29413 the expression of three nitrogenases was reported: the Fe-Mo nitrogenase expressed in heterocytes, another Fe-Mo nitrogenase expressed in vegetative cells under anoxic conditions, and a Fe-V nitrogenase found in heterocytes under Mo-deficiency conditions (Thiel and Pratte, 2014). Regarding the assimilation of organic N sources, cyanobacterial genomes encode for ABC transporters that can mediate the uptake of organic N compounds including amino acids and urea (Montesinos et al., 1995; Pernil et al., 2015; Zhang et al., 2018; Herrero and Flores, 2019). Considering amino acids, glutamine assimilation likely involves the enzyme 'glutamine:oxoglutarate aminotransferase' (GOGAT) while arginine utilization must rely on the arginine decarboxylase pathway, related to the synthesis of polyamines (Burnat and Flores, 2014; Burnat et al., 2018). In addition, the utilization of urea, which can easily diffuse across lipid bilayers, involves the enzyme urease capable of urea hydrolysis, producing CO2 and two ammonium molecules (Valladares et al., 2002). Accordingly, urease has been described as a constitutive enzyme, not regulated by N-containing compounds, with some exceptions. However, urea uptake by the ABC-type transporter, urtABCDE, is subjected to N control by a N control factor of cyanobacteria (NtcA), a transcriptional regulator that regulates N metabolism in Cyanobacteria (Zhang et al., 2018), ensuring that urea uptake system is expressed only under N deficiency (Valladares et al., 2002; Boer et al., 2014). Once inorganic N sources are converted to ammonium, it can be further incorporated into organic compounds by the 'glutamine synthetase' (GS)-glutamate synthase pathway. In this pathway, ammonium is incorporated into glutamate yielding glutamine in an ATP-dependent reaction catalyzed by GS. In sequence, the amino group of glutamine is transferred to '2-oxoglutarate' (2-OG) by GOGAT in a reaction that requires reducing power, provided by reduced ferredoxin. Accordingly, the use of ferredoxin also link the assimilation of ammonium to photosynthesis, as observer for nitrate/nitrite reduction (Martin-Figueroa et al., 2000). As described, 2-OG, which is an intermediate of the 'tricarboxylic acid' (TCA) cycle, is the C-skeleton necessary for N assimilation, via GS-GOGAT. Therefore, ammonium assimilation using 2-OG provides the metabolic basis for coupling between N and C metabolism. This connection highlights the central role of TCA cycle metabolites, which are mainly involved in acetyl-CoA oxidation, producing reducing power (NADH and ATP) and by providing essential precursors required for the biosynthesis of other metabolites (Shiraishi and Savageau, 1992; Steinhauser et al., 2012) (Fig. 15.1).

15.4  Regulation and coordination between C and N metabolic pathways Both C and N metabolisms are tightly coupled in different living organisms and under many levels of control, including the allosteric modulation of protein activities (enzymes, transporters and other proteins) and regulation at transcriptional level, controlling the expression of metabolic related genes (Muro-Pastor et al., 2005; Du et al., 2014; Klahn et al., 2018; Selim et al., 2018). At the molecular level, the protein PII (PII) is one of the key proteins in the regulation of C/N ­metabolism. PII senses the cellular N and C availability as well as the redox power, based on 2-OG levels and by interacting with

250  PART | IV  Algal photosystems and photosynthesis

ATP, ­magnesium (Mg2+) and 2-OG (Forchhammer, 2004; Forcada-Nadal et al., 2018; Selim et al., 2018). Hierarchically, ATP-Mg must first bind on PII, creating the 2-OG binding site that allows 2-OG to interact with PII. Conversely, when ADP binds to PII, the binding site for 2-OG is blocked. Accordingly, when ATP binds to PII the affinity of PII to 2-OG is drastically stimulated, and conformational changes are observed, affecting PII interaction with other proteins and also their activities (Forchhammer, 2004; Fokina et al., 2010). Among the recognized targets of PII are PipX (PII interacting protein X) and NAGK 'N-acetyl-l-glutamate kinase', a key enzyme for arginine synthesis (Espinosa et al., 2014). The protein PII can be found in a phosphorylated and dephosphorylated state and when phosphorylated the association PII-NAGK is impaired, inhibiting NAGK activity. However, basal level of arginine production is observed even in its nonactivated state (Forcada-Nadal et al., 2018). Under N excess, the dephosphorylated form of PII binds to NAGK, enhancing its activity. The association PII-NAGK leads to a higher synthesis of arginine, whose excess is stored as cyanophycin, the main N storage compound found in cyanobacteria (Maheswaran et al., 2006; Fokina et al., 2010; Forcada-Nadal et al., 2018). PipX, when not associated to PII, can interact with NtcA, affecting the transcription of N related genes (Llacer et al., 2010). As consequence, a complex network links 2-OG with PII, PipX, and NtcA, allowing these proteins to regulate the activity and expression of several enzymes related to N and C metabolism (Forchhammer, 2004; Forcada-Nadal et  al., 2018). Accordingly, under high C/N conditions, PII interacts with ATP and 2-OG, and PipX is released, biding to NtcA, leading to the formation of the PipX-NtcA complex and to changes in gene expression (Llacer et al., 2010). In addition, PII coordinates the activities of high-affinity bicarbonate transporters as well as of the nitrate transporter (NrtABCD), nitrate reductase, ABC-type cyanate transporter, which are repressed by ammonium only in the presence of PII (Llacer et al., 2010; Selim et al., 2018). Under low C/N conditions, when PII is dephosphorylated, PipX interacts with PII, forming a complex composed of three PipX and one PII. Under high C/N, PipX is bound with NtcA. Thus, PipX is always associated either with PII or NtcA, depending on the C/N balance (Selim et al., 2018). In its turn, NtcA, a member of CRP (cAMP receptor protein) family, also acts as a transcriptional regulator of N metabolism, being universally distributed in the phylum Cyanobacteria (MuroPastor and Florencio, 2003; Herrero et al., 2004; Zhang et al., 2018; Herrero and Flores, 2019). NtcA binds to palindromic DNA sites, including genes related to N scavenging and assimilation as well as genes related to other diverse functional categories, suggesting cascades of NtcA-dependent regulation (Picossi et al., 2014). The efficiency of the binding of NtcA to a promoter increase following its association to 2-OG, that also contributes to its binding to PipX. Additionally, NtcA can bind itself with DNA, regulating gene transcription (Llacer et al., 2010; Espinosa et al., 2014). It seems reasonable to suggest that among these regulated genes, those related to C uptake and assimilation deserve a special attention, since these processes are providing C and energy for cell growth and maintenance (Esteves-Ferreira et al., 2018). The precise elucidation of those genes will be an important step towards a better comprehension of how cyanobacteria regulate their C/N balance and adjust their growth rate in response to changes in environmental conditions.

15.5  Concluding remarks The significance of cyanobacteria to Earth's ecology is undeniable. Due to their capability in performing both photosynthesis and BNF, they present itself as perfect organism that can be engineered for direct conversion of CO2 and solar energy to biofuels and biochemicals. It seems reasonable to anticipate that by achieving a comprehension of the precise mechanisms involved in the partitioning of C and N towards growth or storage would increase the biotechnological potential of these organisms. To summarize, increasing our understanding on the mechanisms underlying C/N balance in cyanobacteria is of paramount importance not only for basic research but also for biotechnological applications. Researches on bioenergy and biofuels production, greatly rely on changes in the C/N ratios, which can lead to the enhanced production of C-storage compounds used as raw material. In this context, confirmation and further identification of the check points and players involved in C and N metabolisms and their potential link with the ultimate control of growth and metabolic composition may enhance the productivity of specific desired compounds and ultimately allow us achieve economic viability of cyanobacteria-­based biomass and bioproducts, reducing the production costs for large- scale industrial production. We postulate that the successful manipulation of these different characteristics, as discussed above, coupled with the identification of the key regulators of C and N metabolism in distinct cyanobacterial strains should be a significant breakthrough in the field of synthetic biology. Recent advances and ongoing incremental findings suggest that the knowledge obtained in cyanobacteria can be further transferred to crop, once the efficiency of assimilation, use and metabolism of C and N are properly optimized and it could assist towards feeding a growing population in the near future. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c,d, e,f,g,h,i,j,k,l,m,n,o).



Carbon and nitrogen metabolism in cyanobacteria Chapter | 15  251

Acknowledgments This work was made possible through financial support from the Max Planck Society, the CNPq (National Council for Scientific and Technological Development, Brazil) [grant No. 402511/2016-6, and research fellowships to A.N.N., and W.L.A.]; the FAPEMIG (Foundation for Research Assistance of the Minas Gerais State, Brazil) [grant Nos. APQ-01357-14 and RED-00053-16]; CAPES (Coordination for the Improvement of Higher Education Personnel, Brazil) and FAPEMIG [scholarships PNPD-1638006 and BPD-00514-14 to M.G.M.V.V]; CAPES [scholarships to A.V.M.A]; and CNPq [scholarships to N.V.C].

Competing interests The authors declare that they have no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References Aichi, M., Yoshihara, S., Yamashita, M., Maeda, S., Nagai, K., Omata, T., 2006. Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133. Biosci. Biotechnol. Biochem. 70 (11), 2682–2689. Allahverdiyeva, Y., Ermakova, M., Eisenhut, M., Zhang, P., Richaud, P., Hagemann, M., et al., 2011. Interplay between flavodiiron proteins and photorespiration in Synechocystis sp. PCC 6803. J. Biol. Chem. 286 (27), 24007–24014. de Alvarenga, L.V., Vaz, M.G.M.V., Genuario, D.B., Esteves-Ferreira, A.A., Almeida, A.V.M., de Castro, N.V., et al., 2018. Extending the ecological distribution of Desmonostoc genus: proposal of Desmonostoc salinum sp. nov., a novel cyanobacteria from a saline–alkaline lake. Int. J. Syst. Evol. Microbiol. 68 (9), 2770–2782. Arp, D.J., 2000. The nitrogen cycle. In: Triplett, E.W. (Ed.), Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a Biological Process. Horizon Scientific Press, Wymondham, pp. 1–14. Badger, M.R., Price, G.D., 2003. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J. Exp. Bot. 54 (383), 609–622. Badger, M.R., Price, G.D., Long, B.M., Woodger, F.J., 2006. The environmental plasticity and ecological genomics of the cyanobacterial CO2 concentrating mechanism. J. Exp. Bot. 57 (2), 249–265. Bird, C., Wyman, M., 2003. Nitrate/nitrite assimilation system of the marine picoplanktonic cyanobacterium Synechococcus sp. strain WH 8103: effect of nitrogen source and availability on gene expression. Appl. Environ. Microbiol. 69 (12), 7009–7018. Boer, J.L., Mulrooney, S.B., Hausinger, R.P., 2014. Nickel-dependent metalloenzymes. Arch. Biochem. Biophys. 544, 142–152. Burnap, R., Hagemann, M., Kaplan, A., 2015. Regulation of CO2 concentrating mechanism in cyanobacteria. Life 5 (1), 348–371. Burnat, M., Flores, E., 2014. Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena. MicrobiologyOpen 3 (5), 777–792. Burnat, M., Li, B., Kim, S.H., Michael, A.J., Flores, E., 2018. Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth. Mol. Microbiol. 109 (6), 763–780. Castenholz, R.W., Wilmotte, A., Herdman, M., Rippka, R., Waterbury, J.B., Iteman, B., et  al., 2001. Phylum BX. Cyanobacteria. In: Boone, D.R., Castenholz, R.W., Garrity, G.M. (Eds.), Bergey’s Manual® of Systematic Bacteriology. Springer, New York, NY, pp. 473–599. Cohen, Y., Jorgensen, B.B., Revsbech, N.P., Poplawski, R., 1986. Adaptation to hydrogen sulfide of oxygenic and anoxygenic photosynthesis among cyanobacteria. Appl. Environ. Microbiol. 51 (2), 398–407. Daley, S.M.E., Kappell, A.D., Carrick, M.J., Burnap, R.L., 2012. Regulation of the cyanobacterial CO2-concentrating mechanism involves internal sensing of NADP+ and α-ketogutarate levels by transcription factor CcmR. PLoS One 7 (7), e41286. Du, J.H., Forster, B., Rourke, L., Howitt, S.M., Price, G.D., 2014. Characterisation of cyanobacterial bicarbonate transporters in E. coli shows that SbtA homologs are functional in this heterologous expression system. PLoS One 9 (12), e115905. Espinosa, J., Rodriguez-Mateos, F., Salinas, P., Lanza, V.F., Dixon, R., de la Cruz, F., 2014. PipX, the coactivator of NtcA, is a global regulator in cyanobacteria. Proc. Natl. Acad. Sci. 111 (23), E2423–E2430. Esteves-Ferreira, A.A., Cavalcanti, J.H.F., Vaz, M.G.M.V., Alvarenga, L.V., Nunes-Nesi, A., Araujo, W.L., 2017. Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions. Genet. Mol. Biol. 40 (s1), 261–275. Esteves-Ferreira, A.A., Inaba, M., Fort, A., Araujo, W.L., Sulpice, R., 2018. Nitrogen metabolism in cyanobacteria: metabolic and molecular control, growth consequences and biotechnological applications. Crit. Rev. Microbiol. 44 (5), 541–560. Figge, R.M., Cassier-Chauvat, C., Chauvat, F., Cerff, R., 2001. Characterization and analysis of an NAD(P)H dehydrogenase transcriptional regulator critical for the survival of cyanobacteria facing inorganic carbon starvation and osmotic stress. Mol. Microbiol. 39 (2), 455–469. Flaherty, B.L., van Nieuwerburgh, F., Head, S.R., Golden, J.W., 2011. Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation. BMC Genomics 12, 332. Flores, E., Herrero, A., 2005. Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem. Soc. Trans. 33 (1), 164–167. Flores, E., Herrero, A., 2009. Compartmentalized function through cell differentiation in filamentous cyanobacteria. Nat. Rev. Microbiol. 8, 39–50. Fokina, O., Chellamuthu, V.R., Forchhammer, K., Zeth, K., 2010. Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus PII signal transduction protein. Proc. Natl. Acad. Sci. U.S.A. 107 (46), 19760–19765.

252  PART | IV  Algal photosystems and photosynthesis

Forcada-Nadal, A., Llacer, J.L., Contreras, A., Marco-Marin, C., Rubio, V., 2018. The PII-NAGK-PipX-NtcA regulatory axis of cyanobacteria: a tale of changing partners, allosteric effectors and non-covalent interactions. Front. Mol. Biosci. 5, 91. Forchhammer, K., 2004. Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol. Rev. 28 (3), 319–333. Galloway, J.N., 2005. The global nitrogen cycle: past, present and future. Sci. China Ser. C 48 (s2), 669–677. Genuario, D.B., Vaz, M.G.M.V., Santos, S.N., Kavamura, V.N., Melo, I.S., 2019. Cyanobacteria from Brazilian extreme environments. In: Das, S., Dash, H.R. (Eds.), Microbial Diversity in the Genomic Era. Academic Press, New York, NY, pp. 265–284. Giovannoni, S.J., Turner, S., Olsen, G.J., Barns, S., Lane, D.J., Pace, N.R., 1988. Evolutionary relationships among cyanobacteria and green chloroplasts. J. Bacteriol. 170 (8), 3584–3592. Hagemann, M., Bauwe, H., 2016. Photorespiration and the potential to improve photosynthesis. Curr. Opin. Chem. Biol. 35, 109–116. Hagemann, M., Fernie, A.R., Espie, G.S., Kern, R., Eisenhut, M., Reumann, S., et al., 2013. Evolution of the biochemistry of the photorespiratory C2 cycle. Plant Biol. 15 (4), 639–647. Herrero, A., Flores, E., 2019. Genetic responses to carbon and nitrogen availability in Anabaena. Environ. Microbiol. 21 (1), 1–17. Herrero, A., Muro-Pastor, A.M., Flores, E., 2001. Nitrogen control in cyanobacteria. J. Bacteriol. 183 (2), 411–425. Herrero, A., Muro-Pastor, A.M., Valladares, A., Flores, E., 2004. Cellular differentiation and the NtcA transcription factor in filamentous cyanobacteria. FEMS Microbiol. Rev. 28 (4), 469–487. Higgins, C.F., 2001. ABC transporters: physiology, structure and mechanism – an overview. Res. Microbiol. 152 (3-4), 205–210. Hobbie, J.E., Hobbie, E.A., 2013. Microbes in nature are limited by carbon and energy: the starving-survival lifestyle in soil and consequences for estimating microbial rates. Front. Microbiol. 4, 324. Ishii, A., Hihara, Y., 2008. An AbrB-like transcriptional regulator, Sll0822, is essential for the activation of nitrogen-regulated genes in Synechocystis sp. PCC 6803. Plant Physiol. 148 (1), 660–670. Kaneko, Y., Miyagi, A., Hihara, Y., Uchimiya, H., Kaniya, Y., Nishiyama, Y., et al., 2013. Deletion of the transcriptional regulator cyAbrB2 deregulates primary carbon metabolism in Synechocystis sp. PCC 6803. Plant Physiol. 162 (2), 1153–1163. Kaplan, A., Helman, Y., Tchernov, D., Reinhold, L., 2001. Acclimation of photosynthetic microorganisms to changing ambient CO2 concentration. Proc. Natl. Acad. Sci. U.S.A. 98 (9), 4817–4818. Kaplan, A., Reinhold, L., 2002. CO2 concentrating mechanisms in photosynthetic microorganisms. Annu. Rev. Plant Physiol. 50, 539–570. Kerfeld, C.A., Erbilgin, O., 2015. Bacterial microcompartments and the modular construction of microbial metabolism. Trends Microbiol. 23 (1), 22–34. Kerfeld, C.A., Melnicki, M.R., 2016. Assembly, function and evolution of cyanobacterial carboxysomes. Curr. Opin. Plant Biol. 31, 66–75. Khademi, S., O’Connell, J., Remis, J., Robles-Colmenares, Y., Miercke, L.J.W., Stroud, R.M., 2004. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science 305 (5690), 1587–1594. Klahn, S., Bolay, P., Wright, P.R., Atilho, R.M., Brewer, K.I., Hagemann, P., et al., 2018. A glutamine riboswitch is a key element for the regulation of glutamine synthetase in cyanobacteria. Nucleic Acids Res. 46 (19), 10082–10094. Klahn, S., Orf, I., Schwarz, D., Matthiessen, J.K.F., Kopka, J., Hess, W.R., et al., 2015. Integrated transcriptomic and metabolomic characterization of the low-carbon response using an ndhR mutant of Synechocystis sp PCC 6803. Plant Physiol. 169 (3), 1540–1556. Klughammer, B., Sultemeyer, D., Badger, M.R., Price, G.D., 1999. The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp. PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria. Mol. Microbiol. 32 (6), 1305–1315. Knoll, A.H., 2008. Cyanobacteria and earth history. In: Herrero, A., Flores, E. (Eds.), The Cyanobacteria: Molecular Biology, Genomics and Evolution. Caister Academic Press, Poole, pp. 495. Komarek, J., Kastovsky, J., Mares, J., Johansen, J.R., 2014. Taxonomic classification of cyanoprokaryotes (cyanobacterial genera) 2014 using a polyphasic approach. Preslia 86 (4), 295–335. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.



Carbon and nitrogen metabolism in cyanobacteria Chapter | 15  253

Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Lieman-Hurwitz, J., Haimovich, M., Shalev-Malul, G., Ishii, A., Hihara, Y., Gaathon, A., et al., 2009. A cyanobacterial AbrB-like protein affects the apparent photosynthetic affinity for CO2 by modulating low-CO2-induced gene expression. Environ. Microbiol. 11 (4), 927–936. Llacer, J.L., Espinosa, J., Castells, M.A., Contreras, A., Forchhammer, K., Rubio, V., 2010. Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII. Proc. Natl. Acad. Sci. U.S.A. 107 (35), 15397–15402. Maeda, S.I., Badger, M.R., Price, G.D., 2002. Novel gene products associated with NdhD3/D4-containing NDH-1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942. Mol. Microbiol. 43 (2), 425–435. Maeda, S.I., Murakami, A., Ito, H., Tanaka, A., Omata, T., 2015. Functional characterization of the FNT family nitrite transporter of marine picocyanobacteria. Life 5 (1), 432–446. Maeda, S., Omata, T., 2009. Nitrite transport activity of the ABC-type cyanate transporter of the cyanobacterium Synechococcus elongates. J. Bacteriol. 191 (10), 3265–3272. Maheswaran, M., Ziegler, K., Lockau, W., Hagemann, M., Forchhammer, K., 2006. PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp. strain PCC 6803. J. Bacteriol. 188 (7), 2730–2734. Martin-Figueroa, E., Navarro, F., Florencio, F.J., 2000. The GS-GOGAT pathway is not operative in the heterocysts. Cloning and expression of glsF gene from the cyanobacterium Anabaena sp. PCC 7120. FEBS Lett. 476 (3), 282–286. McGinn, P.J., Price, G.D., Badger, M.R., 2004. High light enhances the expression of low-CO2-inducible transcripts involved in the CO2-concentrating mechanism in Synechocystis sp. PCC6803. Plant Cell Environ. 27 (5), 615–626. Montesinos, M.L., Herrero, A., Flores, E., 1995. Amino acid transport systems required for diazotrophic growth in the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 177 (11), 3150–3157. Montesinos, M.L., Muro-Pastor, A.M., Herrero, A., Flores, E., 1998. Ammonium/methylammonium permeases of a cyanobacterium. Identification and analysis of three nitrogen-regulated amt genes in Synechocystis sp. PCC 6803. J. Biol. Chem. 273, 31463–31470. Moroney, J.V., Jungnick, N., DiMario, R.J., Longstreth, D.J., 2013. Photorespiration and carbon concentrating mechanisms: two adaptations to high O2, low CO2 conditions. Photosynth. Res. 117 (1-3), 121–131. Muro-Pastor, M.I., Florencio, F.J., 2003. Regulation of ammonium assimilation in cyanobacteria. Plant Physiol. Biochem. 41 (6-7), 595–603. Muro-Pastor, M.I., Reyes, J.C., Florencio, F.J., 2005. Ammonium assimilation in cyanobacteria. Photosynth. Res. 83 (2), 135–150. Mus, F., Alleman, A.B., Pence, N., Seefeldt, L.C., Peters, J.W., 2018. Exploring the alternatives of biological nitrogen fixation. Metallomics 10 (4), 523–538. Nieves-Morion, M., Flores, E., 2018. Multiple ABC glucoside transporters mediate sugar-stimulated growth in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. Environ. Microbiol. Rep. 10 (1), 40–48. Nishimura, T., Takahashi, Y., Yamaguchi, O., Suzuki, H., Maeda, S., Omata, T., 2008. Mechanism of low CO2-induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942. Mol. Microbiol. 68 (1), 98–109. Ohkawa, H., Pakrasi, H.B., Ogawa, T., 2000. Two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis sp. strain PCC6803. J. Biol. Chem. 275 (41), 31630–31634. Omata, T., Gohta, S., Takahashi, Y., Harano, Y., Maeda, S., 2001. Involvement of a CbbR homolog in low CO2-induced activation of the bicarbonate transporter operon in cyanobacteria. J. Bacteriol. 183 (6), 1891–1898. Omata, T., Price, G.D., Badger, M.R., Okamura, M., Gohta, S., Ogawa, T., 1999. Identification of an ATP-binding cassette transporter involved in bicarbonate uptake in the cyanobacterium Synechococcus sp. strain PCC 7942. Proc. Natl. Acad. Sci. U.S.A. 96 (23), 13571–13576. Paz-Yepes, J., Merino-Puerto, V., Herrero, A., Flores, E., 2008. The amt gene cluster of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 190 (19), 6534–6539. Pernil, R., Picossi, S., Herrero, A., Flores, E., Mariscal, V., 2015. Amino acid transporters and release of hydrophobic amino acids in the heterocystforming cyanobacterium Anabaena sp. Strain PCC 7120. Life 5 (2), 1282–1300. Picossi, S., Flores, E., Herrero, A., 2014. ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium. BMC Genomics 15, 22. Price, G.D., 2011. Inorganic carbon transporters of the cyanobacterial CO2 concentrating mechanism. Photosynth. Res. 109 (1-3), 47–57. Price, G.D., Howitt, S.M., 2010. The cyanobacterial bicarbonate transporter BicA: its physiological role and the implications of structural similarities with human SLC26 transporters. Biochem. Cell Biol. 89 (2), 178–188. Price, G.D., Maeda, S., Omata, T., Badger, M.R., 2002. Modes of active inorganic carbon uptake in the cyanobacterium, Synechococcus sp PCC7942. Funct. Plant Biol. 29 (2-3), 131–149.

254  PART | IV  Algal photosystems and photosynthesis

Price, G.D., Pengelly, J.J.L., Forster, B., Du, J., Whitney, S.M., von Caemmerer, S., et al., 2013. The cyanobacterial CCM as a source of genes for improving photosynthetic CO2 fixation in crop species. J. Exp. Bot. 64 (3), 753–768. Price, G.D., Woodger, F.J., Badger, M.R., Howitt, S.M., Tucker, L., 2004. Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc. Natl. Acad. Sci. U.S.A. 101 (51), 18228–18233. Rae, B.D., Long, B.M., Badger, M.R., Price, G.D., 2013. Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO fixation in cyanobacteria and some proteobacteria. Microbiol. Mol. Biol. R. 77 (3), 357–379. di Rienzi, S.C., Sharon, I., Wrighton, K.C., Koren, O., Hug, L.A., Thomas, B.C., et al., 2013. The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to cyanobacteria. elife 2, e01102. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111 (1), 1–61. Rubio, L.M., Ludden, P.W., 2008. Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu. Rev. Microbiol. 62, 93–111. Sakamoto, T., Inoue-Sakamoto, K., Bryant, D.A., 1999. A novel nitrate/nitrite permease in the marine cyanobacterium Synechococcus sp. strain PCC 7002. J. Bacteriol. 181 (23), 7363–7372. Schirrmeister, B.E., de Vos, J.M., Antonelli, A., Bagheri, H.C., 2013. Evolution of multicellularity coincided with increased diversification of cyanobacteria and the Great Oxidation Event. Proc. Natl. Acad. Sci. U.S.A. 110 (5), 1791–1796. Schneegurt, M.A., Tucker, D.L., Ondr, J.K., Sherman, D.M., Sherman, L.A., 2000. Metabolic rhythms of a diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, heterotrophically grown in continuous dark. J. Phycol. 36 (1), 107–117. Selim, K.A., Haase, F., Hartmann, M.D., Hagemann, M., Forchhammer, K., 2018. PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response. Proc. Natl. Acad. Sci. 115 (21), E4861–E4869. Shelden, M.C., Howitt, S.M., Price, G.D., 2010. Membrane topology of the cyanobacterial bicarbonate transporter, BicA, a member of the SulP (SLC26A) family. Mol. Membr. Biol. 27 (1), 12–22. Shibata, M., Katoh, H., Sonoda, M., Ohkawa, H., Shimoyama, M., Fukuzawa, H., et al., 2002. Genes essential to sodium-dependent bicarbonate transport in cyanobacteria. J. Biol. Chem. 277 (21), 18658–18664. Shiraishi, F., Savageau, M.A., 1992. The tricarboxylic acid cycle in Dictyostelium discoideum. III. Analysis of steady state and dynamic behavior. J. Biol. Chem. 267 (32), 22926–22933. Soo, R.M., Hemp, J., Parks, D.H., Fischer, W.W., Hugenholtz, P., 2017. On the origins of oxygenic photosynthesis and aerobic respiration in cyanobacteria. Science 355 (6332), 1436–1440. Soo, R.M., Skennerton, C.T., Sekiguchi, Y., Imelfort, M., Paech, S.J., Dennis, P.G., et al., 2014. An expanded genomic representation of the phylum cyanobacteria. Genome Biol. Evol. 6 (5), 1031–1045. Stal, L.J., 2015. Nitrogen fixation in cyanobacteria. In: eLS. John Wiley & Sons, Ltd, Chichester, UK, pp. 1–9. Stal, L.J., Moezelaar, R., 1997. Fermentation in cyanobacteria1. FEMS Microbiol. Rev. 21 (2), 179–211. Stanier, R.Y., Cohen-Bazine, G., 1977. Phototrophic prokaryotes: the cyanobacteria. Annu. Rev. Microbiol. 31, 225–274. Stanier, R.Y., Sistrom, W.R., Hansen, T.A., Whitton, B.A., Castenholz, R.W., Pfennig, N., et al., 1978. Proposal to place the nomenclature of the cyanobacteria (blue-green algae) under the Rules of the International Code of Nomenclature of Bacteria. Int. J. Syst. Bacteriol. 28 (2), 335–336. Steinhauser, D., Fernie, A.R., Araujo, W.L., 2012. Unusual cyanobacterial TCA cycles: not broken just different. Trends Plant Sci. 17 (9), 503–509. Tchernov, D., Helman, Y., Keren, N., Luz, B., Ohad, I., Reinhold, L., et al., 2001. Passive entry of CO2 and its energy-dependent intracellular conversion to HCO3- in cyanobacteria are driven by a photosystem I-generated ΔμH+. J. Biol. Chem. 276, 23450–23455. Thiel, T., Pratte, B., 2014. Regulation of three nitrogenase gene clusters in the cyanobacterium Anabaena variabilis ATCC 29413. Life 4 (4), 944–967. Turmo, A., Gonzalez-Esquer, C.R., Kerfeld, C.A., 2017. Carboxysomes: metabolic modules for CO2 fixation. FEMS Microbiol. Lett. 364 (18), https:// doi.org/10.1093/femsle/fnx176. Valladares, A., Montesinos, M.L., Herrero, A., Flores, E., 2002. An ABC-type, high-affinity urea permease identified in cyanobacteria. Mol. Microbiol. 43 (3), 703–715. Wacker, T., Garcia-Celma, J.J., Lewe, P., Andrade, S.L.A., 2014. Direct observation of electrogenic NH4+ transport in ammonium transport (Amt) proteins. Proc. Natl. Acad. Sci. U.S.A. 111 (27), 9995–10000. Wang, H.L., Postier, B.L., Burnap, R.L., 2004. Alterations in global patterns of gene expression in Synechocystis sp. PCC 6803 in response to inorganic carbon limitation and the inactivation of ndhR, a LysR family regulator. J. Biol. Chem. 279 (7), 5739–5751. Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51 (2), 221–271. Woodger, F.J., Bryant, D.A., Price, G.D., 2007. Transcriptional regulation of the CO2-concentrating mechanism in a euryhaline, coastal marine cyanobacterium, Synechococcus sp. strain PCC 7002: role of NdhR/CcmR. J. Bacteriol. 189 (9), 3335–3347. Zehr, J.P., 2011. Nitrogen fixation by marine cyanobacteria. Trends Microbiol. 19 (4), 162–173. Zehr, J.P., Shilova, I.N., Farnelid, H.M., del Carmen Munoz-Marin, M., Turk-Kubo, K.A., 2016. Unusual marine unicellular symbiosis with the nitrogenfixing cyanobacterium UCYN-A. Nat. Microbiol. 2, 16214. Zhang, P.P., Battchikova, N., Jansen, T., Appel, J., Ogawa, T., Aro, E.M., 2004. Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp PCC 6803. Plant Cell 16 (12), 3326–3340. Zhang, S., Bryant, D.A., 2011. The tricarboxylic acid cycle in cyanobacteria. Science 334 (6062), 1551–1553. Zhang, C.C., Zhou, C.Z., Burnap, R.L., Peng, L., 2018. Carbon/nitrogen metabolic balance: lessons from cyanobacteria. Trends Plant Sci. 23 (12), 1116–1130.

Chapter 16

The scientometric analysis of the research on the algal ecology Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

16.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 papers published since 1980. The algal ecology have been among the most-prolific research fronts over time as evidenced with nearly 65,000 papers, comprising nearly 43% of the algal research as a whole, published during the same study period, corresponding to the public concerns about the development of sustainable and environment-friendly bioproducts and bioprocesses at large. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate, the stakeholders should have timely and thorough access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b, 2012a). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal ecology and the underlying research areas as in fields of the algal research (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a,b,c, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012b,c,d,e,f,g,h,i,j,k, 2018a,b,c), energy and fuels (Konur, 2012l,m,n, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g, 2017b,c,d,e,f, 2019d), biomedicine (Konur, 2016h, 2018d), and social sciences (Konur, 2012o,p,q,r,s,t). Although there have been over 1600 literature reviews on the algal ecology, there have been no published scientometric studies in the journal literature. This is contrast to the many published scientometric studies on ecology at large in the journal literature (Chiu and Ho, 2007; Li et al., 2011; Zhang et al., 2010). Therefore, this paper presents the first-ever scientometric study of the research in algal ecology covering the whole range of research fronts as well as whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate.

16.2  Materials and methodology The search for the scientometric analysis of the literature on the algal ecology was carried out in February 2019 using 4 databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI) and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in the Appendix. These keyword sets have been devised in 2 major parts: the keywords related to ecology and keywords related to the algae. There have been 3 distinct keyword sets for the first part: the set of authors related to the ecology, keywords related to the ecology, and selected set of Web of Science subject categories primarily related to ecology. On the other hand, the second part consists of the keywords related to the algae in general, dinoflagellates and coccolithophores, microalgae, macroalgae, diatoms, cyanobacteria, and a selected set of journal titles related to the algae. Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00016-4 © 2020 Elsevier Inc. All rights reserved.

257

258  PART | V  Algal ecology

Additionally, a selected set of keywords and the Web of Science subject categories have been used to eliminate the papers unrelated to algal ecology. The papers located through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subject to the quality control exercise to ensure that these references have been primarily related to the algal ecology. This refined list of papers has formed the core sample for the scientometric and content overview of the literature on the algal ecology. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. Additionally, the search has been carried out for the papers with at least 100 citations and the relevant scientometric data has been collected. These papers have been termed as ‘influential papers’. Furthermore, the data on the scientometric analysis and brief content overview of 20 most-cited papers have also been provided focusing on the determination of the key research fronts for these citation classics, complementing sections on the keywords and research fronts. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

16.3 Results 16.3.1  Documents and indexes The search has resulted in 71,066 papers where there have been 63,148 articles, 3689 meeting abstracts, 1662 reviews, 1119 notes, 528 editorial materials, 431 corrections, 180 letters, and 180 news items. In the first instance, the papers excluding meeting abstracts, news items, and corrections have been selected resulting in 66,637 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 64,370 papers in English. The other major languages have been French, Russian, Japanese, Spanish, and German. This set of 64,370 papers has formed the core sample for the scientometric analysis of the literature on the algal ecology. The articles have formed 94.8% of the final sample whilst reviews, notes, letters, and editorial matters have formed 2.5%, 1.6%, 0.3%, and 0.8% of this sample, respectively. Additionally, 4.1% of these papers have been ‘proceedings papers’ and there have been 6 ‘retracted papers’. On the other hand, 99.4% of these papers have been indexed by the SCI-E whilst only 0.3% of the papers have been indexed by the SSCI and A&HCI. Additionally, 0.5% of the papers have been indexed by the ESCI.

16.3.2 Keywords The most-prolific keywords used in algal ecology have been determined based on the influential papers to locate the hot topics and the primary research fronts in the algal ecology There have been a number of most-prolific keywords for the first set of keywords for the ecology: ‘ecol*, marine, bloom*, *water*, *fish*, sea*, lake, bacter*, climat*, aqua*, feeding, virus*, irradiat*, coast*, sediment*, diet*, *nutrient*, streams, ice, river*, polar, viral, *nitrogen, springs, desert, *ocean*, crust*, *arctic*, ultraviolet, nitrate*, rock, paleo*, forest*, atlantic, cyst, *toxicity, invasive, invasion, predat*, shore*, larva*, harmful’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, phytoplankton, carrageenan*, macroalga*, rhodophyt*, seaweed*, bacillariophy*, diatom, diatoms, and cyanobacter*’. The other prolific keywords for the algae have been ‘coccolith*, dinophycea*, Alexandrium, chlorophycea*, chlorophyt*, ‘green alga*,’ microalga*, ‘micro-alga*,’ Chlamydomonas, *Chlorella, Dunaliella, Euglena, Scenedesmus, ‘brown alga*,’ alginate*, phaeophycea*, kelp*, phaeophyt*, ‘red alga*,’ Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, Ulva, bacillariophyt*, ‘blue green alga*,’ ‘blue-green alga*,’ *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, Synechocystis’.

16.3.3 Authors There have been 90,460 authors contributing to the research on the algal ecology in total. The information on the mostprolific and influential 20 authors is provided in Table 16.1: Authors’ names, gender, institutions, countries, primary research fronts, number of papers produced by these authors, the number of influential papers with at least 100 citations received (I100), and the percentage of the number of influential papers with relative to the number of all the papers published (I-100%).



TABLE 16.1  The most-prolific and influential authors in algal ecology. Gender

Institution

Country

Research fronts

Algae

I-0

I-100

I-100%

1

Paul G. Falkowski

M

Brookhaven Natl. Lab.

US

Photosynthesis

Algae

117

51

43.6

2

Sallie W. Chisholm

F

Massachusetts Inst. Technol.

US

Genomics

Algae

108

41

38.0

3

Francois M.M. Morel

M

Princeton Univ.US

US

Ecology

Algae

89

31

34.8

4

Donald M. Anderson

M

Woods Hole Ocean. Inst.

US

Harmful blooms

Algae

185

30

16.2

5

John A. Raven

M

Univ. Dundee

United Kingdom

Photosynthesis

Algae

131

28

21.4

6

Hans W. Paerl

M

Univ. N. Carolina

US

Harmful blooms

Algae

138

27

19.6

7

Paul J. Harrison

M

Univ. Brit. Columbia

Canada

Ecology

Algae

185

24

13.0

8

Ulf Riebesell

M

Helmholtz Ctr. Ocean. Res.

Germany

Photosynthesis

Algae

106

21

19.8

9

John K. Volkman

M

CSIRO

Australia

Geochemistry

Algae

58

20

34.5

10

Richard J. Geider

M

Univ. Essex

United Kingdom

Photosynthesis

Algae

72

20

27.8

11

Daniel Vaulot

M

Univ. Paris 06

France

Ecology

Algae

78

20

25.6

12

Mark E. Hay

M

Univ. N Carolina

US

Ecology

Algae

75

19

25.3

13

Trevor Platt

M

Fish. Ocean. Canada

Canada

Photosynthesis

Algae

90

17

18.9

14

Patricia M. Glibert

F

Univ. Maryland

US

Harmful blooms

Algae

91

17

18.7

15

Giacomo R. DiTullio

M

Univ. Tennessee

US

Ecology

Algae

51

16

31.4

16

Robert R. Bidigare

M

Texas A&M Univ.

US

Ecology

Algae

57

16

28.1

17

Frederic Partensky

M

Univ. Paris 06

France

Ecology

Algae

71

16

22.5

18

Walker O. Smith

M

Univ. Tennessee

US

Ecology

Algae

75

16

21.3

19

Colin S. Reynolds

M

Freshwater Biol. Assoc.

United Kingdom

Ecology

Algae

82

16

19.5

20

David A. Hutchins

M

Univ. Delaware

US

Ecology

Algae

102

15

14.7

Average

98.1

23.1

Total %

3.0

15.8

M: Male. F: Female. I-0: No. papers, the number of papers for at least 10 papers. I-100: The number of influential papers with at least 100 citations for at least 5 papers, I-100%: The percentage of the number of influential papers with relative to the number of all the papers published.

The scientometric analysis of the research on the algal ecology Chapter | 16  259

Author

260  PART | V  Algal ecology

The data on these authors shows that the most-prolific author with the highest citation impact has been ‘Paul G Falkowski’ of the Brookhaven National Laboratory, working primarily on the ‘photosynthesis’ by algae, with 117 papers. His citation impact is the highest with 51 influential papers. The other most-prolific authors with the high citation impact have been ‘Sallie W Chisholm’, ‘Francois MM Morel’, ‘Donald M Anderson’, ‘John A Raven’, and ‘Hans W Paerl’ with at least 27 influential papers each. The US has been the most-prolific country for these authors with 11 authors whilst United Kingdom, France, and Canada have been the other prolific countries with 3, 2, and 2 authors, respectively. On the other hand, Europe has had only 6 authors as a whole. In total, these top authors have been from 6 countries. There has been a significant gender deficit among these top prolific and influential authors as only 2 of them are females: ‘Sallie W Chisholm’ and ‘Patricia M Glibert’. Similarly, the most-prolific institutions have been ‘University of North Carolina’, ‘University of Paris 06’, and ‘University of Tennessee’ with 2 authors each. In total, these top authors have been affiliated with 17 institutions. The most-prolific research fronts have been the ‘ecology’, ‘photosynthesis’, ‘harmful blooms’ with 10, 5, and 3 authors, respectively. Similarly, the most prolific types of algae studied by these top authors have been ‘algae’ as these authors have studied more than one type of algae. The number of papers published by these authors have ranged from 51 to 185 with 98.1 papers on average. These most-prolific authors have also contributed to nearly 3.0% and 15.8% of all the papers and influential papers, respectively. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the influential papers with relative to the number of all the papers published for each author in the last column (I-100%). These data shows that ‘Paul G Falkowski’, ‘Sallie W Chisholm’, ‘Francois MM Morel’, ‘John K Volkman’, and ‘Giacomo R DiTullio’ have been the top influential authors with at least 31.0% ratios.

16.3.4 Countries Nearly 99.6% of the papers have had country information in their abstract pages and 184 countries and territories have contributed to these papers overall. Table 16.2 provides the information about the most-prolific and influential 20 countries. These 20 most-prolific countries have produced 104.7% and 132.1% of all the papers and influential papers, respectively, as a whole. The most-prolific and influential country has been the US producing 26.9% and 49.6% of all the papers and influential papers, respectively. The other prolific and influential countries have been United Kingdom, Canada, Germany, Australia, and France producing 14.2%, 10.3%, 9.6%, 8.3%, and 7.3% of the influential papers, respectively. The European countries have been relatively dominant in the top-20 country list as they have produced 45.4% and 55.4% of all the papers and influential papers, respectively, as a whole, surpassing significantly the US. Similarly, the Asian countries in this top-20 list, have produced 22.6% and 14.9% of all the papers and influential papers, respectively, as a whole.

16.3.5 Institutions Over 99.6% of the papers have had their institutions listed in their abstract pages. For these papers, 13,712 institutions have contributed to the research on the algal ecology in total. The information about the 20 most-prolific and influential institutions is given in Table 16.3. The most-prolific and influential institution has been the ‘French National Scientific Research Center’ (CNRS) publishing 3.6% and 5.1% of the all and influential papers, respectively. The other prolific and influential institutions have been ‘Helmholtz Association’ of Germany, ‘Woods Hole Oceanographic Institution’ of the US, ‘Sorbonne University’ of France, ‘Fisheries and Oceans Canada’, ‘University of California San Diego’, and ‘Natural Environment Research Council’-NERC of the United Kingdom with over 2.7% of the influential papers each. The most-prolific country for these institutions has been the US with 10 institutions producing 22.9% of the influential papers. Additionally, Canada, France, and Netherlands have had 2 institutions each. On the other hand, Europe has had 7 institutions as a whole, producing 20% of the influential papers. The contribution of these institutions has ranged from 0.4% to 3.6% for all the papers and from 1.6% to 5.1% for the influential papers. Overall, these 20 institutions have contributed to 23.9% and 49.8% of all the papers and influential papers, respectively.

The scientometric analysis of the research on the algal ecology Chapter | 16  261



TABLE 16.2  The most-prolific and influential countries in algal ecology. Country

I-0

I-0%

I-100

I-100%

Surplus %

Europe

29,193

45.4

1592

54.4

9.1

Asia

14,566

22.6

437

14.9

−7.7

1

US

17,340

26.9

1451

49.6

22.7

2

United Kingdom

5859

9.1

415

14.2

5.1

3

Canada

4589

7.1

302

10.3

3.2

4

Germany

5188

8.1

281

9.6

1.5

5

Australia

3828

5.9

242

8.3

2.4

6

France

3873

6.0

213

7.3

1.3

7

Netherlands

1984

3.1

156

5.3

2.2

8

Spain

3287

5.1

101

3.5

−1.6

9

Italy

2218

3.4

86

2.9

−0.5

11

Sweden

1704

2.6

80

2.7

0.1

10

Japan

4216

6.6

74

2.5

−4.1

12

Norway

1274

2.0

72

2.5

0.5

13

Denmark

1237

1.9

70

2.4

0.5

14

China

5328

8.3

62

2.1

−6.2

15

Israel

827

1.3

62

2.1

0.8

16

New Zealand

1194

1.9

59

2.0

0.1

17

Belgium

1047

1.6

43

1.5

−0.1

18

Finland

900

1.4

40

1.4

0.0

19

Switzerland

622

1.0

35

1.2

0.2

20

South Africa

878

1.4

19

0.7

−0.7

Total

67,393

104.7

3863

132.1

27.4

I-0: The number of all the papers. I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations. I-100%: The percentage of the number of influential papers. Europe and Asia values are for only the top-20 countries. Surplus%: I-100%-I-0%.

16.3.6  Research funding bodies Only 38.2% of these papers have had declared any research funding in their abstract pages and overall, 29,051 funding bodies have funded these papers. The corresponding funding rate for the influential papers has been 11.2%. The most-prolific funding body has been the ‘National Natural Science Foundation of China’, funding 3.1% of the papers. The other prolific funding bodies have been ‘Natural Science Foundation’ of the US, ‘Natural Environment Research Council’ of the United Kingdom, ‘Australian Research Council’, ‘Gordon and Betty Moore Foundation’ of the US, and ‘National Basic Research Program of China’.

16.3.7  Publication years Fig. 16.1 shows the number of papers on the algal ecology, published between 1980 and 2018 as of February 2019. The data in this figure shows that the number of papers has risen from 719 papers in 1980 to 3324 papers in 2018. The most prolific decade has been the 2010s with 40.3% of the papers. Additionally, 13.9%, 18.5%, and 27.3% of the papers have been published in the 1980s, 1990s, and 2000s, respectively. Thus, the figure shows that there has been a steadily increasing trend between 1980 and 2018.

TABLE 16.3  The most-prolific and influential institutions in algal ecology. Institutions

Country

I-0

I-0%

I-00

I-100%

Surplus %

US

5376

8.4

671

22.9

14.5

Europe

8322

12.9

585

20.0

7.1

Asia

0

0

0

0

0

1

French Natl. Sci. Ctr.- CNRS

France

2326

3.6

150

5.1

1.5

2

Helmholtz Assoc.

Germany

1639

2.3

109

3.7

1.4

3

Woods Hole Ocean. Inst.

US

637

1.0

105

3.6

2.6

4

Sorbonne Univ.

France

1120

1.7

96

3.3

1.6

5

Fish. Ocean. Canada

Canada

756

1.2

85

2.9

1.7

6

Univ. Calif. San Diego

US

734

1.1

82

2.8

1.7

7

Natrl. Env. Res. Counc.-NERC

England

1045

1.6

79

2.7

1.1

8

Natl. Ocean. Atmosp. Admin.-NOAA

US

808

1.3

74

2.5

1.2

9

Commonwh. Sci. Ind. Res. Org.-CSIRO

Australia

545

0.8

65

2.2

1.4

10

Univ. N Carolina

US

668

1.0

65

2.2

1.2

11

Oregon St. Univ.

US

549

0.9

64

2.2

1.3

12

Univ. Calif. Santa Barbara

US

519

0.8

63

2.2

1.4

13

Univ. Washington

US

512

0.8

63

2.2

1.4

14

Massachusetts Int. Technol.-MIT

US

256

0.4

57

1.9

1.5

15

Utrecht Univ.

Netherlands

607

0.9

56

1.9

1

16

Dalhousie Univ.

Canada

387

0.6

52

1.8

1.2

17

Natl. Aero. Space Admim-NASA.

US

319

0.5

51

1.7

1.2

18

Royal Neth. Inst. Sea Res.-NIOZ

Netherlands

447

0.7

49

1.7

1

19

Univ. Calif. Santa Cruz

US

374

0.6

47

1.6

1

20

Spanish Natl. Res. Counc.-CSIC

Spain

1138

1.7

46

1.6

−0.1

15,386

23.9

1458

49.8

25.9

Total

I-0: The number of all the papers, I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations, I-100%: The percentage of the number of influential papers. The US, Europe, and Asia values are for only the top-20 institutions. Surplus%: I-100%-I-0%.

3500 3000

Number of papers

2500 2000 1500 1000 500 0

Publication years

FIG. 16.1  The number of publications in the algal ecology between 1980 and 2018.

The scientometric analysis of the research on the algal ecology Chapter | 16  263



16.3.8  Source titles Overall, these papers have been published in 2730 journals. Table 16.4 provides the information on the 20 most-prolific and influential journals. These 20 journals have published 26.9% and 56.1% of all the papers and influential papers, respectively, in total. The most-prolific and influential journal has been ‘Limnology and Oceanography’ publishing 2.6% and 11.0% of all the papers and influential papers, respectively. ‘Marine Ecology Progress Series’ has closely followed the top journal with 3.7% and 8.4% of the all the papers and influential papers, respectively.

TABLE 16.4  The most-prolific and influential journals in algal ecology. Journals

Abbr.

Subject

I-0

I-0%

I-100

I- 100%

Surplus %

Ecol.

4749

7.4

454

15.5

8.1

Ocean.

7057

11

722

24.7

13.7

Mar. Fresh. Biol.

13,105

20.4

864

29.5

9.1

Mult. Sci.

520

0.8

249

8.5

7.7

1

Limnology and Oceanography

Limnol. Oceanogr.

Limnol. Ocean.

1694

2.6

324

11.0

8.4

2

Marine Ecology Progress Series

Mar. Ecol. Prog. Ser.

Ecol. Mar. Fresh. Biol. Ocean.

2386

3.7

245

8.4

4.7

3

Journal of Phycology

J. Phycol.

Plant Sci. Mar. Fresh. Biol.

1882

2.9

150

5.1

2.2

4

Nature

Nature

Mult. Sci.

171

0.3

112

3.8

3.5

5

Marine Biology

Mar. Biol.

Mar. Fresh. Biol.

1036

1.6

102

3.5

1.9

6

Science

Science

Mult. Sci.

134

0.2

75

2.6

2.4

7

Ecology

Ecology

Ecol.

258

0.4

71

2.4

2

8

Journal of Experimental Marine Biology and Ecology

J. Exp. Mar. Biol. Ecol.

Ecol. Mar. Fresh. Biol.

1184

1.8

62

2.1

0.3

9

Proceedings of the National Academy of Sciences of the United States of America

Proc. Natl. Acad. Sci. USA

Mult. Sci.

215

0.3

62

2.1

1.8

10

Journal of Plankton Research

J. Plankton Res.

Mar. Fresh. Biol. Ocean.

1329

2.1

55

1.9

−0.2

11

Remote Sensing of Environment

Remote Sens. Environ.

Environ. Sci. Remote Sens. Imaging Sci. Phot. Tech.

289

0.4

51

1.7

1.3

12

Canadian Journal of Fisheries and Aquatic Sciences

Can. J. Fish. Aquat. Sci.

Fish. Mar. Fresh. Biol.

498

0.8

48

1.6

0.8

13

Freshwater Biology

Freshwater Biol.

Ecol. Mar. Fresh. Biol.

655

1.0

42

1.4

0.4

14

Hydrobiologia

Hydrobiologia

Mar. Fresh. Biol.

2071

3.2

39

1.3

−1.9

15

Water Research

Water Res.

Eng. Env. Env. Sci. Water Res.

522

0.8

39

1.3

0.5

16

Deep Sea Research Part II Topical Studies in Oceanography

Deep-Sea Res Pt II

Ocean.

489

0.8

36

1.2

0.4

Continued

264  PART | V  Algal ecology

TABLE 16.4  The most-prolific and influential journals in algal ecology—cont’d Journals

Abbr.

Subject

I-0

I-0%

I-100

I- 100%

Surplus %

17

Journal of Geophysical Research Oceans

J. Geophys. Res.Oceans

Ocean.

400

0.6

34

1.2

0.6

18

Oecologia

Oecologia

Ecol.

266

0.4

34

1.2

0.8

19

Harmful Algae

Harmful Algae

Mar. Fresh. Biol.

1090

1.7

31

1.1

−0.6

20

Estuarine Coastal and Shelf Science

Estuar. Coast. Shelf S.

Mar. Fresh. Biol. Ocean

759

1.2

28

1.0

−0.2

17,328

26.9

1640

56.1

29.2

Total

I-0: The number of all the papers, I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations, I-100%: The percentage of the number of influential papers. Values for 4 subject categories sciences are only for the top-20 journals. Surplus%: I-100%-I-0%.

TABLE 16.5  The most-prolific and influential subject categories in algal ecology. Subject categories

I-0 No. papers

I-0% Papers

I-100 No. papers

I-100% Papers

Surplus %

1

Marine Freshwater Biology

25,186

39.1

1089

37.2

−1.9

2

Oceanography

11,948

18.6

903

30.9

12.3

3

Ecology

10,662

16.6

713

24.4

7.8

4

Limnology

4687

7.3

360

12.3

5.0

5

Plant Sciences

9941

15.4

335

11.5

−3.9

6

Environmental Sciences

8843

13.7

303

10.4

−3.3

7

Multidisciplinary Sciences

2183

3.4

259

8.9

5.5

8

Microbiology

3253

5.1

147

5.0

−0.1

9

Fisheries

3445

5.4

100

3.4

−2.0

10

Geosciences Multidisciplinary

3411

5.3

100

3.4

−1.9

Total

83,559

129.8

4309

147.3

17.5

I-0: The number of all the papers, I-0%: The percentage of the number of all the papers. I-100: The number of influential papers with at least 100 citations, I-100%: The percentage of the number of influential papers. Surplus%: I-100%-I-0%.

The most-prolific subject categories for these journals have been ‘Marine Freshwater Biology’ and ‘Oceanography’ with 10 and 6 journals, respectively. ‘Ecology’ and ‘Multidisciplinary Sciences’ followed these top subjects with 4 and 3 journals, respectively. The other prolific subjects have been ‘Environmental Sciences’, ‘Remote Sensing’, and ‘Water resources’ with 2 papers each.

16.3.9  Subject categories These papers have been indexed by 170 subject categories. The information about the 10 most-prolific and influential subject categories are given in Table 16.5. The most-prolific and influential subject categories have been ‘Marine Freshwater Biology’, ‘Oceanography’, and ‘Ecology’ indexing 37.2% and 30.9%, and 24.4% of the influential papers, respectively. The other prolific and influential subjects have been ‘Limnology’, ‘Plant Sciences’, ‘Environmental Sciences’, and ‘Multidisciplinary Sciences’ with 12.3%, 11.5%, 10.4%, and 8.9% of the influential papers, respectively.

16.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers with at least 100 citations, 4.5% of the research sample of 64,370 papers (Table 16.6).



Research fronts

Algae

Phytoplankton

Microalgae

Cyanobacteria

Diatoms

Dinoflagellates

Coccolithophores

Macroalgae

Total

No.

497

781

161

569

344

299

62

406

3119

%

15.9

25.0

5.2

18.2

11.0

9.6

2.0

13.0

Numbers: The number of influential papers for each research front and type of algae: %: The percentage of influential papers for each research front and type of algae.

The scientometric analysis of the research on the algal ecology Chapter | 16  265

TABLE 16.6  The most-prolific research fronts in algal ecology.

266  PART | V  Algal ecology

The data shows that the field of ‘ecology of phytoplankton’ has been the most prolific research front with 25% of the influential papers. The other key research fronts have been ‘ecology of cyanobacteria’, ‘ecology of algae’, and ‘ecology of macroalgae’, ‘ecology of diatoms’, and ‘ecology of dinoflagellates’ with 18.2%, 15.9%, 13.0%, 11.0%, and 9.6% of the influential papers, respectively.

16.3.11  Citation classics This section provides the information on both the scientometric analysis and brief content overview of the most-cited 20 papers in algal ecology. The information on these papers is given in Table 16.7: authors’ names, publication years, document type, number of authors per paper, lead authors’ names, gender, and paper numbers (lead authors were determined based on the number of papers produced in this field with at least 33 papers in general and 5 influential papers), journals, subject areas, research fronts, types of algae, academic focus of the papers, number of citations received, and the number of citations per year.

16.3.11.1  Scientometric overview of the citation classics These papers have been published between 1980 and 2018. The most-prolific decades have been the 1980s, 1990s, and 2000s with 5, 8, and 7 papers, respectively. The reviews have been overrepresented in these classical papers as there have been 11 articles and 9 reviews. The number of the authors of these papers has ranged from 1 to 44 whilst the mean number of authors has been 8.1. There have been 21 authors with at least 33 papers and 5 influential papers as the lead authors of the citation classics. There has been a significant gender deficit among the lead authors of these classical papers as only 3 authors are female: ‘Sallie W Chisholm’, ‘JoAnn M Burkholder’, and ‘Patricia M Glibert’. The most-prolific and influential lead authors have been ‘Paul G Falkowski’ and ‘Michael J Behrenfeld’ with 3 and 2 citation classics, respectively, working primarily on the ‘algal photosynthesis’. In total, these citation classics have been published by 11 journals. The most-prolific journals have been ‘Nature’, ‘Limnology and Oceanography’, and ‘Science’ with 7, 3, and 2 papers, respectively. In total, these papers have been indexed by 10 subject categories. The most-prolific categories have been ‘Multidisciplinary Sciences’, ‘Oceanography’, ‘Marine Freshwater Biology’, and ‘Limnology’ with 9, 6, 4, and 3 papers, respectively. In total, there have been 9 research fronts. The most-prolific research front has been ‘phytoplankton growth’ with 7 papers. The other prolific research front has been ‘primary production’, ‘harmful algal blooms’, and ‘climate’ with 3, 3, and 2 papers, respectively. There have been 5 types of algae covered by these classical papers. The most prolific type of algae has been ‘phytoplankton’ with 10 papers. In addition, there have been 3, 2, and 2 papers related to ‘cyanobacteria’, ‘microalgae’, and ‘dinoflagellates’, respectively. The most-studied topics have been the ‘iron deficiency’, ‘nutrient limitation’, and ‘harmful algal blooms’ with 5, 2, and 2 papers, respectively, respectively. These papers have received between 831 and 2638 citations each, with a mean value of 1175 citations per paper. On the other hand, the number of citations per year has ranged from 23.7 to 93.7 with a mean value of 57.2 citations per year. The papers by Paerl and Huisman (2008), Hillebrand et al. (1999), and Navarro et al. (2008) have been the most-cited papers on the basis of the number of citations on average, working on the global warming, biovolume calculation, and nanoparticle toxicity, respectively.

16.3.11.2  Brief overview of the content of the citation classics There have been 3 major classes of papers: ‘phytoplankton growth’, ‘primary production’, ‘harmful algal blooms’, ‘climate’, and ‘other research fronts’ with 7, 3, 3, 2, and 5 papers, respectively.

16.3.12  Phytoplankton growth Martin and Fitzwater (1988) study the impact of the iron deficiency on the phytoplankton growth in the northeast Pacific subarctic in a paper with 1313 citations. They find that the addition of dissolved iron resulted in the utilization of excess nitrate (NO3) with an increase in the amounts of chlorophyll. They assert that iron deficiency is limiting phytoplankton growth in these nutrient-rich waters.

TABLE 16.7  The citation classics in algal ecology. Authors

Year

Doc.

N Auths.

Journal

Subject area

Res. fronts

Algae

Topic

Cits.

Av. cits

1

Charlson et al.

1987

R

5

Nature

Mult. Sci.

Climate

Phytoplankton

Climate bioregulation

2638

85.1

2

Hillebrand et al.

1999

A

5

J. Phycol.

Plant. Sci. Mar. Fresh. Biol.

Biovolume

Microalgae

Biovolume calculation

1729

91.0

3

Hallegraeff

1993

R

1

GM Hallegraeff-15

Phycologia

Plant. Sci. Mar. Fresh. Biol.

Harmful blooms

Dinoflagellates

Harmful blooms

1466

58.6

4

Behrenfeld and Falkowski

1997

A

2

MJ Behrenfeld-8; PG Falkowski-51

Limnol. Oceanogr.

Limnol. Oceanogr.

Primary production

Phytoplankton

Carbon fixation

1376

65.5

5

Platt et al.

1980

A

3

T Platt-17

J. Mar. Res.

Oceanogr.

Primary production

Phytoplankton

Photosynthesis inhibition

1337

35.2

6

Martin and Fitzwater

1988

A

2

Nature

Mult. Sci.

Phytoplankton growth

Phytoplankton

Iron deficiency

1313

43.8

7

MendenDeuer and Lessard

2000

A

2

Limnol. Oceanogr.

Limnol. Oceanogr.

Carbon density

Dinoflagellates

Carbon to volume relationships

1253

69.6

8

Martin

1990

A

1

Paleoceanography

Paleo. Oceanogr.

Phytoplankton growth

Phytoplankton

Iron deficiency

1135

40.5

9

Coale et al.

1996

A

19

FP Chavez-9; WP Cochlan-6

Nature

Mult. Sci.

Phytoplankton growth

Phytoplankton

Iron deficiency

1082

49.2

10

Anderson et al.

2002

R

3

DM Anderson-30; PM Glibert-17; JM Burkholder-10

Estuaries

Env. Sci. Mar. Fresh. Biol.

Harmful blooms

Algae

Harmful blooms and eutrophication

1058

66.1

11

Boyd et al.

2000

A

35

PW Boyd-16

Nature

Mult. Sci.

Phytoplankton growth

Phytoplankton

Iron deficiency

1012

56.2

12

Hecky and Kilham

1988

R

2

RE Hecky-5

Limnol. Oceanogr.

Limnol. Oceanogr.

Phytoplankton growth

Phytoplankton

Nutrient limitation

992

33.1

14

Behrenfeld et al.

2006

R

10

MJ Behrenfeld-8; PG Falkowski-47

Nature

Mult. Sci.

Primary production

Phytoplankton

Climate change

978

81.5

14

Martin et al.

1994

R

44

PG Falkowski-47; SW Chisholm-47; RR Bidigare-16

Nature

Mult. Sci.

Phytoplankton growth

Phytoplankton

Iron deficiency

958

39.9

15

Paerl and Huisman

2008

A

2

HW Paerl-23; J Huisman-13

Science

Mult. Sci.

Harmful blooms

Cyanobacteria

Global warming

947

94.7

Lead authors

Continued

TABLE 16.7  The citation classics in algal ecology—cont’d

Authors

Year

Doc.

N Auths.

Journal

Subject area

Res. fronts

Algae

Topic

Cits.

Av. cits

16

Navarro et al.

2008

A

8

Environ. Sci. Technol.

Eng. Env. Env. Sci.,

Toxicity

Microalgae

Nanoparticle toxicity

880

88.0

17

Wright et al.

1991

R

7

Mar. Ecol. Prog. Ser.

Ecol. Mar. Fresh. Biol. Oceanogr.

Pigments

Phytoplankton

Pigment analysis

847

31.4

18

Partensky et al.

1999

R

3

Microbiol. Mol. Biol. R.

Microbiol.

Ecology

Cyanobacteria

Prochlorococcus

837

44.1

19

Smith

1983

A

1

Science

Mult. Sci.

Phytoplankton growth

Cyanobacteria

Nutrient limitation

831

23.7

20

Riebesell et al.

2000

R

6

Nature

Mult. Sci.

Climate

Coccolithophores

CO2 impact

831

46.2

Average

1995

1175

57.2

8.1

Lead authors

F Partensky-16; WR Hess-7; D Vaulot-20

U Riebesell-21; B Rost-5; FMM Morel-31

Doc.: Document. A. Article; R: Review. Gender: gender of lead authors- female authors in italic. N paper: for the authors with at least 33 papers with 0 citations and with at least 5 influential papers -number after the author names. Subject: Web of Science subjects. Topic: primary topic of the papers, Algae: type of algae studied; Res. fronts: primary research fronts studied; Cits.: Number of citations received in total. Av. Cits.: Number of citations per year.



The scientometric analysis of the research on the algal ecology Chapter | 16  269

Martin (1990) presents the ‘iron hypothesis’ in a paper with 1135 citations. He argues that new productivity in southern ocean is limited by iron deficiency where the phytoplankton are unable to take advantage of the excess surface nitrate and phosphate. He asserts that due to the atmospheric dust iron supplies during the last maximum (LGM), phytoplankton growth may have been greatly enhanced with the LGM drawdown of atmospheric CO2. Coale et al. (1996) study the phytoplankton bloom induced by dissolved iron fertilization in the Pacific Ocean in a paper with 1082 citations. They find that the fertilization resulted in massive phytoplankton blooms consuming large quantities of CO2 and NO3. Their findings support the hypothesis that phytoplankton growth in this oceanic region is limited by iron bioavailability. Boyd et al. (2000) study the phytoplankton bloom in the Southern Ocean stimulated by iron fertilization in a paper with 1012 citations. They find that increased iron supply led to elevated phytoplankton biomass and rates of photosynthesis in surface waters, causing a large drawdown of CO2 and NO3, and elevated dimethylsulphide levels. Hecky and Kilham (1988) review the nutrient limitation of phytoplankton in fresh-water and marine environments in a review paper with 992 citations. They argue that dissolved nutrient concentrations are most useful in determining nutrient loading rates of aquatic ecosystems. They assert that the relative proportions of nutrients supplied to phytoplankton can be a strong selective force shaping phytoplankton communities and affecting the biomass yield per unit of limiting nutrient. Martin et al. (1994) test the ‘iron hypothesis’ in the Pacific Ocean in a paper with 958 citations. They find a doubling of plant biomass, a threefold increase in chlorophyll and a fourfold increase in plant production. They assert that that iron limitation can control rates of phytoplankton productivity and biomass in the ocean. Smith (1983) studies the management of cyanobacteria in the lake phytoplankton in a paper with 831 citations. He finds that the relative proportion of cyanobacteria in the phytoplankton is dependent on the ratio of total nitrogen to total phosphorus and the lake water quality can be managed through the modification of this ratio through the control of nutrient additions.

16.3.13  Harmful algal blooms Hallegraeff (1993) reviews harmful algal blooms (HABs) and their global rise in a review paper with 1466 citations. He argues that the prediction of the impact of global climate change on marine HABs is difficult. He asserts that increasing temperature, enhanced surface stratification, alteration of ocean currents, intensification or weakening of local nutrient upwelling, stimulation of photosynthesis by elevated CO2, reduced calcification through ocean acidification, and heavy precipitation and storm events causing changes in land runoff and micronutrient availability may all produce contradictory species- or even strain-specific responses. Anderson et  al. (2002) review HABs and eutrophication focusing on the nutrient sources, composition, and consequences in a review paper with 1058 citations. They note that strong correlations have been demonstrated between total phosphorus inputs and phytoplankton production in freshwaters, and between total nitrogen input and phytoplankton production in estuarine and marine waters. There are also numerous examples in geographic regions where increases in nutrient loading have been linked with the development of large biomass blooms, leading to anoxia and even toxic or harmful impacts on fisheries resources, ecosystems, and human health or recreation. Paerl and Huisman (2008) discuss the relationships between the global warming and proliferation of harmful cyanobacterial blooms in a paper with 947 citations.

16.3.14  Primary production Behrenfeld and Falkowski (1997) study the depth-integrated phytoplankton carbon fixation derived from satellite-based chlorophyll concentration in a paper with 1376 citations. They develop a light-dependent, depth-resolved model for carbon fixation that partitions environmental factors affecting primary production into those that influence the relative vertical distribution of primary production and those that control the optimal assimilation efficiency of the productivity profile. Platt et al. (1980) study the photoinhibition of photosynthesis in marine phytoplankton in a paper with 1337 citations. They develop an empirical equation describing the photosynthesis by phytoplankton as a single, continuous function of available light from the initial linear response through the photoinhibited range at the highest levels. Behrenfeld et al. (2006) study the climate-driven trends in contemporary ocean productivity in a paper with 978 citations. They describe global ocean net primary production (NPP) changes detected from space over the past decade. They assert that the link between the physical environment and ocean biology functions through changes in upper-ocean temperature and stratification, which influence the availability of nutrients for phytoplankton growth.

270  PART | V  Algal ecology

16.3.15 Climate Charlson et al. (1987) discuss the biological regulation of the climate by algae in a review paper with 2638 citations. They note that the major source of cloud-condensation nuclei (CCN) over the oceans is dimethylsulphide, which is produced by planktonic algae in sea water and oxidizes in the atmosphere to form a sulfate aerosol. The biological regulation of the climate is possible through the effects of temperature and sunlight on phytoplankton population and dimethylsulphide production. They argue that to counteract the warming due to doubling of atmospheric CO2, an approximate doubling of CCN would be needed. Riebesell et al. (2000) study the reduced calcification of marine plankton in response to increased atmospheric CO2 in a paper with 831 citations. They find reduced calcite production at increased CO2 concentrations in monospecific cultures of two dominant marine calcifying coccolithophores. Diminished calcification led to a reduction in the ratio of calcite precipitation to organic matter production. They argue that the progressive increase in atmospheric CO2 concentrations may slow down the production of calcium carbonate in the surface ocean.

16.3.16  Other research fronts Hillebrand et al. (1999) study the biovolume calculation for microalgae in a paper with 1729 citations. They present a set of geometric shapes and mathematical equations for calculating biovolumes of microalgae to minimize the effort of microscopic measurement. Menden-Deuer and Lessard (2000) study the carbon to volume relationships for dinoflagellates in a paper with 1253 citations. They find that C and N density in dinoflagellates decreased significantly with increasing cell volume. Partensky et al. (1999) study Prochlorococcus in a paper with 837 citations. They note that its tiny size is an advantage for its adaptation to nutrient-deprived environments. Genetically distinct ecotype are present at depth and in surface waters. They assess the basic knowledge acquired about Prochlorococcus both in the ocean and in the laboratory. Wright et al. (1991) study the improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton in a paper with 847 citations. They find that this method is useful for analysis of phytoplankton pigments in seawater samples. Navarro et al. (2008) study the toxicity of silver nanoparticles (AgNPs) to Chlamydomonas reinhardtii in a paper with 880 citations. They find that the interaction of cysteine with algae influences the toxicity of AgNP, which is mediated by Ag+. These particles contributed to the toxicity as a source of Ag+ which is formed in presence of algae.

16.4 Discussion As there have been over 64,000 core papers related to the algal ecology, comprising over 42% of the papers on the algae as a whole, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts (Konur, 2011, 2012b,c,d,e,f,g,h,i,j,k, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a,b,c,d,e,f,g, 2017a,b,c,d,e,f, 2018a,b,c,d, 2019a,b,c,d, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). The primary mode of scientific communication has been articles whilst reviews have formed 2.5% of the sample. The primary index has been SCI-E indexing more than 99.4% of the papers whilst only 0.3% of the papers have been indexed by the SSCI and A&HCI focusing on the societal aspects of algal ecology. These findings suggest that there is substantial room for the research in social and humanitarian aspects such as policy-related studies as well as scientometric studies in this field. The most-prolific keywords related to the algal ecology have been determined through the detailed examination of the over 3100 influential papers with at least 100 citations. A detailed keyword set has been devised for the search (given in the appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the ecology have been ‘ecol*, marine, bloom*, *water*, *fish*, sea*, lake, bacter*, climat*, aqua*, feeding, virus*, irradiat*, coast*, sediment*, diet*, *nutrient*, streams, ice, river*, polar, viral, *nitrogen, springs, desert, *ocean*, crust*, *arctic*, ultraviolet, nitrate*, rock, paleo*, forest*, atlantic, cyst, *toxicity, invasive, invasion, predat*, shore*, larva*, harmful’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, phytoplankton, carrageenan*, macroalga*, rhodophyt*, seaweed*, bacillariophy**, diatom, diatoms, and cyanobacter*’. These keywords have formed the primary research fronts for the algal ecology. The findings show that although over 90,000 authors have contributed to the research, 20 most-prolific and influential authors have shaped the literature on the algal ecology publishing 3.0% and 15.8% of all the papers and the influential papers, respectively (Table 16.1). The success of these authors, their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988).



The scientometric analysis of the research on the algal ecology Chapter | 16  271

The data provides the evidence for the presence of the significant gender deficit among both the most-prolific authors (Table 16.1) and the lead authors of the citation classics as only 2 and 3 these top authors are female, respectively (Table 16.7) (Bordons et al., 2003). The data on the papers by the most-prolific and influential authors highlight the primary research fronts as the ‘ecology’, ‘photosynthesis’, and ‘harmful blooms’. It has been found during the search process that the author names with 2 or more forenames or surnames have been spelt differently by the databases with significant implications for the recovery of their papers. Similar difficulties have also been observed for the common names such as ‘Wang Y’ or ‘Li Y’ for the recovery of their papers and the related analysis. The data shows that although over 180 countries and territories have contributed to the research in algal ecology, most-prolific 20 countries contributed to 104.7% and 132.1% of all the papers and the influential papers, respectively (Table 16.2). The major producers of the research have been the US, Canada, Australia, and Europe as these countries have had the ‘first-mover advantage’ over the other countries. It is notable that the citation impact of China has been relatively small in relation to other top producers as China has produced 8.3% and 2.1% of all the papers and influential papers, respectively (Guan and Ma, 2007). Similarly, Japan has also had the reduced citation impact. As in the case of countries, although over 13,700 institutions have contributed to the research in algal ecology, the 20 most-prolific institutions, having the first-mover advantages, have published more than 23.9% of all the papers and 49.8% of the influential papers, respectively (Table 16.3). As only 38.2% and 11.2% of all the papers and influential have declared a research funding, respectively, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). It is notable that there has been significant research funding opportunities in China and Latin America in relation to the US and Europe. Thus, there is ample room to extend the research funding opportunities to support the research and development in algal ecology. The steady rise in the number of the publications in this field in the 2000s and 2010s (as of February 2019) provides the strong evidence for the increasing public importance of the algal ecology in recent years (Fig. 16.1). The annual number of publications have risen to over 3300 papers and it is expected that the number of papers would continue to rise in the next decade with at least another 65,000 papers, provided that the opportunities for research funding would increase significantly, corresponding to the increasing importance of the algal ecology to the global society at large. Although over 2700 journals have contributed to the research in algal ecology, the 20 most-prolific journals, having the first-mover advantages, have published over 26.9% and 56.1% of all the papers and influential papers, respectively (Table 16.4). This finding has been most relevant for the top journals. The data on the Web of Science subject categories suggests that the first-3 categories have been the key pillars of the research in algal ecology, indexing together 74.3% and 92.5% of all the papers and influential papers, respectively, forming the scientific basis of the research in this field: ‘Marine Freshwater Biology’, ‘Oceanography’, and ‘Ecology’ (Table 16.5). As the journals related to algae in the top 20 journal list have published only 2.9% and 5.1% of all the papers and influential papers, respectively, the broad search strategy, covering all subject categories and journals, developed for this study, has been justified. Similarly, ‘Ecology’ subject category has indexed only 16.6% and 24.4% of all the papers and influential papers, respectively. The data on the research fronts have confirmed that the major research fronts have been ‘ecology of cyanobacteria’, ‘ecology of algae’, and ‘ecology of macroalgae’, ‘ecology of diatoms’, and ‘ecology of dinoflagellates’ (Table 16.6). The most-studied the types of algae have been ‘phytoplankton’, ‘cyanobacteria’, ‘microalgae’, and ‘dinoflagellates’. The extensive data on the 20 citation classics largely confirm the findings of the earlier sections based on over 64,000 papers (Table 16.7). There has been a significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely overrepresented in the citation classic sample as there have been 9 reviews. Similarly, the most-prolific research fronts have been ‘phytoplankton growth’, ‘primary production’, ‘harmful algal blooms’, and ‘climate’. The most prolific types of algae have been ‘phytoplankton’, ‘cyanobacteria’, ‘microalgae’, and ‘dinoflagellates’. The most-studied topics have been the ‘iron deficiency’, ‘nutrient limitation’, and ‘harmful algal blooms’.

16.5 Conclusion This analytical study of the research in algal ecology at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field.

272  PART | V  Algal ecology

Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as algal research (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a,b,c, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012b,c,d,e,f,g,h,i,j,k, 2018a,b,c), energy and fuels (Konur, 2012l,m,n, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g, 2017b,c,d,e,f, 2019d), biomedicine (Konur, 2016h, 2018d), and social sciences (Konur, 2012o,p,q,r,s,t), complementing over 1600 literature reviews. The data has shown that the annual number of papers in this field has risen to over 3300 papers whilst there have been over 64,000 papers over the study period from 1980 to 2018. It is further expected that the size of the research 65,000 papers in the next decade, corresponding to the increasing public importance of the algal ecology to the global society at large. The provision of the adequate research funding is essential for the further development of the research and practice in this field as only 38.2% and 11.2% of all the papers and influential papers have declared a research funding, respectively. The key research fronts have been ‘ecology of cyanobacteria’, ‘ecology of algae’, and ‘ecology of macroalgae’, ‘ecology of diatoms’, and ‘ecology of dinoflagellates’. The most-studied the types of algae have been ‘phytoplankton’, ‘cyanobacteria’, ‘microalgae’, and ‘dinoflagellates’. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies (Konur, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). It has been found that the detailed keyword set provided in the appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It appears that the structure-processing-property relationships form the basis of the research in algal ecology as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002). It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts.

Appendix: The keyword sets A.1  Keywords related to ecology WC = (ecol* or ocean* or limnol* or meteor* or water* or geol* or geosci* or geochem* or zoo* or soil* or fish* or biodivers* or paleo* or remote* or ‘engineering ocean’ or ‘engineering marine’ or ‘engineering geological’) OR TI = (coral or coralline or plankton* or picoplankton* or marine or ‘food-web*’ or foodweb* or bloom* or *water* or *fish* or *zooplankt* or nanoplankt* or sea* or *invertebrate* or lake or grazing or bacter* or ‘N-2’ or herbivor* or algicid* or climat* or aqua* or feeding or turf or reef or grazer* or lagoon* or virus* or irradiat* or irradiance or coast* or sediment* or diet* or *nutrient* or *lichen* or estuar* or ‘food chain*’ or *tidal or streams or symbiont* or daphn* or ice or *benthic or river* or eutroph* or wetland* or *symbios* or symbiotic* or dmsp or *acclimation or polar or viral or *nitrogen or springs or *phage* or foraging or desert or glacier* or glacial or *ocean* or crust* or *arctic* or mats or ultraviolet or nitrate* or rock or grassland* or copepod* or paleo* or warming or periphytic or lochs or tsunami or forest* or ‘primary product*’ or atlantic or cyst or biogeo* or *toxicity or ‘remote sens*’ or *ecosyst* or *ecophys* or zonation or bivalve* or ‘chemical defen*’ or cladoceran* or fjord* or mussel* or ecotyp* or trout* or crab* or invasive or invasion or mollus* or ecotox* or predat* or shore* or dispersal or amphipod or larva* or harmful or *calanus or gastropod* or mytilus or dimethylsulfonioprop*) or AU = (‘hallegraeff gm’ or ‘paerl hw’ or ‘smol jp’ or ‘anderson dm’ or ‘jeong hj’ or ‘paul vj’ or ‘wiencke c’ or ‘gobler cj’ or ‘vyverman w’ or ‘harrison pj’ or ‘carpenter ej’ or ‘bergman b’ or ‘burkholder jm’ or ‘huisman j’ or ‘hay me’ or ‘olsen jl’ or ‘stevenson rj’ or ‘van etten jl’ or ‘verlaque m’ or ‘graneli e’ or ‘hutchins da’ or ‘raven ja’ or ‘glibert pm’ or ‘riebesell u’ or ‘steinberg pd’ or ‘chisholm sw’ or ‘zehr jp’ or ‘lajeunesse tc’ or ‘pohnert g’ or ‘reed dc’ or ‘ianora a’ or ‘de vernal a’ or ‘flynn kj’ or ‘falkowski p*’ or ‘volkman jk’ or ‘partensky f’ or ‘karsten u’ or ‘sommer u’ or ‘morel fmm’ or ‘platt t’ or ‘hay me’ or ‘smith wo’ or ‘nelson dm’ or ‘reed dc’ or ‘reynolds cs’ or ‘arrigo kr’ or ‘verlaque m’ or ‘qin bq’ or ‘van donk e’ or ‘vaulot d’ or ‘boyd p*’ or ‘geider rj’).

A.2  Keywords related to the algae TI = (alga or algae or algal or phyco* or chlorarachn* or Bigelowiella or phytoplankton or periphyton) OR TI = (chrysophy* or chlorococcal* or chrysophy* or *coccolith* or dinocyst* or dinoflagell* or dinophy* or haptophy* or peridinial* or prymnes* or raphidophy* or ‘red tide*’ or Akashiwo or Amphidinium or Aureococcus or *Ceratium or *Chattonella or Cochlodinium or Crypthecodinium or Dinophysis or Emiliania or Gambierdiscus or *Gonyaulax or *Gymnodinium or Gyrodinium or Hematodinium or Heterocapsa* or Heterosigma or Isochrysis or Karenia* or Karlodinium or Mallomonas



The scientometric analysis of the research on the algal ecology Chapter | 16  273

or Noctiluca* or Ochromonas or Ostreopsis or Oxyrrhis or Peridinium or Pfiesteria* or Phaeocystis or Prorocentrum or Prymnesium or Scrippsiella* or Symbiodinium or Vaucheria* or Zooxanthella*) OR TI = (chlorophycea* or chlorophyt* or cryptomonad* or cryptophy* or euglen* or eustigmatophy* or ‘green alga*’ or microalga* or ‘micro-alga*’ or ‘micro alga*’ or prasinophy* or streptophy* or trebouxiophy* or volvoc* or Acetabularia* or Ankistrodesmus or Asteromonas or Aurantiochytrium or Botryococcus or Chlamydomon* or *Chlorella* or *Chlorococcum or Coccomyx* or Cyanidioschyzon or Cyanidium or Desmodesmus or Dunaliella or Euglen* or Galdieria* or Haematococcus or Micrasterias or Micromonas or Monoraphidium or Nannochloropsis or Neochloris or Ostreococcus or Phormidium or Platymonas or Polytomella or Porphyridium or Prototheca or Pseudokirchneriella or Pyramimonas or Scenedesmus or Schizochytrium or Selenastrum or Tetraselmis or Trebouxia* or Volvox) OR TI = (‘brown alga*’ or ‘macro-alga*’ or ‘macro alga*’ or ‘red alga*’ or agarophy* or characea* or charophy* or cladophora* or cryptonemia* or dictyotales or florideophy* or fucale* or gelidiales or gigartina* or kelp* or macroalga* or phaeophy* or rhodophy* or seaweed* or ulvale* or ulvophy* or zygnematophy* or ‘Chara Vulgaris’ or Alaria or Ascophyllum or Asparagopsis or Asparagopsis or Bangia* or Bostrychia or Bryopsis or Catenella or Caulerpa or Ceramium or Chaetomorpha* or Chondrus or Cladophora or Codium or Corallina or Cystoseira or Ecklonia or Ectocarpus or ‘Eisenia bicyclis’ or Enteromorpha or Eucheuma or Fucus or Gelidium or Gracilaria* or Gracilariopsis or Grateloupia or Halimeda or Halocynthia or Hizikia or Hypnea or Kappaphycus or Laminaria* or Laurencia* or Lessonia* or Lomentaria or Macrocystis or Monostroma* or Mougeotia or Nitella or Nitellopsis or Oedogonium or Padina or Palmaria or Pelvetia or Plocamium or Polysiphonia or Porphyra* or Pyropia or Saccharina or Sargassum or Scytosiphon or Solieria or Spirogyra or Turbinaria or Ulva* or Undaria) or TI = (bacillariophy* or diatom or diatoms or Asterionella or Chaetoceros or Coscinodiscus or Cyclotella or Cylindrotheca or Didymosphenia or Navicula or *Nitzschia or Phaeodactylum or Skeletonema or Stephanodiscus or Thalassiosira*) OR TI = (‘blue green alga*’ or ‘blue-green alga*’ or cyanelle or *cyanobacter* or cyanophy* or prochlorophy* or Acaryochloris or *Anabaena or Anacystis or Aphanizomenon or Aphanothece or Arthrospira or Calothrix or Cyanophora or Cyanothece or Cylindrospermopsis or *Lyngbya* or Macrocystis or Mastigocladus or Microcoleus or Microcystis or Nodularia or Nostoc* or Oscillatoria* or Planktothrix or Plectonema or Prochlorococcus or Prochloron or Prochlorothrix or Scytonema or Spirulina* or *Synechococcus or Synechocystis or Tolypothrix or Trichodesmium) OR SO = (Alga* or ‘British Phycol*’ or ‘Cryptogamie Alg*’ or ‘Diatom Res*’ or ‘European Journal of Phycol*’ or Fottea* or ‘Harmful Algae’ or ‘Journal of Applied Phycol*’ or ‘Journal of Phycol*’ or Phycol*).

A.3  Excluding keywords NOT TI = (*sorp* or *sorb* or waste* or *diesel* or *fuel* or *polymer* or anti* or bioactiv* or scytonemin or *energy or contaminant* or sulfated or classif* or extract* or ‘shewanella alga*’ or *capacitor* or *saccharide* or phylogen* or cultivat* or edible or nuclear or pharm* or galactan* or triacylglycerol or ingredient* or benefit* or fucoidan) OR WC = (food* or pharm* or ‘chemistry med*’ or immunol* or public* or polymer* or physiol* or endocr* or oncol* or med* or biot* or ener* or nutr*).

References Abramo, G., D’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25, 4B. 704–726. Behrenfeld, M.J., Falkowski, P.G., 1997. Photosynthetic rates derived from satellite-based chlorophyll concentration. Limnol. Oceanogr. 42 (1), 1–20. Behrenfeld, M.J., O’Malley, R.T., Siegel, D.A., McClain, C.R., Sarmiento, J.L., Feldman, G.C., et al., 2006. Climate-driven trends in contemporary ocean productivity. Nature 444 (7120), 752–755. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Boyd, P.W., Watson, A.J., Law, C.S., Abraham, E.R., Trull, T., Murdoch, R., et al., 2000. A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization. Nature 407 (6805), 695–702. Brennan, L., Owende, P., 2010. Biofuels from microalgae-a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14 (2), 557–577. Charlson, R.J., Lovelock, J.E., Andreae, M.O., Warren, S.G., 1987. Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate. Nature 326 (6114), 655–661. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Chiu, W.T., Ho, Y.S., 2007. Bibliometric analysis of tsunami research. Scientometrics 73 (1), 3–17. Coale, K.H., Johnson, K.S., Fitzwater, S.E., Gordon, R.M., Tanner, S., Chavez, F.P., et  al., 1996. A massive phytoplankton bloom induced by an ­ecosystem-scale iron fertilization experiment in the equatorial Pacific Ocean. Nature 383 (6600), 495–501.

274  PART | V  Algal ecology

Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Guan, J., Ma, N., 2007. China’s emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32 (2), 79–99. Hecky, R.E., Kilham, P., 1988. Nutrient limitation of phytoplankton in fresh-water and marine environments – a review of recent evidence on the effects of enrichment. Limnol. Oceanogr. 33 (4), 796–822. Hillebrand, H., Durselen, C.D., Kirschtel, D., Pollingher, U., Zohary, T., 1999. Biovolume calculation for pelagic and benthic microalgae. J. Phycol. 35 (2), 403–424. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems – a review. Biotechnol. Adv. 29 (2), 189–198. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books Ltd., Stoke-on-Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energ. 88 (10), 3532–3540. Konur, O., 2012a. The policies and practices for the academic assessment of blind students in higher education and professions. Ener. Educ. Sci. Tech.-B 4 (si1), 240–244. Konur, O., 2012b. Prof. Dr. Ayhan Demirbas’ scientometric biography. Ener. Educ. Sci. Tech.-A 28 (2), 727–738. Konur, O., 2012c. The evaluation of the biogas research: a scientometric approach. Ener. Educ. Sci. Tech.-A 29 (2), 1277–1292. Konur, O., 2012d. The evaluation of the bio-oil research: a scientometric approach. Ener. Educ. Sci. Tech.-A 30 (si1), 379–392. Konur, O., 2012e. The evaluation of the biorefinery research: a scientometric approach. Ener. Educ. Sci. Tech.-A 30 (si1), 347–358. Konur, O., 2012f. The evaluation of the research on the biodiesel: a scientometric approach. Ener. Educ. Sci. Tech.-A 28 (2), 1003–1014. Konur, O., 2012g. The evaluation of the research on the bioethanol: a scientometric approach. Ener. Educ. Sci. Tech.-A 28 (2), 1051–1064. Konur, O., 2012h. The evaluation of the research on the biofuels: a scientometric approach. Ener. Educ. Sci. Tech.-A 28 (2), 903–916. Konur, O., 2012i. The evaluation of the research on the biohydrogen: a scientometric approach. Ener. Educ. Sci. Tech.-A 29 (1), 323–338. Konur, O., 2012j. The evaluation of the research on the microbial fuel cells: a scientometric approach. Ener. Educ. Sci. Tech.-A 29 (1), 309–322. Konur, O., 2012k. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012l. The evaluation of the global energy and fuels research: a scientometric approach. Ener. Educ. Sci. Tech.-A 30 (1), 613–628. Konur, O., 2012m. 100 citation classics in energy and fuels. Ener. Educ. Sci. Tech.-A 30 (si1), 319–332. Konur, O., 2012n. What have we learned from the citation classics in energy and fuels: a mixed study. Ener. Educ. Sci. Tech.-A 30 (si1), 255–268. Konur, O., 2012o. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Ener. Educ. Sci. Tech.-B 4 (4), 1893–1908. Konur, O., 2012p. The evaluation of the research on the arts and humanities in Turkey: a scientometric approach. Ener. Educ. Sci. Tech.-B 4 (3), 1603–1618. Konur, O., 2012q. The evaluation of the educational research: a scientometric approach. Ener. Educ. Sci. Tech.-B 4 (4), 1935–1948. Konur, O., 2012r. The scientometric evaluation of the research on the deaf students in higher education. Ener. Educ. Sci. Tech.-B 4 (3), 1573–1588. Konur, O., 2012s. The scientometric evaluation of the research on the students with ADHD in higher education. Ener. Educ. Sci. Tech.-B 4 (3), 1547–1562. Konur, O., 2012t. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Ener. Educ. Sci. Tech.-B 4 (si1), 250–257. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512.



The scientometric analysis of the research on the algal ecology Chapter | 16  275

Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. Vol. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. Vol. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: The current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O., 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Algal drugs: the state of the research. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019b. Algal genomics. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019c. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019d. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

276  PART | V  Algal ecology

Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Poly. Sci. 37 (1), 106–126. Li, J.F., Wang, M.H., Ho, Y.S., 2011. Trends in research on global climate change: a science citation index expanded-based analysis. Glob. Planet. Chang. 77 (1–2), 13–20. Lieberman, M.B., Montgomery, D.B., 1988. First-mover advantages. Strateg. Manage. J. 9 (S1), 41–58. Martin, J.H., 1990. Glacial-interglacial CO2 change: the iron hypothesis. Paleoceanography 5 (1), 1–13. Martin, J.H., Coale, K.H., Johnson, K.S., Fitzwater, S.E., Gordon, R.M., Tanner, S.J., et al., 1994. Testing the iron hypothesis in ecosystems of the equatorial Pacific Ocean. Nature 71 (6493), 123–129. Martin, J.H., Fitzwater, S.E., 1988. Iron deficiency limits phytoplankton growth in the northeast Pacific subarctic. Nature 331 (6154), 341–343. Menden-Deuer, S., Lessard, E.J., 2000. Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnol. Oceanogr. 45 (3), 569–579. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N., et al., 2008. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii. Environ. Sci. Technol. 42 (23), 8959–8964. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Paerl, H.W., Huisman, J., 2008. Climate – blooms like it hot. Science 320 (5872), 57–58. Partensky, F., Hess, W.R., Vaulot, D., 1999. Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol. Mol. Biol. R. 63 (1), 106–127. Platt, T., Gallegos, C.L., Harrison, W.G., 1980. Photoinhibition of photosynthesis in natural assemblages of marine-phytoplankton. J. Mar. Res. 38 (4), 687–701. Riebesell, U., Zondervan, I., Rost, B., Tortell, P.D., Zeebe, R.E., Morel, F.M.M., 2000. Reduced calcification of marine plankton in response to increased atmospheric CO2. Nature 407 (6802), 364–367. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes-towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Smith, V.H., 1983. Low nitrogen to phosphorus ratios favor dominance by blue-green algae in lake phytoplankton. Science 221 (4611), 669–671. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Wright, S.W., Jeffrey, S.W., Mantoura, R.F.C., Llewellyn, C.A., Bjornland, T., Repeta, D., et al., 1991. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar. Ecol. Prog. Ser. 77 (2–3), 183–196. Zhang, L., Wang, M.H., Hu, J., Ho, Y.S., 2010. A review of published wetland research, 1991-2008: ecological engineering and ecosystem restoration. Ecol. Eng. 36 (8), 973–980.

Chapter 17

Macroalgae as a tool for coastal management in the Mediterranean Sea Charles F. Boudouresque, Aurélie Blanfuné, Sandrine Ruitton, Thierry Thibaut Aix-Marseille University, University of Toulon, CNRS, Marseille, France

17.1 Introduction Macroalgae are worldwide a key component of benthic coastal ecosystems, as primary producers and as canopy-forming species. They encompass a wide range of life history traits (Littler, 1980; Littler and Littler, 1980; Steneck and Dethier, 1994), with diversified responses to natural and human-induced disturbances. In addition, they can exhibit conspicuous and easily visible responses to bottom-up (e.g., pollution) and top-down impacts (e.g., overfishing), by means of cascade effects (Sala et al., 1998). For these reasons, macroalgae have long been used as ecological indicators of the quality of the water bodies, as well as playing a role in multispecies ecological indices of good ecological status (GES), in the framework of the European Union (EU) directives (Belsher and Boudouresque, 1976; Orfanidis et al., 2001; Ballesteros et al., 2007; Ruitton et al., 2014; Boudouresque et al., 2015). Here, we review the past, current and putative uses of macroalgae as powerful tools for coastal management, with special attention to the Mediterranean Sea, a miniature ocean that is well-suited to furthering our understanding and ability to predict the future of the world ocean, in the context of global change (Lejeusne et al., 2010; Boudouresque et al., 2017).

17.2  What are macroalgae? The customary notion of macroalgae actually encompasses a polyphyletic complex within eukaryotes (Boudouresque, 2015; Boudouresque et al., 2015). The species that will be discussed here, which are multicellular photosynthetic organisms, belong to three classes, very far apart in the phylogenetic tree of eukaryotes. (i) The Ulvophyceae (phylum Chlorobionta, subkingdom Viridiplantae) are part of what is popularly called ‘green algae’. (ii) The Florideophyceae (subkingdom Rhodobionta) are part of what is popularly called ‘red algae’. Ulvophyceae and Florideophyceae belong to the kingdom Archaeplastida. (iii) Finally, the Phaeophyceae belong to the phylum Ochrophyta (= Chromobionta), within the kingdom Stramenopiles (= Heterokonta); they are popularly called ‘brown algae’ (Fig. 17.1).

17.3  Why can macroalgae constitute a valuable tool for coastal management? Macroalgae encompass a wide range of life history and morpho-functional traits. One way to distinguish them is to consider annual vs perennial parts (life-history forms) (Feldmann, 1937; Chapman and Chapman, 1976; Garbary, 1976; Meinesz, 1979): (i) epemerophyceae (ephemerals) are annual species occurring throughout the year in the form of successive generations (e.g., Ulva, Ectocarpus, Polysiphonia); (ii) eclipsiophyceae (including hypnophyceae) are annual species, with a single macroscopic generation a year, only present during part of the year (e.g., Porphyra); (iii) phanerophyceae are erect perennial species, of which the entire vegetative apparatus is present throughout the year (e.g., Fucus); (iv) chamaephyceae are encrusting perrennial species, of which the entire vegetative apparatus is present throughout the year (e.g., Ralfsia); (v) hemiphanerophyceae are erect species, of which only a part of the vegetative apparatus is present throughout the year (e.g., Cystoseira amentacea); (vi) pseudo-perennial species are present throughout the year, due to the renewal of the vegetative apparatus, while none of their parts survives more than one year (e.g., Caulerpa taxifolia) (Figs. 17.2 and 17.3). A second way to distinguish macroalgae is to consider morpho-functional groups (Littler, 1980; Littler and Littler, 1980; Littler and Arnold, 1982; Steneck and Dethier, 1994): (i) filamentous uniseriate species (e.g., Cladophora, Bangia); Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00017-6 © 2020 Elsevier Inc. All rights reserved.

277

278  PART | V  Algal ecology

Charophyceae Trentepohliophyceae

Rhodobionta

Ulvophyceae Chlorophyceae Prasinophyceae

Euglyphids Chlorarachniobionta

Glauco -cystobionta

Phytomyxea Haplosporidia

Cen -trohelida?

Foraminifera Radiolaria

Picobiliphyta ?

CRYPTOBIONTA

Dinobionta

Apicomplexa Ciliophora Actinophryda

STRAMENOPILES (= HETEROKONTA) Labyrinthulobionta Bicosoecida

RHIZARIA

Katablepharida

ALVEOLATA

Ellobiopsidae

Oobionta

Cryptophyta

Chromalveolata

ARCHAEPLASTIDA (= PLANTAE)

Cercobionta

Zygnematophyceae

Retaria

Streptobionta Chlorobionta

Viridiplantae

Embryophyta

Ochrophyta LECA

Archamaeba Lobosa

HAPTOBIONTA

Mesomycetozoa

Euglenoidea

Chytridiomycota Fungi

Unikonta

AMOEBOBIONTA (= AMOEBOZOA)

Mycetobionta

Microsporidia Basidiomycota Ascomycota

Parabasalia

Metazoa (= animals)

Choanoflagellata Porifera Placozoa Eumetazoa

OPISTHOKONTA

Diplomonadida

Kinetoplastida Oxymonadida

Retortamonadida

Euglenobionta

Acrasiobionta (= Heterolobosa) Percolobionta

EXCAVATES

DISCICRISTATES

FIG. 17.1  The phylogenetic tree of eukaryotes, much simplified. In the centre, the last common ancestor (Last Eukaryotic Common Ancestor—LECA). The ten kingdoms (Archaeplastida, Alveolata, Opisthokonta, etc.) are enshrined in a rectangle. In green highlighting, the taxa claimed by botanists; in ochre highlighting, the taxa claimed by zoologists; in yellow highlighting, the taxa claimed by the mushroom specialists (botanists); finally, in blue highlighting the taxa claimed by both botanists and zoologists. The macroalgae belong to three groups, framed in red: Ulvophyceae and Rhodobionta (kingdom Archaeplastida) and Ochrophyta (kingdom Stramenopiles). (From Boudouresque, C.F., Caumette, P., Bertrand, J.C., Normand, P., Sime-Ngando T., 2015. Systematic and evolution of microorganisms: general concepts. In: Bertrand, J.C., Caumette, P., Lebaron, P., Matheron, R., Normand, P., SimeNgando, T. (Eds.), Environmental Microbiology: Fundamentals and Applications. Springer, Dordrecht, pp. 107–144. Modified.)

(ii) foliose, single layer or multilayered species (e.g., Ulva, Porphyra); (iii) corticated foliose species (e.g., Padina, Dictyota); (iv) corticated terete species (e.g., Gigartina); (v) leathery species (e.g., Laminaria); (vi) articulated calcareous algae (e.g., Amphiroa, Corallina, Ellisolandia, Jania); and (vii) crustose species (e.g., Litophyllum, Peyssonnelia, Ralfsia). The species classified within a morpho-functional group can belong to very distant taxa; however, they share a common strategy in the fields with regards to e.g., defense and primary production. Macroalgae exhibit a wide range of adaptations to the environmental characteristics: intertidal vs. subtidal, exposed vs. sheltered conditions, well-lit vs. shaded, cold vs. warm water, etc. (e.g., Feldmann, 1937; Lüning, 1990). Some of them are very resistant to organic pollution and even can take benefit from it (e.g., Ulva lactuca), by means of a kind of mixotrophy (Morand and Merceron, 2005; see also Wilce, 1967), while other macroalgae are very sensitive to organic pollution and urbanization, which result in their extirpation from most man-impacted areas (e.g., Cystoseira amentacea) (Mangialajo et al., 2008). Some species can accumulate trace metals in their tissues, allowing their use as biological indicators (Augier et al., 1977). Finally, large and sessile species, which are more or less easy to identify for a trained manager, can constitute tools for routine monitoring of the environmental quality (Arévalo et al., 2007; Ballesteros et al., 2007), provided, of course, that they are correctly identified.

17.4  Take care: Misidentifications can spoil a powerful tool Putting a name to a macroalga is a difficult exercise, even for a specialist. This is not specific to macroalgae: the same remark can be made about bryozoans, hydrozoans, annelids, echinoderms, etc. It is nevertheless an essential prerequisite.

Macroalgae as a tool for coastal management in the Mediterranean Sea Chapter | 17  279



One-year span

Ephemerophyceae

Successive generations

Eclipsiophyceae

A single generation and a resting period

Phanerophyceae

Whole erect vegetative apparatus perennial

Chamaephyceae

Whole encrusting vegetative apparatus perennial

Hemiphanerophyceae

Only a part of the vegetative apparatus present throughout the year

Pseudoperennial

Present throughout the year; not any part survives more than one year

FIG. 17.2  Life history forms of macroalgae.

January – year 1

May – year 1

November – year 1

January – year 2

FIG. 17.3  A pseudoperennial species (e.g., Caulerpa prolifera). The rhizome grows to the right. Dash line: dead or decayed parts of the rhizome.

In one of the first pages of ‘Critica Botanica’, a book written prior to the official starting point of the binomial nomenclature, Linnaeus (1737) wrote: ‘Nomina si nescis, perit cognition rerum’ (translation from Latin: If you know not the names of things, the knowledge of things themselves perishes). Linnaeus was quoting an English lawyer (Coke, 1628), who added ‘Et nomina si perdas, certe distinctio rerum perditur’ (and if you lose the name of things, the distinction between things is certainly lost). Four centuries later, this paradigm has become a hot topic again. As stressed by a number of authors (e.g., Carlton, 1993; Carlton et al., 1999; Boeuf, 2014; Boero, 2016a,b,c; Thibaut et al., 2018; Hutchings, 2019), in the era of biodiversity, species extinctions might be concealed by the extinction of systematists, naturalists and biogeographers, those who would tell the tale of the potential demise of global diversity. Contrary to what the general public believes, the Rio summit in 1992 did not mark the crowning of taxonomy as a key discipline, but the beginning of its decline. Biodiversity then emerged as a source of funding, and all ‘sharks’ on the prowl for funding, no matter whether their objectives are close, very distant from or even completely opposite to, those of biodiversity, pounced on these sources of funding, dismissing along the way, shamelessly but efficiently, the taxonomists. It was all the easier because the taxonomists are often naïve

280  PART | V  Algal ecology

idealists, used to working with little financial means, unschooled in the art of dirty trick and hidden snares. Retirees were therefore no longer being replaced, taxonomy labs were disappearing, skills were being lost. But why is it so important to know about species and to know how to distinguish between them with precision? Two species that are alike (and even belonging to the same genus) can produce very different compounds that may or not be useful for humans. Two similar species may concentrate, or not concentrate, a pollutant. Two similar species may play opposite roles in the functioning of ecosystems. Species characteristics are highly taxon-dependent. In many situations, when the reported species can thrive in the study habitat, and/or are already present in the area or in adjacent areas, misidentification is undetectable in the absence of a description or of available vouchers. In contrast, when a species has been reported in a habitat and/or an area where it is not expected, while a similar-looking species, well-known in the area, and which might be confused with the former, is not mentioned, misidentification may be suspected. Hereafter, we give some examples of doubtful conclusions based upon doubtful determinations. In a study on the effects of organic pollution on benthic communities, near Marseilles (Provence, France) (Jupp, 1977), a third or so of the reported macroalgae are probably misidentified. In particular, two red algae, Ptilota gunneri (as P. ­plumosa) and Palmaria palmata (as Rhodymenia palmata) were mentioned. The former is a North Atlantic species of cold affinity. The latter is also a North Atlantic species, with a wider range distribution. Both species are regarded as not occurring in the Mediterranean Sea (e.g., Gómez-Garreta et al., 2001; Guiry, 2019a). The misidentification is so flagrant that it is not possible to formulate a hypothesis regarding the identity of the misidentified species that were actually present. El Ati-Hellal et al. (2007) selected two macroalgae as biomonitors of contamination by trace metals (Zn, Cu and Cr) in Tunisian coastal waters. The studied seaweeds were identified as Fucus vesiculosus and Enteromorpha spp. Samples were collected off 12 beaches located from the Gulf of Hammamet (northernmost station) to Medenine (southernmost station), along a stretch of shore of > 400 km. The sampling depth was about 2 m (El Ati-Hellal et al., 2007). Fucus vesiculosus is a brown alga (Fucales, Phaeophyceae) of cold to temperate affinities which lives on hard substrates in the intertidal zone. It occurs on both sides of the North Atlantic Ocean. It penetrates into the Mediterranean Sea a few dozen kilometers east of Gibraltar Straits (Alboran Sea), to Malaga (Spain) (Ribera et al., 1992; Flores-Moya et al., 1994; Barcelo-Martí et al., 2000; Benhissoune et al., 2002). The species has never been cited from other Mediterranean areas, including Tunisia (Ben Maiz et al., 1987; Ribera et al., 1992). The presence of F. vesiculosus in Tunisia, as suggested by El Ati-Hellal et al. (2007), is unlikely. Firstly, it is unlikely that such a large and conspicuous species would have gone unnoticed by so many macroalgae specialists (among them, one of the authors of the present chapter, CFB), along such a long stretch of the Tunisian coastline. Secondly, F. vesiculosus is a strictly intertidal species, which cannot thrive in the subtidal zone, at 2 m depth. The question that arises is therefore: what could have been the species that El Ati-Hellal et al. (2007) were dealing with? Two Fucus species are present in the Mediterranean Sea, F. virsoides, endemic to the Adriatic Sea, and F. spiralis. The latter is an Atlantic species which penetrates into the Mediterranean only in the Alboran Sea, close to the Gibraltar Straits (Barcelo-Martí et al., 2000; Benhissoune et al., 2002). Fucus spiralis was observed once in a French coastal lagoon (Gruissan), probably in relation with its utilization as packing material for bait imported from the Atlantic Ocean (Sancholle, 1988); however, the species did not become established. Dictyopteris polypodioides is a brown alga very common in the Mediterranean Sea, in particular along the whole of the Tunisian coast. It thrives in the subtidal zone, down to > 40 m depth. A second species of Dictyopteris has been described from Spain, D. lucida (Ribera Siguan et al., 2005). Since then, D. lucida has been observed in a variety of other Mediterranean localities, in Turkey, Cyprus and Greece (Guiry, 2019b). Although it has not yet been reported from Tunisia, its presence there is probable. Both Dictyopteris species roughly resemble F. vesiculosus in possessing a flattened and dichotomous blade and a midrib. However, they differ from F. vesiculosus in many characteristics, e.g., the far lesser thickness of the blade and the absence of aerocysts (gas vesicles). Dictyopteris polypodioides and/or D. lucida seem to be the best candidates for the seaweed putatively misidentified by El Ati-Hellal et al. (2007) as F. vesiculosus. Apart from bearing a rough morphological resemblance for a nonspecialist, the genera Fucus (order: Fucales) and Dictyopteris (order: Dictyotales) are very distant from a phylogenetic point of view (Phillips et al., 2008). According to current taxonomic criteria, the so-called Enteromorpha species belong to the genus Ulva (Hayden et al., 2003). The genus Ulva encompasses about 40 taxa in the Mediterranean Sea, of which about 30 are ‘enteromorpha-like’ (Gallardo et al., 1993). Distinguishing between these taxa requires close anatomical and genetic observations. It is impossible to think of a definite taxon sampled by El Ati-Hellal et al. (2007). These authors were therefore correct in not ascribing their samples to a definite taxon. Heavy metal concentration by seaweeds is highly species-dependent, as rightly emphasized by El Ati-Hellal et al. (2007). Accurate identification of the sampled species is therefore of paramount importance, e.g., for between-sites and further (i.e., diachronic) comparisons. When comparing the heavy metal content of ‘Enteromorpha spp.’ between Tunisian sites, it is worth noting that the species considered (one species or a mixture of several species) can differ from one site to the next, so that it is difficult to draw operational conclusions.



Macroalgae as a tool for coastal management in the Mediterranean Sea Chapter | 17  281

In the past, misidentification problems were restricted to the field of taxonomic debate and dispute. Nowadays, the picture has radically changed. Accurate species identification is pivotal in the monitoring of good ecological status of the coastal water, via the presence or absence of a taxon, multispecies indices, concentration of contaminants and biotechnology based on natural compounds of the seaweeds. Coastal management strongly depends upon these data: assessment of the quality of a water body, improvement of waste water treatment, eradication or control of exotic species, etc. (e.g., Soltan et al., 2001; Montefalcone, 2009; Mascaró et al., 2013; Thibaut et al., 2017). Management of coastal areas can be expensive, so that errors can result in an unacceptable added cost to the public (tax payers). Errors of determination are therefore no longer just within the domain of scientific debate between taxonomists, but also concern the economic sphere.

17.5  Biological indicators 17.5.1  Delineation of biogeographic provinces A very few species are cosmopolitan, i.e., present in most parts of the world ocean, at least at a given latitude. Among them are some sea turtles and marine mammals. However, most cosmopolitan species are not native to a region and introduced directly or indirectly by means of human activities worldwide, or a complex of cryptic species (Ribera and Boudouresque, 1995; Boudouresque and Verlaque, 2002; Litaker et al., 2009; Zenetos, 2019). In contrast, most species present a natural range restricted to a geographical area. This limited area of distribution is due to their ecological requirements (e.g., salinity, temperature), the context of the ecosystem they inhabit (e.g., competition with other species, presence of herbivores) and biogeographic barriers that prohibit their access to certain areas (Lüning, 1990). Macroalgae can help in delimiting biogeographic provinces in the Mediterranean Sea, the delineation of which thus offering a useful tool for managers. Some biological indicators can be only used within a given province, or must be assessed and calibrated in the context of each province. For example, the use of Fucus virsoides, sensitive to pollution, is restricted to the Adriatic province (Hanel, 2002; Zavodnik et al., 2002). In the Mediterranean Sea, six biogeographic provinces can be distinguished: (i) the northeastern African province, from the Gibraltar Straits and the Sea of Alboran to Sicily, characterized by the flow of relatively low salinity Atlantic waters; (ii) the northwestern province, from the Gulf of Lions to Liguria; (iii) the Tyrrhenian province, from Tuscany to Sardinia; (iv) the Adriatic Sea, characterized by tides at its northern extremity; (v) the eastern Mediterranean, from Tunisia to the Levantine coast, which is the warmest province; tides occur in the Gulf of Gabès, at its westernmost extremity; (vi) the Aegean Sea, between Greece and Turkey. Some authors distinguish only four provinces, namely western, central, eastern and Adriatic, in accordance with the European Union (EU) Marine Framework Strategy Directive (MSFD) (e.g., Zenetos, 2019), while others split these provinces into a larger number of ecoregions, on the basis of currents and the probability of dispersion of organisms across limits (Berline et al., 2014; Ayata et al., 2017).

17.5.2  Indicators of global warming, based on changes in their distribution area Global warming triggers the range extension of species of warm affinities, either native (e.g., the teleosts Thalassoma pavo and Sparisoma cretense, the green alga Caulerpa prolifera), or introduced (e.g., the magnoliophyta Halophila stipulacea and the brown alga Stypocaulon schimperi) (Verlaque and Boudouresque, 1991; Bianchi and Morri, 1994; Francour et al., 1994; Acunto et al., 1995; Guidetti and Boero, 2001; Gambi et al., 2009; Bianchi et al., 2013, 2014; Astruch et al., 2016). At the same time, global warming shrinks the range area of species of cold affinities, such as the magnoliophyta Zostera marina and the teleost Sprattus sprattus (Bianchi, 2007; Perez, 2008; Boudouresque et al., 2009; Pergent et al., 2012, 2014). It is interesting to note that the biological indicators of warming, by the increase of the abundance or northwards spread of indicator species, were the first to demonstrate the warming of the waters of the Mediterranean (Bianchi and Morri, 1994; Francour et al., 1994), at a time when only the measurement of the temperature at depth (> 2000 m) demonstrated the reality of this warming (Bethoux et al., 1990, 1998). In contrast, the sea surface temperature is too widely fluctuating for the differences to have been statistically significant, at that time. A number of Mediterranean macroalgae preferentially dwell in warm waters, and constitute good indicators of the current warming through their range extension, e.g., Anadyomene stellata, Dasycladus vermicularis, Trichosolen myura (Chlorobionta, Archaeplastida), Alsidium corallinum, A. helminthochorton, Digenea simplex, Dipterosiphonia rigens, Halopitys incurva, Hypnea musciformis, Rytiphloea tinctoria, Spyridia filamentosa (Rhodobionta, Archaeplastida), Hydrochlatrus clathratus, Sphacelaria tribuloides (Phaeophyceae, Stramenopiles) (Boudouresque, 1984).

282  PART | V  Algal ecology

17.5.3  Multispecies indices of water quality In contrast to indicator species, the water quality indices are based on the combination of several species. In a general way, they make it possible to get around the problem of irregularities in the abundance of each species, irregularity which can be endogenous (specific to the species) or exogenous (due to the other species present in the habitat). These multispecies indices are therefore more robust than indicator species taken in isolation. In Europe, indices based upon macroalgae had been proposed well before the EU Directives, Water Framework Directive (EC, 2000) and Marine Strategy Framework Directive (EC, 2008). The Bangiophycean Index (BI), proposed by Belsher (1977, see also Hoffmann et al., 1988, Chryssovergis-Bacoyannis, 1995), is based on the sum of percent cover of all species of Bangiophyceae (Rhodobionta) present in a 20 cm × 20 cm quadrat. The percent cover of a species is the percentage of the surface of the substrate that it occupies, in vertical projection. The greater the pollution (nutrients, organic matter), the higher the BI value. The Pollution Index (PI) has also been proposed by Belsher (1977): PI =

Ψ Bangiophyceae Ψ Phaeophyceae + Ψ Cryptonemiales + Ψ Ceramiales

Mean abundance (percent cover) of ESG II

where Ψ is the ratio between quantitative dominance (based on percentage cover) and qualitative dominance (based upon the number of taxa), within 20 cm × 20 cm quadrat (for qualitative and quantitative dominance, see Boudouresque, 1971). As for the BI, the greater the pollution, the higher the BI value. It is important to note that, since the establishment of the PI, the definition of some taxa has changed (e.g., Bangiophyceae and Cryptonemiales), so this index should be redefined. In the framework of the EU EC (2000), Orfanidis et al. (2001, 2003) proposed the Ecological Evaluation Index (EEI), which is based on the morpho-functional groups of benthic macroalgae of Littler (1980) and Littler and Arnold (1982). Macrophytes (including macroalgae) are placed within two Ecological State Groups (ESG). ESG I includes leathery species, articulated calcareous species, crustose species and seagrasses. ESG II includes filamentous species, foliose species and corticated terete species. The EEI is the ratio between ESG I and ESG II abundance (percentage cover); the rationale of the index is to compare K strategists (ESG I) and r strategists (ESG II) (sensu MacArthur and Wilson, 1967), the latter strategists being favored by instability, pollution and disturbances (Simboura et al., 2005; Orfanidis, 2007) (Fig. 17.4). There is a good correlation between EEI and the level of anthropic pressure (Orlando-Bonaca et al., 2008; Orfanidis et al., 2014; but see Iveša et al. (2009) for the northern Adriatic Sea). An improved version of EEI, named EEI-c, has been proposed by Orfanidis et al. (2011). In Mediterranean brackish lagoon habitats, Sfriso et  al. (2007, 2009) have proposed the Macrophyte Quality Index (MaQI). It is based on the presence/absence of some macroalgae (e.g., Chaetomorpha linum, Ceramium spp., Gracilariaceae, Sargassum muticum) and seagrasses (Cymodocea nodosa, Ruppia spp. Zostera marina, Z. noltei), the ratio between the number of Chlorobionta and Rhodobionta, and the variability of some physico-chemical parameters (water transparency, salinity, oxygen saturation), and assessment is done via an original dichotomous key. The CARLIT index is, together with EEI, the most commonly used multispecies biological indicator in the Mediterranean (both EU and non-EU countries). The CARLIT method is based upon the extensive mapping of the mid-littoral and upper infralittoral of the entire rocky coastline (Ballesteros et al., 2007; Mangialajo et al., 2007; Nikolić et al., 2013; Blanfuné et al., 2016a, 2017). Species and communities taken into account for the calculation of the index are (Blanfuné et al., 2017): (i, ii, iii) Cystoseira amentacea/C. mediterranea (respectively continuous belt, abundant patches, scattered plants); (iv) Other shallow Cystoseira species (C. barbata, C. brachycarpa, C. crinita, C. foeniculacea, C. jabukae); (v) Cystoseira compressa; (vi) other soft macroalgae (nonperennial species such as Ulva spp., Cladophora spp. and algal turf), without Cystoseira sp.; (vii) articulated corallines (stands of articulated corallines without Cystoseira species, e.g., Corllina

> 60%

Bad

Poor

Moderate

30 to 60%

Poor

Moderate

Good

0 to 30%

Moderate

Good

High

0 to 30%

30 to 60%

> 60%

Mean abundance (percent cover) of ESG I FIG. 17.4  A matrix based on the mean abundance of ESG I and ESG II to determine the ecological status of transitional and coastal waters, in the framework of the EU Water Framework Directive (EC, 2000). (From Orfanidis, S., Panayotidis, P., Stamatis, N., 2001. Ecological evaluation of transitional and coastal waters: a marine benthic macrophytes-based model. Mediterr. Mar. Sci. 2 (2), 45–65. Modified and redrawn.)



Macroalgae as a tool for coastal management in the Mediterranean Sea Chapter | 17  283

FIG. 17.5  Comparison of Ecological Status (ES) of water bodies (FREC01c through FREC02ab) of northern Corsica (Cape Corse—Capicorsi), according to the CARLIT index, in 2009 and 2015. An improvement of the ecological status is noticeable. (Data from Blanfuné, A., Thibaut, T., Boudouresque, C.F., Mačić, V., Markovic, L., Palomba, L., et al., 2017. The CARLIT method for the assessment of the ecological quality of European Mediterranean waters: relevance, robustness and possible improvements. Ecol. Indic. 72, 249–259.)

­caespitosa, C. officinalis and Ellisolandia elongata); (viiii) encrusting corallines (stands of e.g., Lithophyllum incrustans and Neogoniolithon brassica-florida); (ix) Mytilus galloprovincialis stands without Cystoseira species; (x) stands of green macroalgae (Ulva spp. and Cladophora spp., Chlorobionta); and (xi) Cyanobacteria (communities dominated by Cyanobacteria other than Rivularia sp.). Improved and simplified CARLIT methods have been proposed by Nikolić et al. (2013) and Blanfuné et al. (2017). The CARLIT method makes it possible to accurately compare changes between two successive surveys (Fig. 17.5).

17.5.4  Ecosystem-based quality indices (EBQIs) Until recently, biological indicators were based on one species, or a small number of species. Their role was to be a proxy for the quality of the environment, or the quality of a body of water. However, they did not provide information on the functioning of ecosystems, even though proxy species obviously belong to ecosystems. For example, indices based on the seagrass Posidonia oceanica, e.g., BiPo, PREI, POMI (Gobert et al., 2009; Lopez y Royo et al., 2010, 2011; Mascaró et al., 2012) can show high values, on the basis of the good health of the plant, while the ecosystem is in a dire state, due for example to overfishing (Fig. 17.6). EBQIs are based on a conceptual model of the functioning of the whole ecosystem, from primary producers to top predators (Fig. 17.7) (Personnic et al., 2014; Ruitton et al., 2014; Boudouresque et al., 2015; Thibaut et al., 2017; Astruch et al., 2019). The rationale governing the EBQI is (i) quantifying and assessing some compartments (e.g., compartments 1 through 13 of the Posidonia oceanica ecosystem; Fig. 17.7); (ii) determining their relative weight in the ecosystem functioning; and (iii), using a simple algorithm, calculating a rank for the ecosystem status within a given area, matching the five classes of the ecological status of the EU WFD, from bad to high (Boudouresque et al., 2015).

284  PART | V  Algal ecology

A well-functioning ecosystem

An ecosystem deprived of most of its functional compartments (e.g., via overfishing)

FIG.  17.6  Left: A pristine Posidonia oceanica seagrass ecosystem, with species belonging to all functional compartments: the seagrass in brown (rhizomes) and green (leaves), leaf and rhizomes epibionts in red (primary producers—macroalgae); infauna in black (detritus feeders); the sea urchin Paracentrotus lividus in purple (herbivores) and teleosts (predators, top predators and planktivores). Right: A P. oceanica meadow deprived of most of its functional compartments (e.g., via overfishing), which could be considered as healthy on the basis of biological indicators based upon seagrass descriptors, such as shoot density and meadow coverage, or based upon seagrass and macroalgal epibionts. (From Boudouresque, C.F., Personnic, S., Astruch, P., Ballesteros, E., Bellan-Santini, D., Bonhomme P., et al., 2015. Ecosystem-based versus species-based approach for assessment of the human impact on the Mediterranean seagrass Posidonia oceanica. In: Ceccaldi, H., Henocque, Y., Koike, Y., Komatsu, T., Stora, G., Tusseau-Vuillemin, M.H., (Eds.), Marine Productivity: Perturbations and Resilience of Socio-Ecosystems. Springer, Cham, pp. 235–241. Modified.)

17.6  Management of outstanding species The management of outstanding species of macroalgae (threatened and protected species, canopy-forming species, ecosystem engineers) requires the extensive monitoring of their range and abundance, with the aim of early detection of local or functional extinctions [e.g., the rare and endemic species of the genera Cystoseira and Sargassum (Phaeophyceae, Stramenopiles)]. The exhaustive exploration of the early and recent literature (published articles and books, gray literature) and of voucher specimens housed in herbaria, which enable the confirmation or updating of early identifications, constitutes a powerful tool to establish baselines, dating back to the beginning of the industrial revolution or to the 19th century (Thibaut et al., 2005, 2014, 2015a,b, 2016; Blanfuné et al., 2016b, 2019). Historical data show that the Mediterranean endemic Cystoseira mediterranea is extinct at its northern-limit in Occitania (France) (Thibaut et  al., 2015b; Blanfuné et  al., 2019). Several species, once frequent, have become extinct in French Catalonia: Cystoseira barbata, C. crinita, C. foeniculacea, C. funkii, C. sauvageauana, C. montagnei, Sargassum acinarium, S. hornschuchii and S. vulgare (Thibaut et al., 2005). Along the French Riviera, most Cystoseira and Sargassum species have experienced a severe regression; C. elegans, C. squarrosa, C. montagnei var. montagnei and possibly S. hornschuchii are extinct (Thibaut et al., 2015a). Cystoseira crinita, C. foeniculacea, C. humilis and C. montagnei are extinct at Tremiti Islands (Italy) (Cormaci and Furnari, 1999). Species of the genus Sargassum are those for which the status is the most worrying; for example, S. acinarium is extinct in French Catalonia (see above), Languedoc, western Provence (France) and Linosa Island (Italy) (Serio et al., 2006; Thibaut et al., 2005, 2016). In contrast, while most authors have for decades regarded Cystoseira amentacea as a threatened species (e.g., Bellan-Santini, 1963, 1966; Belsher and Boudouresque, 1976; Soltan et al., 2001; Boudouresque, 2003; Mangialajo et al., 2008), a thorough examination of historical data (literature and herbaria) and the exhaustive mapping of its current distribution have shown that the decline of the species has in fact been slight, very localized, and obviously overestimated; in fact, C. amentacea is one of the most common and healthy Mediterranean species (Thibaut et al., 2014). Finally, in French Catalonia, a suite of data from over a century shows that, despite a general trend of decline, C. mediterranea has experienced at least two episodes of severe regression followed by rapid recovery (Blanfuné et al., 2019).

Macroalgae as a tool for coastal management in the Mediterranean Sea Chapter | 17  285



Pelagic microbial loop

12. Planktivorous teleosts (Spicara, Chromis)

Plankton and POM

4. Filter- and suspensionfeeder leaf epibiota

5 and 6. Benthic filterand suspension-feeders (Pinna, HOM, FOM)

DOC

2. Posidonia leaves (slow recycling)

MPO rhizome epibiota

1. Roots and rhizomes

9. Herbivores 1 (Sarpa, Paracentrotus, Idotea, Pisa, etc.)

Water column

7. Litter detritus (dead leaves, broken rhizomes)

Matte

The matte: a sink for carbon

Organisms leaving the meadow (e.g., teleosts)

11. Piscivorous teleosts (Conger, Scorpaena)

Herbivores 2 (Amphipoda, Jujubinus, Rissoa, etc.)

3. MPO leaf epibiota (rapid recycling)

13. Sea birds (Phalacrocorax, Pandion)

10. Predatory teleosts (Diplodus, Sparus, Labrus, Symphodus, etc.) cephalopods and seastars

Detritus feeders 1 (Amphipoda, Isopoda, Psammechinus, etc.)

8. Detritus feeders 2 and 3 (Amphipoda, Isopoda, Holothuria, etc.)

BAFHS Matte endofauna: annelids, mollusks, etc.

Exported detritus (dead leaves)

Benthic microbial loop

Fragmentation Carbon flux

FIG. 17.7  A conceptual model of the functioning of the Posidonia oceanica seagrass ecosystem. Functional compartments (boxes): primary producers are in green; filter feeders, suspension feeders, litter, detritus feeders, dissolved organic carbon (DOC) and microbial loop are in orange; predators (including herbivores) are in yellow. POM, particulate organic carbon; BAFHS, bacteria, archaea, fungi and heterotrophic Stramenopiles involved in the litter degradation. The width of the arrows roughly represents the volume of the carbon flow. The P. oceanica ecosystem properly speaking is included within the red rectangle. Figs. (1–13, in red) correspond to the compartments taken into account by the EBQI. (Modified from Personnic, S., Boudouresque, C.F., Astruch, P., Ballesteros, E., Blouet, S., Bellan-Santini D., et al., 2014. An ecosystem-based approach to assess the status of a Mediterranean ecosystem, the Posidonia oceanica seagrass meadow. Plos One 9 (6), e98994.)

17.7  Discussion and conclusion The ‘macroalgae’ tool is already widely used for coastal zone management, alone or in association with other descriptors (e.g., physico-chemical, seagrasses and metazoans), in the framework of the EU WFD and MSFD directives. Two multispecies indicators, EEI and CARLIT, constitute the flagships among macroalgal tools. A new type of indices, ecosystem-based Quality Indices (EBQIs), in which macroalgae represent only part of the index, constitute a major innovation, and a new frontier in the development of biological indicators. Indicators based on macroalgae work better than other tools, such as the contaminant content of water and of some organisms, the interpretation of which is sometimes difficult. However, the poor taxonomic knowledge of users of macroalgal-based indicators may be a hindrance to their use, or introduce a bias in their interpretation, despite the fact that the macroalgae chosen are most often relatively easy to identify species. Contrary to what some managers believe, or to what is claimed by those who sell them the molecular tools as ‘the solution’, molecular tools, though very useful and even essential in taxonomy, for the moment engender for management purposes more problems, uncertainties and errors than solutions. The training of managers in the basic taxonomy of the macroalgae used is therefore today a priority objective to improve the efficiency of the use of macroalgae-based tools. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c,d, e,f,g,h,i,j,k,l,m,n,o).

Acknowledgments The authors are indebted to Michèle Perret-Boudouresque, curator of the Plateforme macrophytes of the Mediterranean Institute of Oceanography (MIO), for bibliographical assistance, and to Michael Paul, a native English speaker, for proofreading the paper.

286  PART | V  Algal ecology

References Acunto, S., Maltagliati, F., Rindi, F., Rossi, F., Cinelli, F., 1995. Osservazioni su una prateria di Halophila stipulacea (Forssk.) Aschers. (Hydrocharitaceae) nel Mar Tirreno meridionale. Atti Soc. Tosc. Sci. Nat. Mem. B 102, 19–22. Arévalo, R., Pinedo, S., Ballesteros, E., 2007. Changes in the composition and structure of Mediterranean rocky-shore communities following a gradient of nutrient enrichment: descriptive study and test of proposed methods to assess water quality regarding macroalgae. Mar. Pollut. Bull. 55 (1–6), 104–113. Astruch, P., Bonhomme, P., Goujard, A., Rouanet, E., Boudouresque, C.F., Harmelin, J., et al., 2016. Provence and Mediterranean warming: the parrotfish Sparisoma cretense is coming. Rapp. Comm. Int. Mer Medit. 41, 362. Astruch, P., Goujard, A., Rouanet, E., Boudouresque, C.F., Verlaque, M., Berthier, L., et al., 2019. Assessment of the conservation status of coastal detrital sandy bottoms in the Mediterranean Sea: an ecosystem-based approach in the framework of the ACDSea project. In: Langar, H., Ouerghi, A. (Eds.), Proceedings of the 3rd Symposium on the Conservation of Coralligenous and Other Calcareous Bio-Constructions, Antalya, Turkey, 15–16 January 2019. RAC/SPA, Tunis, pp. 23–29. Augier, H., Gilles, G., Ramonda, G., 1977. L'algue rouge benthique Ceramium ciliatum var. robustum (J. Ag.) G. Mazoyer est un remarquable indicateur biologique de la pollution mercurielle littorale. C.R. Acad. Sci. B Phys. 284, 445–447. Ayata, S.D., Irisson, J.O., Aubert, A., Berline, L., Dutay, J.C., Mayot, N., et al., 2017. Regionalisation of the Mediterranean basin, a MERMEX synthesis. Prog. Oceanogr. 163, 7–20. Ballesteros, E., Torras, X., Pinedo, S., Garcia, M., Mangialajo, L., de Torres, M., 2007. A new methodology based on littoral community cartography dominated by macroalgae for the implementation of the European Water Framework Directive. Mar. Pollut. Bull. 55 (1–6), 172–180. Barcelo-Martí, M.C., Gallardo-García, T., Gómez-Garreta, A., Pérez-Ruzafa, I.M., Ribera-Siguan, M.A., Rull-Lluch, J., 2000. Flora Phycologica Iberica. 1. Fucales. University of Murcia, Murcia. Bellan-Santini, D., 1963. Étude quantitative du peuplement à Cystoseira stricta (Montagne) Sauvageau. Rapp. P.V. Réun. CIESM 17 (2), 133–138. Bellan-Santini, D., 1966. Influence des eaux polluées sur la faune et la flore marines benthiques dans la région marseillaise. Techn. Sci. Municip. 61 (7), 285–292. Belsher, T., 1977. Analyse des répercussions des pollutions urbaines sur le macrophytobenthos de Méditerranée (Marseille, Port-Vendres, Port-Cros). Ph.D Aix-Marseille II University, Marseille. Belsher, T., Boudouresque, C.F., 1976. L’impact de la pollution sur la fraction algale des peuplements benthiques de Méditerranée. In: Atti Tavola Rotonda Internazionale ‘la Biologia Marina per la Difesa e per la Produttività del Mar’, Livorno, pp. 215–260. Ben Maiz, N., Boudouresque, C.F., Ouahchi, F., 1987. Inventaire des algues et phanérogames marines benthiques de la Tunisie. G. Bot. Ital. 121 (5–6), 259–304. Benhissoune, S., Boudouresque, C.F., Perret-Boudouresque, M., Verlaque, M., 2002. A check-list of marine seaweeds of the Mediterranean and Atlantic coasts of Morocco. II. Phaeophyceae. Bot. Mar. 45 (3), 217–230. Berline, L., Rammou, A.M., Doglioli, A., Molcard, A., Petrenko, A., 2014. A connectivity-based eco-regionalization method of the Mediterranean Sea. PLoS One 9 (11), e111978. Bethoux, J.P., Gentili, B., Raunet, J., Tailliez, D., 1990. Warming trend in the western Mediterranean deep water. Nature 347 (6294), 660–662. Bethoux, J.P., Gentili, B., Tailliez, D., 1998. Warming and freshwater budget change in the Mediterranean since the 1940s, their possible relation to the greenhouse effect. Geophys. Res. Lett. 25 (7), 1023–1026. Bianchi, C.N., 2007. Biodiversity issues for the forthcoming tropical Mediterranean Sea. Hydrobiologia 580, 7–21. Bianchi, C.N., Boudouresque, C.F., Francour, P., Morri, C., Parravicini, V., Templado, J., et al., 2013. The changing biogeography of the Mediterranean Sea: from the old frontiers to new gradients. Boll. Mus. Ist. Biol. 75, 81–84. Bianchi, C.N., Corsini-Foka, M., Morri, C., Zenetos, A., 2014. Thirty years after: dramatic change in the coastal marine ecosystems of Kos Island (Greece), 1981-2013. Mediterr. Mar. Sci. 15 (3), 482–497. Bianchi, C.N., Morri, C., 1994. Southern species in the Ligurian Sea (Northern Mediterranean): new records and a review. Bol. Mus. Ist. Biol. 58-59, 181–197. Blanfuné, A., Boudouresque, C.F., Verlaque, M., Beqiraj, S., Kashta, L., Nasto, I., et al., 2016a. Response of rocky shore communities to anthropogenic pressures in Albania (Mediterranean Sea): ecological status assessment through the CARLIT method. Mar. Pollut. Bull. 109 (1), 409–418. Blanfuné, A., Boudouresque, C.F., Verlaque, M., Thibaut, T., 2016b. The fate of Cystoseira crinita, a forest-forming Fucale (Phaeophyceae, Stramenopiles), in France (North Western Mediterranean Sea). Estuar. Coast. Shelf Sci. 181, 196–208. Blanfuné, A., Boudouresque, C.F., Verlaque, M., Thibaut, T., 2019. The ups and downs of a canopy-forming seaweed over a span of more than one century. Sci. Rep. 9, 5250. Blanfuné, A., Thibaut, T., Boudouresque, C.F., Mačić, V., Markovic, L., Palomba, L., et al., 2017. The CARLIT method for the assessment of the ecological quality of European Mediterranean waters: relevance, robustness and possible improvements. Ecol. Indic. 72, 249–259. Boero, F., 2016a. The zoology of babel. Ital. J. Zool. 83 (2), 151–152. Boero, F., 2016b. Teaching zoology in the age of machines. Ital. J. Zool. 83 (3), 283–284. Boero, F., 2016c. Marine biodiversity and ecosystem functioning: the pillars of good environmental status. Biol. Mar. Mediterr. 23 (1), 50–57. Boeuf, G., 2014. La Biodiversité, de l’océan à la cité. Collège de France, Paris. Boudouresque, C.F., 1971. Méthodes d’étude qualitative et quantitative du benthos (en particulier du phytobenthos). Téthys 3 (1), 79–104. Boudouresque, C.F., 1984. Groupes écologiques d’algues marines et phytocénoses benthiques en Méditerranée nord-occidentale. G. Bot. Ital. 118 (S2), 7–42.



Macroalgae as a tool for coastal management in the Mediterranean Sea Chapter | 17  287

Boudouresque, C.F., 2003. The erosion of Mediterranean biodiversity. In: Rodriguez-Prieto, C., Pardinin, G. (Eds.), The Mediterranean Sea: An Overview of its Present State and Plans for Future Protection. Springer, Dordrecht, pp. 53–112. Boudouresque, C.F., 2015. Taxonomy and phylogeny of unicellular eukaryotes. In: Bertrand, J.C., Caumette, P., Lebaron, P., Matheron, R., Normand, P., Sime-Ngando, T. (Eds.), Environmental Microbiology: Fundamentals and Applications. Springer, Dordrecht, pp. 191–257. Boudouresque, C.F., Bernard, G., Pergent, G., Shili, A., Verlaque, M., 2009. Regression of Mediterranean seagrasses caused by natural processes and anthropogenic disturbances and stress: a critical review. Bot. Mar. 52 (5), 395–418. Boudouresque, C.F., Blanfuné, A., Fernandez, C., Lejeusne, C., Perez, T., Ruitton, S., et al., 2017. Marine biodiversity – warming vs. biological invasions and overfishing in the Mediterranean Sea: take care, ‘one train can hide another’. MOJ Ecol. Environ. Sci. 2 (4), 1–13. Boudouresque, C.F., Caumette, P., Bertrand, J.C., Normand, P., Sime-Ngando, T., 2015. Systematic and evolution of microorganisms: general concepts. In: Bertrand, J.C., Caumette, P., Lebaron, P., Matheron, R., Normand, P., Sime-Ngando, T. (Eds.), Environmental Microbiology: Fundamentals and Applications. Springer, Dordrecht, pp. 107–144. Boudouresque, C.F., Personnic, S., Astruch, P., Ballesteros, E., Bellan-Santini, D., Bonhomme, P., et al., 2015. Ecosystem-based versus species-based approach for assessment of the human impact on the Mediterranean seagrass Posidonia oceanica. In: Ceccaldi, H., Henocque, Y., Koike, Y., Komatsu, T., Stora, G., Tusseau-Vuillemin, M.H. (Eds.), Marine Productivity: Perturbations and Resilience of Socio-Ecosystems. Springer, Cham, pp. 235–241. Boudouresque, C.F., Verlaque, M., 2002. Biological pollution in the Mediterranean Sea: invasive versus introduced macrophytes. Mar. Pollut. Bull. 44 (1), 32–38. Carlton, J.T., 1993. Neoextinctions of marine invertebrates. Amer. Zool. 33 (6), 499–509. Carlton, J.T., Geller, J.B., Reaka-Kudla, M.L., Norse, E.A., 1999. Historical extinctions in the sea. Annu. Rev. Ecol. Syst. 30, 515–538. Chapman, V.J., Chapman, D.J., 1976. Life forms in the algae. Bot. Mar. 19 (2), 65–74. Chryssovergis-Bacoyannis, F., 1995. Impact de l’eutrophisation sur les algues benthiques (Chlorophyceae, Fucophyceae, Rhodophyceae) de l’infralittoral supérieur du golfe de Maliakos (mer Égée, Grèce). Ph.D University Aix-Marseille II, Marseille. Coke, E., 1628. The First Part of the Institutes of the Laws of England. Adam Islip, London. Cormaci, M., Furnari, G., 1999. Changes of the benthic algal flora of the Tremiti Islands (Southern Adriatic) Italy. Hydrobiologia 398-399, 75–79. EC, 2000. Council Directive 2008/56/EC of the European Parliament and of the Council of 17 June 2008 establishing a framework for community action in the field of marine environmental policy (Marine Strategy Framework Directive). O.J.L. L 164, 25/06/2008, pp. 19–40. EC, 2008. Council Directive 2000/60/EC of the European parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy. O.J.L., L 327, 22/12/2000, pp. 1–73. El Ati-Hellal, M., Hedhili, A., Dachraoui, M., 2007. Contents of trace metals in water and macroalgae along the Mediterranean coast of Tunisia. Bull. Environ. Contam. Toxicol. 78 (1), 33–37. Feldmann, J., 1937. Recherches sur la végétation marine de la Méditerranée: la côte des Albères. Rev. Algol. 10 (1–4), 1–340. Flores-Moya, A., Conde, F., Sánchez, A., Altamirano, M., 1994. Notas corológicas del macrofitobentos de Andalucía (España) III. Acta Bot. Malacit. 10, 211–213. Francour, P., Boudouresque, C.F., Harmelin, J.G., Harmelin-Vivien, M.L., Quignard, J.P., 1994. Are the Mediterranean waters becoming warmer? Information from biological indicators. Mar. Pollut. Bull. 28 (9), 523–526. Gallardo, T., Gómez-Garreta, A., Ribera, M.A., Cormaci, M., Furnari, G., Giaccone, G., et  al., 1993. Check-list of Mediterranean seaweeds. II. Chlorophyceae Wille s.l. Bot. Mar. 36 (5), 399–421. Gambi, M.C., Barbieri, F., Bianchi, C.N., 2009. New record of the alien seagrass Halophila stipulacea (Hydrocharitaceae) in the western Mediterranean: a further clue to changing Mediterranean Sea biogeography. Mar. Biodiv. Rec. 2, e84. Garbary, D., 1976. Life-forms of algae and their distribution. Bot. Mar. 19 (2), 97–106. Gobert, S., Sartoretto, S., Rico-Raimondino, V., Andral, B., Chery, A., Lejeune, P., et al., 2009. Assessment of the ecological status of Mediterranean French coastal waters as required by the water framework directive using the Posidonia oceanica rapid easy index: PREI. Mar. Pollut. Bull. 58 (11), 1727–1733. Gómez-Garreta, A., Gallardo, T., Ribera, M.A., Cormaci, M., Furnari, G., Giaccone, G., et  al., 2001. Checklist of Mediterranean seaweeds. III. Rhodophyceae Rabenh. 1. Ceramiales Oltm. Bot. Mar. 44 (5), 425–460. Guidetti, P., Boero, F., 2001. Occurrence of the Mediterranean parrotfish Sparisoma cretense (Perciformes: Scaridae) in south-eastern Apulia (south-east Italy). J. Mar. Biol. Assoc. UK 81 (4), 717–718. Guiry, M.D., 2019a. Ptilota gunneri. In: Guiry, M.D., Guiry, G.M. (Eds.), AlgaeBase. http://www.algaebase.org. (Accessed March 20, 2019). Guiry, W., 2019b. Dictyopteris lucida. In: Guiry, M.D., Guiry, G.M. (Eds.), AlgaeBase. http://www.algaebase.org. (Accessed March 20, 2019). Hanel, R., 2002. Recovery of Fucacean associations and associated fish assemblages in the vicinity of Rovinj, Istrian coast, northern Adriatic Sea. Period. Biol. 104 (2), 159–163. Hayden, H.S., Blomster, J., Maggs, C.A., Silva, P.C., Stanhope, M.J., Waaland, J.R., 2003. Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera. Eur. J. Phycol. 38 (3), 277–294. Hoffmann, L., Clarisse, S., Detienne, X., Goffart, A., Renard, R., Demoulin, V., 1988. Evolution of the populations of Cystoseira balearica (Phaeophyceae) and epiphytic Bangiophyceae in the Bay of Calvi (Corsica) in the last eight years. Bull. Soc. Roy. Liège 57 (4–5), 263–273. Hutchings, P., 2019. An advocate for taxonomic research in Australia. Pacific Conserv. Biol. 25 (1), 34–36. Iveša, L., Lyons, D.M., Devescovi, M., 2009. Assessment of the ecological status of north-eastern Adriatic coastal waters (Istria, Croatia) using macroalgal assemblages for the European Union water framework directive. Aquat. Conserv. 19 (1), 14–23. Jupp, B.P., 1977. The effects of organic pollution on benthic organisms near Marseille. Int. J. Environ. Stud. 10 (1), 119–123. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

288  PART | V  Algal ecology

Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Lejeusne, C., Chevaldonné, P., Pergent-Martini, C., Boudouresque, C.F., Perez, T., 2010. Climate change effects on a miniature ocean: the highly diverse, highly impacted Mediterranean Sea. Trends Ecol. Evol. 25 (4), 250–260. Linnaeus, C., 1737. Critica Botanica. Conrad Wishoff, Leiden. Litaker, R.W., Vandersea, R.W., Faust, M.A., Kibler, S.R., Chinain, M., Holmes, M.J., et al., 2009. Taxonomy of Gambierdiscus including four new species, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ruetzleri (Gonyaulacales, Dinophyceae). Phycologia 48 (5), 344–390. Littler, M.M., 1980. Morphological form and photosynthesis performances of marine macroalgae: tests of a functional/form hypothesis. Bot. Mar. 23 (3), 161–165. Littler, M.M., Arnold, K.E., 1982. Primary productivity of marine macroalgal functional form groups from southwestern North America. J. Phycol. 18 (3), 307–311. Littler, M.M., Littler, D.S., 1980. The evolution of thallus form and survival strategies in benthic marine macroalgae: field and laboratory tests of a functional form model. Am. Nat. 116 (1), 25–44. Lopez y Royo, C., Casazza, G., Pergent-Martini, C., Pergent, G., 2010. A biotic index using the seagrass Posidonia oceanica (BiPo), to evaluate ecological status of coastal waters. Ecol. Indic. 10 (2), 380–389. Lopez y Royo, C.L., Pergent, G., Alcoverro, T., Buia, M.C., Casazza, G., Martinez-Crego, B., et al., 2011. The seagrass Posidonia oceanica as indicator of coastal water quality: experimental intercalibration of classification systems. Ecol. Indic. 11 (2), 557–563. Lüning, K., 1990. Seaweeds. In: Their Environment, Biogeography, and Ecophysiology. John Wiley and Sons, New York. MacArthur, R.H., Wilson, E.O., 1967. The Theory of Island Biogeography. Princeton University Press, Princeton. Mangialajo, L., Chiantore, M., Cattaneo-Vietti, R., 2008. Loss of fucoid algae along a gradient of urbanisation, and structure of benthic assemblages. Mar. Ecol. Prog. Ser. 358, 63–74. Mangialajo, L., Ruggieri, N., Asnaghi, V., Chiantore, M., Povero, P., Cattaneo-Vietti, R., 2007. Ecological status in the Ligurian Sea: the effect of coastline urbanisation and the importance of proper reference sites. Mar. Pollut. Bull. 55 (1–6), 30–41. Mascaró, O., Alcoverro, T., Dencheva, K., Díez, I., Gorostiaga, J.M., Krause-Jensen, D., et al., 2013. Exploring the robustness of macrophyte-based classification methods to assess the ecological status of coastal and transitional ecosystems under the water framework directive. Hydrobiologia 704 (1), 279–291. Mascaró, O., Bennett, S., Marbà, N., Nikolić, V., Romero, J., Duarte, C.M., et al., 2012. Uncertainty analysis along the ecological quality status of water bodies: the response of the Posidonia oceanica multivariate index (POMI) in three Mediterranean regions. Mar. Pollut. Bull. 64 (5), 926–931. Meinesz, A., 1979. Contribution à l’étude de Caulerpa prolifera (Forsskål) Lamouroux (Chlorophycée – Caulerpale). I. Morphogénèse et croissance dans une station des côtes continentales françaises de la Méditerranée. Bot. Mar. 22 (1), 27–39. Montefalcone, M., 2009. Ecosystem health assessment using the seagrass Posidonia oceanica: a review. Ecol. Indic. 9 (4), 595–604. Morand, P., Merceron, M., 2005. Macroalgal population and sustainability. J. Coastal Res. 21 (5), 1009–1020.



Macroalgae as a tool for coastal management in the Mediterranean Sea Chapter | 17  289

Nikolić, V., Žuljević, A., Mangialajo, L., Antolić, B., Kušpilić, G., Ballesteros, E., 2013. Cartography of littoral rocky-shore communities (CARLIT) as a tool for ecological quality assessment of coastal waters in the eastern Adriatic Sea. Ecol. Indic. 34, 87–93. Orfanidis, S., 2007. Comments on the development of new macroalgal indices to assess water quality within the Mediterranean. Mar. Pollut. Bull. 54 (5), 626–627. Orfanidis, S., Dencheva, K., Nakou, K., Tsioli, S., Papathanasiou, V., Rosati, I., 2014. Benthic macrophyte metrics as bioindicators of water quality: towards overcoming typological boundaries and methodological tradition in Mediterranean and Black Seas. Hydrobiologia 740 (1), 61–78. Orfanidis, S., Panayotidis, P., Stamatis, N., 2001. Ecological evaluation of transitional and coastal waters: a marine benthic macrophytes-based model. Mediterr. Mar. Sci. 2 (2), 45–65. Orfanidis, S., Panayotidis, P., Stamatis, N., 2003. An insight to the ecological evaluation index (EEI). Ecol. Indic. 3 (1), 27–33. Orfanidis, S., Panayotidis, P., Ugland, K.I., 2011. Ecological evaluation index continuous formula (EEI-c) application: a step forward for functional groups, the formula and reference condition values. Mediterr. Mar. Sci. 12 (1), 199–231. Orlando-Bonaca, M., Lipej, L., Orfanidis, S., 2008. Benthic macrophytes as a tool for delineating, monitoring and assessing ecological status: the case of the Slovenian coastal waters. Mar. Pollut. Bull. 56 (4), 666–676. Perez, T., 2008. Impact des Changements Climatiques sur la Biodiversité en Mer Méditerranée. CAR/ASP, Tunis. Pergent, G., Bazairi, H., Bianchi, C.N., Boudouresque, C.F., Buia, M.C., Clabaut, P., et al., 2012. Les Herbiers de Magnoliophytes Marines de Méditerranée. Résilience et Contribution à l’Atténuation des Changements Climatiques. IUCN, Malaga. Pergent, G., Bazairi, H., Bianchi, C.N., Boudouresque, C.F., Buia, M.C., Clabaut, P., et al., 2014. Climate change and Mediterranean seagrass meadows: a synopsis for environmental managers. Mediterr. Mar. Sci. 15 (2), 462–473. Personnic, S., Boudouresque, C.F., Astruch, P., Ballesteros, E., Blouet, S., Bellan-Santini, D., et al., 2014. An ecosystem-based approach to assess the status of a Mediterranean ecosystem, the Posidonia oceanica seagrass meadow. PLoS One 9 (6), e98994. Phillips, N., Burrowes, R., Rousseau, F., de Reviers, B., Saunders, G.W., 2008. Resolving evolutionary relationships among the brown algae using chloroplast and nuclear genes. J. Phycol. 44 (2), 394–405. Ribera, M.A., Boudouresque, C.F., 1995. Introduced marine plants, with special reference to macroalgae: mechanisms and impact. In: Round, F.E., Chapman, D.J. (Eds.), Progress in Phycological Research. Biopress Ltd, pp. 187–268. Ribera, M.A., Gómez-Garreta, A., Gallardo, T., Cormaci, M., Furnari, G., Giaccone, G., 1992. Check-list of Mediterranean seaweeds. 1. Fucophyceae (Warming, 1884). Bot. Mar. 35 (2), 109–130. Ribera Siguan, M.A., Gómez-Garreta, A., Pérez Ruzafa, I., Barcelò-Martí, M.C., Rull Lluch, J., 2005. A new species of Dictyopteris (Dictyotales, Phaeophyceae) from the Iberian Peninsula: Dictyopteris lucida sp. nov. Phycologia 44 (6), 651–657. Ruitton, S., Personnic, S., Ballesteros, E., Bellan-Santini, D., Boudouresque, C.F., Chevaldonné, P., et al., 2014. An ecosystem-based approach to assess the status of the Mediterranean coralligenous habitat. In: Bouafif, C., Langar, H., Ouerghi, A. (Eds.), Proceedings of the 2nd Mediterranean Symposium on the Conservation of Coralligenous and Other Calcareous Bio-Concretions. RAC/SPA, Tunis, pp. 153–158. Sala, E., Boudouresque, C.F., Harmelin-Vivien, M., 1998. Fishing, trophic cascade and the structure of algal assemblages: evaluation of an old but untested paradigm. Oikos 82 (3), 425–439. Sancholle, M., 1988. Présence de Fucus spiralis (Phaeophyceae) en Méditerranée occidentale. Cryptog. Algol. 9 (2), 157–161. Serio, D., Alongi, G., Catra, M., Cormaci, M., Furnari, G., 2006. Changes in the benthic algal flora of Linosa Island (Straits of Sicily, Mediterranean Sea). Bot. Mar. 49 (2), 135–144. Sfriso, A., Facca, C., Ghetti, P.F., 2007. Rapid quality index (R-MaQI), based mainly on macrophyte associations, to assess the ecological status of Mediterranean transitional environments. Chem. Ecol. 23 (6), 493–503. Sfriso, A., Facca, C., Ghetti, P.F., 2009. Validation of the Macrophyte quality index (MaQI) set up to assess the ecological status of Italian marine transitional environments. Hydrobiologia 617 (1), 117–141. Simboura, N., Panayotidis, P., Papathanassiou, E., 2005. A synthesis of the biological quality elements for the implementation of the European water framework directive in the Mediterranean ecoregion: the case of Saronikos Gulf. Ecol. Indic. 5 (3), 253–266. Soltan, D., Verlaque, M., Boudouresque, C.F., Francour, P., 2001. Changes in macroalgal communities in the vicinity of a Mediterranean sewage outfall after the setting up of a treatment plant. Mar. Pollut. Bull. 42 (1), 59–70. Steneck, R.S., Dethier, M.N., 1994. A functional group approach to the structure of algal-dominated communities. Oikos 69 (3), 476–498. Thibaut, T., Blanfuné, A., Boudouresque, C.F., 2018. Une idée neuve, la taxonomie (Nomina si nescis, perit cognitio rerum). Tropezien 99, 14–15. Thibaut, T., Blanfuné, A., Boudouresque, C.F., Cottalorda, J.M., Hereu, B., Susini, M.L., et al., 2016. Unexpected temporal stability of Cystoseira and Sargassum forests in Port-Cros, one of the oldest Mediterranean marine National Parks. Cryptog. Algol. 37 (1), 61–90. Thibaut, T., Blanfuné, A., Boudouresque, C.F., Verlaque, M., 2015a. Decline and local extinction of Fucales in the French Riviera: the harbinger of future extinctions? Mediterr. Mar. Sci. 16 (1), 206–224. Thibaut, T., Blanfuné, A., Boudouresque, C.F., Verlaque, M., 2015b. Loss of the habitat-forming Cystoseira mediterranea at its northern-limit of distribution in the Mediterranean Sea. Eur. J. Phycol. 50, 106. Thibaut, T., Blanfuné, A., Boudouresque, C.F., Verlaque, M., Ruitton, S., 2016. The Sargassum conundrum: very rare, threatened or locally extinct in the NW Mediterranean and still lacking protection. Hydrobiologia 781 (1), 3–23. Thibaut, T., Blanfuné, A., Markovic, L., Verlaque, M., Boudouresque, C.F., Perret-Boudouresque, M., et al., 2014. Unexpected abundance and long-term relative stability of the brown alga Cystoseira amentacea, hitherto regarded as a threatened species, in the north-western Mediterranean Sea. Mar. Pollut. Bull. 89 (1–2), 305–323. Thibaut, T., Blanfuné, A., Boudouresque, C.F., Personnic, S., Ruitton, R., Ballesteros, E., et al., 2017. An ecosystem-based approach to assess the status of Mediterranean algae-dominated shallow rocky reefs. Mar. Pollut. Bull. 117 (1–2), 311–329.

290  PART | V  Algal ecology

Thibaut, T., Pinedo, S., Torras, X., Ballesteros, E., 2005. Long-term decline of the populations of Fucales (Cystoseira spp. and Sargassum spp.) in the Albères coast (France, North-western Mediterranean). Mar. Pollut. Bull. 50 (12), 1472–1489. Verlaque, M., Boudouresque, C.F., 1991. Stypopodium schimperi (Dictyotales, Fucophyceae), algue de mer Rouge récemment apparue en Méditerranée. Cryptog. Algol. 12, 195–211. Wilce, R.T., 1967. Heterotrophy in Arctic sublittoral seaweeds: a hypothesis. Bot. Mar. 10 (3–4), 185–197. Zavodnik, N., Iveša, L., Travizi, A., 2002. Note on recolonization by fucoid algae Cystoseira spp. and Fucus virsoides in the North Adriatic Sea. Acta Adriat. 43 (1), 25–32. Zenetos, A., 2019. Mediterranean Sea: 30  years of biological invasions (1988-2017). In: Langar, H., Ouerghi, A. (Eds.), Proceedings of the 1st Mediterranean Symposium on the Non-indigenous Species. Antalya, Turkey, 18 January 2019. RAC/SPA Publ, Tunis, pp. 13–19.

Chapter 18

Linking phytoplankton community structure to aquatic ecosystem functioning: A mini-review of the current status and future directions Tatenda Dalua,b, Daniel A. Lemleyc, Gavin C. Snowd, Naicheng Wue,f a

University of Venda, Thohoyandou, South Africa, bSouth African Institute for Aquatic Biodiversity, Grahamstown, South Africa, cNelson Mandela University, Port Elizabeth, South Africa, dUniversity of the Witwatersrand, Johannesburg, South Africa, eKiel University, Kiel, Germany, f Xi’an Jiaotong-Liverpool University, Suzhou, China

18.1  Phytoplankton use in biomonitoring activities Aquatic ecosystems degradation and water quality changes have been the focus for many decades mostly due to anthropogenic impacts such as urbanization, agriculture, mining and industrialization (Dudgeon et al., 2006; Vorosmarty et al., 2010; Ekelund and Hader, 2018). Water quality problems are one of the greatest sustainable water management challenges. Rapid urbanization, land use change and agricultural activities (i.e., increased fertilizer, herbicides, pesticides and chemical compounds use) pollute and contaminate aquatic ecosystems which have detrimental impacts on the environment. Thus, the important role of phytoplankton, particularly diatoms and flagellates, is of great interest in environmental monitoring due to the growing demand for effective ecosystem condition evaluation and monitoring approaches (Xu et al., 2014; Tan et al., 2017; Guo and Xu, 2019; Mangadze et al., 2019). These two groups represent an important aquatic ecosystem component (freshwater, estuarine and marine) and respond quickly to environmental change. Diatoms are abundant and widespread in various aquatic ecosystems and are one of the most important primary producer groups (Biggs et al., 1990; Dixit et al., 1992; Stevenson et al., 1996; Soininen, 2007). Diatoms are a highly diverse group with a relatively narrow preference for several environmental variables, culminating in a rapid community structure response to changing environmental conditions (Teittinen et al., 2015; Mangadze et al., 2019). Several factors affect diatom assemblage composition, including spatio-temporal variation such as the water body physico-chemical variables, biological interactions and dispersal history, although diatoms primarily reflect the chemical quality of the water (Korhonen et al., 2013; Teittinen et al., 2015). At least three factors affect the magnitude of temporal variation in diatom assemblage composition and these are (1) temporal variation degree may be related to assemblage diversity, i.e., highly diverse assemblages, species abundances vary strongly but total biomass and assemblage composition may vary less, (2) temporal turnover is affected by ecosystem productivity, i.e., highly productive systems, assemblage composition varies more due to multiple stable states in assemblages, and (3) assemblage variation is affected by ecosystem size (Korhonen et al., 2013). Studies have demonstrated that flagellates functional traits (e.g., body-size spectrum, feeding types) are considerably associated with water quality and have many bioassessment-related advantages in aquatic ecosystems due to their short life cycles, plus quick response to environmental change compared to any metazoa (Xu et al., 2014; Guo and Xu, 2019). As a primary microfauna component, diatoms and flagellates play important roles in microbial food web functioning (Teittinen et  al., 2015). With relative immobility, ease of sampling, easy taxonomic identification and standardized methods for spatio-temporal comparisons, flagellates have widely been accepted as better indicators to assess freshwater, estuarine and marine ecosystems environmental stress and anthropogenic impacts. Measures of trait-based ecological factors (i.e., functional diversity and/or distinctness) have distinct advantages for environmental bioassessment due to a statistical framework for testing deviation from expectation (Guo and Xu, 2019). Several approaches for measuring functional diversity have been suggested based on species multiple ecological and ­morphological Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00018-8 © 2020 Elsevier Inc. All rights reserved.

291

292  PART | V  Algal ecology

traits (Shi et al., 2012; Jiang et al., 2013). Thus, species specific traits are considered good ecosystem functioning indicators as they are directly or indirectly related to ecosystem functioning and processes (Xu et al., 2014).

18.2  Why use phytoplankton community structure as an index of ecosystem functioning? Any changes in the environment over space or time within aquatic ecosystems are likely to cause measurable changes in phytoplankton community composition and biomass. It is worth noting that the term community does convey some beneficial interactions between species so it is probably more accurate to refer to assemblage, which can include species that are present incidentally (Reynolds et al., 2002). Conditions that are likely to cause these changes include variations in climate (rainfall and temperature), dissolved gases, concentrations of nutrients associated with eutrophication (e.g., nitrogen and phosphorus), competition and herbivory (Cupertino et  al., 2019). Of particular interest are the changes associated with cultural eutrophication, altered river discharge, changes in temperature associated with climate change, and the response to pollution-related chemicals (e.g., plastics and herbicides). It is inefficient and expensive to attempt to measure all possible environmental variables making the use of ecological indicators a more viable and focused option, providing synthesized evidence of any change (Cupertino et al., 2019).

18.3  Stressor-response relationships within aquatic ecosystems Phytoplankton community structure varies across environmental gradients and these may be altered directly or indirectly by local and regional disturbances (e.g., species invasions, climate change, pollution) (Reynolds, 1998; Herrero et al., 2018). Predicting anthropogenic stressor effects on phytoplankton biomass and community is fundamental to the effective conservation, management and restoration of water quality (Cottingham, 1999; Mangadze et al., 2019). Stressor-response relationships are widely used in phytoplankton-based water quality assessments to predict and characterize effects of acidification (i.e., acid mine drainage), contaminant loading (i.e., organic, inorganic), water abstraction (i.e., water overexploitation), and climate change (Dalu et al., 2017; Rosenfeld, 2017; Larned and Schallenberg, 2018). Stressor-response relationships are mostly used to predict and characterize ecological anthropogenic ecological degradation and/or pressure and stressor levels increase or decrease (Mangadze et al., 2017; Larned and Schallenberg, 2018). Four types of stressor-response relationships are widely used in nature and these are exponential, linear, logistic, and logarithmic (Fig. 18.1). These stressor-response relationships highlight Maximum change in slope

Response

Maximum change in slope

(A)

(B)

(C) Hysteresis offset

Maximum change in slope

c Re

Inflection point

g De

er ov

d ra

y

on ati

Maximum change in slope

(D)

Bifurcation points

(E)

(F) Stressor

FIG. 18.1  Different types of theoretical stressor-relationships that are typically observed in nature: (A) linear with constant sensitivity over a stressor range, (B) logarithmic with high sensitivity at low stressor levels and high resistance at high stressor levels, (C) exponential with high resistance at low stressor levels and high sensitivity at high stressor levels, (D) logistic with high resistance at high and low stressor levels, and high sensitivity near the inflection point, (E) logistic with degradation and recovery trajectories offset by an interval of the stressor range, and (F) logistic with bifurcation. The dotted arrows indicate stressor thresholds. (Reproduced with permission from Larned, S.T., Schallenberg, M., 2018. Stressor-response relationships and the prospective management of aquatic ecosystems. New Zeal. J. Mar. Freshw. Res. 53, 489–512. https://doi.org/10.1080/00288330.2018.1524388.)



Linking phytoplankton community structure to aquatic ecosystem functioning Chapter | 18  293

S-R relationships based on naturally occurring stressor ranges

Response

S-R relationships based on observed stressor ranges

Response

S-R relationships based theoretical models

(B)

Response

(A)

Stressor FIG. 18.2  Sequences of stressor-response relationships based on theoretical models for naturally occurring relationships due to natural stressors (dashed lines) to observed relationships as a result of monitoring over a limited stressor (dotted lines): (A) logistic stressor-response relationship is logarithmic when observations exclude low stressor levels and this is typically observed for phytoplankton as environmental impact increases, and (B) exponential stressor-response relationship is linear when observations exclude high stressor levels and this is typically observed for other aquatic organisms such as fish and invertebrates and also for ecosystem integrity and impact with increasing environmental impact (see Stevenson, 2014; Dalu and Froneman, 2016). (Reproduced with permission from Larned, S.T., Schallenberg, M., 2018. Stressor-response relationships and the prospective management of aquatic ecosystems. New Zeal. J. Mar. Freshw. Res. 53, 489–512. https://doi.org/10.1080/00288330.2018.1524388.)

both degradation (low to high stressor levels) and recovery trajectories (high to low stressor levels). A particular type of curve that corresponds to different stressor-response relationships may well be identified and selected based on theoretical models (e.g., rapid declines in contaminant-sensitive species populations, saturable nutrient-limited phytoplankton growth). At any particular stressor level, a response variable can occupy two or more different states within the bifurcation zone (i.e., indicated by dotted arrows) where responses to stressor level changes are unstable and can oscillate (Figs. 18.1 and 18.2). The system stabilizes in one state when stressor levels are increasingly divergent from the bifurcation zone. Phytoplankton normally exhibits a logistic relationship with increasing stressor and/or pressure levels (i.e., environmental impact) (Figs. 18.1D and E and 18.2A; Stevenson, 2014; Dalu and Froneman, 2016). For instance, along a nutrient pollution gradient, e.g., one can expect a shift from high phytoplankton biological support at low nutrient concentrations to a low phytoplankton support at high nutrient concentrations. At high nutrient concentrations, harmful phytoplankton growth (e.g., cyanobacteria) and oxygen depletion risk increases greatly with dire impacts on aquatic biota. An example highlighting the effects of multiple stressors on biological quality in Ebro River, north-east Spain is highlighted in Example 1.

Example 1. Assessing multiple stressor effects on biological quality: A case of the Ebro River Multiple stressors affect the ecological status of aquatic ecosystems and stressors are defined as anthropogenic disturbances, which could cause possible harmful effects to organisms and communities (Crain et al., 2008; Segner et al., 2014), with a potential to drive evolutionary processes (Parsons, 2005). In this example, Herrero et al. (2018) assessed the status of Ebro catchment (north-east Spain) waterbodies using three biological quality elements (BQEs; macrophytes, invertebrates, diatoms) based on available catchment data. The BQEs responses were analyzed based on a conceptual model (Fig. 18.3) that relates to the ecological status of Ebro River aquatic ecosystems, with most significant stressors (i.e., discharge reduction, hydromorphological alterations, nutrient enrichment, riparian cover loss) being included. The conceptual model identified the main drivers (i.e., climate change, flood ­protection, agriculture, energy and hydropower, industry), pressures (i.e., direct effects of these drivers, that create these ecosystem modifications; Feld et al., 2016), and stressors (i.e., measurable variables resulting from anthropogenic pressure that significantly affect biological integrity; Segner et al., 2014) were identified and selected according to Sabater et al. (2009) and Barcelo and Petrovic (2011) as they significantly influenced the ecological status of Ebro River aquatic ecosystems (Fig. 18.3). Changes in land use, mean discharge, nitrate, rainfall, total phosphorus (TP) and water temperature were modeled according to the future scenarios of climate change and land use and highlighted a stressor evolution that could cause a decrease in the Ebro catchment ecosystem quality. The decline was observed for diatoms and invertebrates, mainly due to expected TP concentration increases. Headwater waterbodies were the most sensitive to future land use and climate changes. Analysis highlighted that the Ebro River basin biological communities represented by the three BQEs were affected by high nutrient concentrations (i.e., TP),

294  PART | V  Algal ecology

DRIVERS

PRESSURES

STRESSORS

POINT PRESSURES

TOTAL PHOSPHORUS

AGRICULTURE

CLIMATE CHANGE

INDUSTRY

ENERGY HYDROPOWER

FLOOD PROTECTION

NITRATES

DIFFUSE PRESSURES

CHANGE IN MEAN DISCHARGE

ABSTRACTION/ FLOW DIVERSION OF SURFACE WATERS PHYSICAL AND HYDROLOGICAL ALTERATION

CHANGE IN WATER TEMPERATURE

BIOTIC INDICATORS

DIATOMS (IPS)

MACROPHYTES (IVAM)

INVERTEBRATES (IBMWP)

HYDROMORPHOLOGICAL ALTERATIONS (IHF)

FIG. 18.3  Conceptual model for the Ebro basin highlighting the relationship that exists between stressors and ecological indicators. (Reproduced with permission from Herrero, A., Gutierrez-Canovas, C., Vigiak, O., Lutz, S., Kumar, R., Gampe, D., et al., 2018. Multiple stressor effects on biological quality elements in the Ebro River: present diagnosis and predicted responses. Sci. Total Environ. 630, 1608–1618.)

agriculture and population growth. These pressures showed negative additive effects on the biological indices, which suggest multistressor interactive effects. Agriculture and population numbers accounted for a higher variance than nutrients mostly due to being linked to other impacts such as pesticides and other pollutants and riparian degradation which affect the BQEs. Furthermore, predictions based on climate scenarios showed a steady decrease in water discharge and increasing TP concentrations, which were linked to a mean decrease in the diatom and invertebrate based BQEs. Results highlighted that population growth and agriculture can be managed independently, but efforts to achieve a good ecological integrity will need to consider all these factors combined for effective management to be successful (Herrero et al., 2018).

18.4  Traits in river phytoplankton biomonitoring In addition to species composition, experiments, reviews and metaanalyses have started investigating phytoplankton trait composition. Wu et al. (2017) summarized the latest proposed traits of river microphytoplankton, which included a total of twelve algal traits with 79 categories (Table 18.1), and have been widely used recently (e.g., B-Beres et al., 2016; Colina et al., 2016; Lange et al., 2016; Sun et al., 2018; Wu et al., 2019). A case study of river phytoplankton functional groups response to multiple stressors in a German lowland catchment is highlighted in Example 2. TABLE 18.1  Phytoplankton traits, descriptions, and expected responses to different environmental stressors, e.g., flow regulation, light, nutrient enrichment, and grazing. Expected responses to stressors Traits

Categories

Abbreviations

Resource

Disturbance

1. Functional diversity

Functional richness

FRic

Functional evenness

FEve

Disturbances will reduce while high resource supply will increase functional diversity

Functional divergence

FDiv

Functional attribute diversity

FAD

Functional dispersion

FDis

Functional redundancy

FRed

TABLE 18.1  Phytoplankton traits, descriptions, and expected responses to different environmental stressors, e.g., flow regulation, light, nutrient enrichment, and grazing—cont’d Expected responses to stressors Traits 2. Cell size

Categories 3

Nano (5–100 μm ) 3

Micro (100–300 μm )

Abbreviations

Resource

BioVol_c1

Smaller cells have higher nutrient uptake rates and growth rates that allow greater resilience to disturbance making them advantage under nutrient-limiting and high disturbance conditions; Larger cells show converse trend

BioVol_c2

3

Meso (300–600 μm )

BioVol_c3

Disturbance

3

BioVol_c4

3

Very large (>1500 μm )

BioVol_c5

Low profile

LowPro

High profile

HigPro

Motile taxa

MotTax

Planktonic taxa

PlaTax

Colonial

LifFor_col

Filamentous

LifFor_fil

Flagellate

LifFor_fla

Unicellular

LifFor_uni

Guild +Cell sizea



Combination between diatom guilds and cell size classes

Life form +Cell sizeb



Combination between life forms and cell size classes

6. Nitrogen fixation

Yes (1) or no (0)

NitFix_1

N-fixer algae has advantage under nutrient-limiting condition but their relation to disturbances varies

7. Attachment to substratum

Non attached

AttSub_non

Medium attached

AttSub_med

Algae with stronger attachment are more likely to retain under high disturbance condition

Tightly attached

AttSub_hig

Motile attached

Motile_att

Motile gliding

Motile_gli

Motile drift

Motile_dri

9. Reproductive strategies

Fission

RepStr_fis

Fragmentation

RepStr_fra

10. Spore formation

No spore formation

SpoFor_non

Zoospores

SpoFor_zoo

Akinetes

SpoFor_aki

Oospores and zygospores

SpoFor_oos.zyg

Optimum temperature for growth

Topt

Maximum persistence temperature

Tmax

Minimum persistence temperature

Tmin

Saturated fatty acids

SAFA

Monounsaturated fatty acids

MUFA

Polyunsaturated fatty acids

FUFA

Highly unsaturated fatty acid

HUFA

Macro (600–1500 μm )

3. Diatom guild

4. Life form

5. Ecomorphology

8. Motility

11. Temperature traits

12. Algal quality

Advantage at lower resources and high disturbance; Favor higher resources and low disturbance; Advantage in resource gathering and low-flow depositional condition (MotTax and PlaTax)

LifFor_fil has advantage in resource gathering but susceptible to high disturbance regimes; LifFor_uni has advantage in under depositional and high resource conditions

Actively motile algae have advantage in resource gathering and low-flow depositional condition

RepStr_fis has advantage for dispersal and recolonization after disturbance SpoFor_aki and SpoFor_oos.zyg have advantage in unfavorable conditions

Topt Tmax Tmin decline with latitude increasing

Nutrient enrichment, low light and low temperature increases PUFA%, which in turn affect growth and PUFA composition in stream grazers

Note: Resource acquisition includes nutrient enrichment (e.g., global land use change) and light (global warming); disturbance includes flow regulation and grazing. a

A simple combination between 4 guilds and 5 cell size classes, resulting in 20 combinations.

b

A simple combination between 4 life forms and 5 cell size classes, resulting in 20 combinations.

Source: Wu, N.C., Dong, X.H., Liu, Y., Wang, C., Baattrup-Pedersen, A., Riis, T., 2017. Using river microalgae as indicators for freshwater biomonitoring: review of published research and future directions. Ecol. Indic. 81, 124–131.

296  PART | V  Algal ecology

Example 2. River phytoplankton functional groups response to multiple stressors in a German lowland catchment To help understand the key ecological processes that govern the river phytoplankton under multiple pressures, Qu et al. (2019) carried out an interdisciplinary study in a German lowland catchment of Treene by linking catchment hydrological processes to riverine phytoplankton functional groups (PFGs) with the aim of examining their responses to hydrological regime (H), physicochemical condition (P) and land-use pattern (L) across two contrasting hydrological periods. The hydrological regime was calculated based on the outputs of a well-established ecohydrological model (SWAT) within the catchment. The variation partitioning analysis results indicated that P and H dominated during the dry period and P also during high flows. Structural equation models (SEM, Fig. 18.4) demonstrated that the skewness of 7 days discharge was considered as a key driver of H, which always had an indirect effect on the functional group TB (benthic diatoms) during both hydrological periods. Phosphorus had direct impacts on the functional group M (mainly composed by the genus Microcystis) in both periods, while urban area had an indirect affect during the high flow period, and water bodies a direct impact during low flow periods on M. The results emphasized that climate change and anthropogenic activities such as shifting flow regime and land-use change directly or indirectly affect riverine phytoplankton via physicochemical conditions. In addition, their findings highlighted not only the suitability of phytoplankton functional groups as robust bio-indicators, but also the importance of hydrological periods in biomonitoring activities. Dec. 2014 –0.482

0.471

H20

0.617

PO4

0.558 0.276 –0.378

M

R2 = 0.219

TP

SO4

0.321

–0.448

TB

URMD

R2 = 0.103 0.245

H36

0.193

NMDS1

–0.318

Sep. 2015

NPR

H20

–0.157

R2 = 0.728

–0.465

DO

–0.224

M

0.503

R2 = 0.375

0.525

WATR

TB

R2 = 0.276 –0.410

WT

NMDS1 0.217

0.308

R2 = 0.310

FIG.  18.4  The causal relationships between hydrological regime (H), land-use pattern (L), physicochemical condition (P) and phytoplankton bioindicators by structural equation models. Solid arrows represent direct paths while dashed arrows are indirect paths. M and TB: phytoplankton functional groups, n-MDS1: 1st n-MDS axis community composition.

18.5  Estuarine phytoplankton community shifts: What’s in a life form? Niche differentiation is a key factor shaping the structure of estuarine phytoplankton communities and is driven by preferences for specific environmental conditions (e.g., salinity, temperature) (Klais et al., 2011; Spilling et al., 2018). The overlapping requirements for essential growth-limiting resources (e.g., light and nutrient availability) has facilitated the adoption of unique traits that shape ecological niches and, ultimately, enable the survival, and occasional dominance, of co-occurring phytoplankton functional groups and taxa. One of the classifications that can be made in this regard is the separation of the phytoplankton community into two broad groups, i.e., diatoms and flagellates. Both life forms have similar nutrient requirements, however, since flagellates are predominantly nonsiliceous in nature, the reliance of diatoms



Linking phytoplankton community structure to aquatic ecosystem functioning Chapter | 18  297

on d­ issolved silicate (DSi) availability for growth is one of the key distinctions between these two groups. Furthermore, the characteristic motility of flagellates serves as a behavioral trait that confers a competitive advantage over diatoms, particularly in stratified estuarine waters (Wasmund et al., 2017; Spilling et al., 2018). The ability of flagellates to undertake vertical migration through the water column facilitates grazer avoidance, nutrient uptake from deeper waters and the maintenance of populations in desired environmental conditions (e.g., mesohaline waters) (Lemley et al., 2018). The varying life-history strategies broadly employed by diatoms and flagellates (r- and K-strategists, respectively) typically promotes co-existence and succession between the two groups, with diatoms generally replaced by flagellates (typically dinoflagellates) during spring bloom periods. This successional pattern has been partially ascribed to an indirect synergistic relationship (Heisler et al., 2008 and references therein), whereby the decomposition of spring diatom blooms fuels subsequent flagellate-dominated accumulations by increasing nitrogen availability, i.e., remineralisation of ammonium. However, the inherently dynamic hydrological nature of estuaries acting together with the stressors of global changes (e.g., cultural eutrophication and warming) often confound expected successional patterns, culminating in less predictable phytoplankton community shifts (e.g., Xiao et al., 2018). Despite global change issues being a relatively modern problem, the effects of cultural eutrophication are prevalent in aquatic ecosystems globally. The process of cultural eutrophication is encapsulated by the initial increase in anthropogenic nutrient loading—particularly nitrogen (N) and phosphorus (P)—which, in turn, facilitates the accelerated growth of primary producers and causes an ‘undesirable disturbance’ to the balance of organisms in an aquatic ecosystem (OSPAR, 2003). In terms of phytoplankton, the most notable responses to eutrophication include high-biomass proliferations and/or harmful algal blooms (HABs). While the role of increased nutrient loading in triggering such responses is largely undisputed, it is the nutrient composition and proportions of these inputs that are responsible for more subtle alterations to phytoplankton community dynamics (e.g., composition, diversity, succession patterns) (Glibert, 2017). For example, if a scenario in which N and P loading increases disproportionately to DSi is assumed, then a shift towards flagellate-dominated communities can be expected, given that diatoms tend to favor reduced N:DSi ( brown seaweed > green seaweed. The optimum dosage of enzyme was 2.0 mL per 10 g of seaweed. The optimal fermentation conditions for bioethanol production using seaweed included a commercial yeast dosage of 30 wt% and a fermentation time of 3 days. Hamouda et al. (2016) study the enhancement of bioethanol production from Ulva fasciata by biological and chemical hydrolysis in a paper with eight citations. U. fasciata was converted to sugars by acid and base hydrolysis (NaOH, ammonium oxalate, HCl, and H2SO4) at different concentrations (1%, 3%, 5%, 7%) at 121 °C for one, two and three rounds in autoclave. Biological hydrolysis of alga was also done by bacterial growth and enzymatic hydrolysis of alga. The maximum amount of sugars was 700 mg sugar/g alga at one round of autoclave with 3% sulfuric acid. The sugar ratio was 641 mg sugar/g alga that treated by a bacterial strain (Bacillus subtilis SH04). Sugar yields from U. fasciata by amylase partially purified from Bacillus subtilis SH04 (B4) and Bacillus cereus SH06 (B6) followed by autoclaving for one round gave 458.3 and 516.1 mg sugar/g algal biomass, respectively. The ethanol efficiency using Saccharomyces cerevisiae SH02 was 78.3% with 5% sugar concentration that produced by acid hydrolysis. Ethanol production was 55.9% after enzymatic hydrolysis of alga and fermentation by Saccharomyces cerevisiae SH02. Jiang et al. (2016) study the thermochemical hydrolysis of Ulva in a paper with eight citations. They investigate the impact of change and the comparative significance of thermochemical process temperature, treatment time, % acid and % solid load on carbohydrates release from Ulva. They compared the potential fermentation yields of these hydrolysate products using metabolic models of Escherichia coli, Saccharomyces cerevisiae wild type, Saccharomyces cerevisiae RN1016 with xylose isomerase and Clostridium acetobutylicum. % Acid plays the most significant role and treatment time the least significant role in affecting the monosaccharaides released from Ulva. They also found that within the tested range of parameters, hydrolysis with 121 °C, 30 min 2% acid, 15% solids could lead to the highest yields of conversion: 54.1–57.5 g ethanol kg−1 Ulva dry weight by S. cerevisiae RN1016 with xylose isomerase. Hu et al. (2017) study the effect of different pretreatments on the thermal conversion of Enteromorpha in a paper with seven citations. They use water-wash and acid-wash (7%, 10% of hydrochloric acid, 7% of sulfuric acid, 7% of phosphoric acid) pretreatments. The removal capacity of H2SO4 was the most obvious. During the pretreatments, OH, CO/ CH, SO and CO functional groups in Enteromorpha were cleaved. Phosphoric acid and hydrochloric acid made the intensities of the absorption peaks of OH and SO de-crease the most obvious, respectively. The volatile components of Enteromorpha were easy to evolve after the pretreatment. Khan and Hussain (2015) study the ethanol production from Ulva fasciata in a paper with seven citations. The carbohydrates were extracted by distilled water and was hydrolyzed by various methods including acidic and basic as well as by using ammonia (NH3), hydrogen peroxide and mixture of organic solvents in the presence of aluminum chloride (AlCl3). The comparative study of these methods revealed their efficiency order as sodium hydroxide NaOH (10%)/heating (method-2) > NaOH (10%)/H2O2 in microwave oven (method-5) > ammonia (50%)/reflux (method-6). The presence of glucose was confirmed. The hydrolyzed samples were fermented by Saccharomyces cerevisiae. Chudnovsky et  al. (2018) monitor complex monosaccharide mixtures derived from Ulva by combined optical and microelectromechanical techniques in a paper with five citations. They tested two orthogonal techniques for rapid phenotyping of the green macroalga Ulva based on its glucose, rhamnose, xylose and glucuronic acid contents as derived for reference by acid hydrolysis. They use two complementary methods: ‘near infrared reflection spectroscopy’ (NIRS) and ‘microelectromechanical systems’ (MEMS) ‘resonating membrane vibrometry’. The best estimation was found for rhamnose and glucose contents, whereas xylose and uronic acid content predictions were found to be less accurate.



The pioneering research on the bioethanol production from green macroalgae Chapter | 25  391

25.3.4  Pioneering research on the enzymatic hydrolysis of green macroalgae for bioethanol production There have been 10 papers primarily related to the enzymatic hydrolysis of the green macroalgae (Table 25.3). Yanagisawa et al. (2011a) study the enzymatic hydrolysis of Ulva in a paper with 71 citations. They find that glucans were the only polysaccharides that could be hydrolyzed to fermentable sugars. Successive saccharification with an enzyme was effectively used to obtain high concentrations of glucose and an ethanol yield of more than 3% was obtained. Trivedi et al. (2013) study the enzymatic hydrolysis and production of bioethanol from Ulva fasciata Delile in a paper with 61 citations. Among the different cellulases investigated for efficient saccharification, cellulase 22,119 showed the highest conversion efficiency of biomass into reducing sugars than Viscozyme L, Cellulase 22,086 and 22,128. Preheat treatment of biomass in aqueous medium at 120 °C for 1 h followed by incubation in 2% (v/v) enzyme for 36 h at 45 °C gave a maximum yield of sugar 206.8 mg/g. The fermentation of hydrolysate gave ethanol yield of 0.45 g/g reducing sugar accounting for 88.2% conversion efficiency. Enzyme can be used twice without compromising on the saccharification efficiency. Trivedi et al. (2015) study the enzymatic hydrolysis of Ulva fasciata using cellulase produced from the marine fungus Cladosporium sphaerospermum through solid state fermentation in a paper with 27 citations. They find that the hydrolysis of U. fasciata with enzyme (10 U/g) for 24 h at 40 °C and pH 4 gave maximum yield of sugar 112 mg/g dry weight. On fermentation, they obtain an ethanol yield of 0.47 g/g reducing sugar, corresponding to 93.81% conversion efficiency. Harshvardhan et al. (2013) develop a cellulase from a marine Bacillus sp. H1666 and use it for the enzymatic hydrolysis of Ulva lactuca in a paper with 16 citations. They obtain 450 mg/g increase in glucose yield after saccharification. Kiyohara et al. (2006) study the structure of ‘β-1,3-xylooligosaccharides’ generated from Caulerpa racemosa var. laete-virens β-1,3-xylan by the action of β-1,3-xylanase in a paper with 12 citations. They report the structural analysis of oligosaccharides generated from β-1,3-xylan of this macroalga by the action of β-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different Rfs on TLC (EX2-EX5). Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. This macroalga is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a β-1,3-Xyl chain and linked via covalent bonds. Li et al. (2015) develop a novel enzyme produced by Catenovulum sp. LP to use it in the enzymatic hydrolysis of Ulva prolifera in a paper with 10 citations. They obtain high efficiency for reducing sugar production, which reached 50.2% yield in 6 h. The viscosity of 1.2% U. prolifera noticeably declined from initially 1127 to 7.2 mPa's in 95 min of hydrolysis even in the room temperature. Yanagisawa et al. (2011b) obtain enzyme mixtures (cellulase and amylase) from the scallop and use them for the enzymatic hydrolysis and fermentation of Ulva (sea lettuce) with the use of Saccharomyces cerevisiae in a paper with eight citations. They obtain an ethanol yield as high as 7.2 g/L, which corresponded to ~37% of the conversion of glucans in sea lettuce. The process did not require any additional nutrients, such as yeast extract or peptone, the use of which increases the cost of fermentation to a high level. Cioroiu et al. (2017) study the rheological characterization of Cladophora vagabond for bioethanol processing in the presence of cellulase enzyme in a paper with six citations. They evaluate the effects of operation temperature (t = 25, 50 °C), cellulase/dried algae ratio (R = 0, 16 U/mgda), and algal suspension mass concentration (c = 5–15%) on rheological behavior and parameters. Algal suspensions behaved as non-Newtonian fluids obeying either a Bingham plastic linear relationship or an Ostwald-de Waele power law corresponding to a pseudoplastic fluid. Jmel et al. (2016) study the enzymatic functionalization of Enteromorpha sp. cellulose in a paper with six citations. The cellulose content was about 21.4% (w/w). The 36% crystallinity index of the extracted cellulose revealed a high amorphous character. The moisture adsorption isotherms and specific surface measurements were respectively 42.9 g/100 g and 8.3 m2/g. The Enteromorpha sp. cellulose was first enzymatically saccharified by a commercial cellulase preparation from Aspergillus niger with a hydrolysis yield of 70.4%. Neifar et al. (2016) study the enzymatic hydrolysis of Chaetomorpha linum for bioethanol production in a paper with six citations. The hydrothermally pretreated C. linum biomass was treated with Aspergillus niger cellulase at various liquid to solid ratios (50–100 mL/g), enzyme concentrations (10–60 U/g) and incubations times (4–44 h). The optimum saccharification conditions were: L/S ratio 100 mL/g; enzyme concentration 52 U/g; and time 44 h. Their application led to a maximum sugar yield of 30.2 g/100 g dry matter. Saccharomyces cerevisiae fermentation of the algal hydrolysate provided 8.6 g ethanol/100 g dry matter.

Macroalgae

Treatment and hydrolysis

Monosugar yield

1

Ulva (Sea lettuce)

Enzymatic

2

Ulva fasciata Delile

3

Fermenting bacteria

Ethanol yield

References

Cits.

Glucose

More than 3%

Yanagisawa et al. (2011a)

71

Enzymatic, heat

206.8 mg/g

0.45 g/g reducing sugar, 88.2% conversion efficiency.

Trivedi et al. (2013)

61

Ulva fasciata

Enzymatic

112±10 mg/g sugar

0.47 g/g reducing sugars

Trivedi et al. (2015)

27

4

Ulva lactuca

Enzymatic

450 mg/g glucose

Harshvardhan et al. (2013)

17

5

Caulerpa racemosa var.

Enzymatic

Sugars

Kiyohara et al. (2006)

12

67

Ulva prolifera

Enzymatic

50.2% reducing sugar

Li et al. (2015)

10

8

Cladophora vagabunda

Enzymatic

Sugars

Cioroiu et al. (2017)

6

9

Enteromorpha sp.

Enzymatic

70.4%

Jmel et al. (2016)

6

10

Chaetomorpha linum

Enzymatic, hydrothermal

32.2% sugar

Neifar et al. (2016)

6

Saccharomyces cerevisiae

8.6 g ethanol/100 g dry matter

392  PART | VI  Algal bioenergy and biofuels

TABLE 25.3  The pioneering research on the enzymatic hydrolysis of green macroalgae.



The pioneering research on the bioethanol production from green macroalgae Chapter | 25  393

25.3.5  Pioneering research on the fermentation of green macroalgae for bioethanol production There have been 13 papers primarily related to the fermentation of the green macroalgae (Table 25.4). Kim et al. (2011) study the ethanol production from Ulva lactuca using Escherichia coli KO11 after acid and enzymatic hydrolysis in a paper with 143 citations. They note that the strategy of acid hydrolysis followed by simultaneous enzyme treatment and inoculation with E. coli KO11 could be a viable strategy to produce ethanol from Ulva. van der Wal et al. (2013) study the production of ‘acetone, butanol, and ethanol’ (ABE) from Ulva lactuca in a paper with 83 citations. They obtain solubilization of over 90% of sugars by hot-water treatment followed by hydrolysis using commercial cellulases. They obtain ABE by Clostridium beijerinckii. Hydrolysate-based media were fermentable without nutrient supplementation. C. beijerinckii utilized all sugars in the hydrolysate and produced ABE at high yields (0.35 g ABE/g sugar consumed). They further obtain 1,2 propanediol was the main fermentation product (9.7 g/L) in control cultures of C. beijerinckii on rhamnose and glucose. Bikker et al. (2016) study the production of ethanol together with animal feed and propanediol, butanol, and acetone in a biorefinery from Ulva lactuca containing 225 g protein (N × 4.6) kg−1 dry matter (DM) in a paper with 40 citations. The sugars in the biomass were solubilized by hot water treatment followed by enzymatic hydrolysis and centrifugation resulting in a sugar-rich hydrolysate (38.8 g/L sugars) containing glucose, rhamnose and xylose, and a protein-enriched (343 g kg−1 in DM) extracted fraction. The hydrolysate was used successfully for the production of acetone, butanol, ethanol and 1,2-propanediol by clostridial fermentation. Korzen et al. (2015c) study the ethanol production through the ‘simultaneous hydrolysis and fermentation’ (SSF) process from Ulva rigida using sonication treatment and Saccharomyces cerevisiae in a paper with 27 citations. Sonication provided a faster way for the simultaneous release of glucose from U. rigida and its conversion into bioethanol. Within 3 h, they obtained 196 mg glucose per gram of dry weight of biomass and 333.3 mg bioethanol per gram of glucose. Golberg et  al. (2014) develop a biorefinery design for the production of ethanol from Ulva for an average town in rural India, combining thermodynamic, metabolic, and sustainability analyses in a paper with 26 citations. They include sustainability and legislation factors, intensive macro algae Ulva farming, and metabolic modeling of the biological twostep conversion of Ulva feedstock by a yeast (Saccharomyces cerevisiae), and then by a bacterium (Escherichia coli), into bioethanol. Uchida and Murata (2004) study the fermentation of Ulva spp. focusing on the fermenting bacteria in a paper with 23 citations. They determine them as Lactobacillus brevis, Debaryomyces hanseni var. hansenii, and a Candida zeylanoides, suggesting that the observed fermentation along with enzymatic hydrolysis can be categorized to lactic acid and ethanol fermentation. Inoculating the individually cultured cell suspensions of the three kinds of bacteria with cellulase induced the fermentation in various kinds of seaweed. Ben Yahmed et al. (2016) develop an integrated biorefinery approach using Chaetomorpha linum for the co-production of bioethanol and biogas in a paper with 18 citations. Among three pretreatments of C. linum, consisting of acidic, neutral and alkali ones, 3% NaOH pretreatment gave the best results. The hydrolysis of C. linum feedstock with a crude specific enzyme preparation gave the maximum yield of fermentable sugar of 0.22 g/g dry substrate. An ethanol yield of 0.41 g/g reducing sugar corresponding to about 0.09 g/g pretreated algae was obtained after alcoholic fermentation by Saccharomyces cerevisiae. In the integrated proposed process, mycelium issued from the fungal fermentation, liquid issued from alkali pretreatment, residual from the non-hydrolysable biomass and all effluents and co-products represent a heterogeneous substrate that feed an anaerobic digester for biogas production. The biomethane yield reached 0.26 L/gVS with single waste (0.3 g/g). Trivedi et al. (2016) develop an integrated process for Ulva fasciata for the production of mineral rich liquid extract (MRLE), lipid, ulvan, and cellulose in a paper with 16 citations. The main benefits of this process are its simplicity and the consistent yields obtained from the residual biomass after each successive extraction step. For example, dry Ulva biomass yields 26% of its starting mass as MRLE, 3% as lipid, 25% as ulvan, and 11% as cellulose. The enzymatic hydrolysis and fermentation of the final cellulose fraction under optimized conditions produced ethanol at a competitive 0.45 g/g reducing sugar. These yields are comparable to those obtained by direct processing of the individual components from primary biomass. Kostas et al. (2016) study the fermentation of Ulva screening 24 fermenting yeast strains following the acid hydrolysis in a paper with 16 citations. They select the strains of Saccharomyces spp., Pichia sp., Candida sp.). These selected strains converted the monomeric sugars into bioethanol. Chirapart et al. (2014) study the ethanol production from algae Ulva intestinalis and Rhizoclonium riparium in a paper with 11 citations. The carbohydrate contents were relatively low: R. riparium (29.53%). Glucose obtained from R. riparium (6.52%) was higher than in U. intestinalis (2.78%). The ethanol yields were relatively low: R. riparium (0.09 × 10−3 g ethanol g−1 sugars; 33.8 μg ethanol g−1 glucose), U. intestinalis (0.07 × 10−3 g ethanol g−1 sugars; 9.98 μg ethanol g−1 glucose).

Macroalgae

Treatment and hydrolysis

Monosugar yield

Fermenting bacteria

Ethanol yield

SSF

1

Ulva lactuca

Acid, enzymatic

Sugars

Escherichia coli

Ethanol

*

2

Ulva lactuca

Hot-water, enzymatic

Sugars

Clostridium beijerinckii

0.35 g ABE/g sugar

3

Ulva lactuca

Hot water, enzymatic

38.8 g/L sugars, protein extract

Clostridia

Ethanol

4

Ulva rigida

Sonication

196 mg glucose/g algae

Saccharomyces cerevisiae

333 mg ethanol/g glucose

5

Ulva

Saccharomyces cerevisiae, Escherichia coli

Ethanol

6

Ulva spp.

Enzymatic

Sugars

Lactobacillus brevis, Debaryomyces hanseni var. hansenii, Candida zeylanoides

Ethanol

7

Chaetomorpha linum

Base (NaOH), enzymatic

0.22 g/g dry algae

Saccharomyces cerevisiae

0.41 g/g reducing sugar

8

Ulva

Acid

Sugars

Saccharomyces spp., Pichia sp., Candida sp.

13 g/L sugar

9

Ulva fasciata

Enzymatic

Sugars

0.45 g/g reducing sugar

10

Ulva intestinalis, Rhizoclonium riparium

2.78%, 6.52%

0.074 × 10 (−3) g ethanol g (−1) sugars, 0.086 × 10 (−3) g ethanol g (−1) sugars

11

Ulva rigida

Sugars

16 wt% (based on the dry weight of algae)

12

Ulva rigida

13

Ulva pertusa Kjellman

*, have SSF process.

Sonication

Ethanol Saccharomyces cerevisiae, Pichia stipitis

37% reducing sugar

Co-products

References

Cits.

Kim et al. (2011)

143

Propanediol, acetone, butanol

van der Wal et al. (2013)

83

Animal feed, propanediol, butanol, acetone

Bikker et al. (2016)

40

Korzen et al. (2015c)

27

Golberg et al., 2014

26

Lactic acid

Uchida and Murata (2004)

23

Biogas

Ben Yahmed et al. (2016)

18

Kostas et al. (2016)

16

Trivedi et al. (2016)

16

Chirapart et al. (2014)

11

Korzen et al. (2015b)

10

Korzen et al. (2015a)

8

Lee et al. (2013)

6

*

Chemicals

* Animal feed

394  PART | VI  Algal bioenergy and biofuels

TABLE 25.4  The pioneering research on the fermentation of green macroalgae.



The pioneering research on the bioethanol production from green macroalgae Chapter | 25  395

Korzen et al. (2015b) study the marine integrated culture of carbohydrate rich Ulva rigida for enhanced production of bioethanol in a paper with 10 citations. They report the optimized production of Ulva rigida co-cultured with fed-fish in an offshore mariculture (fish cages) system. Enhanced production of biomass with elevated content of desired carbohydrates is achieved. The farmed biomass was further converted to bioethanol by a one-step sonication assisted SSF process. An ethanol yield of 16 wt% (based on the dry weight of algae) is obtained. Korzen et al. (2015a) carry out the economic analysis of bioethanol production from Ulva rigida in a paper with eight citations. Ulva was co-cultured with fish in an intensive offshore aquaculture unit. By simultaneously producing ethanol with valuable ‘Dried Distillers Grains with Solubles’ (DDGS) by-products such as animal feed, the economic viability of this system is plausible over a production range of 77–240 dry tons of seaweed per day. Sensitivity analyses place profitability as mainly dependent on DDGS prices and on the daily growth rate (biomass yield) of the macroalga. These two are key factors to achieve profitability at the 600-ha scenario. Lee et al. (2013) study the ethanol production from Ulva pertusa Kjellman using a two-step process with immobilized Saccharomyces cerevisiae and Pichia stipites in a paper with six citations. The first step of the process included a continuous column reactor using immobilized S. cerevisiae, and the second step included a repeated-batch reactor using immobilized P. stipitis. The glucose and xylose in 20 L of medium containing the U. pertusa Kjellman hydrolysate was converted completely to about 5.0 g/L ethanol through the two-step process, in which the overall ethanol yield from total reducing sugar was 0.37 and the volumetric ethanol productivity was 0.13 g/L/h. The volumetric ethanol productivity of the two-step process was about 2.7 times greater than that when P. stipitis was used alone for ethanol production from the hydrolysate. In addition, the overall ethanol yield from glucose and xylose was superior to that when P. stipitis was used alone for ethanol production.

25.4 Discussion 25.4.1  The research landscape The section on the research landscape highlights the most-prolific authors, institutions, journals, countries, publication years, and subject categories. The authors and institutions from Israel, South Korea, and India have primarily contributed to the research in this field. The data on the publication years show that this research field has primarily developed in recent years. It is notable that 35 of these papers have been indexed by the subject categories of ‘Biotechnology Applied Microbiology’ and ‘Energy Fuels’. The citation impact of these papers has been relatively significant. There have been 13, 10, 10, and 13 papers related to the pretreatment, acid hydrolysis, enzymatic hydrolysis, and fermentation of the green macroalgae, respectively.

25.4.2  Pioneering research on the pretreatments of green macroalgae for bioethanol production The research on the pretreatments of green macroalgae has formed a significant part of the research on the bioethanol production from green macroalgae as 21 papers have covered various pretreatments. Of them, 13 papers have primarily focused on the pretreatment of the green macroalgae (Table 25.1). Instead of acids or enzymes, a number of pretreatments degrading the polysaccharides have been used in the pretreatments of the green macroalgae. The most-prolific pretreatments have been hydrothermal treatment, ultrasonication, microwave irradiation, and hydrogen peroxide with 9, 4, 3, and 3 papers, respectively. van der Wal et al. (2013), Bikker et al. (2016), Kim et al. (2014, 2016), Okuda et al. (2008), Neifar et al. (2016), Choi et al. (2012, 2013), and Schultz-Jensen et al. (2013) have used the hydrothermal pretreatment to make the green macroalgal biomass accessible to the following steps of acid hydrolysis, enzymatic hydrolysis, or fermentation. Korzen et  al. (2015b,c), Zhao and Ruan (2011), and Woods et  al. (2011) have used the ultrasonication treatment. Tsubaki et al. (2014, 2017) and Khan and Hussain (2015) have used microwave irradiation treatment. Li et al. (2016), Khan and Hussain (2015), and Zhao and Ruan (2011) have used the hydrogen peroxide treatment. The other pretreatment used with green macroalgae have been ball milling (Schultz-Jensen et al., 2013), hot-compress (Jang et al., 2012), silicon beads or sand (Woods et al., 2011), water-wash (Hu et al., 2017), and ionic liquids (Pezoa-Conte et al., 2015). The pioneering research on the pretreatment of the green macroalgae has provided ample evidence for the value of these pretreatments for the following steps of acid hydrolysis, enzymatic hydrolysis, and fermentation for the green macroalgae. The optimization of the pretreatments emerges as a strategic point in these studies.

396  PART | VI  Algal bioenergy and biofuels

25.4.3  Pioneering research on the acid hydrolysis of green macroalgae for bioethanol production The research on the acid hydrolysis of green macroalgae has formed a significant part of the research on the bioethanol production from green macroalgae as 15 papers have covered various acids. Of them, 10 papers have primarily focused on the acid hydrolysis of the green macroalgae (Table 25.2). Instead of enzymes or pretreatments, a number of acids degrading the polysaccharides have been used in the acid hydrolysis of the green macroalgae. A number of papers have focused on the thermal acid treatment of green macroalgae in general (Kim et al., 2011; Yoza and Masutani, 2013; Kostas et al., 2016; Hong et al., 2014; Jiang et al., 2016; Chudnovsky et al., 2018). The most prolific acids have been sulfuric acid (Jang et al., 2012; Hamouda et al., 2016; Hu et al., 2017; Zhao and Ruan, 2011; Feng et al., 2011), hydrochloric acid (Yang and Huang, 2016; Hamouda et al., 2016; Hu et al., 2017; Feng et al., 2011), sodium hydroxide (Ben Yahmed et al., 2016; Hamouda et al., 2016; Khan and Hussain, 2015), and ammonia (Hamouda et al., 2016; Khan and Hussain, 2015). The other acids have been trifluoroacetic acid (Quemener et al., 1997), phosphotungstic acid (Tsubaki et al., 2014, 2017), maleic acid (Feng et al., 2011), and phosphoric acid (Hu et al., 2017). The pioneering research on the acid hydrolysis of the green macroalgae has provided ample evidence for the value of this hydrolysis for the following steps of enzymatic hydrolysis and fermentation for the green macroalgae. The optimization of the hydrolysis emerges as a strategic point in these studies.

25.4.4  Pioneering research on the enzymatic hydrolysis of green macroalgae for bioethanol production The research on the enzymatic hydrolysis of green macroalgae has formed a significant part of the research on the bioethanol production from green macroalgae as 27 papers have covered this hydrolysis. Of them, 10 papers have primarily focused on the enzymatic hydrolysis of the green macroalgae (Table 25.3). Instead of acids or pretreatments, a number of enzymes degrading the polysaccharides have been used in the enzymatic hydrolysis of the green macroalgae. These papers are Ben Yahmed et al. (2016), Bikker et al. (2016), Choi et al. (2012, 2013), Cioroiu et al. (2017), Hamouda et al. (2016), Harshvardhan et al. (2013), Hong et al. (2014), Hu et al. (2017), Jiang et al. (2016), Jmel et al. (2016), Kim et al. (2011, 2014), Kiyohara et al. (2006), Li et al. (2015, 2016), Neifar et al. (2016), Okuda et al. (2008), Quemener et al. (1997), Trivedi et al. (2013, 2015, 2016), Uchida and Murata (2004), van der Wal et al. (2013), Yanagisawa et al. (2011a,b), and Yoza and Masutani (2013). The pioneering research on the enzymatic hydrolysis of the green macroalgae has provided ample evidence for the value of this hydrolysis for the following step of fermentation for the green macroalgae. The optimization of the hydrolysis emerges as a strategic point in these studies.

25.4.5  Pioneering research on the fermentation of green macroalgae for bioethanol production The research on the fermentation of hydrolysates of green macroalgae has formed a significant part of the research on the bioethanol production from green macroalgae as 28 papers have covered this field. Of them, 13 papers have primarily focused on the fermentation of the green macroalgae (Table 25.4). A number of papers have covered the fermentation of the green macroalgae in general (Chirapart et al., 2014; Choi et al., 2013; Feng et al., 2011; Hong et al., 2014; Korzen et al., 2015a,b; Schultz-Jensen et al., 2013; Trivedi et al., 2013, 2015, 2016; Yanagisawa et al., 2011a; Yoza and Masutani, 2013). The most-prolific studied fermentation bacterium has been Saccharomyces cerevisiae (Ben Yahmed et al., 2016; Golberg et al., 2014; Hamouda et al., 2016; Jiang et al., 2016; Khan and Hussain, 2015; Korzen et al., 2015c; Kostas et al., 2016; Lee et al., 2013; Li et al., 2016; Neifar et al., 2016; Yanagisawa et al., 2011b). The other prolific fermentation bacteria have been Escherichia coli (Kim et al., 2011; Golberg et al., 2014; Jiang et al., 2016); Clostridia (van der Wal et al., 2013; Bikker et al., 2016; Jiang et al., 2016); Pichia (Kostas et al., 2016; Lee et al., 2013); and Candida (Uchida and Murata, 2004; Kostas et al., 2016). Lactobacillus brevis, and Debaryomyces hanseni var. hansenii have also been studied (Uchida and Murata, 2004). It is notable that there have been only three papers on the ‘simultaneous saccharification and fermentation’ process in contrast to ‘separate saccharification and fermentation’ process (Kim et al., 2011; Korzen et al., 2015b,c). There have been a number of studies covering other co-products besides bioethanol production (Ben Yahmed et al., 2016; Kim et al., 2016; Korzen et al., 2015a; Trivedi et al., 2016; Uchida and Murata, 2004; van der Wal et al., 2013). These have been propanediol, acetone, butanol, animal feed, lactic acid, biogas, chemicals, levulinic acid, HMF, and furfural.



The pioneering research on the bioethanol production from green macroalgae Chapter | 25  397

The pioneering research on the fermentation of the green macroalgae has provided ample evidence for the value of fermentative processes using fermenting bacteria for the production of bioethanol from green macroalgae. The optimization of the fermentative processes emerges as a strategic point in these studies.

25.5 Conclusion This study of the pioneering research on the bioethanol production from green macroalgae at the global scale covering the whole range of research fronts as well as all types of green macroalgae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. The research in this field have progressed in four subfields: pretreatments, acid hydrolysis, enzymatic hydrolysis of green macroalgae and fermentation of hydrolysates of green macroalgae (Tables 25.1–25.4). The most-prolific pretreatments have been hydrothermal treatment, ultrasonication, microwave irradiation, and hydrogen peroxide with nine, four, three, and three papers, respectively. The other pretreatments used with green macroalgae have been ball milling, hot-compress, silicon beads or sand, and ionic liquids. The most prolific acids have been sulfuric acid, hydrochloric acid, sodium hydroxide, and ammonia. The other acids have been trifluoroacetic acid, phosphotungstic acid, maleic acid, and phosphoric acid. The most-studied fermentation bacterium has been Saccharomyces cerevisiae. The other prolific fermentation bacteria have been Escherichia coli, Clostridia, Pichia, and Candida. Lactobacillus brevis, and Debaryomyces hanseni var. hansenii have also been studied. It is notable that there have been only three papers on the ‘simultaneous saccharification and fermentation’ process in contrast to ‘separate saccharification and fermentation’ process. There have been a number of studies covering other co-products besides bioethanol production. These have been propanediol, acetone, butanol, animal feed, lactic acid, biogas, chemicals, levulinic acid, HMF, and furfural. These studies highlight the need for the production of bioethanol from green macroalgae in a biorefinery together with high-value coproducts to reduce the costs of the production. The most-studied green macrolgae have been the various species of Ulva with 28 papers. The other prolific species of green macroalgae have been Chaetomorpha, Cladophora, Enteromorpha, and Monostroma. The other studied species have been Codium and Caulerpa. It has been found that the detailed keyword set provided in the Appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. The citation impact of these pioneering studies has been significant, contributing to the wider research fields of algal polysaccharides, plant biology, bioenergy and biofuels, bioethanol, algal ecology, algal bioenergy and biofuels, and medical applications of macroalgae among others. It is notable that the applications of nanotechnology in this field has not been significant. It is expected the applications of nanotechnology in this field would accelerate to further optimize the methodology for the determination of structures of photosystems and photosynthesis-related structures and processes (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e,f). It appears that the structure-processing-property relationships form the basis of the research in this field as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002). These pioneering studies with at least five citations each in this field have formed the basis for the rapid expansion of this research field with the promising results for the bioethanol production from green macroalgae. It is recommended that the complementary studies should be carried out in other related fields such as bioethanol production from brown macroalgae, red macroalgae, and macroalgae in general. It is further recommended that a full scientometric study of these fields should be carried out.

Appendix. The keyword sets A.1  Ethanol-related keywords TI = (Ethanol or bioethanol or ethanogen* or ethanologen* or *ferment* or *saccharification or hydrolys* or hydrolyz* or ‘pretreatment*’ or pretreatment* or hydrolytic or ‘ionic liquid*’ or Ultrasound or ‘acid hydrolysis’ or ‘acidic hydrolysis’

398  PART | VI  Algal bioenergy and biofuels

or ‘hydrogen peroxide’ or enzymatic or sonication or ‘microwave irradiation’ or ‘hydroxyl radicals’ or ‘hot water’ or fractination or ‘fermentable sugar*’ or ‘reducing sugar*’ or cellulase* or pichia or mannose or saccharomyces or clostridium or candida or lactobacillus or detoxification or ‘uronic acid’ or rhamnose or ‘manuronic acid’ or ‘gluronic acid’ or glucosidase* or glucuronidase) NOT TI = (Extract* or supercritical or dehydration or pervaporation or membrane* or immobil* or water or solvent* or separation or entrap* or encaps* or *hydrogen or molass* or lipid* or ‘bio-oil*’ or hydrothermal or biogas or *methane or ch4 or h2 or transester* or oil* or liquefaction or beads or *crude or cultivat* or *sorp* or binding).

A.2  Green macrolgae-related keywords TS = (Caulerpa or Chaetomorpha or Cladophora or Codium or Enteromorpha or Hydrodictyon or Monostroma or Rhizoclonium or ‘sea lettuce’ or Spirogyra or Ulva or ‘green macroalga*’ or ‘green saeweed*’).

References Ben Yahmed, N., Jmel, M.A., Ben Alaya, M., Bouallagui, H., Marzouki, M.N., Smaali, I., 2016. A biorefinery concept using the green macroalgae Chaetomorpha linum for the coproduction of bioethanol and biogas. Energy Convers. Manag. 119, 57–265. Bikker, P., van Krimpen, M.M., van Wikselaar, P., Houweling-Tan, B., Scaccia, N., van Hal, J.W., et al., 2016. Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels. J. Appl. Phycol. 28 (6), 3511–3525. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sust. Energ. Rev. 14 (2), 557–577. Chirapart, A., Praiboon, J., Puangsombat, P., Pattanapon, C., Nunraksa, N., 2014. Chemical composition and ethanol production potential of Thai seaweed species. J. Appl. Phycol. 26 (2), 979–986. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Choi, W.Y., Han, J.G., Lee, C.G., Song, C.H., Kim, J.S., Seo, Y.C., et al., 2012. Bioethanol production from Ulva pertusa Kjellman by high-temperature liquefaction. Chem. Biochem. Eng. Q. 26 (1), 15–21. Choi, W.Y., Kang, D.H., Lee, H.Y., 2013. Enhancement of the saccharification yields of Ulva pertusa Kjellman and rape stems by the high-pressure steam pretreatment process. Biotechnol. Bioproc. E 18 (4), 728–735. Chudnovsky, A., Golberg, A., Linzon, Y., 2018. Monitoring complex monosaccharide mixtures derived from macroalgae biomass by combined optical and microelectromechanical techniques. Process Biochem. 68, 136–145. Cioroiu, D.R., Parvulescu, O.C., Koncsag, C.I., Dobre, T., Raducanu, C., 2017. Rheological characterization of algal suspensions for bioethanol processing. Rev. Chim. (Bucharest, Rom.) 68 (10), 2311–2316. Feng, D.W., Liu, H.Y., Li, F.C., Jiang, P., Qin, S., 2011. Optimization of dilute acid hydrolysis of Enteromorpha. Chin. J. Oceanol. Limnol. 29 (6), 1243–1248. Golberg, A., Vitkin, E., Linshiz, G., Khan, S.A., Hillson, N.J., Yakhini, Z., et al., 2014. Proposed design of distributed macroalgal biorefineries: thermodynamics, bioconversion technology, and sustainability implications for developing economies. Biofuels Bioprod. Biorefin. 8 (1), 67–82. Hamouda, R.A., Sherif, S.A., Dawoud, G.T.M., Ghareeb, M.M., 2016. Enhancement of bioethanol production from Ulva fasciata by biological and chemical saccharification. Rend. Lincei. Sci. Fis. 27 (4), 665–672. Harshvardhan, K., Mishra, A., Jha, B., 2013. Purification and characterization of cellulase from a marine Bacillus sp H1666: a potential agent for single step saccharification of seaweed biomass. J. Mol. Catal. B Enzym. 93, 51–56. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol. Adv. 29 (2), 189–198. Hong, I.K., Jeon, H., Lee, S.B., 2014. Comparison of red, brown and green seaweeds on enzymatic saccharification process. J. Ind. Eng. Chem. 20 (5), 2687–2691. Hu, Y.M., Wang, S.A., Wang, Q.A., He, Z.X., Lin, X.C., Xu, S.N., 2017. Effect of different pretreatments on the thermal degradation of seaweed biomass. Proc. Combust. Inst. 36 (2), 2271–2281. Jang, S.S., Shirai, Y., Uchida, M., Wakisaka, M., 2012. Production of mono sugar from acid hydrolysis of seaweed. Afr. J. Biotechnol. 11 (8), 1953–1963. Jiang, R., Linzon, Y., Vitkin, E., Yakhini, Z., Chudnovsky, A., Golberg, A., 2016. Thermochemical hydrolysis of macroalgae Ulva for biorefinery: Taguchi robust design method. Sci. Rep. 6, 27761. Jmel, M.A., Ben Messaoud, G., Marzouki, M.N., Mathlouthi, M., Smaali, I., 2016. Physico-chemical characterization and enzymatic functionalization of Enteromorpha sp. cellulose. Carbohydr. Polym. 135, 274–279. Khan, A.M., Hussain, M.S., 2015. Production of biofuels from marine macroalgae Melanothamnus afaqhusainii and Ulva fasciata. J. Chem. Soc. Pak. 37 (2), 371–379. Kim, N.J., Li, H., Jung, K., Chang, H.N., Lee, P.C., 2011. Ethanol production from marine algal hydrolysates using Escherichia coli KO11. Bioresour. Technol. 102 (16), 7466–7469. Kim, D.H., Lee, S.B., Jeong, G.T., 2014. Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis. Bioresour. Technol. 161, 348–353. Kim, D.H., Lee, S.B., Kim, S.K., Park, D.H., Jeong, G.T., 2016. Optimization and evaluation of sugars and chemicals production from green macro-algae Enteromorpha intestinalis. Bioenergy Res. 9 (4), 1155–1166.



The pioneering research on the bioethanol production from green macroalgae Chapter | 25  399

Kiyohara, M., Hama, Y., Yamaguchi, K., Ito, M., 2006. Structure of β-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens β-1,3xylan by the action of β-1,3-xylanase. J. Biochem. 140 (3), 369–373. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2019a. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

400  PART | VI  Algal bioenergy and biofuels

Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Korzen, L., Peled, Y., Shamir, S.Z., Shechter, M., Gedanken, A., Abelson, A., et al., 2015a. An economic analysis of bioethanol production from the marine macroalga Ulva (Chlorophyta). Technology 3 (2–3), 114–118. Korzen, L., Pulidindi, I.N., Israel, A., Abelson, A., Gedanken, A., 2015b. Marine integrated culture of carbohydrate rich Ulva rigida for enhanced production of bioethanol. RSC Adv. 5 (73), 59251–59256. Korzen, L., Pulidindi, I.N., Israel, A., Abelson, A., Gedanken, A., 2015c. Single step production of bioethanol from the seaweed Ulva rigida using sonication. RSC Adv. 5 (21), 16223–16229. Kostas, E.T., White, D.A., Du, C.Y., Cook, D.J., 2016. Selection of yeast strains for bioethanol production from UK seaweeds. J. Appl. Phycol. 28 (2), 1427–1441. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Lee, S.E., Kim, Y.O., Choi, W.Y., Kang, D.H., Lee, H.Y., Jung, K.H., 2013. Two-step process using immobilized Saccharomyces cerevisiae and Pichia stipitis for ethanol production from Ulva pertusa Kjellman hydrolysate. J. Microbiol. Biotechnol. 23 (10), 1434–1444. Li, Y.P., Huang, Z.J., Qiao, L.K., Gao, Y., Guan, H.S., Hwang, H.M., 2015. Purification and characterization of a novel enzyme produced by Catenovulum sp. LP and its application in the pre-treatment to Ulva prolifera for bio-ethanol production. Process Biochem. 50 (5), 799–806. Li, Y.P., Cui, J.F., Zhang, G.L., Liu, Z.K., Guan, H.S., Hwang, H.M., et al., 2016. Optimization study on the hydrogen peroxide pretreatment and production of bioethanol from seaweed Ulva prolifera biomass. Bioresour. Technol. 214, 144–149. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal‐free organic dyes for dye‐sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Neifar, M., Chatter, R., Chouchane, H., Genouiz, R., Jaouani, A., Masmoudi, A.S., et al., 2016. Optimization of enzymatic saccharification of Chaetomorpha linum biomass for the production of macroalgae-based third generation bioethanol. AIMS Bioeng. 3 (3), 400–411. Okuda, K., Oka, K., Onda, A., Kajiyoshi, K., Hiraoka, M., Yanagisawa, K., 2008. Hydrothermal fractional pretreatment of sea algae and its enhanced enzymatic hydrolysis. J. Chem. Technol. Biotechnol. 83 (6), 836–841. Pezoa-Conte, R., Leyton, A., Anugwom, I., von Schoultz, S., Paranko, J., Maki-Arvela, P., et al., 2015. Deconstruction of the green alga Ulva rigida in ionic liquids: closing the mass balance. Algal Res. 12, 262–273. Quemener, B., Lahaye, M., Bobin-Dubigeon, C., 1997. Sugar determination in ulvans by a chemical-enzymatic method coupled to high performance anion exchange chromatography. J. Appl. Phycol. 9 (2), 179–188. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes—towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Schultz-Jensen, N., Thygesen, A., Leipold, F., Thomsen, S.T., Roslander, C., Lilholt, H., et al., 2013. Pretreatment of the macroalgae Chaetomorpha linum for the production of bioethanol—comparison of five pretreatment technologies. Bioresour. Technol. 140, 36–42. Trivedi, N., Gupta, V., Reddy, C.R.K., Jha, B., 2013. Enzymatic hydrolysis and production of bioethanol from common macrophytic green alga Ulva fasciata Delile. Bioresour. Technol. 150, 106–112. Trivedi, N., Reddy, C.R.L., Radulovich, R., Jha, B., 2015. Solid state fermentation (SSF)-derived cellulase for saccharification of the green seaweed Ulva for bioethanol production. Algal Res. 9, 48–54.



The pioneering research on the bioethanol production from green macroalgae Chapter | 25  401

Trivedi, N., Baghel, R.S., Bothwell, J., Gupta, V., Reddy, C.R.K., Lali, A.M., et al., 2016. An integrated process for the extraction of fuel and chemicals from marine macroalgal biomass. Sci. Rep. 6, 30728. Tsubaki, S., Oono, K., Hiraoka, M., Ueda, T., Onda, A., Yanagisawa, K., et al., 2014. Hydrolysis of green-tide forming Ulva spp. by microwave irradiation with polyoxometalate clusters. Green Chem. 16 (4), 2227–2233. Tsubaki, S., Oono, K., Onda, A., Ueda, T., Mitani, T., Hiraoka, M., 2017. Microwave-assisted hydrolysis of biomass over activated carbon supported polyoxometalates. RSC Adv. 7 (20), 12346–12350. Uchida, M., Murata, M., 2004. Isolation of a lactic acid bacterium and yeast consortium from a fermented material of Ulva spp. (Chlorophyta). J. Appl. Microbiol. 97 (6), 1297–1310. van der Wal, H., Sperber, B.L.H.M., Houweling-Tan, B., Bakker, R.R.C., Brandenburg, W., Lopez-Contreras, A.M., 2013. Production of acetone, butanol, and ethanol from biomass of the green seaweed Ulva lactuca. Bioresour. Technol. 128, 431–437. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Woods, L., Riccobono, M., Mehan, N., Hestekin, J., Beitle, R., 2011. Synergistic effect of abrasive and sonication for release of carbohydrate and protein from algae. Sep. Sci. Technol. 46 (4), 601–604. Yanagisawa, M., Nakamura, K., Ariga, O., Nakasaki, K., 2011a. Production of high concentrations of bioethanol from seaweeds that contain easily hydrolyzable polysaccharides. Process Biochem. 46 (11), 2111–2116. Yanagisawa, M., Ojima, T., Nakasaki, K., 2011b. Bioethanol from sea lettuce with the use of crude enzymes derived from waste. J. Mater. Cycles Waste Manage. 13, 321–326. Yang, C.F., Huang, C.R., 2016. Biotransformation of 5-hydroxy-methylfurfural into 2,5-furan-dicarboxylic acid by bacterial isolate using thermal acid algal hydrolysate. Bioresour. Technol. 214, 311–318. Yoza, B.A., Masutani, E.M., 2013. The analysis of macroalgae biomass found around Hawaii for bioethanol production. Environ. Technol. 34 (13–14), 1859–1867. Zhao, C., Ruan, L.W., 2011. Biodegradation of Enteromorpha prolifera by mangrove degrading micro-community with physical-chemical pretreatment. Appl. Microbiol. Biotechnol. 92 (4), 709–716.

Further reading Konur, O., 2019b. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham.

Chapter 26

The scientometric analysis of the research on the algal biomedicine Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

26.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 papers published since 1980. The algal biomedicine has been among the most-prolific research fronts over time as evidenced with over 30,000 papers published during the same study period, corresponding to the public concerns about the development of sustainable and environment-friendly bioproducts and bioprocesses in medicine. In line with the teachings of North's New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate, the stakeholders should have timely and thorough access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b, 2012a,b). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal biomedicine and the underlying research areas as in fields of the algal research (Konur, 2011a, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a,b,c, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012c,d,e,f,g,h,i,j,k,l, 2018a,b,c), energy and fuels (Konur, 2012m,n,o, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e, 2019d), biomedicine (Konur, 2016i, 2018d), and social sciences (Konur, 2011b, 2012p,q,r,s,t,u,v,x,y,z, 2013a,b,c,d,e,f,g,h,i,j,k,l). Although there have been nearly 800 literature reviews on the algal biomedicine, there has been no published scientometric studies in the journal literature. However, there has been three recent book chapters on the various aspects of the algal biomedicine (Konur, 2017a, 2019a, 2020k). This is contrast to over 2500 published scientometric studies on biomedicine in the journal literature (Garfield, 1984, 1987; Paladugu et al., 2002). Therefore, this paper presents the first-ever scientometric study of the research in algal biomedicine covering the whole range of research fronts as well as whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate.

26.2  Materials and methodology The search for the scientometric analysis of the literature on the algal biomedicine was carried out in January 2019 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI) and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in the Appendix. These keyword sets have been devised in three major parts: the keywords related to biomedicine and keywords related to the algae as well as the cross-subject keywords related to algal biomedicine. There have been three distinct keyword sets for the first part: the set of core subject categories related to the biomedicine, the set of core journal titles related to biomedicine and keywords related to the biomedicine. Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00026-7 © 2020 Elsevier Inc. All rights reserved.

405

406  PART | VII  Algal biomedicine

On the other hand, the second part consists of the keywords related to the algae in general, dinoflagellates and coccolithophores, microalgae, macroalgae, diatoms, cyanobacteria, and journal titles related to the algae. A detailed set of cross-subject keywords related to the both biomedicine and algae has formed the third keyword set. The papers located through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subject to the quality control exercise to ensure that these references have been primarily related to the algal biomedicine. This refined list of papers has formed the core sample for the scientometric and content overview of the literature on the algal biomedicine. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. Additionally, the search has been carried out for the papers with at least 100 citations and the relevant scientometric data has been recovered. These papers have been termed as ‘influential papers’. Furthermore, the data on the scientometric analysis and brief content overview of 20 most-cited papers have also been provided focusing on the determination of the key research fronts for these citation classics, complementing sections on the keywords and research fronts. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

26.3 Results 26.3.1  Documents and indexes The search has resulted in 34,516 papers where there have been 28,895 articles, 3399 meeting abstracts, 817 reviews, 716 notes, 278 letters, 166 corrections, 144 editorial materials, and 67 news items. In the first instance, the papers excluding meeting abstracts, news items, and corrections have been selected resulting in 30,850 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 30,015 papers in English. The other major languages have been Japanese, Russian, French, Chinese, French, and German. This set of 30,015 papers has formed the core sample for the scientometric analysis of the literature on the algal biomedicine. The articles have formed 93.8% of the final sample while reviews, notes, letters, and editorial matters have formed 2.6%, 2.3%, 0.9%, and 0.4% of this sample, respectively. Additionally, 3.2% of these papers have been ‘proceedings papers’ and there have been four ‘retracted papers’. On the other hand, 98.5% of these papers have been indexed by the SCI-E while only 0.3% of the papers have been indexed by the SSCI and A&HCI focusing on the societal and humanitarian aspects of algal biomedicine. Additionally, 1.5% of the papers have been indexed by the ESCI.

26.3.2 Keywords The most-prolific keywords used in algal biomedicine have been determined based on the influential papers to locate the hot topics and the primary research fronts in the algal biomedicine There have been a number of most-prolific keywords for the first set of keywords for the biomedicine: ‘*polysaccharide*, antioxidant*’ at the first instance and ‘pharm*, “*active compound*”, “polyunsaturated fatty acid*”, bioactiv*, “docosahexaenoic acid*”, antimicrobial*, “natural product*”, inflam*, *cancer, astaxanthin*, antibiotic*, immuno*, *proliferative, antibacter*’ as the other prolific keywords. On the other hand, there have been a number of prolific journals related to algal biomedicine: ‘“Marine Drugs”, “Journal of Natural Products”, Biomaterials, “International Journal of Pharmaceutics”, “Journal of Biomedical Materials Research Part A”, Virology, “Journal of Biomaterials Science Polymer Edition”, “Chemical Pharmaceutical Bulletin”, “Acta Biomaterialia”, Biopolymers, “Journal of Materials Science Materials in Medicine”, and “Biomed Research International”. Similarly, the most-prolific subject categories related to the algal biomedicine have been “Pharmacology Pharmacy”, “Engineering Biomedical”, “Materials Science Biomaterials”, “Chemistry Medicinal”, Biophysics, Neurosciences, Immunology, Oncology, Orthopedics, “Medicine Research Experimental”, Virology, “Cell Tissue Engineering”, “Endocrinology Metabolism”, Surgery, Hematology, Pathology, Transplantation, and “Infectious Diseases”’.



Algal biomedicine research Chapter | 26  407

Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, macroalga*, phytoplankton, rhodophyt*, seaweed*, bacillariophycea*, diatom, diatoms, and cyanobacter*’. The other prolific keywords for the algae have been ‘coccolith*, dinophycea*, Alexandrium, chlorophycea*, chlorophyt*, “green alga*”, microalga*, “micro-alga*”, Chlamydomonas, *Chlorella, Dunaliella, Euglena, Scenedesmus, “brown alga*”, phaeophycea*, kelp*, phaeophyt*, “red alga*”, Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, Ulva, bacillariophyt*, “blue green alga*”, “blue-green alga*”, *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, Synechocystis’. On the other hand, the most-prolific cross-subject keywords have been ‘alginate*, agar, fucoidan, “sulfated polysaccharide*”, carrageenan*, agarose’ at the first instance and ‘alginic, channelrhodopsin*, cell, culture’ as the other prolific keywords. Additionally, the key words of ‘tissue, antibiotic*, fucan*, “sulfated galactan*”, “sulphated polysaccharide*”, fucoxanthin*, ulvan*, griffithsin, laminarin, laminaran, fucoidin*, fucosterol, calothrixin*, cryptophycin*, curacin, cyanovirin*, scytonemin’ formed the other research fronts.

26.3.3 Authors There have been 71,563 authors contributing to the research on the algal biomedicine in total. The information on the most-prolific and influential 20 authors is provided in Table 26.1: authors’ names, gender, institutions, countries, primary research fronts, number of papers produced by these authors, the number of influential papers with at least 100 citations received (I-100), and the percentage of the number of influential papers with relative to the number of all the papers published (I-100%). The data on these authors shows that the most-prolific author with the highest citation impact has been ‘Karl Deisseroth’ of the Stanford University, working primarily on the ‘channelrhodopsins’ from microalgae, with 71 papers. His citation impact is highest with 32 influential papers. The other most-prolific authors with the high citation impact have been ‘David J Mooney’, ‘Peter Hegemann’ ‘Gudmund Skjak-Braek’, and ‘Olav Smidsrod’ with 15 or more influential papers each. The United States and Germany have been the most-prolific countries for these authors with eight and three authors, respectively while China, Norway, and South Korea have been the other prolific countries with two authors each. On the other hand, Europe has had five authors as a whole. Similarly, the most-prolific institutions have been ‘Chinese Academy of Sciences’, ‘Norwegian University of Sciences and Technology’, ‘Stanford University’, and ‘University of Hawaii Manoa’ with two authors each. The most-prolific research fronts have been the ‘sulfated polysaccharides’, ‘alginates’, and ‘channelrhodopsins’ with six, five, and five authors, respectively. The other minor research fronts have been ‘cryptophycins’, ‘curacins’, and ‘cyanovirins’. The number of papers published by these authors have ranged from 26 to 137. These most-prolific authors have also contributed to nearly 3.8% and 24.3% of all the papers and influential papers, respectively. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the influential papers with relative to the number of all the papers published for each author in the last column (I-100%). These data shows that ‘David J Mooney’ has been the top influential author with 50.0% ratio. The other most-influential authors have been ‘Karl Deisseroth’, ‘Edward S Boyden’, ‘Smadar Cohen’, and ‘Feng Zhang’ with over 35% ratio each.

26.3.4 Countries Nearly 99.7% of the papers have had country information in their abstract pages and 133 countries and territories have contributed to these papers overall. Table 26.2 provides the information about the most-prolific and influential 20 countries. These 20 most-prolific countries have produced 95.3% and 111.2% of all the papers and influential papers, respectively, as a whole. The most-prolific and influential country has been the US producing 17.5% and 32.2% of all the papers and influential papers, respectively. Additionally, Japan, the United Kingdom, China, and Germany have emerged as the other most-prolific and influential countries following the US producing 16.9%, 5.5%, 13.2%, and 5.4% of all the papers, respectively. These countries have also produced 10.0%, 7.3%, 7.2%, and 6.7% of the influential papers, respectively. The European countries have been dominant in the top-20 country list as they have produced 29.8% and 38.2% of all the papers and influential papers, respectively, as a whole, surpassing significantly the United States, Japan, and China. Similarly, the Asian countries in this top-20 list, have produced 37.8% and 27.5% of all the papers and influential papers, respectively, as a whole.

Authors

Gender

Institution

Country

Research fronts

Algae

I-0

I-100

I-100%

1

Karl Deisseroth

M

Stanford Univ.

United States

Channelrhodopsins

Microalgae

71

32

45.1

2

David J Mooney

M

Univ. Michigan

United States

Alginates

Macroalgae

36

18

50.0

3

Peter Hegemann

M

Humboldt Univ.

Germany

Channelrhodopsins

Microalgae

70

17

24.3

4

Gudmund Skjak-Braek

M

Norw. Univ. Sci. Technol.

Norway

Alginates

Macroalgae

93

16

17.2

5

Olav Smidsrod

M

Norw. Univ. Sci. Technol.

Norway

Alginates

Macroalgae

50

15

30.0

6

Smadar Cohen

M

Ben Gurion Univ.

Israel

Alginates

Macroalgae

35

14

40.0

7

Feng Zhang

M

Stanford Univ.

United States

Channelrhodopsins

Microalgae

36

13

36.1

8

Edward S Boyden

M

Massachusetts Inst. Technol.

United States

Channelrhodopsins

Microalgae

31

13

41.9

9

Richard E Moore

M

Univ. Hawaii Manoa

United States

Cryptophycins

Cyanobacteria

54

12

22.2

10

Michael R Boyd

M

Natl. Cancer Inst.

United States

Cyanovirins

Cyanobacteria

40

12

30.0

11

You-Jin Jeong

M

Cheju Natl. Univ.

S. Korea

Polysaccharides

Macroalgae

137

11

8.0

12

Anatolii I Usov

M

Russian Acad. Sci.

Russia

Polysaccharides

Macroalgae

59

11

18.6

13

Ernst Bamberg

M

Max Planck Inst.

Germany

Channelrhodopsins

Microalgae

31

10

32.3

14

William H Gerwick

M

Univ. Calif. San Diego

United States

Curacins

Cyanobacteria

114

9

7.9

15

Gregory ML Patterson

M

Univ. Hawaii Manoa

United States

Cryptophycins

Cyanobacteria

26

9

34.6

16

Paulo AS Mourao

M

Univ. Fed. Rio de Janeiro

Brazil

Polysaccharides

Macroalgae

53

8

15.1

17

Se-Kwon Kim

M

Pukyong Natl. Univ.

S. Korea

Polysaccharides

Macroalgae

77

7

9.1

18

Quanbin Zhang

M

Chinese Acad. Sci.

China

Polysaccharides

Macroalgae

74

7

9.5

19

Zhien Li

M

Chinese Acad. Sci.

China

Polysaccharides

Macroalgae

27

7

25.9

20

Ulrich Zimmermann

M

Univ. Wurzburg

Germany

Alginates

Macroalgae

34

6

17.6

Average

57.4

12.4

25.8

Total

1148

247

Total %

3.8

24.3

M, male; F, female; I-0, the number of papers for at least 17 papers; I-100, the number of influential papers with at least 100 citations for at least 2 papers; I-100%, the percentage of the number of influential papers with relative to the number of all the papers published.

408  PART | VII  Algal biomedicine

TABLE 26.1  The most-prolific and influential authors in algal biomedicine.

Algal biomedicine research Chapter | 26  409



TABLE 26.2  The most-prolific and influential countries in algal biomedicine. Country

I-0

I-0%

I-100

I-100%

Europe

8952

29.8

388

38.2

Asia

11,338

37.8

280

27.5

1

United States

5242

17.5

329

32.2

2

Japan

2884

6.9

102

10.0

3

United Kingdom

1664

5.5

74

7.3

4

China

3969

13.2

73

7.2

5

Germany

1619

5.4

68

6.7

6

France

1526

5.1

56

5.5

7

South Korea

1854

6.2

52

5.1

8

Canada

938

3.1

49

4.8

9

Spain

1023

3.4

42

4.1

11

India

2092

7.0

39

3.8

10

Norway

460

1.5

35

3.4

12

Israel

376

1.3

31

3.0

13

Australia

732

2.4

30

3.0

14

Italy

995

3.3

27

2.7

15

Switzerland

301

1.0

26

2.6

16

Portugal

453

1.5

25

2.5

17

Brazil

1014

3.4

24

2.4

18

Netherlands

394

1.3

19

1.9

19

Sweden

517

1.7

16

1.6

20

Taiwan

539

1.8

14

1.4

Total

28,592

95.3

1131

111.2

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Europe and Asia values are for only the top-20 countries.

26.3.5 Institutions Over 99.6% of the papers have had their institutions listed in their abstract pages. For these papers, 10,367 institutions have contributed to the research on the algal biomedicine in total. The information about the 20 most-prolific and influential institutions is given in Table 26.3. The most-prolific and influential institution has been the ‘Stanford University’ publishing 0.4% and 3.4% of the all and influential papers, respectively. ‘Centre National de la Recherche Scientifique’—CNRS, ‘Howard Hughes Medical Institute’, ‘Massachusetts Institute of Technology’, and ‘Chinese Academy of Sciences’ have been the other influential institutions as they have produced 2.8%, 2.8%, 2.5%, and 2.4% of the influential papers, respectively. The most-prolific countries for these institutions have been the United States and Germany with eight and three institutions, respectively. On the other hand, Europe has had seven institutions as a whole where these European institutions produced 7.0% and 10.5% of the all the papers and influential papers, respectively. Similarly, the Asian institutions have produced 4.0% and 4.7% the all the papers and influential papers, respectively while the US institutions have produced 3.1% and 17.5% of all the papers and influential papers, respectively. The contribution of these institutions has ranged from 0.2% to 2.4% for all the papers and from 1.1% to 3.4% for the influential papers. Overall, these 20 institutions have contributed to 14.5% and 37.7% of all the papers and influential ­papers, respectively.

410  PART | VII  Algal biomedicine

TABLE 26.3  The most-prolific and influential institutions in algal biomedicine. Institutions

Country

I-0

I-0%

I-00

I-100% papers

United States

924

3.1

178

17.5

Europe

2093

7.0

107

10.5

Asia

1191

4.0

48

4.7

1

Stanford Univ.

United States

104

0.4

35

3.4

2

Ctr. Natl. Rech. Sci.—CNRS

France

722

2.4

28

2.8

3

Howard Hughes Med. Inst.

United States

69

0.2

28

2.8

4

Massachusetts Inst. Technol.

United States

112

0.4

25

2.5

5

Chin. Acad. Sci.

China

723

2.4

24

2.4

6

Harvard Univ.

United States

156

0.5

24

2.4

7

Norw. Univ. Sci. Technol.

Norway

210

0.7

22

2.2

8

Natl. Cancer Inst.—NCI

United States

129

0.4

20

2.0

9

Max Planck Soc.

Germany

142

0.5

19

1.9

10

Ben Gurion Univ.

Israel

139

0.5

19

1.9

11

Univ. Michigan

United States

119

0.4

19

1.9

12

Cons. Super. Invest. Cient.—CSIC

Spain

316

1.1

18

1.8

13

Univ. Calif. San Diego

United States

170

0.6

15

1.5

14

Humboldt Univ.

Germany

91

0.3

14

1.4

15

Russian Acad. Sci.

Russia

406

1.4

13

1.3

16

Univ. Porto

Portugal

142

0.5

13

1.3

17

Hokkaido Univ.

Japan

242

0.8

12

1.2

18

Jeju Natl. Univ.

S. Korea

226

0.8

12

1.2

19

Univ. Washington

United States

65

0.2

12

1.2

20

Univ. Wurzburg

Germany

64

0.2

11

1.1

4347

14.5

383

37.7

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Europe and Asia values are for only the top-20 institutions.

26.3.6  Research funding bodies Only 46.4% of these papers have had declared any research funding in their abstract pages and overall, 14,410 funding bodies have funded these papers. The most-prolific funding bodies have been the ‘National Natural Science Foundation of China’, ‘National Institute of General Medical Sciences’ of the United States, and ‘National Cancer Institute’ of the US funding 4.7%, 0.9%, and 0.9% of the papers, respectively. The other prolific funding bodies have been the ‘National Council for Scientific and Technological Development’— (CNPQ) of Brazil, ‘Fundamental Research Funds for the Central Universities’ of China, ‘National Science Foundation’ of the United States, and ‘Natural Science Foundation of China’ with at least 0.5% of the papers each.

26.3.7  Publication years Fig. 26.1 shows the number of papers on the algal biomedicine, published between 1980 and 2018 as of January 2019.



Algal biomedicine research Chapter | 26  411

FIG. 26.1  The number of publications in the algal biomedicine between 1980 and 2018.

The data in this figure shows that the number of papers has risen from 272 papers in 1980 to 2386 papers in 2018. The most prolific decade has been the 2010s with 51.1% of the papers. Additionally, 10.6%, 15.0%, and 23.3% of the papers have been published in the 1980s, 1990s, and 2000s, respectively. Thus, the Figure shows that there has been a steadily increasing trend between 1980 and January 2019.

26.3.8  Source titles Overall, these papers have been published in 3530 journals. Table 26.4 provides the information on the 20 most-prolific and influential journals. These 20 journals have published 17.0% and 36.4% of all the papers and influential papers, respectively, in total. The most-prolific and influential journals have been ‘Biomaterials’ and ‘Carbohydrate Polymers’ publishing 6.7% and 3.5% of the influential papers, respectively and 0.7% and 3.4% of all the papers, respectively. ‘Carbohydrate Research’, ‘International Journal of Pharmaceutics’, ‘Journal of Controlled Release’, ‘Journal of Applied Phycology’, and ‘Journal of Biological Chemistry’ have followed these top journals as the other most prolific and influential journals with at least 1.8% of the influential papers each. The most-prolific subject categories for these journals have been ‘Chemistry Applied’ and ‘Biochemistry and Molecular Biology’ with five journals each, followed by ‘Food Science and Technology’, ‘Polymer Science’, and ‘Pharmacology Pharmacy’ with three journals each. The other prolific subjects have been ‘Biotechnology and Applied Microbiology’, ‘Chemistry Organic’, ‘Engineering Biomedical’, ‘Multidisciplinary Sciences’ and ‘Materials Science Biomaterials’ with two journals each.

26.3.9  Subject categories These papers have been indexed by 170 subject categories. The information about the 10 most-prolific and influential subject categories are given in Table 26.5. As expected, the most-prolific and influential subject categories have been ‘Biochemistry & Molecular Biology’, ‘Pharmacology Pharmacy’, and ‘Chemistry Applied’ indexing 14.3%, 11.6%, and 11.0% of all the papers and 18.7%, 12.1%, and 12.1% of the influential papers, respectively. The other prolific and influential subjects have been ‘Biotechnology Applied Microbiology’, ‘Engineering Biomedical’, ‘Materials Science Biomaterials’, ‘Polymer Science’, and ‘Chemistry Organic’ with at least 9.2% of the influential papers each. Thus, the first-five categories have been the key pillars of the research in algal biomedicine, indexing together 64.6% of the influential papers.

Journals

Abbr.

Subject

I-0

I-0%

I-100

I-100%

Biomedical Sci.

1187

4.0

155

15.2

1

Biomaterials

Biomaterials

Eng. Biomed., Mats. Sci. Biomats.

204

0.7

68

6.7

2

Carbohydrate Polymers

Carbohyd. Polym.

Chen. Appl., Chem. Org., Polym. Sci.

1011

3.4

36

3.5

3

Carbohydrate Research

Carbohyd. Res.

Bioch. Mol. Biol., Chem. Appl., Chem. Org.

280

0.9

22

2.2

4

International Journal of Pharmaceutics

Int. J. Pharmaceut.

Pharm. Pharm.

180

0.6

21

2.0

5

Journal of Controlled Release

J. Control. Release

Chem. Mult., Pharm. Pharm.

74

0.2

19

1.9

6

Journal of Applied Phycology

J. Appl. Phycol.

Biot. Appl. Microb., Mar. Fresh. Biol.

526

1.8

18

1.8

7

Journal of Biological Chemistry

J. Biol. Chem.

Bioch. Mol. Biol.

92

0.3

18

1.8

8

Journal of Agricultural and Food Chemistry

J. Agr. Food Chem.

Agr. Mult., Chem. Appl., Food sci. Technol.

201

0.7

15

1.5

9

Analytical Biochemistry

Anal. Biochem.

Bioch. Res. Meth., Bioch. Mol. Biol., Chem. Anal.

199

0.7

15

1.5

10

Biomacromolecules

Biomacromolecules

Bioch. Mol. Biol., Chem. Org., Polym. Sci.

186

0.6

15

1.5

11

International Journal of Biological Macromolecules

Int. J. Biol. Macromol.

Bioch. Mol. Biol., Chem. Appl., Polym. Sci.

603

2.0

14

1.4

12

Biotechnology and Bioengineering

Biotechnol. Bioeng.

Biot. Appl. Microb.

155

0.5

14

1.4

13

Toxicon

Toxicon

Pharm. Pharm., Toxic.

137

0.5

14

1.4

14

Food Chemistry

Food Chem.

Chem. Appl., Food Sci. Technol., Nutr. Diet.

211

0.7

13

1.3

15

Nature

Nature

Mult. Sc.

20

0.1

13

1.3

16

Proceedings of the National Academy of Sciences of the United States of America

P. Natl. Acad. Sci. USA

Mult. Sci.

41

0.1

12

1.2

17

Journal of Natural Products

J. Nat. Prod.

Plant Sci., Chem. Med., Pharm. Pharm.

416

1.4

11

1.1

18

Journal of Biomedical Materials Research

J. Biomed. Mater. Res.

Eng. Biomed., Mats. Sci. Biomats.

162

0.5

11

1.1

19

Nature Neuroscience

Nat. Neurosci.

Neurosci.

14

0.1

11

1.1

20

Food Hydrocolloids

Food Hydrocolloid.

Chem. Appl., Food Sci. Technol.

394

1.3

10

1.0

5106

17.0

370

36.4

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Values for biomedical sciences are only for the top-20 journals.

412  PART | VII  Algal biomedicine

TABLE 26.4  The most-prolific and influential journals in algal biomedicine.

Algal biomedicine research Chapter | 26  413



TABLE 26.5  The most-prolific and influential subject categories in algal biomedicine. Subject categories

I-0 no. papers

I-0% papers

I-100 no. papers

I-100% papers

Biomed. Sci.

6195

20.6

333

32.7

1

Biochemistry Molecular Biology

4298

14.3

190

18.7

2

Pharmacology Pharmacy

3480

11.6

127

12.1

3

Chemistry Applied

3315

11.0

123

12.1

4

Biotechnology Applied Microbiology

3694

12.3

116

11.4

5

Engineering Biomedical

1194

4.0

105

10.3

6

Materials Science Biomaterials

1521

5.1

101

9.9

7

Polymer Science

3184

10.6

100

9.8

8

Chemistry Organic

1973

6.6

94

9.2

9

Food Science Technology

2805

9.3

92

9.0

10

Plant Sciences

1748

5.8

49

4.8

Total

27,212

90.7

1097

107.9

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Values for biomedical sciences are only for the top-10 subject categories.

26.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers with at least 100 citations, 3.1% of the research sample of 30,015 papers, Table 26.6. The records in this dataset has been refined from 1017 papers to 923 papers to focus on the core papers for the field of algal biomedicine. The data shows that the field of ‘alginates’ has been the most prolific research front with 372 papers, forming 40.3% of these influential papers. The other key research fronts have been ‘sulfated polysaccharides in general’, ‘agaroses’, ‘cyanobacterial biomedicine’, ‘macroalgal biomedicine’, ‘microalgal biomedicine’, and ‘channelrhodopsins’ with 10.7%, 10.6%, 8.7%, 8.0%, 6.9%, and 6.3% of the influential papers, respectively. The most-studied types of algae have been ‘macroalgae’ with 76.2% of the influential papers. Additionally, the papers on the ‘microalgae’ and ‘cyanobacteria’ have formed 13.2% and 8.7% of these papers, respectively.

26.3.11  Citation classics This section provides the information on both the scientometric analysis and brief content overview of the most-cited 20 papers in algal biomedicine. The information on these papers is given in Table 26.7: authors' names, publication years, document type, number of authors per paper, lead authors' names, gender, and paper numbers (lead authors were determined based on the number of papers produced in this field with at least 17 papers in general and 5 influential papers), journals, subject areas, research fronts, types of algae, academic focus of the papers, number of citations received, and the number of citations per year.

26.3.11.1  Scientometric overview of the citation classics These papers have been published between 1982 and 2012. The most-prolific decade has been the 2000s with 11 papers. Additionally, there have been three, four, and two papers published in the 1980s, 1990s, and 2010s, respectively. The reviews have been over-represented in these classical papers as there have been 13 articles and 7 reviews. The number of the authors of these papers has ranged from 2 to 9 while the mean number of authors has been 3.8. The most-prolific and influential lead authors have been ‘Karl Deisseroth’ and ‘David J Mooney’ with three citation classics each working primarily on the channelrhodopsins and alginates, respectively. The other prolific lead authors have

414  PART | VII  Algal biomedicine

TABLE 26.6  The most-prolific research fronts in algal biomedicine. Research fronts

Algae

Microalgae

Cyanobacteria

Diatoms

Dinoflagellates

Macroalgae

Total

1

General

12

64

80

4

2

74

236 (25.6%)

2

Channelrhodopsins

3

Alginates

372

372 (40.3%)

4

Agaroses

98

98 (10.6%)

5

Carrageenans

40

40 (4.3%)

6

Sulfated polysaccharides

99

99 (10.7%)

7

Agars

20

20 (2.2%)

Total

58

12 (1.2%)

122 (13.2%)

58 (6.3%)

80 (8.7%)

4 (0.4%)

2 (0.2%)

703 (76.2%)

Numbers, the number of influential papers for each research front and type of algae; %, the percentage of influential papers for each research front and type of algae; The number in bold, the most-prolific research fronts with at least 50 influential papers; Dinoflagellates, it includes coccolithophores.

TABLE 26.7  The citation classics in algal biomedicine. Subject area

Res. fronts

Algae

Topic

Cits.

Av. cits.

Nat. Neurosci.

Neurosci.

Channelrhodopsins

Microalgae

Optical control of neural activity

2020

155

2

Nucleic Acids Res.

Bioch. Mol. Biol.

Agaroses

Macroalgae

DNA transfer

1762

53

A

2

Cell

Bioch. Mol. Biol., Cell Biol.

Agaroses

Macroalgae

Cultures of chondrocytes

1755

49

2012

R

2

Prog. Polym. Sci.

Polym. Sci.

Alginates

Macroalgae

Applications

1602

267

Wiegand et al.

2008

A

3

Nat. Protoc.

Bicoh. Res. Meth.

Agars

Macroalgae

Microbial substance concentration

1328

133

6

Rowley et al.

1999

A

3

DJ Mooney-18

Biomaterials

Eng. Biomed., Mats. Sci. Biomats.

Alginates

Macroalgae

Hydrogels

1259

66

7

Nagel et al.

2003

A

9

P Hegemann-17; E Bamberg-10

P. Natl. Acad. Sci. USA

Mult. Sci.

Channelrhodopsins

Microalgae

Channelrhodopsin-2

1240

83

8

Smidsrod and SkjakBraek

1990

R

2

O Smidsrod-15; G Skjak-Braek-16

Trends Biotechnol.

Biot. Appl. Microb.

Alginates

Macroalgae

Cell immobilization

1125

40

9

George and Abraham

2006

R

2

J. Control. Release

Chem. Mult., Pharm. Pharm.

Alginates

Macroalgae

Intestinal drug delivery

952

94

10

Dretzen et al.

1991

A

4

Anal. Biochem.

Biochem. Res. Meth.; Bioch. Mol. Biol.; Chem. Anal.

Agaroses

Macroalgae

DNA transfer

883

24

11

Gombotz and Wee

1998

R

2

Adv. Drug Deliver. Rev.

Pharm. Harm.

Alginates

Macroalgae

Drug delivery

839

42

Authors

Year

Doc.

N auths.

1

Boyden et al.

2005

A

5

2

Reed and Mann

1985

A

3

Benya and Shaffer

1982

4

Lee and Mooney

5

Lead authors

Journal

ES Boyden-13; F Zhang-13; E Bamberg-10; Deisseroth-32

KY Lee-6; DJ Mooney-18

Continued

2004

2000

Kuo and Ma

Guerin et al.

Adamantidis et al.

Mauck et al.

Tonnesen and Karlsen

Martinsen et al.

Chen et al.

Average

14

15

16

17

18

19

20

A

A

R

A

A

R

A

A

R

Doc.

3.8

6

3

2

8

5

3

2

7

3

N auths.

O Smidsrod-15; G Skjak-Braek-16

F Zhang-13; K Deisseroth-32

K Deisseroth-32

DJ Mooney-18

Lead authors

J. Control. Release

Biotechnol. Bioeng.

Drug Dev. Ind. Pharm.

J. Biomech. Eng.-T. ASME

Nature

Trends Biotechnol.

Biomaterials

Nature

Macromol. Biosci.

Journal

Chem. Mult., Pharm. Pharm.

Biot. Appl. Microb.

Chem. Med., Pharm. Pharm.

Biophys., Eng. Biomed.

Mult. Sci.

Biot. Appl. Microb.

Eng. Biomed., Mats. Sci. Biomats.

Mult. Sci.

Bioch. Mol. Biol., Mats. Sci. Biomats., Polym. Sci.

Subject area

Alginates

Alginates

Alginates

Agaroses

Channelrhodopsins

Astaxanthin

Alginates

Channelrhodopsins

Alginates

Res. fronts

Macroalgae

Macroalgae

Macroalgae

Macroalgae

Microalgae

Microalgae

Macroalgae

Microalgae

Macroalgae

Algae

Drug delivery

Immobilization

Drug delivery

Tissue engineering

Hypocretin neurons

Applications

10,30

552

569

597

612

620

648

674

772

Basal ganglia circuitry Tissue engineering

793

Cits.

Hydrogels

Topic

71.4

34

20

32

34

56

43

40

97

66

Av. cits.

Doc., document; A, article; R, review; gender, gender of lead authors—female authors in italic; N paper, for the authors with at least 17 papers with 0 citations and with at least 2 influential papers—number after the author names; Subject, web of science subjects; Topic, primary topic of the papers; Algae, type of algae studied; Res. fronts, primary research fronts studied; Cits., number of citations received in total; Av. cits., number of citations per year.

1989

2002

2008

2007

2003

2001

2010

Kravitz et al.

13

2006

Augst et al.

Year

12

Authors

TABLE 26.7  The citation classics in algal biomedicine—cont’d



Algal biomedicine research Chapter | 26  417

been ‘Feng Zhang’, ‘Ernst Bamberg’, ‘Olav Smidsrod’, and ‘Gudmund Skjak-Braek’ with two papers each. The other lead authors have been ‘Edward S Boyden’, ‘Kuen Yong Lee’, and ‘Peter Hegemann’ with one paper each. In total, nine lead authors have contributed to these citation classics and there has been a significant gender deficit among the lead authors of these classical papers as all these authors are male. In total, these citation classics have been published by only 16 journals. The most-prolific journals have been ‘Biomaterials’, ‘Journal of Controlled Release’, ‘Nature’, and ‘Trends in Biotechnology’ with two papers each. In total, these papers have been indexed by 14 subject categories. The most-prolific category has been ‘Biochemistry and Molecular Biology’ with four papers, closely followed by ‘Biotechnology Applied Microbiology’, ‘Engineering Biomedical’, ‘Materials Science Biomaterials’, ‘Multidisciplinary Sciences’, ‘Nature’, ‘Pharmacology Pharmacy’, ‘Polymer Science’, with three papers each. The fields of ‘Neuroscience’ and ‘Chemistry Multidisciplinary’ have followed these top subjects with two papers each. In total, there have been four primary research fronts. The most-prolific research front has been ‘alginates’ with 10 papers. The other prolific research fronts have been ‘agaroses’ and ‘channelrhodopsins’ with four papers each. There have been also one paper each for ‘agars’ and ‘astaxanthin’. There have been two types of algae covered by these classical papers. The most prolific type of algae has been ‘macroalgae’ with 15 papers. The other prolific type of algae has been ‘microalgae’ with five papers. There have been no papers related to ‘diatoms’, ‘cyanobacteria’, ‘dinoflagellates and coccolithophores’, and ‘algae in general’. The most-studied topic has been the ‘drug delivery’ by alginates with four papers. The other prolific topics have been ‘DNA transfer’ by agaroses, ‘applications’ of alginates, alginate ‘hydrogels’, and ‘immobilization’ by alginates with two papers each. These papers have received between 552 and 2020 citations each, with a mean value of 1030 citations per paper. On the other hand, the number of citations per year has ranged from 20 to 267 with a mean value of 71.4 citations per year. The paper by Lee and Mooney (2012) on the alginates with 1602 total citations and 267 citations per year has been the mostcited paper.

26.3.11.2  Brief overview of the content of the citation classics There have been four major classes of papers: ‘alginates’, ‘agaroses’, ‘channelrhodopsins’, and ‘other research fronts’ with 10, 4, 4, and 2 papers, respectively. Alginates Lee and Mooney (2012) review the properties and biomedical applications of alginates in a review paper with 1602 citations. They provide a comprehensive overview of general properties of alginate and its hydrogels, their biomedical applications, and propose new perspectives for future studies with these biopolymers. Rowley et al. (1999) study the modification of alginate hydrogels in a paper with 1259 citations. They covalently modify alginates with ‘Arg-Gly-Asp’ (RGD)-containing cell adhesion ligands and culture mouse skeletal myoblasts on alginate hydrogel surfaces coupled with ‘Gly-Arg-Gly-Asp-Tyr’ (GRGDY) peptides. They find that myoblasts adhere to GRGDYmodified alginate surfaces, proliferate, fuse into multinucleated myofibrils, and express heavy-chain myosin which is a differentiation marker for skeletal muscle. Smidsrod and Skjak-Braek (1990) review the Ca2+ alginates as immobilization matrix for living cells in a review paper with 1125 citations. They evaluate the potential of this method on the basis of the structural and functional relationships in alginate gels. George and Abraham (2006) review the alginates and chitosan for the intestinal delivery of protein drugs in a review paper with 952 citations. The incorporation of protein into these two matrices can be done under relatively mild environment with minimum protein denaturation. Alginate is a good mucoadhesive agent. The pore size of alginate gel microbeads is between 5 and 200 nm and coated beads and microspheres are better oral delivery vehicles. Cross-linked alginate has more capacity to retain the entrapped drugs and mixing of alginate with other polymers solve the problem of drug leaching. Gombotz and Wee (1998) review the controlled release of proteins from alginate matrices in a review paper with 839 citations. They focus on the chemistry of alginate, its gelation mechanisms, and the physical properties of alginate gels where biomolecules have been incorporated into and released from alginate systems. Augst et al. (2006) review applications of the alginate hydrogels in a review paper with 793 citations. They note that these hydrogels have been used as scaffolds for tissue engineering, as delivery vehicles for drugs, and as model extracellular matrices for basic biological studies. These applications require tight control of mechanical stiffness, swelling, degradation, cell attachment, and binding or release of bioactive molecules. They explain that the control over these properties can be achieved by chemical or physical modifications of the polysaccharide itself or the gels formed from alginate.

418  PART | VII  Algal biomedicine

Kuo and Ma (2001) study the ionically cross-linked alginate hydrogels as scaffolds for tissue engineering focusing on the structure, gelation rate, and mechanical properties in a paper with 674 citations. They control gelation rate and formulate homogeneous alginate gels as scaffolds with defined dimensions for tissue engineering applications. They find that the mechanical properties of the alginate gels were controlled by the compositional variables. Their results show how alginate gel and gel/cell systems could be formulated with controlled structure, gelation rate, and mechanical properties for tissue engineering and other biomedical applications. Tonnesen and Karlsen (2002) review the drug delivery systems by alginates in a review paper with 597 citations. They discuss the present use and future possibilities of alginate as a tool in drug formulation. They note that the ability of alginate to form two types of gel dependent on pH (an acid gel and an ionotropic gel) gives this biopolymer unique properties compared to neutral macromolecules. They assert that alginates would make an important contribution in the development of polymeric delivery systems. Martinsen et al. (1989) study the alginates as immobilization material focusing on the chemical and physical properties of alginate gel beads in a paper with 569 citations. They prepare calcium alginate gel beads and find that the physical properties of beads depended strongly on the composition, sequential structure, and molecular size of the polymers. They further find that beads with the highest mechanical strength, lowest shrinkage, best stability toward monovalent cations, and highest porosity were made from alginate with a content of l-guluronic acid higher than 70% and an average length of the G-blocks higher than 15. Chen et al. (2004) study the development of a novel pH-sensitive hydrogel composed of ‘N,O-carboxymethyl chitosan’ (NOCC) and alginate cross-linked by genipin for protein drug delivery in the intestine in a paper with 552 citations. They focus on the swelling characteristics of these hydrogels as a function of pH values. Additionally, they study release profiles of a model protein drug from these hydrogels in simulated gastric and intestinal media. Their results show that the genipin-crosslinked NOCC/alginate hydrogel could be a suitable polymeric carrier for site-specific protein drug delivery in the intestine. Agaroses Reed and Mann (1985) study the capillary DNA transfer from agarose gels to nylon membranes in a paper with 1762 citations. They find that the alkaline solvent induces covalent fixation of DNA to the membrane. They observe saving in time and materials, an improved resolution, and substantial increase in sensitivity of subsequent hybridization analyses. Benya and Shaffer (1982) study the evolution of the differentiated collagen phenotype in the dedifferentiated chondrocytes when cultured in agarose gels in a paper with 1755 citations. They show a complete return to the differentiated collagen phenotype. Their results emphasize the primary role of cell shape in the modulation of the chondrocyte phenotype and show a reversible system for the study of gene expression. Dretzen et  al. (1981) develop a method for the recovery of biologically active DNA fragments from both agarose and acrylamide gels in a paper with 883 citations. The procedure involves electrophoresis of the fragments onto strips of diethylaminoethyl-cellulose paper inserted in the gel between bands. The electrophoretic transfer is achieved in the original resolving gel, thereby enabling purification of DNA fragments with a yield of 60–80%. Mauck et  al. (2000) study the production of functional engineered tissue construct through dynamic deformational loading of chondrocyte-seeded agarose scaffolds at physiological strain levels in a paper with 612 citations. They find that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading. They further find that sulfated glycosaminoglycan content was greater in dynamically loaded disks compared to free swelling controls at day 21. Channelrhodopsins Boyden et al. (2005) study the genetically targeted optical control of neural activity in a paper with 2020 citations. They adapt Channelrhodopsin-2 (ChR2), a rapidly gated light-sensitive cation channel, by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons. They show reliable, millisecond-­timescale control of neuronal spiking, as well as control of excitatory and inhibitory synaptic transmission. Nagel et al. (2003) study Channelrhodopsin-2 in a paper with 1240 citations. They show that ChR2 is a directly lightswitched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. Kravitz et  al. (2010) study the regulation of Parkinsonian motor behaviors by optogenetic control of basal ganglia circuitry in a paper with 772 citations. They directly activate basal ganglia circuitry in vivo, using optogenetic control of



Algal biomedicine research Chapter | 26  419

direct- and indirect-pathway medium spiny projection neurons (MSNs), achieved through Cre-dependent viral expression of Channelrhodopsin-2. They find that bilateral excitation of indirect-pathway MSNs elicited a Parkinsonian state, distinguished by increased freezing, bradykinesia, and decreased locomotor initiations. In contrast, activation of direct-pathway MSNs reduced freezing and increased locomotion. Their findings establish a critical role for basal ganglia circuitry in the bidirectional regulation of motor behavior and show that modulation of direct pathway circuitry may represent an effective therapeutic strategy for ameliorating Parkinsonian motor deficits. Adamantidis et al. (2007) study the neural substrates of awakening probed with optogenetic control of hypocretin (Hcrt) neurons in a paper with 620 citations. They directly probe the impact of Hcrt neuron activity on sleep state transitions with in vivo neural photostimulation, genetically targeting Channelrhodopsin-2 to Hcrt cells and using an optical fiber to deliver light deep in the brain, directly into the lateral hypothalamus, of freely moving mice. They find that direct, selective, photostimulation of Hcrt neurons increased the probability of transition to wakefulness from either slow wave sleep or rapid eye movement sleep. Other research fronts Wiegand et al. (2008) study both the agar and broth dilution methods to determine the ‘minimal inhibitory concentration’ (MIC) of antimicrobial substances, inhibiting the growth the aerobic bacteria in a paper with 1328 citations. The agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. This protocol can be completed in 3 days. Guerin et al. (2003) review the applications of astaxanthin from Haematococcus pluvialis for human health and nutrition in a paper with 648 citations. As the astaxanthin is a strong coloring agent and a potent antioxidant, they focus on the antioxidant, UV-light protection, anti-inflammatory and other biomedical properties of astaxanthin. They assert that protecting body tissues from oxidative damage with daily ingestion of natural astaxanthin might be a practical and beneficial strategy in health management.

26.4 Discussion As there have been over 30,000 core papers related to the algal biomedicine, comprising more than 20% of the papers on the algae as a whole, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts (Konur, 2011a, 2012c,d,e,f,g,h,i,j,k,l, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a,b,c,d,e,f,g,h,i, 2017a,b,c,d,e, 2018a,b,c, 2019a,b,c,d, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). The primary mode of scientific communication has been articles while reviews have formed 2.6% of the sample. The primary index has been SCI-E indexing more than 98.5% of the papers while only 0.3% of the papers have been indexed by the SSCI and A&HCI focusing on the societal and humanitarian aspects of algal biomedicine. These findings suggest that there is substantial room for the research in these aspects such as policy-related studies as well as scientometric and consumer studies in this field. The most-prolific keywords related to the algal biomedicine have been determined through the detailed examination of the 923 influential papers with at least 100 citations. A detailed keyword set has been devised for the search (given in the Appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the biomedicine have been ‘*polysaccharide*, antioxidant*’ at the first instance and ‘pharm*, “*active compound*”, “polyunsaturated fatty acid*”, bioactiv*, “docosahexaenoic acid*”, antimicrobial*, “natural product*”, inflam*, *cancer, astaxanthin*, antibiotic*, immuno*, *proliferative, antibacter*’ as the other prolific keywords. On the other hand, there have been a number of prolific journals related to algal biomedicine: ‘“Marine Drugs”, “Journal of Natural Products”, Biomaterials, “International Journal of Pharmaceutics”, “Journal of Biomedical Materials Research Part A”, Virology, “Journal of Biomaterials Science Polymer Edition”, “Chemical Pharmaceutical Bulletin”, “Acta Biomaterialia”, Biopolymers, “Journal of Materials Science Materials in Medicine”, and “Biomed Research International”. Similarly, the most-prolific subject categories related to the algal biomedicine have been “Pharmacology Pharmacy”, “Engineering Biomedical”, “Materials Science Biomaterials”, “Chemistry Medicinal”, “Biophysics”, Neurosciences, Immunology, Oncology, Orthopedics, “Medicine Research Experimental”, Virology, “Cell Tissue Engineering”, “Endocrinology Metabolism”, Surgery, Hematology, Pathology, Transplantation, and “Infectious Diseases”’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, macroalga*, rhodophyt*, seaweed*, bacillariophycea*, diatom, diatoms, cyanobacter*’. These keywords have formed the primary research fronts for the algal biomedicine.

420  PART | VII  Algal biomedicine

On the other hand, the most-prolific cross-subject keywords have been ‘alginate*, agar, fucoidan, “sulfated polysaccharide*”, carrageenan*, agarose’ at the first instance and ‘alginic, channelrhodopsin*, cell, culture’ as the other prolific keywords. Additionally the key words of ‘tissue, antibiotic*, fucan*, “sulfated galactan*”, “sulphated polysaccharide*”, fucoxanthin*, ulvan*, griffithsin, laminarin, laminaran, fucoidin*, fucosterol, calothrixin*, cryptophycin*, curacin, cyanovirin*, scytonemin’ formed the other research fronts. The findings show that although over 71,500 authors have contributed to the research, 20 most-prolific and influential authors have shaped the literature on the algal biomedicine publishing 3.8% and 24.3% of all the papers and the influential papers, respectively (Table 26.1). The success of these authors, their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988). The data provides the evidence for the presence of the significant gender deficit among both the lead authors (Table 26.1) and the lead authors of the citation classics as all these top authors are male (Table 26.7; Bordons et al., 2003). The data on the papers by the most-prolific and influential authors highlight the primary research fronts as the ‘sulfated polysaccharides’, ‘alginates’, and ‘channelrhodopsins’. The other minor research fronts have been ‘cryptophycins’, ‘curacins’, and ‘cyanovirins’. The data in Table 26.1 provides information on the most-prolific and influential authors, institutions, countries, journals, topics, and their citation impact in terms of the I-100 and I-100% by these authors. It has been found during the search process that the author names with two or more forenames or surnames have been spelt differently by the databases with significant implications for the recovery of their papers. For example, there have been 63 and 30 papers for ‘Skjak-Braek G’ and ‘SkjakBraek G’, respectively. Similar difficulties have also been observed for the common names such as ‘Wang Y’ or ‘Li Y’ for the recovery of their papers and the related analysis. The data shows that although over 130 countries and territories have contributed to the research in algal biomedicine, most-prolific 20 countries contributed to 95.3% and 111.2% of all the papers and the influential papers, respectively (Table 26.2). The major producers of the research have been the United States, Japan, China, and Europe as these countries have had the ‘first-mover advantage’ over the other countries. It is notable that the citation impact of China has been relatively small in relation to other top producers as China has produced 13.2% and 7.2% of all the papers and influential papers, respectively (Guan and Ma, 2007). As in the case of countries, although over 10,300 institutions have contributed to the research in algal biomedicine, the 20 most-prolific institutions mainly from the United States and Europe, having the first-mover advantages, have published more than 14.5% of all the papers and 37.7% of the influential papers, respectively (Table 26.3). As only 46.4% of the papers have declared a research funding, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). It is notable that there has been significant research funding opportunities in China in relation to the United States and Europe. Thus, there is ample room to extend the research funding opportunities to support the research and development in algal biomedicine. The steady rise in the number of the publications in this field in the 2000s and 2010s (as of January 2019) provides the strong evidence for the increasing public importance of the algal biomedicine in recent years (Fig. 26.1). The annual number of publications have risen to nearly 2400 papers and it is expected that the number of papers would continue to rise in the next decade with at least another 30,000 papers, provided that the opportunities for research funding would increase significantly, corresponding to the increasing importance of the algal biomedicine to the global society at large. Although nearly 3300 journals have contributed to the research in algal biomedicine, the 20 most-prolific journals, having the first-mover advantages, have published over 17.0% and 36.4% of all the papers and influential papers, respectively (Table 26.4). This finding has been most relevant for ‘Biomaterials’ and ‘Carbohydrate Polymers’ publishing 6.7% and 3.5% of the influential papers, respectively. The data on the Web of Science subject categories suggests that the first-five categories have been the key pillars of the research in algal biomedicine, indexing together 64.6% of the influential papers, forming the scientific basis of the research in this field: ‘Biochemistry & Molecular Biology’, ‘Pharmacology Pharmacy’, ‘Chemistry Applied’, ‘Biotechnology Applied Microbiology’, and ‘Engineering Biomedical’ (Table 26.5). As the journals related to algae and biomedicine have published only a small part of both all the papers and influential papers, the broad search strategy, covering all subject categories and journals, developed for this study, has been justified. For example, all the subject categories and journals related to the biomedicine presented in the search set have covered only 17.5% of the papers. Similarly, all the journals on the algae have produced only 2.4% of the papers. The data on the research fronts have confirmed that the major research fronts have been ‘alginates’, ‘sulfated polysaccharides in general’, ‘agaroses’, ‘cyanobacterial biomedicine’, ‘macroalgal biomedicine’, ‘microalgal biomedicine’, and ‘channelrhodopsins’ (Table 26.6).



Algal biomedicine research Chapter | 26  421

The most-studied the types of algae has been ‘macroalgae’ with 76.2% of the papers. The other prolific types of algae have been ‘microalgae’ and ‘cyanobacteria’. The extensive data on the 20 citation classics largely confirm the findings of the earlier sections based on over 30,000 papers (Table 26.7). There has been significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely over-represented in the citation classic sample as there have been seven reviews. Similarly, the most-prolific research fronts have been ‘alginates’, ‘agaroses’ and ‘channelrhodopsins’ with 10, 4, and 4 papers, respectively. The most prolific types of algae have been ‘macroalgae’ and ‘microalgae’ with 15 and 5 papers, respectively. The moststudied topics have been ‘drug delivery’ by alginates, ‘DNA transfer’ by agaroses, ‘applications’ of alginates, ‘alginate hydrogels’, and ‘immobilization’ by alginates. It appears that the structure-processing-property relationships form the basis of the research in algal biomedicine as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

26.5 Conclusion This analytical study of the research in algal biomedicine at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as bioenergy and biofuels (Konur, 2012c,d,e,f,g,h,i,j,k,l, 2018a,b,c), energy and fuels (Konur, 2012m,n,o, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e, 2019d), biomedicine (Konur, 2016i, 2018d), complementing nearly 800 literature reviews. The data has shown that the annual number of papers in this field has risen to nearly 2400 papers while there have been over 30,000 papers over the study period from 1980 to 2018. It is further expected that the size of the research output would continue to increase in the incoming years and decades, with at least another 20,000 papers in the next decade, corresponding to the increasing public importance of the algal biomedicine to the global society at large. The provision of the adequate research funding is essential for the further development of the research and practice in this field as only 46.4% of the papers have declared a research funding. The key research fronts have been ‘alginates’, ‘sulfated polysaccharides in general’, ‘agaroses’, ‘cyanobacterial biomedicine’, ‘macroalgal biomedicine’, ‘microalgal biomedicine’, and ‘channelrhodopsins’. The most-studied types of algae has been ‘macroalgae’ with 76.2% of the papers. The other prolific types of algae have been ‘microalgae’ and ‘cyanobacteria’. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies (Konur, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). It has been found that the detailed keyword set provided in the Appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts.

Appendix. The keyword sets A.1  Biomedicine related keywords A.1.1  Keywords related to the subject categories WC = (‘chemistry med*’ or ‘clinical neur*’ or ‘critical care*’ or ‘engineering biomed*’ or ‘materials science biomat*’ or ‘primary health*’ or ‘radiology nuclear*’ or ‘social sciences biomed*’ or allergy or anatomy* or andrology or anest* or audiology* or cardiac* or ‘cell & tissue*’ or dentistry* or dermatology or emergency* or endocr* or gastro* or geriatr* or health* or hemat* or immuno* or infectious* or integrative* or medic* or nursing or neuro* or obstet* or oncol* or opht* or orthoped* or otorhin* or pathol* or pediatr* or peripheral* or pharm* or psychiatry or rehabil* or reproductive* or respir* or rheum* or substance* or surgery or transplantation or tropical* or urol* or virol*).

422  PART | VII  Algal biomedicine

A.1.2  Keywords related to the journal titles SO = (Antimicrobial* or Antiviral* or Chemotherapy or ‘International Journal of Antimicrobial*’ or ‘Journal of Antibiotics’ or ‘Journal of antimicrobial*’ or ‘Journal of Chemotherapy’ or ‘Journal of Microencapsulation’ or ‘Journal of Antimicrobial*’ or Peptides or Probiotics*).

A.1.3  Keywords related to the paper titles TI = (pharm* or ‘*active compound*’ or ‘eicosapentaenoic acid*’ or ‘omega-3*’ or ‘polyunsaturated fatty acid*’ or bioactiv* or ‘docosahexaenoic acid*’ or antioxidant* or antioxidative or *polysaccharide* or therapeutic* or ‘recombinant protein*’ or angiotensin* or ‘value compound*’ or antimicrobial* or pufa or *medic* or ‘natural product*’ or inflam* or *cancer or chemoprev* or cardio* or ‘biologically active’ or epa or ‘functional food*’ or nutraceutical* or astaxanthin* or ‘beta carotene’ or antiviral or galactan* or cholesterol or anticoagula* or antitumor* or antitumour* or *hiv or *therapy or antibiotic* or hypertens* or ‘alpha-glucosidase*’ or immuno* or *drug* or *diabet* or ‘high value’ or polyphenol* or carcinogen* or *glycem* or *proliferative or cosmetic* or vaccine* or prebiot* or probiot* or antibacter* or skin* or macrolide* or sunscreen* or influenza or bone).

A.2  Keywords related to the algae A.2.1  Algae in general [TI = (alga or algae or algal or phycolog* or chlorarachn* or Bigelowiella or periphyton*) OR SO = (Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘European Journal of Phycology’ or Fottea* or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Research’)] NOT TI = (*gaas or algaln* or algan* or algarve or algasb or palga* or ‘shewanella algae’ or threshold*).

A.2.2  Dinoflagellates and coccolithophores TI = (chrysophycea* or chlorococcales or chrysophyt* or *coccolith* or dinocyst* or dinoflagell* or dinophycea* or dinophyt* or haptophyt* or peridiniales or prymnesiophycea* or raphidophycea* or raphidophyt* or zooxanthella* or Akashiwo or Amphidinium or Aureococcus or *Ceratium or *Chattonella or Cochlodinium or Crypthecodinium or Dinophysis or Emiliania or Gambierdiscus or *Gonyaulax or *Gymnodinium or Gyrodinium or Hematodinium or Heterocapsa or Heterosigma or Isochrysis or Karenia* or Karlodinium or Mallomonas or Noctiluca or Ochromonas or Ostreopsis or Oxyrrhis or Peridinium or Pfiesteria or Phaeocystis or Prorocentrum or Prymnesium or Scrippsiella or Symbiodinium or Vaucheria) NOT TI = (*toxin* or *toxic* or poison*).

A.2.3 Microalgae TI = (chlorophycea* or chlorophyt* or cryptomonad* or cryptophycea* or cryptophyt* or euglen* or eustigmatophycea* or ‘green alga*’ or microalga* or ‘micro-alga*’ or ‘micro alga*’ or prasinophycea* or streptophyt* or Trebouxiophycea* or volvocales or Acetabularia or Ankistrodesmus or Asteromonas or Aurantiochytrium or Botryococcus or Chaetoceros or Chlamydomonas or *Chlorella or *Chlorococcum or Coccomyxa or Cyanidioschyzon or Cyanidium or Desmodesmus or Dunaliella or Euglena or Galdieria or Haematococcus or Micrasterias or Micromonas or Monoraphidium or Nannochloropsis or Neochloris or Ostreococcus or Phormidium or Platymonas or Polytomella or Porphyridium or Prototheca or Pseudokirchneriella or Pyramimonas or Scenedesmus or Schizochytrium or Selenastrum or Tetraselmis or Trebouxia or Volvox).

A.2.4 Macroalgae TI = (‘brown alga*’ or ‘macro-alga*’ or ‘macro alga*’ or ‘red alga*’ or agarophyt* or characea* or charophyt* or cladophorales or cryptonemiales or dictyotales or florideophycea* or fucale* or fucoid or gelidiales or gigartinale* or gracilariales or kelp* or laminariale* or macroalga* or phaeophycea* or phaeophyt* or rhodophyce* or rhodophyt* or seaweed* or ulvale* or ulvophycea* or zygnematophycea* or ‘Chara vulgaris’ or Alaria or Ascophyllum or Asparagopsis or Asparagopsis or Bangia* or Bostrychia or Bryopsis or Catenella or Caulerpa or Ceramium or Chaetomorpha or Chondrus or Cladophora or Codium or Corallina or Cystoseira or Ecklonia or Ectocarpus or ‘Eisenia bicyclis’ or Enteromorpha or Eucheuma or Fucus or Gelidium or Gigartina* or Gracilaria or Gracilariopsis or Grateloupia or Halimeda or Halocynthia or Hizikia or Hypnea or Kappaphycus or Laminaria or Laurencia* or Lessonia or Lomentaria or Macrocystis or Monostroma or Mougeotia or Nitella or Nitellopsis or Oedogonium or Padina or Palmaria or Pelvetia or Plocamium or Polysiphonia or Porphyra or Pyropia or Saccharina or Sargassum or Scytosiphon or Solieria or Spirogyra or Turbinaria or Ulva or Undaria).



Algal biomedicine research Chapter | 26  423

A.2.5 Diatoms [TI = (bacillariophycea* or bacillariophyt* or diatom or diatoms or Asterionella or Chaetoceros or Coscinodiscus or Cyclotella or Cylindrotheca or Didymosphenia or Navicula or Nitzschia or Phaeodactylum or Skeletonema or Stephanodiscus or Thalassiosira) OR SO = (‘Diatom Research’)] NOT [TI = (diatomic* or atom* or *molecule*or amphorae or diatomyid* or dissociation or ‘rare gas*’ or *silica) OR SO = (‘Journal of Chemical Physics’)].

A.2.6 Cyanobacteria TI = (‘blue green alga*’ or ‘blue-green alga*’ or cyanelle or *cyanobacter* or cyanophyt* or cyanophycea* or nostocales or oscillatoriales or prochlorophyt* or Acaryochloris or *Anabaena or Anacystis or Aphanizomenon or Aphanothece or Arthrospira or Calothrix or Cyanophora or Cyanothece or *Lyngbya* or Mastigocladus or Microcoleus or Moorea or Nostoc or Oscillatoria or Planktothrix or Plectonema or Prochlorococcus or Prochloron or Prochlorothrix or Scytonema or Spirulina or *Synechococcus or Synechocystis or ThermoSynechococcus or Tolypothrix or Trichodesmium) NOT TI = (*toxin* or dms or microcyst* or *toxic* or poison* or nodularin or cylindros*).

A.2.7 Journals SO = (Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘Cryptogamie Algologie’ or ‘Diatom Research’ or ‘European Journal of Phycology’ or Fottea* or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Research’).

A.3  Cross-subject keywords [TI = (algin or alginic* or alginate*) NOT TI = (capacit* or batteries or *diesel* or pyroly* or fuel* or *sorp* or *removal or *fouling or pseudomonas or azotobacter or cataly* or bacterial or pesticide*)] OR TI = (channelrhodopsin*) or [AU = (‘deisseroth k’ or ‘svoboda k’ or ‘boyden es’ or ‘hegemann p’) and TS = (channelrhodopsin*)] or TI = [agar and (tissue* or cancer* or cell* or culture* or antibiotic* or antimicrobial*)] OR TI = (fucoidan* or fucan* or ‘sulfated polysaccharide*’ or ‘sulfated galactan*’ or ‘sulphated polysaccharide*’ or fucoxanthin* or ulvan* or griffithsin or laminarin or laminaran or fucoidin* or fucosterol or siphonaxanthin or agaran or agaroid*) OR TI = (patellamide* or calothrixin* or microginin or *hapalosin* or microcolin or dendroamide* or spirulan or cryptophycin* or curacin or cyanovirin* or scytonemin or furcellaran* or scytovirin* or halocynthiaxanthin or phycocolloid* or cyanobactin*) OR [TI = (carrageenan* or carrageenin*) NOT TI = (inflam* or hyperalges* or pleur* or *paw or odema)] OR [TI = (agarose*) NOT TI = (‘affinity chromatography’)].

References Abramo, G., D'Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Adamantidis, A.R., Zhang, F., Aravanis, A.M., Deisseroth, K., De Lecea, L., 2007. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450 (7168), 420–424. Augst, A.D., Kong, H.J., Mooney, D.J., 2006. Alginate hydrogels as biomaterials. Macromol. Biosci. 6 (8), 623–633. Benya, P.D., Shaffer, J.D., 1982. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30 (1), 215–224. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K., 2005. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8 (9), 1263–1268. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sust. Energ. Rev. 14 (2), 557–577. Chen, S.C., Wu, Y.C., Mi, F.L., Lin, Y.H., Yu, L.C., Sung, H.W., 2004. A novel pH-sensitive hydrogel composed of N,O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. J. Control. Release 96 (2), 285–300. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Dretzen, G., Bellard, M., Sassone-Corsi, P., Chambon, P., 1981. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem. 112 (2), 295–298. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 1984. 101 citation classics from annals of internal medicine. Curr. Contents (47), 3–13. Garfield, E., 1987. 100 citation-classics from the journal of the American Medical Association. JAMA, J. Am. Med. Assoc. 257 (1), 52–59. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127.

424  PART | VII  Algal biomedicine

George, M., Abraham, T.E., 2006. Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review. J. Control. Release 114 (1), 1–14. Gombotz, W.R., Wee, S.F., 1998. Protein release from alginate matrices. Adv. Drug Deliv. Rev. 31 (3), 267–285. Guan, J., Ma, N., 2007. China's emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21 (5), 210–216. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol. Adv. 29 (2), 189–198. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books, Stoke on Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011a. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2011b. The scientometric evaluation of the research on the students with disabilities in higher education. Soc. Polit. Econ. Cult. Res. 3 (2), 81–148. Konur, O., 2012a. The gradual improvement of disability rights for the disabled tenants in the UK: the promising road is still ahead. Soc. Polit. Econ. Cult. Res. 4 (3), 71–112. Konur, O., 2012b. The policies and practices for the academic assessment of blind students in higher education and professions. Energ. Educ. Sci. Technol. B 4 (si1), 240–244. Konur, O., 2012c. Prof. Dr. Ayhan Demirbas' scientometric biography. Energ. Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012d. The evaluation of the biogas research: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012e. The evaluation of the bio-oil research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 379–392. Konur, O., 2012f. The evaluation of the biorefinery research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 347–358. Konur, O., 2012g. The evaluation of the research on the biodiesel: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012h. The evaluation of the research on the bioethanol: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012i. The evaluation of the research on the biofuels: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012j. The evaluation of the research on the biohydrogen: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012k. The evaluation of the research on the microbial fuel cells: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012l. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012m. The evaluation of the global energy and fuels research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (1), 613–628. Konur, O., 2012n. 100 citation classics in energy and fuels. Energ. Educ. Sci. Technol. A 30 (si1), 319–332. Konur, O., 2012o. What have we learned from the citation classics in energy and fuels: a mixed study. Energ. Educ. Sci. Technol. A 30 (si1), 255–268. Konur, O., 2012p. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1893–1908. Konur, O., 2012q. The evaluation of the research on the arts and humanities in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (3), 1603–1618. Konur, O., 2012r. The evaluation of the educational research: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012s. The scientometric evaluation of the research on the deaf students in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1573–1588. Konur, O., 2012t. The scientometric evaluation of the research on the students with ADHD in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2012u. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Energ. Educ. Sci. Technol. B 4 (si1), 250–257. Konur, O., 2012v. The evaluation of the research on the doctoral education: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (si1), 593–600. Konur, O., 2012x. The scientometric evaluation of the institutional research: the Karadeniz Universities—part 3. Energ. Educ. Sci. Technol. B 4 (si1), 850–856. Konur, O., 2012y. The scientometric evaluation of the institutional research: the Karadeniz Universities—part 2. Energ. Educ. Sci. Technol. B 4 (si1), 844–849. Konur, O., 2012z. The scientometric evaluation of the institutional research: the Karadeniz Universities—part 1. Energ. Educ. Sci. Technol. B 4 (si1), 836–843. Konur, O., 2013a. The scientometric evaluation of the institutional research. The Marmara Universities—part  4. Energ. Educ. Sci. Technol. B 5 (2), 365–380.



Algal biomedicine research Chapter | 26  425

Konur, O., 2013b. The scientometric evaluation of the institutional research. The Marmara Universities—part  3. Energ. Educ. Sci. Technol. B 5 (2), 349–364. Konur, O., 2013c. The scientometric evaluation of the institutional research. The Marmara Universities—part  2. Energ. Educ. Sci. Technol. B 5 (2), 333–348. Konur, O., 2013d. The scientometric evaluation of the institutional research. The Marmara Universities—part  1. Energ. Educ. Sci. Technol. B 5 (2), 317–332. Konur, O., 2013e. The scientometric evaluation of the institutional research. The Ege Universities—part 3. Energ. Educ. Sci. Technol. B 5 (1), 83–98. Konur, O., 2013f. The scientometric evaluation of the institutional research. The Ege Universities—part 2. Energ. Educ. Sci. Technol. B 5 (1), 67–82. Konur, O., 2013g. The scientometric evaluation of the institutional research. The Ege Universities—part 1. Energ. Educ. Sci. Technol. B 5 (1), 51–66. Konur, O., 2013h. The scientometric evaluation of the institutional research. The Akdeniz Universities—part  3. Energ. Educ. Sci. Technol. B 5 (1), 151–166. Konur, O., 2013i. The scientometric evaluation of the institutional research. The Akdeniz Universities—part  2. Energ. Educ. Sci. Technol. B 5 (1), 135–150. Konur, O., 2013j. The scientometric evaluation of the institutional research. The Akdeniz Universities—part  1. Energ. Educ. Sci. Technol. B 5 (1), 119–134. Konur, O., 2013k. The scientometric evaluation of the institutional research. The inner Anatolian Universities—part 4. Energ. Educ. Sci. Technol. B 5 (2), 267–282. Konur, O., 2013l. The scientometric evaluation of the institutional research. The inner Anatolian Universities—part 3. Energ. Educ. Sci. Technol. B 5 (2), 251–266. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–626. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–682. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–108. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–556. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–422. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–302. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–370. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2016c. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016d. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016f. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016g. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016h. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895.

426  PART | VII  Algal biomedicine

Konur, O., 2016i. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Algal drugs: the state of the research. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019b. Algal genomics. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019c. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019d. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Kravitz, A.V., Freeze, B.S., Parker, P.R.L., Kay, K., Thwin, M.T., Deisseroth, K., et al., 2010. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466 (7306), 622–626. Kuo, C.K., Ma, P.X., 2001. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties. Biomaterials 22 (6), 511–521. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Lieberman, M.B., Montgomery, D.B., 1988. First‐mover advantages. Strateg. Manag. J. 9 (S1), 41–58.



Algal biomedicine research Chapter | 26  427

Martinsen, A., Skjak-Braek, G., Smidsrod, O., 1989. Alginate as immobilization material: I. correlation between chemical and physical properties of alginate gel beads. Biotechnol. Bioeng. 33 (1), 79–89. Mauck, R.L., Soltz, M.A., Wang, C.C.B., Wong, D.D., Chao, P.H.G., Valhmu, W.B., et  al., 2000. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J. Biomech. Eng. T. ASME 122 (3), 252–260. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal‐free organic dyes for dye‐sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., et al., 2003. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100 (24), 13940–13945. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Paladugu, R., Schein, M., Gardezi, S., Wise, L., 2002. One hundred citation classics in general surgical journals. World J. Surg. 26 (9), 1099–1105. Reed, K.C., Mann, D.A., 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13 (20), 7207–7221. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes—towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Smidsrod, O., Skjak-Braek, G., 1990. Alginate as immobilization matrix for cells. Trends Biotechnol. 8 (3), 71–78. Tonnesen, H.H., Karlsen, J., 2002. Alginate in drug delivery systems. Drug Dev. Ind. Pharm. 28 (6), 621–630. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Wiegand, I., Hilpert, K., Hancock, R.E.W., 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3 (2), 163–175.

Chapter 27

Microalgal biotechnology applied in biomedicine Jorge Alberto Vieira Costa, Juliana Botelho Moreira, Letícia Schneider Fanka, Roberta da Costa Kosinski, Michele Greque de Morais Federal University of Rio Grande, Rio Grande, Brazil

27.1 Introduction Microalgae are photosynthetic organisms capable of converting solar energy into chemical energy by fixing CO2, and their cell growth is about 100 times faster than terrestrial plants (Lam and Lee, 2012). These microorganisms have the ability for rapid development using natural or artificial light. By manipulation of the cultivation conditions, such as nutrient concentration, environmental factors (light intensity and temperature), medium pH, and cell growth phase, the microalgae can be stimulated to synthesize and accumulate specifics biocompounds (Costa et al., 2019). Moreover, microalgae cultivation can be carried out on non-arable land, avoiding competition with land use for plant cultivation and/or food production (Rajneesh et al., 2017). The microalgal biomass is considered a source of metabolites with biological activity. Some of the high-value bioactive compounds produced by microalgae are pigments such as phycocyanin (Morais et  al., 2018a; Prates et  al., 2018), β-carotene and astaxanthin (Rammuni et al., 2019), polyunsaturated fatty acids (Kumar et al., 2019), phenolic compounds (Christ-Ribeiro et al., 2019; Kuntzler et al., 2018), polysaccharides (Ekelhof and Melkonian, 2017), vitamin B12 (Grossman, 2016), vitamin E (Mudimu et al., 2017) and vitamin K1 (Tarento et al., 2018), and lutein (Florez-Miranda et al., 2017). These metabolites present nutraceutical, antimicrobial, anti-inflammatory, anti-aging, hypocholesterolemic, antioxidant, immunosuppressive, photoprotective, and neurotransmitter properties (Sathasivam et al., 2017), conferring promising and sustainable potential for the application of microalgae and their metabolites in the biomedical and pharmaceutical fields. In this context, the production of bioactive compounds from microalgae consists of selecting the producer species, defining the better culture conditions, isolating and purifying the molecule of interest. Knowledge of the biochemical composition of each microalgae species is essential to explore its potential use in different sectors. Furthermore, it is necessary to confirm the biological activity of metabolites by clinical trials to obtain the approval of the regulatory agencies (Garcia et al., 2017; Matos et al., 2016). In this way, this chapter addresses the bioactive compounds constituent in microalgae, as well as their characteristics and applications in the biomedical field, elucidating recent studies and future perspectives.

27.2 Microalgae Microalgae are photosynthetic, microscopic and unicellular organisms, which may or may not form colonies (Brand et al., 2013). The ability of these microorganisms to adapt to adverse environmental conditions, such as temperature and nutrient supply, makes microalgae widely used for the discovery of new drugs. These characteristics may be related to microalgal metabolites, which may act to aid in the defense mechanism of the human organism (Morais et al., 2015). The classification of microalgae is given according to pigments, chemical nature of the reserve products, and constituents of the cell wall. Microalgae are classified in: diatoms or Bacillariophyta, which are found predominantly in oceans and freshwater environments; green algae or Chlorophyceae, found both in marine and freshwater environments; blue-green algae or Cyanophyta, important in the fixation of nitrogen and carbon dioxide, that can be found in different environments; golden algae or Chrysophyceae are grown in habitats such as rivers and lakes (Kunjapur and Eldridge, 2010).

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00027-9 © 2020 Elsevier Inc. All rights reserved.

429

430  PART | VII  Algal biomedicine

FIG. 27.1  Top view of raceway-type reactor (A) and front view of the vertical tubular reactor (B).

Carbon, nitrogen and phosphorus are the main nutrients for the development of microalgae. In this way, these organisms can be grown in their natural environments, chemically defined (or synthetic) culture media, industrial effluents and sewage treatment systems (Chang et al., 2013). Microalgae can be grown in both open and closed systems. Cultivation in open systems can be performed in natural ponds or open bioreactors such as raceway-type (Fig. 27.1A). However, closed systems, such as tubular photobioreactors (Fig. 27.1B), provide a more safe environment to avoid contamination by unwanted cultures (Tredici, 2010). Due to the possibility of manipulation of the cultivation conditions and the high cellular multiplication rate of the microalgae, the obtaining biomass, from the cultivation of these microorganisms, becomes an alternative to the use of metabolites produced by plants. Moreover, the microalgae do not compete for arable land for their growth (Stephens et al., 2013). The microalgal biomass presents applications in several areas, such as biofertilizers (Renuka et al., 2018), biodiesel (Morais et al., 2018b), biopolymers (Silva et al., 2018; Moreira et al., 2016), food supplementation (Lucas et al., 2018; Santos et al., 2016), pharmaceutical industry (Manirafasha et al., 2016), as well as in biomedicine (Sathasivam et al., 2017). This wide applicability can be attributed to the composition of the biomass, rich in carbohydrates, proteins, lipids, pigments, vitamins, and minerals. The presence of these bioactive compounds in microalga biomass causes an increasing interest in research in the field of biomedicine since they are correlated with their efficiency in the fight against diseases (Patil and Kim, 2017).

27.3  Bioactive compounds and their properties Bioactive compounds are physiologically active substances with functional properties in the human body. There is a growing enthusiasm, by researchers and industries, in developing different biocompounds that can potentially be used as functional ingredients and drugs (Morais et al., 2015). In recent years, there has been interest in the production of bioactive compounds from natural sources, especially microalgae, driven by a growing number of scientific studies demonstrating the beneficial effects of these compounds on health (Zhang et al., 2019; Sathasivam et al., 2017; Park et al., 2017; Manirafasha et al., 2016; Raposo et al., 2015).

27.3.1 Proteins Proteins have been widely used as food ingredients, providing healthy food for humans (Capriotti et al., 2016). The constituent proteins of various foods are formed by peptides that are in an inactive state in the parent protein and can be released during enzymatic digestion (Toldra et al., 2018). Several peptides have bioactivity characteristics, including antioxidant, antibacterial, antithrombotic and antihypertensive effects (Nandan and Nampoothiri, 2017). Moreover, peptides exhibit angiotensin I-converting enzyme (ACE) inhibitory activity, preventing the risk of cardiovascular disease (Tu et al., 2018). Meat, milk, and egg proteins provide about 34% of the world's protein intake and represent 14% of global greenhouse gas emissions (FAO, 2017). Thus, there is a need for sustainable alternative protein sources (Van der Spiegel et al., 2013). Microalgae have high protein content, therefore are considered to be an alternative ingredient added to food. Moreover, microalgal biomass has the potential to reduce malnutrition rates, which has been increasing with population rise and food shortages caused by limited agricultural land, soil erosion, and climate change (Hulsen et al., 2018). Spirulina platensis can reach up to 74% of protein, standing out between foods like meat and soy (Andrade and Costa, 2008). In addition to antioxidant and antimicrobial properties, proteins of microalgal origin may exhibit anti-inflammatory activity. Microalgae also produce bioactive compounds capable of killing cancer cells through the apoptosis process or by altering cell signaling due to activation of the protein kinase C (Sigamani et al., 2016). Dolastatin 10 and dolastatin 15 are composed of amino acid residues that have activity against cancer (Llopiz, 2016). Proteins such as lectins can be applied in promising formulations to replace inefficient conventional antibiotics (Martinez-Frances and Escudero-Onate, 2018).



Microalgal biomedicine Chapter | 27  431

Microalgae are also composed of pigments that participate in photosynthesis reactions, among them are phycobiliproteins, such as phycocyanin and phycoerythrin. Phycocyanin has an effect on oxidative stress and can be applied as a pharmaceutical and nutraceutical compound due to its antioxidant activity (Moreira et  al., 2019; Morais et  al., 2018a). Furthermore, phycobiliproteins have been used as fluorescent markers (Morais et al., 2018a), representing an innovative material to be investigated in biomedical studies. Liu et  al. (2018) found that the application of photodynamic therapy with selenium-enriched phycocyanin promoted a tumor inhibition rate of 75%, indicating a new perspective for treatments against cancer.

27.3.2 Lipids Several diseases can be treated by the ingestion of polyunsaturated fatty acids. When added to the diet, these biocompounds affect physiological processes, preventing cardiovascular disease, cancer, asthma, arthritis, renal and cutaneous disorders, depression and schizophrenia (Sharma and Sharma, 2017). The microalgae have in their composition fatty acids such as lauric acid, palmitic acid, linoleic acid, oleic acid, stearic acid, which can be used as antibiotics, also having antibacterial activity (Yoon et al., 2018). Among the bioactive compound constituents in microalgae, the production of ‘polyunsaturated fatty acids’ (PUFAs) or long chain fatty acids are widely known (Guiheneuf and Stengel, 2013). Studies are focused on the interest in long chain fatty acids with unsaturation in the omega-3 and omega-6 position, due to the numerous health benefits. The omega-6 strengthens the nervous and immune systems and prevents depression, multiple sclerosis, bipolarity, schizophrenia, and attention deficit. The omega-3 reduces the level of cholesterol and triglycerides in the blood, increases the capacity of learning and memory, has anti-inflammatory action and helps control blood pressure (Saini and Keum, 2018). ‘Docosahexaenoic acid’ (DHA) and ‘eicosapentaenoic acid’ (EPA) are PUFAs produced in significant quantities by different species of microalgae, which has increased along the trophic levels in the marine food chain. Moreover, these compounds are of particular interest because of their bioactivity (Chauton et al., 2015). Regular consumption of supplements with EPA and DHA can reduce inflammation and prevent cardiovascular disease (Adarme-Vega et al., 2012). DHA is important for maintaining the membrane fluidity of the brain and retina. Some of the mediators derived from DHA are involved in reducing inflammation and protecting against injury (Saini and Keum, 2018). Astaxanthin and β-carotene are liposoluble organic compounds found in microalgae. Astaxanthin can be used as a food supplement and pharmaceutical product, while α-carotene and β-carotene, which are precursors of vitamin A, and violaxanthin, neoxanthin, lutein, and zeaxanthin, have antioxidant activity. Moreover, β-carotene is considered anticarcinogenic, and is used for the control of cholesterol, preventing the onset of heart disease (Sharma and Sharma, 2017). Dunaliella salina biomass constitutes about 14% of β-carotene (Blanco et al., 2007), and Haematococcus pluvialis is a major source of astaxanthin (1–8%) (Hejazi and Wijffels, 2004).

27.3.3 Carbohydrates Carbohydrates are natural compounds that consist of carbon, hydrogen, and oxygen, and range from simple sugars to complex polysaccharides such as starch and dietary fiber. In health terms, carbohydrate is the main macromolecule responsible for providing energy to the human body. Moreover, the carbohydrates are used to compensate for the energy content acquired by the lipids. This lipid replacement is a response to public health guidelines to avoid coronary heart disease (Chambers et al., 2019). The connections between the different components of carbohydrates and the way the molecules are organized indicate how these compounds affect the physiology of the gastrointestinal tract. Due to the physical properties of the different carbohydrates, they interact with the microbiota and the mucosa of the gastrointestinal tract (Knudsen et al., 2012). Carbohydrate constituent of the microalgal biomass may present antiviral, antibacterial, antitumor and anti-­inflammatory activities. Microalgae are capable of producing various carbohydrates, including galacturonic acid, glucuronic acid, mannose, ribose, fructose, glucose, xylose, galactose, and sulfated polysaccharide (Dewi et al., 2018). Sulfated polysaccharides have activity on several viruses, such as herpes simplex (Lopes et al., 2017), human cytomegalovirus, and measles virus (Rizwan et al., 2018). Calcium Spirulan inhibits the reverse transcriptase of HIV-1, HIV-2, HSV and influenza viruses. The structure of this carbohydrate may consist of mannose, ribose, fructose, glucose, xylose, galactose, galacturonic acid, glucuronic acid, and calcium and sulfate ions. Another sulfated polysaccharide is Nostoflan, which has antiviral activity on HSV-1, HSV-2, HCMV, influenza, adeno (type 2) and coxsackie (Conn-5) (Llopiz, 2016). Moreover, the bioactivity of the polysaccharides enables its use as fibers, probiotics, and prebiotics (Sathasivam et al., 2017).

432  PART | VII  Algal biomedicine

27.3.4  Phenolic compounds The phenolic compounds have hydroxyl groups attached to the aromatic ring. These phytochemicals are grouped as a function of the number of phenolic rings they contain and the radicals linking those rings to each other. The antioxidant activity of phenolic compounds is attributed to their ability to act as reducing agents for free radicals. Phenolic compounds have received attention because their intake is related to the lower incidence of degenerative diseases, such as cancer, diabetes, Alzheimer, and cardiovascular diseases (Gutierrez-Grijalva et al., 2016). Catechin, epicatechin, benzoic acid, gallic acid, and cinnamic acid are found in microalgae that have antioxidant potential, acting in the free radical scavenging (Jerez-Martel et al., 2017). The main factor in the antioxidant activity of phenolic compounds is their number and position of the hydroxyl groups. Flavonoids have more hydroxyl groups, therefore have higher antioxidant activity (Bravo, 1998). Moreover, phenolic compounds have also been attributed to their anti-­ inflammatory properties (Ambriz-Perez et al., 2016). In addition to antioxidant and anti-inflammatory activities, it has been demonstrated that microalgae phenolic compounds have antimicrobial properties against Staphylococcus aureus, Escherichia coli (Kuntzler et  al., 2018), and Aspergillus ­flavus (Souza et al., 2011).

27.3.5 Vitamins The microalgae are capable of producing vitamin A, B1, B2, B3, B5, B6, B7, B9, B12, C, D2, D3, E, and K (Buono et al., 2014; Mudimu et  al., 2017). These structures present antioxidant property, and the ability to inhibit the inflammatory process (Chew et al., 2017). Vitamin B12 is obtained in greater concentrations by the microalga Spirulina platensis when compared to food of vegetal and animal origin. This vitamin act in improving the immune system (Sathasivam et al., 2017). The presence of vitamin B12 in the microalgae biomass is of particular interest to vegetarians since without the ingestion of meat it is not possible to reach the recommended daily intake of this vitamin (Nazih and Bard, 2018). Tocopherols, also known as vitamin E, are produced by photosynthetic organisms and can be applied as food supplements. Vitamin E presents antioxidant properties, eliminating lipophilic radicals, as well as acting in the development of tissues and organs, such as the brain. Moreover, vitamin E beneficially influence on treatment of Alzheimer's disease (Gugliandolo et al., 2017) and acts against heart disease and atherosclerosis (Raposo et al., 2015). Taking vitamin E from microalgae is beneficial since the α-tocopherol levels are much higher than other sources, such as food crops for example (Shintani and DellaPenna, 1998; Carballo-Cardenas et al., 2003). According to Mudimu et al. (2017), the genus Microchloropsis was among the highest producers of α-tocopherol. Vitamin K plays an essential role in several biochemical processes, including blood coagulation, bone maintenance, prevention of arterial hardening and neuroprotection. Moreover, vitamin K prevents chronic diseases such as osteoporosis (Vermeer, 2012; Tarento et al., 2018).

27.4  Bioengineering of microalgae for the synthesis of bioactive compounds The composition of the microalgae biomass depends on several factors involved in the cultivation, such as nutrient concentrations in the medium, bioreactors, environmental conditions (temperature and illumination), agitation, pH and growth phase. By altering any of these factors, cellular metabolism is directed to produce compounds of interest (Table 27.1) and alter the rate of cell growth according to the purpose of cultivation. Thus, the cultivation conditions must be optimized aiming to obtain specific biocompounds and achieve maximum productivity of the microalgal biomass (Rizwan et al., 2018; Costa et al., 2019). For the accumulation of proteins, the nitrogen source is extremely important in cultures of microalgae that aim at the production of this biocompound. Cultivation of Spirulina to obtain phycocyanin is generally performed in medium containing nitrates, sodium bicarbonate, phosphates, sulfates, and microelements. The high alkalinity of the cultivation medium, generally above pH 9.5, inhibits the growth of contaminating microorganisms, facilitating large-scale cultivation. Phycocyanin production also occurs in heterotrophic and mixotrophic cultures in the absence of light using, for example, glucose, fructose or glycerol as the energy source (Morais et al., 2018a). Nutrient limitation, particularly nitrogen, is often used to increase specific biocompounds in microalgae biomass. In cultures under nitrogen stress, these microorganisms have the potential to accumulate lipids or carbohydrates (Braga et al., 2018, 2019; Ikaran et al., 2015), or vitamins (Tarento et al., 2018; Mudimu et al., 2017). Nitrogen-limited cultures can accelerate the degradation of pigments and proteins to provide intracellular nitrogen to maintain higher biomass productivity while accumulating carbohydrates (Li et al., 2018). Studies have also shown a greater accumulation of lipids in microalgae

Microalgal biomedicine Chapter | 27  433



TABLE 27.1  Cultivation conditions of microalgae for the production of bioactive compounds. Microalgae

Parameters

Bioactive compounds

References

Anabaena cylindrical CS-172

Light intensity; concentration of NaNO3 and K2HPO4

Vitamin K1

Tarento et al. (2018)

Chlorella fusca LEB 111

Concentration of NaNO3; fly ashes

Proteins, lipids, and carbohydrates

Braga et al. (2019)

Chlorella kessleri UTEX 2229

Autotrophic, heterotrophic and mixotrophic systems; photoperiod

Proteins, lipids, carbohydrates, pigments and fatty acids

Deng et al. (2019)

Chlorella vulgaris FACHB-8

Concentration of NaNO3

Proteins and amino acids

Xie et al. (2017)

Dunaliella salina SAG 184.80

Photoperiod

Protein

Sui et al. (2019)

Nannochloropsis oculata ASM15

Temperature; concentration of NaNO3

α-Tocopherol

Mudimu et al. (2017)

Netrium digitus CCAC 3119

pH; concentration of NaNO3

Extracellular polysaccharide

Ekelhof and Melkonian (2017)

Parachlorella sp. JD-076

Carbon source; light intensity

Lutein

Heo et al. (2018)

Phaeodactylum tricornutu, Tetraselmis suecica, Chlorella vulgaris

Concentration of NaNO3 and K2HPO4

Carotenoids and chlorophyll

Goiris et al. (2015)

Porphyridium marinum CCAP 1380/10

Concentration of NaNO3 and K2HPO4; light intensity; metal solution

Phycoerythrin

Gargouch et al. (2018)

Scenedesmus incrassatulus CLHESi01

Agitation

Lutein

Florez-Miranda et al. (2017)

Spirulina sp. LEB 18

Injection time of CO2; flow rate

Carbohydrates

Braga et al. (2018)

Spirulina sp. LEB 18

Concentration of glycerol

Fatty acids

Morais et al. (2019)

Trachydiscus minutus CCALA 838

Nitrogen source; salinity; light intensity; temperature

Fatty acids

Cepak et al. (2014)

grown under nitrogen and phosphorus limitation (Moussa et al., 2017). Furthermore, the increase in lipid content in microalgae can be performed using the dual-stage strategy. The first stage involves culturing under sufficient nutrient conditions to stimulate its growth, and in the second stage, the cells are exposed to nutrient deprivation for the accumulation of the biocompound (Mathimani et al., 2018). Regarding environmental factors, the main parameters affecting the lipid and carbohydrate productivities in microalgae are temperature and light intensity (Hindersin et al., 2014). Light is a factor that affects productivity, photosynthesis, cell composition, metabolic pathway, and the economic efficiency of the microalgae cultivation process (Deng et al., 2019). Studies have suggested that the contents of protein (Sui et al., 2019), pigments such as phycocyanin (Dejsungkranont et al., 2017), fatty acids (Li et al., 2014), phenolic compounds (Gomez et al., 2016), and vitamins (Tarento et al., 2018) have changed in response to light intensity. In addition, the wavelength of light used in cultivation also affects the production of fatty acids (Duarte et al., 2019) and phycocyanin (Prates et al., 2018). Photoperiod changes may also affect the content of this pigment (Prates et al., 2018), proteins and fatty acids (Matos et al., 2017) during microalgae cultivation. The production of the polyunsaturated fatty acids docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) by microalgae can be performed in autotrophic, heterotrophic and mixotrophic systems (Adarme-Vega et al., 2012), depending on factors such as pH, glucose, nitrogen and NaCl (Kumar et al., 2019). Increasing the biomass and lipids productivities through the creation of lineages with the genetic engineering approach is a strategy to improve the productivity of EPA and DHA in microalgae (Chauton et al., 2015). Microalgae when exposed to high concentrations of oxygen and free radicals tend to develop efficient protective systems against ‘reactive oxygen species’ (ROS) and free radicals, producing pigments such as chlorophylls, carotenoids,

434  PART | VII  Algal biomedicine

xanthophylls, and phycobiliproteins (Petruk et al., 2018; Goiris et al., 2015). Moreover, when cultivated in the presence of UV radiation, microalgae may increase the production of chlorophyll, carotenoids (Sharma and Sharma, 2015), DHA and EPA contents (Chauton et al., 2015).

27.5  Microalgal biomass applied in biomedicine The antioxidant potential of metabolites of microalgal origin is of extreme importance for the pharmaceutical industry. Thus, several researches have been carried out, relating these compounds to the treatment of different human diseases (David et al., 2015). Coronary heart disease and cancer are the leading cause of death around the world. In this way, it is important that there be an elaboration of alternative treatments that aim to contribute to the existing ones. The study by Takahashi et al. (2015) relates the use of microalgal metabolites with mechanisms that induce apoptosis of cancer cells, as well as actions that disintegrate malignant tumors. Regarding coronary diseases, studies show the microalgal biomass, as well as their pigments, vitamins, lipids, polysaccharides and amino acids, have an antioxidant character, as auxiliaries in the prevention of cardiovascular diseases. Some positive effects of the use of these compounds are in the increase of HDL ‘high-density lipoprotein’, as well as the reduction of cholesterol and triglyceride levels (Ku et al., 2015). The use of microalgal compounds is also directly related to antiviral (Wijesekara et  al., 2011), anti-inflammatory (Albuquerque et al., 2013), anticancer, antimicrobial (Hassouani et al., 2017), anticoagulant (Wang et al., 2013) and immunomodulatory (Carballo et al., 2018) activities. These beneficial characteristics to human health were found in different species of microalgae, as well as several metabolites extracted from these microorganisms. Table 27.2 correlates studies of the use of microalgae biomass, their metabolites and macromolecules for diseases' treatment. In a study performed by Zhang et al. (2019), with the microalgae Chlorella pyrenoidosa FACHB-9, Chlorococcum sp. and Scenedesmus sp., it was verified the effectiveness of the use of exopolysaccharides extracted from these microalgae against cancerous cells of the uterine cervix. Park et al. (2017) extracted exopolysaccharides from the marine microalga

TABLE 27.2  Microalgal metabolites produced by different species of microalgae and their potential applications for biomedicine. Microalgae

Metabolites and/or bioproducts

Applications

References

Arthronema africanum

Phycocyanin

Antitumoral

Gardeva et al. (2014)

Chlorella vulgaris

Biomass extract

Tumor inhibition

Balaji et al. (2017)

Chlorococcum humicola

Biomass extract

Antioxidant

Balaji et al. (2017)

Chlorococcum humicola

β-Carotene

Inhibition of pathogenic microorganisms

Bhagavathy et al. (2011)

Gyrodinium impudium KG03

Polysaccharide

Inhibition of influenza A virus

Kim et al. (2012)

Limnothirix sp. 37-2-1

Phycocyanin

Apoptosis of prostate cancer cells

Gantar et al. (2012)

Scytosiphon lomentaria

Biomass

Inhibition of Salmonella typhimurium proliferation

Taskin et al. (2010)

Spirulina máxima

Phycobiliproteins

Reduction of preeclampsia manifestations

Castro-Garcia et al. (2018)

Spirulina sp. LEB 18

Phenolic compounds and polyhydroxybutyrate

Inhibition of Staphylococus aureus and Escherichia coli

Kuntzler et al. (2018)

Spirulina platensis

Biomass and polysaccharide

Inhibition of HSV 1 virus

Mader et al. (2016)

Spirulina platensis

Phenolic compounds

Antioxidant

Souza et al. (2015)

Synechococcus spp. PCC7942

Biomass extract

Inhibition of Staphylococus aureus

Fatima et al. (2017)



Microalgal biomedicine Chapter | 27  435

Thraustochytriidae sp., in which were tested in different cancer cell lines, such as BG-1 ovarian, MCF-7 breast, and SW-620 colon cancer cell lines, and had positive effects on reduction of proliferation of these cells, besides assisting in immunomodulation. A study with Spirulina platensis related the use of their biomass as well as the use of phycocyanin and polysaccharides as auxiliaries in the prevention of cardiovascular diseases (Park et al., 2017). Spirulina has also been studied for antibacterial (Shishido et al., 2015) and antiviral (Farag et al., 2016) activities, aiding in the treatment of different cancer types, such as cancer of the breast, ovary, skin, uterine cervix, kidney and stomach (El-Hack et al., 2019). Moreover, Spirulina biomass was used in the development of scaffolds for applications in epithelial tissue regeneration (Steffens et al., 2014). Supplementation of Chlorella reduced anemia in pregnant women (Sonada, 1972), possibly due to the high content of iron, and constituents such as folate and vitamin B12. Nakano et al. (2010) also confirmed this effect from this microalga. Furthermore, Chlorella also promoted other beneficial effects, including enhancement of the immune system, and improvement of ulcerative colitis (Merchant and Andre, 2001).

27.6  Future perspectives The interest in the bioactive molecules from microalgae occurs due to the specificity in their chemical conformation, acting directly on the disease, without affecting other aspects of health. The protein potential of microalgal cells makes possible the exploitation of recombinant proteins, in order to express specific characteristics of different strains (Kwon et al., 2019). The development of gene transfer technologies in microalgae opens new opportunities for the use of these systems. In this way, it is possible to use microalgal biotechnology for different purposes in the clinical sector, such as the development of enzymes, antibodies, growth factors, drugs, and vaccines. It is believed that new studies must be performed, which may increasingly assist in the treatment of different diseases. Furthermore, the possibility of large-scale production should be explored, aiming at the feasibility of using bioactive compounds from microalgae in the pharmaceutical and biomedical industries. The increase of the quality of life expectation, coupled with advances in medicine, has enabled the development of new technologies. Nanobiotechnology can be used for the development of temporary matrices (scaffolds) for application in injured tissues. Scaffolds can be produced from biopolymers and biocompounds derived from microalgae. The greatest advantage of the use of the nanobiotechnology is the degradability and biocompatibility with human cells and tissues (Schmatz et al., 2016). Therefore, it is necessary that the research in the biomedicine field be continuous and specific for the development of new methods of treatments of diseases.

27.7 Conclusion The ability of microalgae to produce bioactive compounds such as proteins, lipids, carbohydrates, phenolic compounds, pigments, and vitamins, makes their biomass stand out for applications in the biomedicine area. The microalgal metabolites present different biological activities, being proven that their use helps in functions beneficial to the human organism, stimulating the defense mechanisms, preventing chronic diseases, besides helping to reduce free radicals. In this context, it is interesting that there is continuity of studies that aim at the improvement of microalgae strains for its application in biomedicine. Moreover, the optimization of the conditions of cultivation and extraction of the biocompounds should be considered for that microalgal biomass is a promising alternative, aiming for the improvement of the biocompatibility with cells and tissues.

References Adarme-Vega, T.C., Lim, D.K.Y., Timmins, M., Vernen, F., Li, Y., Schenk, P.M., 2012. Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production. Microb. Cell Factories 11, 96. Albuquerque, I.R.L., Cordeiro, S.L., Gomes, D.L., Dreyfuss, J.L., Filgueira, L.G.A., Leite, E.L., et al., 2013. Evaluation of anti-nociceptive and antiinflammatory activities of a heterofucan from Dictyota menstrualis. Mar. Drugs 11 (8), 2722–2740. Ambriz-Perez, D.L., Leyva-Lopez, N., Gutierrez-Grijalva, E.P., Heredia, J.B., Yildiz, F., 2016. Phenolic compounds: natural alternative in inflammation treatment. A review. Cogent Food Agric. 2 (1), 1131412. Andrade, M.R., Costa, J.A.V., 2008. Cultivation of Spirulina platensis microalgae in alternative sources of nutrients. Cienc. Agrotecnol. 32 (5), 1551–1556. Balaji, M., Thamilvanan, D., Vinayagam, C.S., Balakumar, B.S., 2017. Anticancer, antioxidant activity and GC-MS analysis of selected micro algal members of Chlorophyceae. Int. J. Pharm. Sci. Res. 13, 3302–3314. Bhagavathy, S., Sumathi, P., Jancy, S.B., 2011. Green algae Chlorococcum humicola—a new source of bioactive compounds with antimicrobial activity. Asian Pac. J. Trop. Biomed. 1 (1), S1–S7.

436  PART | VII  Algal biomedicine

Blanco, A.M., Moreno, J., del Campo, J.A., Rivas, J., Guerrero, M.G., 2007. Outdoor cultivation of lutein-rich cells of Muriellopsis sp. in open ponds. Appl. Microbiol. Biotechnol. 73 (6), 1259–1266. Braga, V.S., Mastrantonio, D.J.S., Costa, J.A.V., Morais, M.G., 2018. Cultivation strategy to stimulate high carbohydrate content in Spirulina biomass. Bioresour. Technol. 269, 221–226. Braga, V.S., Moreira, J.B., Costa, J.A.V., Morais, M.G., 2019. Potential of Chlorella fusca LEB 111 cultivated with thermoelectric fly ashes, carbon dioxide and reduced supply of nitrogen to produce macromolecules. Bioresour. Technol. 277, 55–61. Brand, J.J., Andersen, R.A., Nobles, D.R., 2013. Maintenance of microalgae in culture collections. In: Richmond, A., Hu, Q. (Eds.), Handbook of Microalgal Culture: Applied Phycology and Biotechnology, second ed. Blackwell Science, New Delhi, pp. 80–89. Bravo, L., 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 56 (11), 317–333. Buono, S., Langellotti, A.L., Martello, A., Rinna, F., Fogliano, V., 2014. Functional ingredients from microalgae. Food Funct. 5 (8), 1669–1685. Capriotti, A.L., Cavaliere, C., Piovesana, S., Samperi, R., Lagana, A., 2016. Recent trends in the analysis of bioactive peptides in milk and dairy products. Anal. Bioanal. Chem. 408 (11), 2677–2685. Carballo, C., Chronopoulou, E.G., Letsiou, S., Maya, C., Labrou, N.E., Infante, C., et al., 2018. Antioxidant capacity and immunomodulatory effects of a chrysolaminarin-enriched extract in Senegalese sole. Fish Shellfish Immunol. 82, 1–8. Carballo-Cardenas, E.C., Tuan, P.M., Janssen, M., Wijffels, R.H., 2003. Vitamin E (α-tocopherol) production by the marine microalgae Dunaliella tertiolecta and Tetraselmis suecica in batch cultivation. Biomol. Eng. 20 (4–6), 139–147. Castro-Garcia, S.Z., Chamorro-Cevallos, G., Quevedo-Corona, L., McCarty, M.F., Bobadilla-Lugo, R.A., 2018. Beneficial effects of phycobiliproteins from Spirulina maxima in a preeclampsia model. Life Sci. 211, 17–24. Cepak, V., Pribyl, P., Kohoutkova, J., Kastanek, P., 2014. Optimization of cultivation conditions for fatty acid composition and EPA production in the eustigmatophycean microalga Trachydiscus minutus. J. Appl. Phycol. 26 (1), 181–190. Chambers, E., Byrne, C.S., Frost, G., 2019. Carbohydrate and human health: is it all about quality? Lancet 393 (10170), 384–386. Chang, Y.Y., Wu, Z.C., Bian, L., Feng, D.L., Leung, D.Y.C., 2013. Cultivation of Spirulina platensis for biomass production and nutrient removal from synthetic human urine. Appl. Energy 102, 427–431. Chauton, M.S., Reitan, K.I., Norsker, N.H., Tveteras, R., Kleivdal, H.T., 2015. A technoeconomic analysis of industrial production of marine microalgae as a source of EPA and DHA-rich raw material for aquafeed: research challenges and possibilities. Aquaculture 436, 95–103. Chew, K.W., Yap, J.Y., Show, P.L., Suan, N.H., Juan, J.C., Ling, T.C., et al., 2017. Microalgae biorefinery: high value products perspectives. Bioresour. Technol. 229, 53–62. Christ-Ribeiro, A., Graca, C.S., Kupski, L., Badiale-Furlong, E., de Souza-Soares, L.A., 2019. Cytotoxicity, antifungal and anti mycotoxins effects of phenolic compounds from fermented rice bran and Spirulina sp. Process Biochem. 80, 190–196. Costa, J.A.V., de Freitas, B.C.B., Lisboa, C.R., Santos, T.D., de Fraga Brusch, L.R., Morais, M.G., 2019. Microalgal biorefinery from CO2 and the effects under the blue economy. Renew. Sust. Energ. Rev. 99, 58–65. David, B., Wolfender, J.L., Dias, D.A., 2015. The pharmaceutical industry and natural products: historical status and new trends. Phytochem. Rev. 14 (2), 299–315. Dejsungkranont, M., Sirisansaneeyakul, S., Chisti, Y., 2017. Simultaneous production of C-phycocyanin and extracellular polymeric substances by photoautotrophic cultures of Arthrospira platensis. J. Chem. Technol. Biotechnol. 92 (10), 2709–2718. Deng, X.Y., Chen, B., Xue, C.Y., Li, D., Hu, X.L., Gao, K., 2019. Biomass production and biochemical profiles of a freshwater microalga Chlorella kessleri in mixotrophic culture: effects of light intensity and photoperiodicity. Bioresour. Technol. 273, 358–367. Dewi, I.C., Falaise, C., Hellio, C., Bourgougnon, N., Mouget, J.L., 2018. Anticancer, antiviral, antibacterial, and antifungal properties in microalgae. In: Levine, I.A., Fleurence, J. (Eds.), Microalgae in Health and Disease Prevention. Academic Press, San Diego, pp. 235–262. Duarte, J.H., de Souza, C.O., Druzian, J.I., Costa, J.A.V., 2019. Light emitting diodes applied in Synechococcus nidulans cultures: effect on growth, pigments production and lipid profiles. Bioresour. Technol. 280, 511–514. Ekelhof, A., Melkonian, M., 2017. Enhanced extracellular polysaccharide production and growth by microalga Netrium digitus in a porous substrate bioreactor. Algal Res. 28, 184–191. El-Hack, M.E.A., Abdelnour, S., Alagawany, M., Abdo, M., Sakr, M.A., Khafaga, A.F., et al., 2019. Microalgae in modern cancer therapy: current knowledge. Biomed. Pharmacother. 111, 42–50. FAO, 2017. Livestock Solutions for Climate Change. Food and Agriculture Organization, Rome. Farag, M.R., Alagawany, M., El-Hack, M.E.A., Kuldeep, D., 2016. Nutritional and healthical aspects of Spirulina (Arthrospira) for poultry, animals and human. Int. J. Pharmacol. 12 (1), 36–51. Fatima, N., Ahmad, I.Z., Chaudhry, H., 2017. Alterations in the antibacterial potential of Synechococcus spp. PCC7942 under the influence of UV-B radiations on skin pathogens. Saudi J. Biol. Sci. 24 (7), 1657–1662. Florez-Miranda, L., Canizares-Villanueva, R.O., Melchy-Antonio, O., Martinez-Jeronimo, F., Flores-Ortiz, C.M., 2017. Two stage heterotrophy/photoinduction culture of Scenedesmus incrassatulus: potential for lutein production. J. Biotechnol. 262, 67–74. Gantar, M., Dhandayuthapani, S., Rathinavelu, A., 2012. Phycocyanin induces apoptosis and enhances the effect of topotecan on prostate cell line LNCaP. J. Med. Food 15 (12), 1091–1095. Garcia, J.L., de Vicente, M., Galan, B., 2017. Microalgae, old sustainable food and fashion nutraceuticals. Microb. Biotechnol. 10 (5), 1017–1024. Gardeva, E.G., Toshkova, R.A., Yossifova, L.S., Minkova, K., Ivanova, N., Gigova, L., 2014. Antitumor activity of C-phycocyanin from Arthronema africanum (Cyanophyceae). Braz. Arch. Biol. 57 (5), 675–684. Gargouch, N., Karkouch, I., Elleuch, J., Elkahoui, S., Michaud, P., Abdelkafi, S., et al., 2018. Enhanced B-phycoerythrin production by the red microalga Porphyridium marinum: a powerful agent in industrial applications. Int. J. Biol. Macromol. 120 (B), 2106–2114.



Microalgal biomedicine Chapter | 27  437

Goiris, K., van Colen, W., Wilches, I., Leon-Tamariz, F., de Cooman, L.D., Muylaert, K., 2015. Impact of nutrient stress on antioxidant production in three species of microalgae. Algal Res. 7, 51–57. Gomez, A.L., Lopez, J.A., Rodriguez, A., Fortiz, J., Martinez, L.R., Apolinar, A., et al., 2016. Producción de compuestos fenólicos por cuatro especies de microalgas marinas sometidas a diferentes condiciones de iluminación (Production of phenolic compounds by four species of marine microalgae under different light conditions). Lat. Am. J. Aquat. Res. 44 (1), 137–143. Grossman, A., 2016. Nutrient acquisition: the generation of bioactive vitamin B12 by microalgae. Curr. Biol. 26 (8), R319–R321. Gugliandolo, A., Bramanti, P., Mazzon, E., 2017. Role of vitamin E in the treatment of Alzheimer's disease: evidence from animal models. Int. J. Mol. Sci. 18 (12), 1–21. Guiheneuf, F., Stengel, D.B., 2013. LC-PUFA-enriched oil production by microalgae: accumulation of lipid and triacylglycerols containing n-3 LC-PUFA is triggered by nitrogen limitation and inorganic carbon availability in the marine haptophyte Pavlova lutheri. Mar. Drugs 11 (11), 4246–4266. Gutierrez-Grijalva, E.P., Ambriz-Pere, D.L., Leyya-Lopez, N., Castillo-Lopes, R.I., Heredia, J.B., 2016. Review: dietary phenolic compounds, health benefits and bioaccessibility. Arch. Latinoam. Nutr. 66 (2), 87–100. Hassouani, M., Sabour, B., Belattmania, Z., El Atouani, S., Reani, A., Ribeiro, T., et al., 2017. In vitro anticancer, antioxidant and antimicrobial potential of Lyngbya aestuarii (cyanobacteria) from the Atlantic coast of Morocco. J. Mater. Environ. Sci. 8 (S), 4923–4933. Hejazi, M.A., Wijffels, R.H., 2004. Milking of microalgae. Trends Biotechnol. 22 (4), 189–194. Heo, J., Shin, D.S., Cho, K., Cho, D.H., Lee, Y.J., Kim, H.S., 2018. Indigenous microalga Parachlorella sp. JD-076 as a potential source for lutein production: Optimization of lutein productivity via regulation of light intensity and carbon source. Algal Res. 33, 1–7. Hindersin, S., Leupold, M., Kerner, M., Hanelt, D., 2014. Key parameters for outdoor biomass production of Scenedesmus obliquus in solar tracked photobioreactors. J. Appl. Phycol. 26 (6), 2315–2325. Hulsen, T., Hsieh, K., Lu, Y., Tait, S., Batstone, D.J., 2018. Simultaneous treatment and single cell protein production from agri–industrial wastewaters using purple phototrophic bacteria or microalgae—a comparison. Bioresour. Technol. 254, 214–223. Ikaran, Z., Suarez-Alvarez, S., Urreta, I., Castanon, S., 2015. The effect of nitrogen limitation on the physiology and metabolism of Chlorella vulgaris var L3. Algal Res. 10, 134–144. Jerez-Martel, I., García-Poza, S., Rodriguez-Martel, G., Rico, M., Afonso-Olivares, C., Gomez-Pinchetti, J.L., 2017. Phenolic profile and antioxidant activity of crude extracts from microalgae and cyanobacteria strains. J. Food Qual. 2017, 2924508. Kim, M., Yim, J.H., Kim, S.Y., Kim, H.S., Lee, W.G., Kim, S.J., et al., 2012. In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03. Antivir. Res. 93 (2), 253–259. Knudsen, K.E.B., Hedemann, M.S., Laerke, H.N., 2012. The role of carbohydrates in intestinal health of pigs. Anim. Feed Sci. Technol. 173 (1–2), 41–53. Ku, C.S., Kim, B., Pham, T.X., Yang, Y., Weller, C.L., Carr, T.P., et al., 2015. Hypolipidemic effect of a blue-green alga (Nostoc commune) is attributed to its nonlipid fraction by decreasing intestinal cholesterol absorption in C57BL/6J mice. J. Med. Food 18 (11), 1214–1222. Kumar, B.R., Deviram, G., Mathimani, T., Duc, P.A., Pugazhendhi, A., 2019. Microalgae as rich source of polyunsaturated fatty acids. Biocatal. Agric. Biotechnol. 17, 583–588. Kunjapur, A.M., Eldridge, R.B., 2010. Photobioreactor design for commercial biofuel production from microalgae. Ind. Eng. Chem. Res. 49 (8), 3516–3526. Kuntzler, S.G., de Almeida, A.C.A., Costa, J.A.V., Morais, M.G., 2018. Polyhydroxybutyrate and phenolic compounds microalgae electrospun nanofibers: a novel nanomaterial with antibacterial activity. Int. J. Biol. Macromol. 113, 1008–1014. Kwon, K.C., Lamb, A., Fox, D., Jegathese, S.J.P., 2019. An evaluation of microalgae as a recombinant protein oral delivery platform for fish using green fluorescent protein (GFP). Fish Shellfish Immunol. 87, 414–420. Lam, M.K., Lee, K.T., 2012. Microalgae biofuels: a critical review of issues, problems and the way forward. Biotechnol. Adv. 30 (3), 673–690. Li, T.T., Zheng, Y.B., Yu, L.A., Chen, S.L., 2014. Mixotrophic cultivation of a Chlorella sorokiniana strain for enhanced biomass and lipid production. Biomass Bioenergy 66, 204–213. Li, X.T., Li, W., Zhai, J., Wei, H.X., 2018. Effect of nitrogen limitation on biochemical composition and photosynthetic performance for fed-batch mixotrophic cultivation of microalga Spirulina platensis. Bioresour. Technol. 263, 555–561. Liu, Z.J., Fu, X.A., Huang, W., Li, C.X., Wang, X.Y., Huang, B., 2018. Photodynamic effect and mechanism study of selenium-enriched phycocyanin from Spirulina platensis against liver tumours. J. Photochem. Photobiol. B 180, 89–97. Llopiz, A., 2016. Compuestos bioactivos aislados de cianobacterias y microalgas: propiedades y aplicaciones potenciales en la biomedicina (Active compounds from cyanobacteria and microalgae: properties and potential applications in biomedicine). Bionatura 1 (2), 79–87. Lopes, N., Ray, S., Espada, S.F., Bomfim, W.A., Ray, B., Faccin–Galhardi, L.C., et al., 2017. Green seaweed Enteromorpha compressa (Chlorophyta, Ulvaceae) derived sulphated polysaccharides inhibit herpes simplex virus. Int. J. Biol. Macromol. 102, 605–612. Lucas, B.F., Morais, M.G., Santos, T.D., Costa, J.A.V., 2018. Spirulina for snack enrichment: nutritional, physical and sensory evaluations. LWT-Food Sci. Technol. 90, 270–276. Mader, J., Gallo, A., Schommartz, T., Handke, W., Nagel, C.H., Guther, P., et al., 2016. Calcium spirulan derived from Spirulina platensis inhibits herpes simplex virus 1 attachment to human keratinocytes and protects against herpes labialis. J. Allergy Clin. Immunol. 137 (1), 197–203. Manirafasha, E., Ndikubwimana, T., Zeng, X.H., Lu, Y.H., Jing, K.J., 2016. Phycobiliprotein: potential microalgae derived pharmaceutical and biological reagent. Biochem. Eng. J. l09, 282–296. Martinez-Frances, E., Escudero-Onate, C., 2018. Cyanobacateria and microalgae in the production of valuable bioactive compounds. In: Jacob-Lopes, E., Zepka, L.Q., Queiroz, M.I. (Eds.), Microalgal Biotechnology. IntechOpen, London, pp. 68–84. Mathimani, T., Uma, L., Prabaharan, D., 2018. Formulation of low-cost seawater medium for high cell density and high lipid content of Chlorella vulgaris BDUG 91771 using central composite design in biodiesel perspective. J. Clean. Prod. 198, 575–586.

438  PART | VII  Algal biomedicine

Matos, A.P., Feller, R., Moecke, E.H.S., de Oliveira, J.V., Junior, A.F., Derner, R.B., et al., 2016. Chemical characterization of six microalgae with potential utility for food application. J. Am. Oil Chem. Soc. 93 (7), 963–972. Matos, A.P., Cavanholi, M.G., Moecke, E.H.S., Sant'Anna, E.S., 2017. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. 224, 490–497. Merchant, R.E., Andre, C.A., 2001. A review of recent clinical trials of the nutritional supplement Chlorella pyrenoidosa in the treatment of fibromyalgia, hypertension, and ulcerative colitis. Altern. Ther. Health Med. 7 (3), 79–91. Morais, M.G., Vaz, B.S., Morais, E.G., Costa, J.A.V., 2015. Biologically active metabolites synthesized by microalgae. BioMed. Res. Int. 2015, 1–15. Morais, M.G., Prates, D.F., Moreira, J.B., Duarte, J.H., Costa, J.A.V., 2018a. Phycocyanin from microalgae: properties, extraction and purification, with some recent applications. Ind. Biotechnol. 14, 30–37. Morais, E.G., Cassuriaga, A.P.A., Callejas, N., Martinez, N., Vieitez-Osorio, I., Jachmanian-Alpuy, I., et al., 2018b. Evaluation of CO2 biofixation and biodiesel production by Spirulina (Arthospira) cultivated in air-lift photobioreactor. Braz. Arch. Biol. Technol. 61, 1–11. Morais, E.G., Druzian, J.I., Nunes, I.L., Morais, M.G., Costa, J.A.V., 2019. Glycerol increases growth, protein production and alters the fatty acids profile of Spirulina (Arthrospira) sp. LEB 18. Process Biochem 76, 40–45. Moreira, J.B., Terra, A.L.M., Costa, J.A.V., Morais, M.G., 2016. Utilization of CO2 in semi-continuous cultivation of Spirulina sp. and Chlorella fusca and evaluation of biomass composition. Braz. J. Chem. Eng. 33 (3), 691–698. Moreira, J.B., Lim, L., da Rosa Zavareze, E., Dias, A.R.G., Costa, J.A.V., Morais, M.G., 2019. Antioxidant ultrafine fibers developed with microalga compounds using a free surface electrospinning. Food Hydrocoll. 93, 131–136. Moussa, I.D.B., Chtourou, H., Karray, F., Sayadi, S., Dhouib, A., 2017. Nitrogen or phosphorus repletion strategies for enhancing lipid or carotenoid production from Tetraselmis marina. Bioresour. Technol. 238, 325–332. Mudimu, O., Koopmann, I.K., Rybalka, N., Friedl, T., Schulz, R., Bilger, W., 2017. Screening of microalgae and cyanobacteria strains for α-tocopherol content at different growth phases and the influence of nitrate reduction on α-tocopherol production. J. Appl. Phycol. 29 (6), 2867–2875. Nakano, S., Takekoshi, H., Nakano, M., 2010. Chlorella pyrenoidosa supplementation reduces the risk of anemia, proteinuria and edema in pregnant women. Plant Food Hum. Nutr. 65 (1), 23–30. Nandan, A., Nampoothiri, K.M., 2017. Molecular advances in microbial aminopeptidases. Bioresour. Technol. 245 (B), 1757–1765. Nazih, H., Bard, J.M., 2018. Microalgae in human health: interest as a functional food. In: Levine, I.A., Fleurence, J. (Eds.), Microalgae in Health and Disease Prevention. Academic Press, San Diego, pp. 211–226. Park, G.T., Go, R.E., Lee, H.M., Lee, G.A., Kim, C.W., Seo, J.W., et al., 2017. Potential anti-proliferative and immunomodulatory effects of marine microalgal exopolysaccharide on various human cancer cells and lymphocytes in vitro. Mar. Biotechnol. 19 (2), 136–146. Patil, M.P., Kim, G.D., 2017. Eco-friendly approach for nanoparticles synthesis and mechanism behind antibacterial activity of silver and anticancer activity of gold nanoparticles. Appl. Microbiol. Biotechnol. 101 (1), 79–92. Petruk, G., Gifuni, I., Illiano, A., Roxo, M., Pinto, G., Amoresano, A., et al., 2018. Simultaneous production of antioxidants and starch from the microalga Chlorella sorokiniana. Algal Res. 34, 164–174. Prates, D.F., Radmann, E.M., Duarte, J.H., Morais, M.G., Costa, J.A.V., 2018. Spirulina cultivated under different light emitting diodes: enhanced cell growth and phycocyanin production. Bioresour. Technol. 256, 38–43. Rajneesh, Singh, S.P., Pathak, J., Sinha, R.P., 2017. Cyanobacterial factories for the production of green energy and value-added products: an integrated approach for economic viability. Renew. Sust. Energ. Rev. 69, 578–595. Rammuni, M.N., Ariyadasa, T.U., Nimarshana, P.H.V., Attalage, R.A., 2019. Comparative assessment on the extraction of carotenoids from microalgal sources: astaxanthin from H. pluvialis and β-carotene from D. salina. Food Chem. 277, 128–134. Raposo, M.F.J., Miranda, A.M., Morais, B., 2015. Microalgae for the prevention of cardiovascular disease and stroke. Life Sci. 125, 32–41. Renuka, N., Guldhe, A., Prasanna, R., Singh, P., Bux, F., 2018. Microalgae as multi-functional options in modern agriculture: current trends, prospects and challenges. Biotechnol. Adv. 36 (4), 1255–1273. Rizwan, M., Mujtaba, G., Memon, S.A., Lee, K., Rashid, N., 2018. Exploring the potential of microalgae for new biotechnology applications and beyond: a review. Renew. Sust. Energ. Rev. 92, 394–404. Saini, R.K., Keum, Y.S., 2018. Omega-3 and omega-6 polyunsaturated fatty acids: dietary sources, metabolism, and significance—a review. Life Sci. 203, 255–267. Santos, T.D., de Freitas, B.C.B., Moreira, J.B., Zanfonato, K., Costa, J.A.V., 2016. Development of powdered food with the addition of Spirulina for food supplementation of the elderly population. Innov. Food Sci. Emerg. 37 (B), 216–220. Sathasivam, R., Radhakrishnan, R., Hashem, A., Abd-Allah, E.F., 2017. Microalgae metabolites: a rich source for food and medicine. Saudi J. Biol. Sci. https://doi.org/10.1016/j.sjbs.2017.11.003. Schmatz, D.A., da Silva Uebel, L., Kuntzler, S.G., Dora, C.L., Costa, J.A.C., Morais, M.G., 2016. Scaffolds containing Spirulina sp. LEB 18 biomass: development, characterization and evaluation of in vitro biodegradation. J. Nanosci. Nanotechnol. 16 (1), 1050–1059. Sharma, R., Sharma, V.K., 2015. Effect of ultraviolet-B radiation on growth and pigments of Chlorella vulgaris. J. Indian Bot. Soc. 94 (1–2), 81–88. Sharma, N., Sharma, P., 2017. Industrial and biotechnological applications of algae: a review. J. Adv. Plant Biol. 1 (1), 1534. Shintani, D., DellaPenna, D., 1998. Elevating the vitamin E content of plants through metabolic engineering. Science 282 (5396), 2098–2100. Shishido, T.K., Humisto, A., Jokela, J., Liu, L., Wahlsten, M., Tamrakar, A., et al., 2015. Antifungal compounds from cyanobacteria. Mar. Drugs 13 (4), 2124–2140. Sigamani, S., Ramamurthy, D., Natarajan, H., 2016. A review on potential biotechnological applications of microalgae. J. Appl. Pharm. Sci. 6 (8), 179–184.



Microalgal biomedicine Chapter | 27  439

Silva, C.K., Costa, J.A.V., Morais, M.G., 2018. Polyhydroxybutyrate (PHB) synthesis by Spirulina sp. LEB 18 using biopolymer extraction waste. Appl. Biochem. Biotechnol. 185 (3), 822–833. Sonada, M., 1972. Effect of Chlorella extract on pregnancy anemia. Jpn. J. Nutr. Diet 30 (5), 218–225. Souza, M.M., Prietto, L., Ribeiro, A.C., Souza, T.D., Badiale-Furlong, E., 2011. Assessment of the antifungal activity of Spirulina platensis phenolic extract against Aspergillus flavus. Ciênc. Agrotecnol. 35, 1050–1058. Souza, T.D., Prietto, L., Souza, M.M., Furlong, E.B., 2015. Profile, antioxidant potential, and applicability of phenolic compounds extracted from Spirulina platensis. Afr. J. Biotechnol 14 (41), 2903–2909. Steffens, D., Leonardi, D., da Luz Soster, P.R., Lersch, M., Rosa, A., Crestani, T., et al., 2014. Development of a new nanofiber scaffold for use with stem cells in a third degree burn animal model. Burns 40 (8), 1650. Stephens, E., de Nys, R., Ross, I.L., Hankamer, B., 2013. Algae fuels as an alternative to petroleum. J. Pet. Environ. Biotechnol. 4, 148. Sui, Y., Muys, M., Vermeir, P., D'Adamo, S., Vlaeminck, S.E., 2019. Light regime and growth phase affect the microalgal production of protein quantity and quality with Dunaliella salina. Bioresour. Technol. 275, 145–152. Takahashi, K., Hosokawa, M., Kasajima, H., Hatanaka, K., Kudo, K., Shimoyama, N., et al., 2015. Anticancer effects of fucoxanthin and fucoxanthinol on colorectal cancer cell lines and colorectal cancer tissues. Oncol. Lett. 10 (3), 1463–1467. Tarento, T.D.C., McClure, D.D., Vasiljevski, E., Schindeler, A., Dehghani, F., Kavanagh, J.M., 2018. Microalgae as a source of vitamin K1. Algal Res. 36, 77–87. Taskin, E., Caki, Z., Ozturk, M., 2010. Assessment of in  vitro antitumoral and antimicrobial activities of marine algae harvested from the eastern Mediterranean Sea. Afr. J. Biotechnol. 9 (27), 4272–4277. Toldra, F., Reig, M., Aristoy, M.C., Mora, L., 2018. Generation of bioactive peptides during food processing. Food Chem. 267, 395–404. Tredici, M.R., 2010. Photobiology of microalgae mass cultures: understanding the tools for the next green revolution. Biofuels 1 (1), 143–162. Tu, M.L., Wang, C., Chen, C., Zhang, R.Y., Liu, H.X., Lu, W.H., et al., 2018. Identification of a novel ACE-inhibitory peptide from casein and evaluation of the inhibitory mechanisms. Food Chem. 256, 98–104. Van der Spiegel, M., Noordam, M.Y., van der Fels-Klerx, H.J., 2013. Safety of novel protein sources (insects, microalgae, seaweed, duckweed, and rapeseed) and legislative aspects for their application in food and feed production. Compr. Rev. Food Sci. Food Saf. 12 (6), 662–678. Vermeer, C., 2012. Vitamin K: the effect on health beyond coagulation—an overview. Food Nutr. Res. 56, 5329. Wang, X.M., Zhang, Z.S., Yao, Z.Y., Zhao, M.X., Qi, H.M., 2013. Sulfation, anticoagulant and antioxidant activities of polysaccharide from green algae Enteromorpha linza. Int. J. Biol. Macromol. 58, 225–230. Wijesekara, I., Pangestuti, R., Kim, S.K., 2011. Biological activities and potential health benefits of sulfated polysaccharides derived from marine algae. Carbohydr. Polym. 84 (1), 14–21. Xie, T.H., Xia, Y., Zeng, Y., Li, X.R., Zhang, Y.K., 2017. Nitrate concentration-shift cultivation to enhance protein content of heterotrophic microalga Chlorella vulgaris: over-compensation strategy. Bioresour. Technol. 233, 247–255. Yoon, B.K., Jackman, J.A., Valle-Gonzalez, E.R., Cho, N.J., 2018. Antibacterial free fatty acids and monoglycerides: biological activities, experimental testing, and therapeutic applications. Int. J. Mol. Sci. 19 (4), 1–40. Zhang, J.Z., Liu, L., Ren, Y.Y., Chen, F., 2019. Characterization of exopolysaccharides produced by microalgae with antitumor activity on human colon cancer cells. Int. J. Biol. Macromol. 128, 761–767.

Chapter 28

Applications of cyanobacteria in biomedicine Bahareh Nowruzia, Gisoo Sarvarib, Saúl Blancoc,* a

Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran, bDepartment of Pharmaceutical Chemistry, Faculty of Pharmaceutical Chemistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran, cDepartamento de Biodiversidad y Gestión Ambiental, Facultad de Ciencias Biológicas y Ambientales,Universidad de León, León, Spain

28.1  Introduction (scope of the review) Antibiotics, the so-called ‘miracle drugs’, came to existence half a century ago, but over the last decade, the efficiency of antibiotics is dropping due to the overuse and growth of pathogen resistance; moreover, unraveling this resistance is not straightforward since antibiotic resistance is actually produced through multiple ways. Considering the urgency of the issue, efforts to develop new antibiotics are being carried out by pharmaceutical companies (Saleem et  al., 2010). Cyanobacteria, being one of the oldest known organisms inhabiting the earth with unique structural features, produce various compounds with diverse biological activities. These oxygen-producing photosynthetic microorganisms, which have been maintaining the oxygen levels on the earth for ca. 3.5 billion years, cytologically resemble Gram-negative bacteria despite their photoautotrophic physiology. They contain water-soluble red and blue phycobiliproteins and chlorophyll a in addition to the photosystems I and II (Nowruzi et al., 2012a). The adaptation mechanisms shown by cyanobacteria allows them to survive in severe climate conditions and tolerate limiting factors such as heat, drought, salinity, nitrogen starvation, cold, photo-oxidation, osmotic and ultraviolet stress (Moghadam and Nowruzi, 2008; Wase and Wright, 2008; Nowruzi and Moghadam, 2006). Certain molecules such as the anti-microtubule agents curacin A and dolastatin 10, have gone through preclinical and/or clinical trials as potential anticancer drugs (Tan, 2007). Cyanobacterial natural products can be classified according to different structural typologies including peptides, polyketides, alkaloids, lipids, and terpenes (Dittmann et al., 2015). Besides, each cyanobacterial strain produces a category of bioactive compounds, so that new drugs are being constantly discovery from these sources (Nowruzi et al., 2018a). Cyanobacteria are also known to produce toxins (Nowruzi et al., 2012c, 2013b), particularly hepatotoxins and neurotoxins (Wase and Wright, 2008), which act either as blockers (e.g., kalkitoxin and jamaicamide A) or activators (e.g., antillatoxin) of the eukaryotic voltage-gated sodium (Nav) channels and, besides their potential analgesics and neuroprotectant features, they are useful molecular tools to characterize functionally Nav channels (Tan, 2007). Over the past 30 years, many large pharmaceutical companies have reduced the use of natural products and drug discovery screening due to (a) problems related to strain availability, (b) difficulties related to natural products chemistry (e.g., substantial slower working paces) and (c) concerns about intellectual property rights (Harvey, 2008; Lam, 2007; Singh and Barrett, 2006; Baker et al., 2007; Rishton, 2008). Finally, the use of compound collections prepared by combinatorial chemistry methods has been also influential but not very successful in delivering new drug in significant numbers (Molinski et al., 2009).

28.2  Improving access to natural products as biomedical compounds It is now evident that the chemical diversity of natural products is a better option than the variety of available synthetic compounds for drug discovery (Grabowski and Schneider, 2007). Therefore, the use of natural chemical diversity is becoming increasingly frequent (Harvey, 2008). Early publications showed that only a small number of cyanobacteria taxa were * Current address: Laboratorio de diatomología y calidad de aguas, Instituto deInvestigación de Medio Ambiente, Recursos Naturales y Biodiversidad, León, Spain Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00028-0 © 2020 Elsevier Inc. All rights reserved.

441

442  PART | VII  Algal biomedicine

accessible for screening (Baker et al., 2007). Now, extensive cyanobacteria collections, together with better cyanobacteria culture techniques and new improvements in spectroscopy, analytical technologies and high-throughput screening (Jarvis, 2007), are providing new chemicals for use in drug discovery assays (Harvey, 2008) leading to advances in producing compound analogs and derivatives of natural products, with enhanced pharmacological or pharmaceutical characteristics (Sunazuka et al., 2008). Another interesting feature is their ability to be used as cores of compound (alkaloids, polyketides, terpenoids, and flavonoids) libraries produced through combinatorial techniques (Yao et al., 2007).

28.3  New approaches to the value of biomedical natural products With the advancements in analytical spectroscopy, many observations are now available so that the detection of new molecules requires only a few micrograms, a fraction of the amount needed only 10 years ago (Molinski et al., 2009). The progress in fractionation techniques used for isolating and purifying natural products (Yao et al., 2007), analytical structure determination (Harvey, 2007) has led to screening natural product mixtures with timescales suitable for high-throughput screening campaigns. Complex structures can be resolved now with much 1100 secondary metabolites identified, with complex chemical structures, reported from 39 different genera (Dittmann et al., 2015). Cyanobacterial compounds are mainly obtained from the Lyngbya, Symploca, Microcystis, Nostoc and Hapalosiphon (Dittmann et al., 2015; Williams, 2009) (Table 28.1).

28.6  Role of systems biology in cyanobacterial drug discovery and biomedical compounds After the successful outcome of the Human Genome Project in 2001 (Wolkenhauer, 2001), resulting investigations led to more integrative studies encompassing genomic, transcriptomic, proteomic, and metabolite behavior aspects (Wase and Wright, 2008). Systems biology which is defined as the study of the correlation between different interacting or cooperating components of a living system such as cells, tissues and metabolic pathways, can help us with gaining awareness on the ways living systems operate using computational power (Kitano, 2002). In order to study some particular phenomenon in a specific biosynthetic pathway, some information about both the responsible gene and the proteins in charge of that occurrence is needed. The function of linking the chemical diversity of natural products and genomes in addition to modeling and prediction by incorporating such biological information could provide substantial knowledge for comprehension of such complicated biological system (Wase and Wright, 2008). According to the comprehensive microbial resource declaration the genome sequences of 89 human pathogenic bacteria, non-homologous in humans, have been recognized. This could be a suitable method for the identification of new drug targets (Sakharkar et al., 2004) and the developments in synthetic biology now supply a solution to cyanobacteria being refractory to genetic manipulation, opening up cyanobacteria as a rich source of new natural products and novel enzymes.

28.7  Model for systems biology based cyanobacterial drug discovery Pharmaceutical industry is now focused on high throughput screening systems, bioinformatics and genomics tools, including rational design and combinatorial chemistry for the identification of novel bioactive compounds (Singh et al., 2011). Identifying sets of secondary metabolite biosynthetic gene clusters with potential therapeutic efficiency intervention is a primary step, which is guided by the chemical structure of the known compounds in cyanobacteria (Dittmann et al., 2015). Sequence data, as raw material for in silico drug target discovery, can be either a protein or a nucleic acid sequence (Wase and Wright, 2008). Software packages such as GENEMARK, GLIMMER, fgenesB (Oliynyk et  al., 2007) (Pattern/Markov chain-based bacterial operon and gene prediction), BPROM (Budde et al., 2007) (Prediction of bacterial promoters), and FindTerm (Wagschal et al., 2007) (Finding Terminators in bacterial genomes) are commonly used in this regard. Cyanobacteria showing positive screening outcomes are then selected for genomic characterization and proteome mining in order to identify biosynthetic gene clusters responsible for biologically active secondary metabolites, and the proteins associated to the production of these active components (Wase and Wright, 2008). This is possible because databases of cyanobacterial chemicals and biosynthetic gene clusters have been collected during decades through laborious screening of gene libraries (e.g., National Cancer Database, DrugBank, NCBI, Pubchem, ChemIDPlus, ANTIMIC, minimizing the pace for drug discovery by both reducing the number of compounds used in real screens and the costs of screening (Wase and Wright, 2008). Recently, genomic projects have been implemented aiming at locating biosynthetic pathways of natural

444  PART | VII  Algal biomedicine

TABLE 28.1  Current status of potential cyanobacteria therapeutics. Cyanobacterial compounds (class)

Cyanobacterial strain

Apratoxin A

Biological target

Potential therapeutic uses

References

Lyngbya bouillonii

STAT3, KB and LoVo cell lines cytotoxic against human tumor cell lines (0.36–0.52 nM)

Oncology, early stage adenocarcinoma (induction of G-1 phase cell cycle arrest)

Williams (2009) and Singh et al. (2011)

Apratoxin D

Lyngbya sp.

Antiproliferative

Oncology

Gutierrez et al. (2008)

Coibamide A

Leptolyngbya

Antiproliferative

Oncology

Medina et al. (2008)

Curacin A-D

Lyngbya majuscula 19L

Colon, renal and breast cancer cell lines. Involvement of HMGCoA in formation of cyclopropyl ring

Oncology, antimitotic, inhibits microtubule assembly, antiinflammatory, antiproliferative, immunosuppressant, herbicidal

Williams (2009), Tan (2007), Singh et al. (2011), Edwards and Gerwick (2004), and Burja et al. (2001)

Cryptophycin

Nostoc sp.

Tubulin polymerization antiproliferative and antimitotic agents, cytotoxicity against human tumor cell lines and human solid tumors

Oncology, destabilization of microtubule dynamics and the induction of hyperphosphorylation of the anti-apoptotic protein B-cell leukemia/lymphoma 2 (BCl-2), triggering programmed cell death

Williams (2009), Lu et al. (2001), and Mozzachiodi et al. (2001)

Largazole

Symploca sp.

Histone deacetylase

Oncology, anti-epileptics, neurological disorders, mood stabilizer

Taori et al. (2008)

Microcystin

Microcystis aeruginosa PCC 7806, M aeruginosa K-139, Planktothrix agardhii CYA126

Lymphocytes

Cytotoxic, inhibit membranebound leucine aminopeptidase enzyme inhibitor, cytotoxic, tumor promoter, anticancer

Christiansen et al. (2003), Becker et al. (2004), and Nowruzi et al. (2013b)

Hassallidins

Anabaena sp. SYKE 748A

Antifungal activity

Neuhof et al. (2006)

Sulfoglycolipid

Scytonema sp.

HIV-1

Inhibit reverse transcriptase and DNA polymerases

Singh et al. (2011)

Dolastatin 10

Symploca sp.

Binds to tubulin on rhizoxin-binding site

Affects microtubule assembly in P388 lymphocytic leukemia cell line (NCI)

Horgen et al. (2002) and Luesch et al. (2002)

Dolastatin 15

Lyngbya sp.

Binds to vinca alkaloid site of tubulin

Breast cancers treatment

Sato et al. (2007)

Jamaicamides (A-C)

L. majuscula

H-460 human lung cell carcinoma, neuro-2Aneuroblastoma cell line

Neurotoxic, cytotoxic against H-460 human lung and neuro-2a mouse neuroblastoma cell lines

Lautru and Challis (2004)

Kalkitoxin

L. majuscula

Block voltage sensitive Na+ channel

Neurotoxic, neural necrosis through N-methyl-d-asparate receptor mechanisms

LePage et al. (2005)

Astaxanthin

Haematococcus pluvialis

Colon cancer cell lines

Expression decrease of cyclin D1, increase of p53 and some cyclin kinase inhibitors (p21WAF-1/CIP-1, p27)

Palozza et al. (2009)

Polysaccharide

Navicula directa

HSV1, 2, influenza A virus

Inhibition of hyaluronidase

Guedes et al. (2011)

Hectochlorin

L. majuscula

Colon, melanoma, ovarian

Actin binding compounds

Marquez et al. (2002)

Pheophorbide a-, b-like compounds

Dunaliella primolecta

HSV1

Inhibition of cytopathic effect

Arun and Singh (2016)



Applications of cyanobacteria in biomedicine Chapter | 28  445

products. Here, biosynthetic gene clusters are essayed in microbial genomes as surrogate hosts and subsequently the encoded peptidic secondary metabolites are expressed in order to predict the biosynthetic pathways (Leikoski et al., 2010). Eventually, these modifications all intensify the pharmacokinetic attributes and supply essential initial information concerning the structure-activity relationships (SARs) of the leads (Williams, 2009).

28.8  Identification of drug targets A couple of probable targets in microbial cells are surface-exposed adhesions, cell-wall polypeptides, and membranebound enzymes (Saleem et al., 2010). These drug targets can be explored identifying virulence-related genes in the pathogen via DNA microarrays, which should include ‘pathogenicity islands’, genes related to virulence factors and multi-drug transporters (Palka-Santini et al., 2007). Virulence genes in pathogenic bacteria producing adhesions, toxins or virulence exist on plasmids or bacteriophages and in ‘pathogenicity islands’ (specific regions in the bacterial chromosomes), especially in pathogenic strains of either Gram-negative or Gram-positive bacteria (Wase and Wright, 2008). The analogs enzymes present in parasitic or free-living organisms with small genomes are another potential drug target. Inhibiting bacterial multi-drug transporters could be very useful since these export antibiotic molecules causing antibiotic resistance. Protein-based approaches are also essential because proteins are way closer to the biological function than mRNA molecules (Wase and Wright, 2008). For instance, protein synthesis is one of the best antibacterial targets, and studies in this area have led to the development of a number of highly successful clinical drugs. The final step would be a virtual screening to identify cyanobacterial molecules and then docking the binding site of cyanobacterial organic molecule or the biologically active sites so that potential targets can be detected. This screening could be carried out via commercially available conventional databases: whereas the Dictionary of Natural Products provides structural information on 150,000 different compounds, these should be physically available to be tested for any predicted activity (Harvey, 2008).

28.9  Virtual screening Drug discovery involves both random screening and rational design, that is, the use of data from previous publications for drug design and the subsequent analysis of the correlation between structure and properties. In practice, the lack of experimental data on this subject makes this approach unviable, despite the emergence of large information sources such as Human Genome Project and the Online Mendelian Inheritance in Man (OMIM), which provide data about the molecular basis of several diseases (Wase and Wright, 2008). In summary, the main problems involved in drug discovery are the identification of potential active compounds against specific targets, and its optimization to obtain specific physicochemical properties such as enhanced efficacy, pharmacokinetics or toxicological profiles (Wase and Wright, 2008; Nowruzi et al., 2012b). High-throughput screening Facilities used for in labs cannot always be beneficial and cost effective for very large collections of compounds so using an in silico approach makes more sense (Stockwell, 2004). Virtual screening, which is a very economical and productive technology, is used for high-throughput screening (thanks to the support of computational analysis) to choose appropriate compounds for specific receptor molecules. In virtual screening, compound selection is based either on (a) the bidimensional properties of the compound, (b) target-specific pharmacophores, or (c) three-dimensional modeling such as receptor-ligand docking (Singh and Barrett, 2006). In order to combine the benefits of virtual screening of natural products (and their combinatorial analogs) with a ready access to physical samples for testing, the Drug Discovery Portal (see http://www.ddp.strath.ac.uk/) has gathered a broad range of compounds from various laboratories in a unique database which is free to access (Harvey, 2008).

28.10  Cyanobacteria involvement in production of biomedical metabolites The natural products used for testing various biological activities were isolated from a broad variety of taxa including the cyanobacteria which are shown to be a novel and rich source of bioactive compounds (Nowruzi and Blanco, 2019; Burja et al., 2001; Tan, 2007). Multiple bioactive compounds extracted from cyanobacteria showed new and diverse activities in addition to the various chemical structures such as peptides (lipopeptides, cyclic peptides and cyclic depsipeptides), polyketides, alkaloids, terpenes, fatty acids, amides, carbohydrates and other organic chemicals (Burja et al., 2001; Ramaswamy et al., 2006; Tan, 2007; Medina et al., 2008; Tidgewell et al., 2010). These compounds are applicable factors in agriculture (Biondi et al., 2004), industry (Rastogi and Sinha, 2009), drug discovery and pharmacy (Tan, 2007). The complex structure of cyanobacterial bioactive compounds makes them difficult to be produced synthetically (Kaushik and Chauhan, 2008).

446  PART | VII  Algal biomedicine

Cyanobacterial chemical diversity can be compared to those of Actinomycetes which have been used in many crucial drugs. Many different chemo types produced by a single species is not uncommon. Lyngbya majuscule is a proper example because of the incredible diversity of structures found in this ubiquitous filamentous cyanobacterium. Amino acids, alkaloids, amides, fatty acids, lipopeptides (cyclic or linear) and multiple other compounds are isolated from this strains (McPhail et al., 2007; Burja et al., 2001; Jones et al., 2009, 2010; Tan et al., 2008). The microbial diversity of cyanobacteria, broad range of chemical and biologically active compounds with antimicrobial, anticancer, antiviral, immunosuppressant, insecticidal and anti-inflammatory activities has made cyanobacteria an attractive source of novel drugs for use in diverse therapeutic areas and progression in drug discovery (Nowruzi et al., 2012b; Wase and Wright, 2008; Luesch et al., 2002; Tan, 2006) (Table 28.2).

TABLE 28.2  Bioactive compounds from cyanobacteria. Species of cyanobacteria

Bioactive compounds

Biological activity

Class

References

Lyngbya lagerheimii, Phormidium tenue

Sulfolipid

Anti HIV-1 activity

Fatty acid (sulfo)

Skulberg (2000)

Lyngbya majuscula 19L

Barbamide

Antimolluscicidal

Chlorinated lipopeptide

Chang (2004)

L. majuscula

Antillatoxin B

Neurotoxic Ichthyotoxic, activator of voltage-gated sodium channel

Cyclic lipopeptide

Yokokawa et al. (2000)

Synechocystis trididemni

Didemnin

Anticancer, antiviral, immunosuppressive

Lipopeptide

Mitchell et al. (2000)

Cylindrospermum licheniforme

Cylindrocyclophane

Anticancer, cytotoxic

Alkaloid, macrocycle, chloro

Nakamura (2012) and Burja et al. (2001)

Cylindrospermopsis raciborskii

Cylindrospermopsin

Cytotoxic

Alkaloid

Li et al. (2001)

Prochloron didemni

Patellamide A, B, C and D

Cytotoxic, biological activity against multi-drug resistant UO-31 renal cell lines

Cyclic lipopeptide

Schmidt et al. (2005) and Ramaswamy et al. (2006)

L. majuscula

Lyngbyabellins A and B,

Cytotoxic, anticancer, cytoskeleton disruption

Lipopeptides

Yokokawa et al. (2002) and Milligan (2000)

L. majuscula

Antillatoxin B

Neurotoxic ichthyotoxic, activator of voltage-gated sodium channel, with sodium channel-activating

Lipopeptide

Yokokawa et al. (2002) and Tan (2007)

L. semiplena

Semiplenamides A-G

All displayed weak to moderate toxicity in brine shrimp assay; and 38 showed weak affinity for the rat cannabinoid CB1 receptor; showed moderate inhibitor of anandamide membrane transporter

Lipopeptide

Tan (2007)

N. ellipsosporum

Cyanovirin

Anti-HIV, antiviral HIV1 (interacts with high mannose groups of envelope glycoproteins, gp120 and blocks its interaction with target cell receptors) HIV-2 HSV-6 Mesles virus SIV FIV

Peptide and proteins

Singh et al. (2011)

Applications of cyanobacteria in biomedicine Chapter | 28  447



TABLE 28.2  Bioactive compounds from cyanobacteria—cont’d Species of cyanobacteria

Bioactive compounds

Biological activity

Class

References

P. tenue

Monogalactopyranosyl glycerol digalactopyranosyl glycerol

Anti-HIV, anticancer

Sulfolipids

Hayashi (2006)

Calothrix sp.

Calothrixin

Antimalarial, anticancer against HeLa epithelial carcinoma

Indoles

Vijayakumar and Menakha (2015)

Symploca hydnoides, Symploca sp. VP453

Symplostatin 1 Symplostatin 3

Against Murine colon 38 and murine mammary 16/C cell lines against microtubule depolymerization

Analog of dolastatin 10

Luesch et al. (2002)

Lyngbya majuscula

Lyngbyatoxins A-C

Cytotoxic, ichthyotoxic, tumor promoter, protein kinase C activator, skin irritant

L. majuscula

Hectochlorin

Against colon, melanoma, ovarian and renal and lung cancer cell lines, promote actin polymerization

L. majuscula

Hermitamides A and B

Ichthyotoxic, brine shrimp toxicity, cytotoxic

Lyngbya majuscula

Somocystinamide A

Cytotoxic against neuro2a neuroblastoma cells (IC50 = 1.4 lg mL−1), pluripotent inhibitor of angiogenesis and tumor cell proliferation, Induces apoptosis in endothelial cells.

Lipopeptide

Wrasidlo et al. (2008) and Tan (2007)

Oscillatoria nigroviridis

Oscillatoxin

Anticancer, toxic general

Aromaic

Burja et al. (2001)

Microcystis aeruginosa

Microviridin

Antibiotic, anticancer

Tricyclic depsipeptides

Han et al. (2006) and SoriaMercado et al. (2009)

L. majuscula

Hermitamides AeB

Against lung cancer, potent blockers of the hNav1.2 voltage-gated sodium channel., Ichthyotoxic

Lipopeptide

Burja et al. (2001)

L. majuscula

Lyngbyatoxin A-C

Ichthyotoxic, cytotoxic, tumor promoter, protein kinase C activator, skin irritant

Burja et al. (2001)

Caulerpa taxifolia, Green algae

Caulerpenyne

Cytotoxicity; anticancer, antitumor, and antiproliferative activities

Li and Tius (2002)

L. majuscula

Carmabin A-B

Anticancer, antiproliferative

N-Methylated peptide

Burja et al. (2001)

N. linckia and N. spongiaeforme var. tenue

Boromycin

Cytotoxicity against human epidermoid carcinoma (LoVo), human colorectal adenocarcinoma activity, potent cytotoxicity against drug-resistant murine and human solid tumors

Polyether-macrolide antibiotic

Vijayakumar and Menakha (2015)

Burja et al. (2001) and Wase and Wright (2008) Lipopeptide

Marquez et al. (2002) and Tan (2007) Burja et al. (2001)

Continued

448  PART | VII  Algal biomedicine

TABLE 28.2  Bioactive compounds from cyanobacteria—cont’d Species of cyanobacteria

Bioactive compounds

Biological activity

L. majuscula

Microcolin A-C

Antiproliferative, anticancer, cytotoxic, immunosuppressive

L. majuscula

Malyngamide A-U

Antimicrobial, antifeedant, cytotoxic, immunosuppressive

Lipopeptide

Burja et al. (2001)

L. majuscula

Pitiamide A-B

Antifeedant

Fatty acid amides

Osborne et al. (2001)

L. majuscula

Yanucamides A and B

Brine shrimp toxicity

Depsipeptides

Burja et al. (2001)

Nodularia spumigena

Nodularia toxin

Enzyme inhibition

Lipopeptide

Wase and Wright (2008)

Aulosira fertilissima

Aulosirazole

Anticancer

Aromatic

Bernardo et al. (2004)

Oscillatoria acutissima

Acutiphycin and 20, 21-didehydroacutiphycin

Antineoplastic agent

Lipopeptide

Burja et al. (2001)

Class

References McPhail et al. (2007)

Spirulan and Ca-spirulan derived from Spirulina sp. are regarded as the most notable antiviral polysaccharide compounds provided their broad-spectrum activity against HIV-1, HIV-2, H, influenza and other enveloped viruses. Another interesting compound is nostoflan from Nostoc flagelliforme, an acidic polysaccharide showing potent viricidal activity against herpes simplex virus-1 (Singh et al., 2011). Ichthyopeptins A and B, derived from Microcystis ichthyoblabe, are potential agents against the influenza a virus, with an IC50 value of 12.5 mg mL−1 (Zainuddin et al., 2007). Cyanovirin-N and scytovirin are also potent viricidal drugs that interfere several steps of viral fusion process. Cyanovirin-N, for example, shows both in vitro and in vivo activity against HIV and other lentiviruses in nanomolar concentrations (Bokesch et al., 2003). Natural cyanobacterial sulfoglycolipids show confirmed HIV-reverse transcriptase and DNA polymerase inhibitory effects (Singh et al., 2011). New attempts to find antibacterial activity via screening of cyanobacterial extracts have started (Biondi et al., 2004), although very few cyanobacteria-related antibacterial compounds have been detected to date. Noscomin57, from Nostoc commune. An antibacterial activity of Anabaena extracts against vancomycin-resistant S. aureus with a MIC of 32–64 mg mL−1 has been reported by (Bhateja et al., 2006). In a recent project operated by the Panamanian International Co-operative Biodiversity Group, five classes of antiprotozoal compounds were isolated from Cyanobacteria. Nostocarboline showed activity against Trypanosoma brucei, T. cruzi, Leishmania donovani and Plasmodium falciparum with IC50 values ranging from 0.5 to 0.194 mM (Barbaras et al., 2008). The aerucyclamide C68 isolated from Microcystis aeruginosa PCC 7806 has been also found to be active against T. brucei (Simmons et al., 2008). More than 120 cyanobacterial alkaloids with various biological activities were introduced. Some of these compounds, such as microginins (used for the treatment of high blood pressure), aeruginosins and cyanopeptolins (a serine inhibitor used for asthma and viral infections) are described (Singh et al., 2011). Kempopeptin B, kempopeptin A and chymotrypsin are other group of protease inhibitory products (Taori et al., 2008; Liu et al., 2014). The immunomodulatory activity of cyanobacteria exhibits diverse effects on immune systems, such as the increase of phagocytic activity in macrophages, the stimulation of antibody and cytokine production and the accumulation of natural killer cells into tissues, or the activation of T and B cells (Singh et al., 2011). Shen et al. (2003) demonstrated the immunotoxicity of a cyanobacterial bloom extract containing microcystin that showed clinical efficiency in the lipopolysaccharideinduced lymph proliferation, together with a dose-dependent reduction in the amount of antibody-forming cells in mice vaccinated with sheep T-dependent antigen red blood cells, thus showing immunosuppression. A considerable number of highly active cyanobacterial compounds target tubulin or actin filaments in eukaryotic cells and have exhibited potent antimitotic properties, which makes them a noteworthy source of potential anticancer agents (Tan, 2007).



Applications of cyanobacteria in biomedicine Chapter | 28  449

Coibamide A, extracted from a Leptolyngbya strain, shows a novedous action mechanism targeting tubulin or actin filaments. Notable cytotoxical properties against breast, central nervous system, colon and ovary cancers has been observed (Medina et al., 2008). Cryptophycins are examples of cyanobacteria-derived tubulin-binding compounds with potent anticancer activity. Cryptophycin A was first isolated from Nostoc sp. strains ATCC 53789 and GSV224 (Molinski et al., 2009). Microtubule dynamics suppression and blocking of G2/M phases are features making this molecule a potent anti-carcinoma metabolite (Singh et al., 2011). Cryptophycins 249 and 309 show better water solubility and stability. According to a study by (Becker et al., 2004) the thioesterase derived from the cryptophycin biosynthetic pathway through the macrocyclization of a series of linear synthetic forerunners generating 16-membered cyclic depsipeptides, showed significant efficiency as anticancer agents (Nowruzi et al., 2018b). Largazole is extracted from Symploca sp. (Taori et al., 2008) and shows a considerable histone deacetylase (HDAC) inhibitory activity (Ying et al., 2008). The FDA ratification of HDAC inhibitor suberoylanilide hydroxamic acid as a treatment for dermal T-cell lymphomas, besides its mood stability properties and anti-epileptic characteristics, confirms this compound for cancer treatment (Marks and Breslow, 2007). Apratoxins, were initially isolated from a chemically rich Lyngbya boulloni strain of and demonstrated a unique action pattern against a panel of 60 cancer cell lines (Shoemaker, 2006). Limited findings until now indicate that the induction of G1-phase cell-cycle arrest and apoptosis is how Apratoxins function as anticancer agents (Luesch et al., 2002). Apratoxin A showed moderate cytotoxicity in multiple human tumor cell lines (e.g., lovo cell lines and KB cancer cells). Other analogs, especially Apratoxin D, have been studied in order to develop a lead structure (Tan, 2007). A group of cyclic glycosylated lipopeptide cyanobacteria metabolites are the Hassallidins A (Neuhof et al., 2005) and B (Neuhof et al., 2006) which are isolated from cyanobacterium of the genus Hassallia; Hassallidins are a type of broadspectrum compounds with activity against opportunistic human pathogenic fungi. Because of their extra hydrophilic unit, they also have high water solubility; a feature that makes them a very interesting basis for new antifungal drugs (Saleem et al., 2010).

28.11 Conclusions In conclusion, the results in this review highlighted the importance of potential therapeutic activities of natural products isolated from cyanobacteria such as antitumor, antibacterial and antiviral effects and protease inhibition activity, and emphasized the need to continue to explore natural sources, using faster and cheaper techniques such as virtual screening and system biology for metabolite purification, characterization and assessment in cyanobacterial compounds that have not entered the clinical trials yet. Additionally, cyanobacteria are economically beneficial microbial source for drug discovery because their cultivation requires simple inorganic nutrients for growth. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c, d,e,f,g,h,i,j,k,l,m,n,o).

References Arun, N., Singh, D.P., 2016. A review on pharmacological applications of halophilic alga Dunaliella. Indian J. Mar. Sci. 45 (3), 440–447. Baker, D.D., Chu, M., Oza, U., Rajgarhia, V., 2007. The value of natural products to future pharmaceutical discovery. Nat. Prod. Rep. 24 (6), 1225–1244. Barbaras, D., Kaiser, M., Brun, R., Gademann, K., 2008. Potent and selective antiplasmodial activity of the cyanobacterial alkaloid nostocarboline and its dimers. Bioorg. Med. Chem. Lett. 18 (15), 4413–4415. Becker, J.E., Moore, R.E., Moore, B.S., 2004. Cloning, sequencing, and biochemical characterization of the nostocyclopeptide biosynthetic gene cluster: molecular basis for imine macrocyclization. Gene 325, 35–42. Bernardo, P.H., Chai, C.L., Heath, G.A., Mahon, P.J., Smith, G.D., Waring, P., et al., 2004. Synthesis, electrochemistry, and bioactivity of the cyanobacterial calothrixins and related quinones. J. Med. Chem. 47 (20), 4958–4963. Bhateja, P., Mathur, T., Pandya, M., Fatma, T., Rattan, A., 2006. Activity of blue green microalgae extracts against in vitro generated Staphylococcus aureus with reduced susceptibility to vancomycin. Fitoterapia 77 (3), 233–235. Biondi, N., Piccardi, R., Margheri, M.C., Rodolfi, L., Smith, G.D., Tredici, M.R., 2004. Evaluation of Nostoc strain ATCC 53789 as a potential source of natural pesticides. Appl. Environ. Microbiol. 70 (6), 3313–3320. Bokesch, H.R., O’Keefe, B.R., McKee, T.C., Pannell, L.K., Patterson, G.M., Gardella, R.S., et al., 2003. A potent novel anti-HIV protein from the cultured cyanobacterium Scytonema varium. Biochemistry 42 (9), 2578–2584. Budde, P.P., Davis, B.M., Yuan, J., Waldor, M.K., 2007. Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae. J. Bacteriol. 189 (2), 491–500. Burja, A.M., Banaigs, B., Abou-Mansour, E., Burgess, J.G., Wright, P.C., 2001. Marine cyanobacteria—a prolific source of natural products. Tetrahedron 57 (46), 9347–9377.

450  PART | VII  Algal biomedicine

Chang, Z., Sitachitta, N., Rossi, J.V., Roberts, M.A., Flatt, P.M., Jia, J., et al., 2004. Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 67 (8), 1356–1367. Christiansen, G., Fastner, J., Erhard, M., Borner, T., Dittmann, E., 2003. Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J. Bacteriol. 185 (2), 564–572. Dittmann, E., Gugger, M., Sivonen, K., Fewer, D.P., 2015. Natural product biosynthetic diversity and comparative genomics of the cyanobacteria. Trends Microbiol. 23 (10), 642–652. Edwards, D.J., Gerwick, W.H., 2004. Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase. J. Am. Chem. Soc. 126 (37), 11432–11433. Freiberg, C., Brotz-Oesterhelt, H., Labischinski, J., 2004. The impact of transcriptome and proteome analyses on antibiotic drug discovery. Curr. Opin. Microbiol. 7 (5), 451–459. Grabowski, K., Schneider, G., 2007. Properties and architecture of drugs and natural products revisited. Curr. Chem. Biol. 1 (1), 115–127. Guedes, A.C., Amaro, H.M., Malcata, F.X., 2011. Microalgae as sources of high added-value compounds—a brief review of recent work. Biotechnol. Prog. 27 (3), 597–613. Gutierrez, M., Suyama, T.L., Engene, N., Wingerd, J.S., Matainaho, T., Gerwick, W.H., 2008. Apratoxin D, a potent cytotoxic cyclodepsipeptide from Papua New Guinea collections of the marine cyanobacteria Lyngbya majuscula and Lyngbya sordida. J. Nat. Prod. 71 (6), 1099–1103. Han, B., Gross, H., Goeger, D.E., Mooberry, S.L., Gerwick, W.H., 2006. Aurilides B and C, cancer cell toxins from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 60 (4), 572–575. Harvey, A.L., 2007. Natural products as a screening resource. Curr. Opin. Chem. Biol. 11 (5), 480–484. Harvey, A.L., 2008. Natural products in drug discovery. Drug Discov. Today 13 (19–20), 894–901. Hayashi, O., Ono, S., Ishii, K., Shi, Y., Hirahashi, T., Katoh, T., 2006. Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice. J. Appl. Phycol. 18 (1), 47–56. Horgen, F.D., Kazmierski, E.B., Westenburg, H.E., Yoshida, W.Y., Scheuer, P.J., 2002. Malevamide D: isolation and structure determination of an isodolastatin H analogue from the marine cyanobacterium Symploca hydnoides. J. Nat. Prod. 65 (4), 487–491. Ishida, K., Welker, M., Christiansen, G., Cadel-Six, S., Bouchier, C., Dittmann, E., et al., 2009. Plasticity and evolution of aeruginosin biosynthesis in cyanobacteria. Appl. Environ. Microbiol. 75 (7), 2017–2026. Jarvis, L.M., 2007. Liquid Gold Mine: scientists in Norway are plumbing the seas for the next blockbuster medicine. Chem. Eng. News 85 (41), 22–28. Jones, A.C., Gerwick, L., Gonzalez, D., Dorrestein, P.C., Gerwick, W.H., 2009. Transcriptional analysis of the jamaicamide gene cluster from the marine cyanobacterium Lyngbya majuscula and identification of possible regulatory proteins. BMC Microbiol. 9, 247. Jones, A.C., Monroe, E.A., Eisman, E.B., Gerwick, L., Sherman, D.H., Gerwick, W.H., 2010. The unique mechanistic transformations involved in the biosynthesis of modular natural products from marine cyanobacteria. Nat. Prod. Rep. 27 (7), 1048–1065. Kaushik, P., Chauhan, A., 2008. In vitro antibacterial activity of laboratory grown culture of Spirulina platensis. Indian J. Microbiol. 48 (3), 348–352. Kennedy, J., 2008. Mutasynthesis, chemobiosynthesis, and back to semi-synthesis: combining synthetic chemistry and biosynthetic engineering for diversifying natural products. Nat. Prod. Rep. 25 (1), 25–34. Kitano, H., 2002. Systems biology: a brief overview. Science 295 (5560), 1662–1664. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.



Applications of cyanobacteria in biomedicine Chapter | 28  451

Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Kopp, F., Marahiel, M.A., 2007. Where chemistry meets biology: the chemoenzymatic synthesis of nonribosomal peptides and polyketides. Curr. Opin. Biotechnol. 18 (6), 513–520. Lam, K.S., 2007. New aspects of natural products in drug discovery. Trends Microbiol. 15 (6), 279–289. Lautru, S., Challis, G.L., 2004. Substrate recognition by nonribosomal peptide synthetase multi-enzymes. Microbiology 150 (6), 1629–1636. Leikoski, N., Fewer, D.P., Jokela, J., Wahlsten, M., Rouhiainen, L., Sivonen, K., 2010. Highly diverse cyanobactins in strains of the genus Anabaena. Appl. Environ. Microbiol. 76 (3), 701–709. LePage, K.T., Goeger, D., Yokokawa, F., Asano, T., Shioiri, T., Gerwick, W.H., et al., 2005. The neurotoxic lipopeptide kalkitoxin interacts with voltagesensitive sodium channels in cerebellar granule neurons. Toxicol. Lett. 158 (2), 133–139. Li, L.H., Tius, M.A., 2002. Stereospecific synthesis of cryptophycin 1. Org. Lett. 4 (10), 1637–1640. Li, R., Carmichael, W.W., Brittain, S., Eaglesham, G.K., Shaw, G.R., Mahakhant, A., et  al., 2001. Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria). Toxicon 39 (7), 973–980. Littleton, J., Rogers, T., Falcone, D., 2005. Novel approaches to plant drug discovery based on high throughput pharmacological screening and genetic manipulation. Life Sci. 78 (5), 467–475. Liu, L., Jokela, J., Wahlsten, M., Nowruzi, B., Permi, P., Zhang, Y.Z., et al., 2014. Nostosins, trypsin inhibitors isolated from the terrestrial cyanobacterium Nostoc sp. strain FSN. J. Nat. Prod. 77 (8), 1784–1790. Lu, K., Dempsey, J., Schultz, R.M., Shih, C., Teicher, B.A., 2001. Cryptophycin-induced hyperphosphorylation of Bcl-2, cell cycle arrest and growth inhibition in human H460 NSCLC cells. Cancer Chemother. Pharmacol. 47 (2), 170–178. Luesch, H., Yoshida, W.Y., Moore, R.E., Paul, V.J., Mooberry, S.L., Corbett, T.H., 2002. Symplostatin 3, a new dolastatin 10 analogue from the marine cyanobacterium Symploca sp. VP452. J. Nat. Prod. 65 (1), 16–20. Marks, P.A., Breslow, R., 2007. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat. Biotechnol. 25 (1), 84–90. Marquez, B.L., Watts, K.S., Yokochi, A., Roberts, M.A., Verdier-Pinard, P., Jimenez, J.I., et al., 2002. Structure and absolute stereochemistry of hectochlorin, a potent stimulator of actin assembly. J. Nat. Prod. 65 (6), 866–871. McAlpine, J.B., Bachmann, B.O., Piraee, M., Tremblay, S., Alarco, A.M., Zazopoulos, E., et al., 2005. Microbial genomics as a guide to drug discovery and structural elucidation: ECO-02301, a novel antifungal agent, as an example. J. Nat. Prod. 68 (4), 493–496. McPhail, K.L., Correa, J., Linington, R.G., Gonzalez, J., Ortega-Barria, E., Capson, T.L., et al., 2007. Antimalarial linear lipopeptides from a Panamanian strain of the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 70 (6), 984–988. Medina, R.A., Goeger, D.E., Hills, P., Mooberry, S.L., Huang, N., Romero, L.I., et al., 2008. Coibamide A, a potent antiproliferative cyclic depsipeptide from the Panamanian marine cyanobacterium Leptolyngbya sp. J. Am. Chem. Soc. 130 (20), 6324–6325. Milligan, K.E., Marquez, B.L., Williamson, R.T., Gerwick, W.H., 2000. Lyngbyabellin B, a toxic and antifungal secondary metabolite from the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 63 (10), 1440–1443. Mitchell, S.S., Faulkner, D.J., Rubins, K., Bushman, F.D., 2000. Dolastatin 3 and two novel cyclic peptides from a palauan collection of Lyngbya majuscula. J. Nat. Prod. 63 (2), 279–282. Moghadam, A.A., Nowruzi, B., 2008. A new report of N fixation by two species of cyanobacteria. Iran. J. Sci. Technol. Trans. A Sci. 32 (2), 147–151. Molinski, T.F., Dalisay, D.S., Lievens, S.L., Saludes, J.P., 2009. Drug development from marine natural products. Nat. Rev. Drug Discov. 8 (1), 69–85. Mozzachiodi, R., Scuri, R., Roberto, M., Brunelli, M., 2001. Caulerpenyne, a toxin from the seaweed Caulerpa taxifolia, depresses afterhyperpolarization in invertebrate neurons. Neuroscience 107 (3), 519–526. Nakamura, H., Hamer, H.A., Sirasani, G., Balskus, E.P., 2012. Cylindrocyclophane biosynthesis involves functionalization of an unactivated carbon center. J. Am. Chem. Soc. 134 (45), 18518–18521. Neuhof, T., Schmieder, P., Preussel, K., Dieckmann, R., Pham, H., Bartl, F., et al., 2005. Hassallidin A, a glycosylated lipopeptide with antifungal activity from the cyanobacterium Hassallia sp. J. Nat. Prod. 68 (5), 695–700. Neuhof, T., Schmieder, P., Seibold, M., Preussel, K., von Dohren, H., 2006. Hassallidin B—second antifungal member of the Hassallidin family. Bioorg. Med. Chem. Lett. 16 (16), 4220–4222. Newman, D.J., Hill, R.T., 2006. New drugs from marine microbes: the tide is turning. J. Ind. Microbiol. Biotechnol. 33 (7), 539–544. Nowruzi, B., Blanco, S., 2019. In silico identification and evolutionary analysis of candidate genes involved in the biosynthesis methylproline genes in cyanobacteria strains of Iran. Phytochem. Lett. 29, 199–211. Nowruzi, B., Moghadam, A.A., 2006. Two new records of heterocystus cyanobacteria (Nostocaceae) from paddy fields of Golestan Province. Iran. J. Bot. 11 (2), 169–173. Nowruzi, B., Khavari-Nejad, R.A., Sivonen, K., Kazemi, B., Najafi, F., Nejadsattari, T., 2012a. Phylogenetic and morphological evaluation of two species of Nostoc (Nostocales, Cyanobacteria) in certain physiological conditions. Afr. J. Agric. Res. 7 (27), 3887–3897. Nowruzi, B., Khavari-Nejad, R.A., Sivonen, K., Kazemi, B., Najafi, F., Nejadsattari, T., 2012b. A gene expression study on strains of Nostoc (Cyanobacteria) revealing antimicrobial activity under mixotrophic conditions. Afr. J. Biotechnol. 11 (51), 11296–11308. Nowruzi, B., Khavari-Nejad, R.A., Sivonen, K., Kazemi, B., Najafi, F., Nejadsattari, T., 2012c. Identification and toxigenic potential of a Nostoc sp. Algae 27 (4), 303–313.

452  PART | VII  Algal biomedicine

Nowruzi, B., Khavari-Nejad, R.A., Kazemi, B., Najafi, F., Nejadsattari, T., 2013a. Optimization of cultivation conditions to maximize extracellular investments of two Nostoc strains. Algol. Stud. 142 (2013), 63–76. Nowruzi, B., Khavari-Nejad, R.A., Sivonen, K., Kazemi, B., Najafi, F., Nejadsattari, T., 2013b. Identification and toxigenic potential of a cyanobacterial strain (Stigomena sp.). Prog. Biol. Sci. 3 (1), 79–85. Nowruzi, B., Fahimi, H., Ordodari, N., Assareh, R., 2017a. Genetic analysis of polyketide synthase and peptide synthase genes of cyanobacteria as a mining tool for new pharmaceutical compounds. J. Pharm. Health Sci. 5 (2), 139–150. Nowruzi, B., Fahimi, H., Ordodari, N., 2017b. Molecular phylogenetic and morphometric evaluation of Calothrix sp. N42 and Scytonema sp. N11. Rostaniha 18 (2), 210–221. Nowruzi, B., Haghighat, S., Fahimi, H., Mohammadi, E., 2018a. Nostoc cyanobacteria species: a new and rich source of novel bioactive compounds with pharmaceutical potential. J. Pharm. Health Serv. Res. 9 (1), 5–12. Nowruzi, B., Blanco, S., Nejadsattari, T., 2018b. Chemical and molecular evidences for the poisoning of a duck by anatoxin-a, nodularin and cryptophycin at the coast of Lake Shoormast (Mazandaran Province, Iran). Int. J. Algae 20 (4), 359–376. Nowruzi, B., Wahlsten, M., Jokela, J., 2019. A report on finding a new peptide aldehyde from cyanobacterium Nostoc sp. Bahar M by LC-MS and Marfey’s analysis. Iran. J. Biotechnol. 17 (2), 71–78. Oliynyk, M., Samborskyy, M., Lester, J.B., Mironenko, T., Scott, N., Dickens, S., et al., 2007. Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338. Nat. Biotechnol. 25 (4), 447–453. Osborne, N.J.T., Webb, P.M., Shaw, G.R., 2001. The toxins of Lyngbya majuscula and their human and ecological health effects. Environ. Int. 27 (5), 381–392. Palka-Santini, M., Putzfeld, S., Cleven, B.E., Kronke, M., Krut, O., 2007. Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing. J. Microbiol. Methods 68 (3), 468–477. Palozza, P., Torelli, C., Boninsegna, A., Simone, R., Catalano, A., Mele, M.C., et  al., 2009. Growth-inhibitory effects of the astaxanthin-rich alga Haematococcus pluvialis in human colon cancer cells. Cancer Lett. 283 (1), 108–117. Quinn, R.J., Carroll, A.R., Pham, N.B., Baron, P., Palframan, M.E., Suraweera, L., 2008. Developing a drug-like natural product library. J. Nat. Prod. 71 (3), 464–468. Ramaswamy, A.V., Flatt, P.M., Edwards, D.J., Simmons, T.L., Han, B., 2006. The secondary metabolites and biosynthetic gene clusters of marine cyanobacteria. Applications in biotechnology. In: Proksch, P., Muller, W.E.G. (Eds.), Frontiers in Marine Biotechnology. Horizon Bioscience, Norfolk, pp. 175–224. Rastogi, R.P., Sinha, R.P., 2009. Biotechnological and industrial significance of cyanobacterial secondary metabolites. Biotechnol. Adv. 27 (4), 521–539. Rishton, G.M., 2008. Natural products as a robust source of new drugs and drug leads: past successes and present day issues. Am. J. Cardiol. 101 (10A), 43D–49D. Sakharkar, K.R., Sakharkar, M.K., Chow, V.T., 2004. A novel genomics approach for the identification of drug targets in pathogens, with special reference to Pseudomonas aeruginosa. In Silico Biol. 4 (3), 355–360. Saleem, M., Nazir, M., Ali, M.S., Hussain, H., Lee, Y.S., Riaz, N., et al., 2010. Antimicrobial natural products: an update on future antibiotic drug candidates. Nat. Prod. Rep. 27 (2), 238–254. Sato, M., Sagawa, M., Nakazato, T., Ikeda, Y., Kizaki, M., 2007. A natural peptide, dolastatin 15, induces G2/M cell cycle arrest and apoptosis of human multiple myeloma cells. Int. J. Oncol. 30 (6), 1453–1459. Schmidt, E.W., Nelson, J.T., Rasko, D.A., Sudek, S., Eisen, J.A., Haygood, M.G., et al., 2005. Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. Proc. Natl. Acad. Sci. U.S.A. 102 (20), 7315–7320. Shen, P.P., Zhao, S.W., Zheng, W.J., Hua, Z.C., Shi, Q., Liu, Z.T., 2003. Effects of cyanobacteria bloom extract on some parameters of immune function in mice. Toxicol. Lett. 143 (1), 27–36. Shoemaker, R.H., 2006. The NCI60 human tumour cell line anticancer drug screen. Nat. Rev. Cancer 6 (10), 813–823. Simmons, T.L., Engene, N., Urena, L.D., Romero, L.I., Ortega-Barria, E., Gerwick, L., et al., 2008. Viridamides A and B, lipodepsipeptides with antiprotozoal activity from the marine cyanobacterium Oscillatoria nigro-viridis. J. Nat. Prod. 71 (9), 1544–1550. Singh, S.B., Barrett, J.F., 2006. Empirical antibacterial drug discovery—foundation in natural products. Biochem. Pharmacol. 71 (7), 1006–1015. Singh, R.K., Tiwari, S.P., Rai, A.K., Mohapatra, T.M., 2011. Cyanobacteria: an emerging source for drug discovery. J. Antibiot. (Tokyo) 64 (6), 401–412. Skulberg, O.M., 2000. Microalgae as a source of bioactive molecules–experience from cyanophyte research. J. Appl. Phycol. 12 (3–5), 341–348. Soria-Mercado, I.E., Pereira, A., Cao, Z., Murray, T.F., Gerwick, W.H., 2009. Alotamide A, a novel neuropharmacological agent from the marine cyanobacterium Lyngbya bouillonii. Org. Lett. 11 (20), 4704–4707. Stockwell, B.R., 2004. Exploring biology with small organic molecules. Nature 432 (7019), 846–854. Sunazuka, T., Hirose, T., Omura, S., 2008. Efficient total synthesis of novel bioactive microbial metabolites. Acc. Chem. Res. 41 (2), 302–314. Tan, L.T., 2006. Biomedical potential of marine cyanobacteria. J. Coast. Dev. 9 (3), 129–136. Tan, L.T., 2007. Bioactive natural products from marine cyanobacteria for drug discovery. Phytochemistry 68 (7), 954–979. Tan, L.T., Chang, Y.Y., Ashootosh, T., 2008. Besarhanamides A and B from the marine cyanobacterium Lyngbya majuscula. Phytochemistry 69 (10), 2067–2069. Taori, K., Paul, V.J., Luesch, H., 2008. Structure and activity of largazole, a potent antiproliferative agent from the Floridian marine cyanobacterium Symploca sp. J. Am. Chem. Soc. 130 (6), 1806–1807. Tidgewell, K., Clark, B.R., Gerwick, W.H., 2010. The natural products chemistry of cyanobacteria. In: Liu, H.-W.(.B.)., Mander, L. (Eds.), Comprehensive Natural Products Chemistry II. vol. 2. Elsevier, Amsterdam, pp. 141–188. Vijayakumar, S., Menakha, M., 2015. Pharmaceutical applications of cyanobacteria—a review. J. Acute Med. 5 (1), 15–23.



Applications of cyanobacteria in biomedicine Chapter | 28  453

Wagschal, K., Franqui-Villanueva, D., Lee, C., Kibblewhite, R., Robertson, G., Wong, D., 2007. Genetic and biochemical characterization of a novel bacterial A-L-Arabinofuranosidease. Enzym. Microb. Technol. 40, 747–753. Wase, N.V., Wright, P.C., 2008. Systems biology of cyanobacterial secondary metabolite production and its role in drug discovery. Expert Opin. Drug Discovery 3 (8), 903–928. Williams, P.G., 2009. Panning for chemical gold: marine bacteria as a source of new therapeutics. Trends Biotechnol. 27 (1), 45–52. Wolkenhauer, O., 2001. Systems biology: the reincarnation of systems theory applied in biology? Brief. Bioinform. 2 (3), 258–270. Wrasidlo, W., Mielgo, A., Torres, V.A., Barbero, S., Stoletov, K., Suyama, T.L., et al., 2008. The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8. Proc. Natl. Acad. Sci. U.S.A. 105 (7), 2313–2318. Yao, N., Song, A., Wang, X., Dixon, S., Lam, K.S., 2007. Synthesis of flavonoid analogues as scaffolds for natural product-based combinatorial libraries. J. Comb. Chem. 9 (4), 668–676. Ying, Y., Taori, K., Kim, H., Hong, J., Luesch, H., 2008. Total synthesis and molecular target of largazole, a histone deacetylase inhibitor. J. Am. Chem. Soc. 130 (26), 8455–8459. Yokokawa, F., Fujiwara, H., Shioiri, T., 2000. Total synthesis and revision of absolute stereochemistry of antillatoxin, an ichthyotoxic cyclic lipopeptide from marine cyanobacterium Lyngbya majuscule. Tetrahedron 56 (12), 1759–1775. Yokokawa, F., Sameshima, H., Katagiri, D., Aoyama, T., Shioiri, T., 2002. Total syntheses of lyngbyabellins A and B, potent cytotoxic lipopeptides from the marine cyanobacterium Lyngbya majuscule. Tetrahedron 58 (46), 9445–9458. Zainuddin, E.N., Mentel, R., Wray, V., Jansen, R., Nimtz, M., Lalk, M., et al., 2007. Cyclic depsipeptides, ichthyopeptins A and B, from Microcystis ichthyoblabe. J. Nat. Prod. 70 (7), 1084–1088.

Further reading Nowruzi, B., Khavari-Nejad, R.A., Nejadsattari, T., Sivonen, K., Fewer, D.P., 2016. A proposal for the unification of two cyanobacterial strains of Nostoc as the same species. Rostaniha 17 (2), 161–172.

Chapter 29

Health benefits of bioactive seaweed substances Yimin Qin State Key Laboratory of Bioactive Seaweed Substances, Qingdao, China

29.1 Introduction In recent years, the importance of marine biomass as a source of novel bioactive substances is growing rapidly. It is estimated that more than 20,000 new compounds have been isolated from marine biomass, with a wide range of applications from pharmaceutical products to functional foods (Hu et al., 2015). Seaweeds are important marine organisms and their commercial applications date back to ancient times. In China, where homology of medicine and food has been recognized for well over 2000 years, the health benefits of seaweeds were recognized in ancient medicinal books such as ‘Sheng Nong’s Herbal Classic’, ‘Supplementary Records of Famous Physicians’, ‘Marine Herbal’, ‘Compendium of Materia Medica’, etc. (Xia and Abbott, 1987). Modern science and technology have uncovered the many bioactive seaweed substances and allowed their separation and purification into high valued bio-products, which are now widely used in a wide range of health related industries (Zemke-White and Ohno, 1999; Das, 2015). Bioactive seaweed substances (BASS) are a group of chemical components extracted from seaweed biomass, which can influence the biological processes of living organisms through chemical, physical, biological and other mechanisms. These substances include biomass components in the extracellular matrix, cell wall, plasma and other parts of the seaweed cells generated through primary and secondary metabolism, of which, the primary metabolites are generated when the seaweed cells process nutrients through bio-degradation or bio-synthesis, such as amino acids, nucleotides, polysaccharides, lipids, vitamins, etc., while secondary metabolites are those chemicals modified from primary metabolites, including genetic materials, medicinal materials, biotoxins, functional materials and other seaweed based substances (Qin, 2018). Based on their roles in the seaweed cell, these many bioactive seaweed substances can be divided into structural components such as alginate, carrageenan and agar, physiological substances such as the many halogenated chemicals evolved as part of the defense mechanism for seaweeds, and metabolic compounds such as β-carotene, astaxanthin, eicosapentaenoic acid, etc. According to their chemical structures, they can be divided into polysaccharides, polypeptides, amino acids, lipids, sterols, terpenoids, glycosides, non-peptide nitrous compounds, enzymes, pigments, and other chemical species with defined chemical structures. According to their bioactivities, they include many substances with novel health benefits such as anti-tumor, immune regulation, blood sugar reduction, radiation resistance, reduction of hematic fat, anticoagulant, antithrombotic, anti-inflammatory, anti-allergic, antibacterial, antiviral, oxidation resistance, resistance to UV radiation, inhibition of enzyme activities, anti-aging, anti-HIV, deodorant, fatigue resistance and other functions (Nomura et al., 2013; Conde et al., 2015). This chapter summarizes the health benefits of the many types of bioactive seaweed substances in their applications in functional foods, nutraceuticals, drugs, cosmetics, biomedical materials and other health products.

29.2  Applications of bioactive seaweed substances in health products Seaweed biomass represents a vast amount of structurally diverse natural resources (Hill, 2012; Blunt et al., 2013). They are versatile products widely used for food in direct human consumption, also as ingredients for the global food and cosmetics industries, as fertilizers and as animal feed additives. Because of the many highly bioactive substances, seaweeds are now increasingly used as a source for drugs, nutraceuticals and other high valued products (Xu et al., 2004; Jimenez-Escrig et al., 2012; Murphy et al., 2014). Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00029-2 © 2020 Elsevier Inc. All rights reserved.

455

456  PART | VII  Algal biomedicine

Bioactive seaweed substances are valuable in a group of industries including pharmaceuticals, nutraceuticals, functional foods, biomedical materials, cosmetics, fertilizers, in addition to traditional applications in textiles, chemicals and environmental protection. In order to fully explore the commercial potential of seaweed biomass, new and advanced technologies need to be developed to separate seaweeds into their purified components. Once extracted and purified, the various types of bioactive seaweed substances can be screened for their bioactivities and utilized in appropriate applications. Fig. 29.1 illustrates the screening of bioactive seaweed substances for specific applications. In recent years, much attention has been paid to marine derived biomaterials for biological, biomedical, and environmental applications. Marine bioactive substances for healthcare are the most important and fastest growing sector among marine biomaterials. Seaweeds offer an abundant source of polysaccharides such as alginate, agar and carrageenan, of which, alginate has found numerous applications in biomedical science and engineering due to its favorable properties such as biocompatibility and ease of gelation. Alginate hydrogels have been particularly attractive in wound healing, drug delivery and tissue engineering applications, as these gels retain structural similarity to the extracellular matrices in tissues and can be manipulated to play several critical roles. Sodium alginate solutions can be extruded into aqueous calcium chloride bath to form calcium alginate fibers for the production of nonwoven wound dressings that possess unique gel forming capabilities when on contact with wound exudate. As shown in Fig. 29.2, nonwoven fabrics made from alginate fibers can form a soft hydrogel when calcium ions in the fiber exchange with sodium ions in the wound fluid, a property highly valuable in wound dressings, face masks, absorbent pads and other medical and hygiene materials (Qin, 2005, 2006, 2008, 2016).

29.3  Health benefits of bioactive seaweed substances The development of modern science and technology has allowed the separation of the individual bioactive seaweed substances into purified compounds for detailed scientific evaluation of their bioactivities. A large volume of scientific literature is now available to explore the scientific rationale behind the chemical composition and the pharmacological effects of the many bioactive seaweed substances. Some of the many health benefits are summarized below.

29.3.1  Anti-tumor effects The anti-tumor effects of marine bioactive substances have received much attention in recent years both in terms of the wide varieties of substances that can be extracted, and due to the high potency of some novel compounds. Although it is known that dietary seaweeds have anti-tumor effect, past research has primarily been conducted using partially purified fractions or crude extracts, with many early studies using mouse models. Since the 1980s, research on seaweed extracts has focused more on isolated fractions, particularly of polysaccharides and also small molecule extracts including terpenes, sterols, macrolides, alkaloids, halogenated phenols and carotenoids. Extracted peptides and proteins have also been studied, but

Seaweed Biomass Extraction, separation, purification

Bioactive Seaweed Substances Screening

High Activity

Chemical Modifications

Drug Applications

New Drugs

Herb Medicine

Low Activity

Health Applications

Health Products

Biomedical Materials

FIG. 29.1  The screening of bioactive seaweed substances for specific applications.

Functional Foods

Cosmetics



Health benefits of bioactive seaweed substances Chapter | 29  457

FIG. 29.2  The nonwoven fabrics made from alginate fibers can form a soft hydrogel when calcium ions in the fiber exchange with sodium ions in the wound fluid.

to a more limited extent than other chemical groups. More recently, there has been increased interest in the bioactivity of polyphenols. From a medical perspective, the anti-tumor mechanism is due to a number of bioactivities, such as: 1. Kill cancer cells through interfering their mitosis and inhibiting microtubule assembly. 2. Inhibit synthesis of protein kinase C. 3. Inhibit protein synthesis. 4. Enhance body self-defense capabilities through inducing secretion of interleukin, tumor necrosis factor, interferon and other cytokines. 5. Inhibit new blood capillaries in the tumor. Murphy et al. (2014) carried out a detailed review of the studies on marine macro algae for their potential use as a source of novel anti-cancer drugs. It showed that a large number of studies of crude, partially purified and purified seaweed extracts, collected from many locations, have shown potential as sources of potent anti-cancer drugs when tested in vitro and/or in vivo. In particular, polysaccharides, polyphenols, proteinaceous molecules, carotenoids, alkaloids, terpenes and other seaweed derived substances were investigated for their anti-tumor activities, among which, specific inhibitory activity on a number of key cellular processes was identified, including apoptosis pathways, telomerase and tumor angiogenesis. Seaweed derived polysaccharides are the most widely studied cytotoxic agents, and the most common source of these polysaccharides is from brown seaweeds, which are the sources of the sulfated polysaccharide variants such as fucoidans. One of the main groups of polysaccharides present in green seaweeds is the sulfated hetero-polysaccharides known as ulvans, which are composed mainly of sulfate, rhamnose, xylose and glucuronic acid. In red seaweeds, the major polysaccharides are sulfated galactans including agar and carrageenans, with the latter more highly sulfated than agars. Porphyran is another type of sulfated polysaccharide found in red seaweeds. Sulfation is important to the anti-tumor effect of seaweed derived polysaccharides. For example, the importance of fucoidan sulfation to cytotoxic activity has been well established. In vitro and in vivo studies have compared the activity of native fucoidan with that of fractions which have either been artificially over-sulfated or contain more sulfate by means of the fractionation technique used (Yamamoto et al., 1984; Koyanagi et al., 2003; Teruya et al., 2007). An over-sulfated

458  PART | VII  Algal biomedicine

fucoidan fraction, but not a native fraction, had an anti-tumor effect against L-1210 leukemic cells in mice (Yamamoto et al., 1984). Because of their anti-tumor properties, seaweed derived polysaccharides were shown to be capable of extending survival time for tumor bearing mice, which is a result of reduced tumor size in association with improved immune defense (Itoh et al., 1993). The effect of fucoidan in prolonging the survival of tumor-bearing mice was associated with a significant enhancement of the activity of natural killer (NK) lymphocytes and increased production of interferon gamma (IFN-γ) by T cells (Maruyama et al., 2003). In addition to polysaccharides, seaweed derived carotenoids and polyphenols also have anti-tumor effects. For example, fucoxanthin is considered to have cytotoxicity against cancer cells through a tubulin binding action (Januar et al., 2012). In addition, carotenoids can generate anti-tumor effect through a variety of apoptotic pathways and the inhibition of tumor angiogenesis. Polyphenols are known to have anti-cancer properties through their cytotoxic and anti-oxidant activities.

29.3.2  Immunoregulation properties The immune system is responsible for body’s defense against bacteria and virus, and for the prevention of cancer. Seaweeds can improve the immunoregulation properties through the various bioactive substances such as carotenes and sulfated polysaccharides. It is known that carotene rich foods can help prevent lung cancer and enhance the body’s immunocompetence. In a study where scientists follow up 2000 people with 195 specified foods in their diet, there was clear correlation between carotene rich food and the reduction of lung cancer. Phycobiliproteins are also known to enhance immunocompetence (Li and Yang, 2005). Sulfated polysaccharides such as fucoidan can regulate the immune response through activating or inhibiting the activities of macrophages and other immune cells to either enhance or suppress the production of specific antibodies. While macrophages can release a number of cytokines such as interleukin (IL), tumor necrosis factor (TNF), interferon (INF), and other chemokines which are important elements of the immune response, sulfated polysaccharides can interact with the macrophage cells to stimulate its immune response.

29.3.3  Anti-oxidant properties Anti-oxidants can protect cells against oxidative damage by oxygen, peroxide, hydrogen peroxide radicals and hydroxyl radicals. In the human body, once the delicate balance between active oxygen and anti-oxidant substances are disrupted, the associated oxidative stress is known to be related to cancer, aging and inflammatory diseases. Among the many bioactive seaweed substances, phlorotannins are excellent anti-oxidant agents. Ahn et al. (2007) used electron spin resonance spectrometry (ESR) to evaluate the ability of phlorotannins to remove free radicals and found that these seaweed derived substances can protect cells against hydrogen peroxide induced damages. Li et al. (2009) also confirmed the antioxidant properties of extracts from an edible marine brown alga, E. cava. In addition to phlorotannins, fucothanxin is also shown to have good anti-oxidant properties which can offer protection to cells against oxidation induced damage (Heo et al., 2008). Other studies also confirmed seaweed extracts had good anti-oxidant activities (Chandini et al., 2008; O’Sullivan et al., 2011). The free radical scavenging properties of phlorotannins, sulfated polysaccharides, fucosterol, and carotenoid pigments such as fucoxanthin and astaxanthin from marine algae and their byproducts can be used as functional ingredients to reduce chronic diseases in the human body. In addition, antioxidants from natural sources such as seaweeds can be used as ingredients to enhance the quality and shelf-life of food products. Collectively, the wide range of biological activities associated with the anti-oxidative ingredients derived from seaweeds has potential applications not only in the functional food industry, but also in the pharmaceutical and cosmeceutical industries (Li and Kim, 2011). Since skin aging is closely related to free radical activities, the anti-oxidant properties of seaweed extracts is important in the development of skin care products (Masaki, 2010), where health benefits can be derived from a wide variety of natural anti-oxidant substances, including enzymes such as oxide enzyme, superoxide dismutase and catalase, glutathione, vitamins A, C and E, etc., in addition to the seaweed derived polysaccharides and oligosaccharides.

29.3.4  Reduction of blood pressure Saturated fatty acids are known to increase blood pressure while poly-unsaturated fatty acids can help reduce blood pressure. Seaweeds are rich in linolenic acid and its derivatives which can reduce blood viscosity and smooth the interaction between blood vessels and vasoconstrictor substance. It is known that when linolenic acid concentration increases by 1%, blood pressure can reduce by 5 mmHg.



Health benefits of bioactive seaweed substances Chapter | 29  459

29.3.5  Reduction of blood sugar Dietary alginate is known to be able to reduce blood sugar level. In a study where a 5-g supplement of sodium alginate was added to test meals containing similar levels of digestible carbohydrates, fats and proteins, a cohort of diabetes type II patients showed a reduction in blood peak glucose and plasma insulin rise by 31% and 42%, respectively. The addition of sodium alginate in a liquid drink showed similar results in blunting postprandial plasma glucose and insulin elevation. Snack bars including alginate also reduced postprandial peak glucose concentrations and total glucose uptake over 3 h. when compared with snack bars containing guar gum. These results suggest that glucose absorption rates can be reduced in the presence of alginate (Brownlee et al., 2009; Dettmar et al., 2011).

29.3.6  Reduction of blood fat Hyperlipidemia is a result of abnormal fat metabolism, resulting in elevated levels for total cholesterol, total triglyceride and low density lipoprotein cholesterol in the plasma, while the concentration of high density lipoprotein cholesterol (HDL-C) is low. Animal studies have shown that the presence of alginate in the small intestinal lumen decreases uptake of fats and reduces plasma cholesterol under a range of different diets. The reason is likely due to the increased levels of fecal bile and cholesterol excretion. In general, viscous dietary fibers tend to reduce plasma cholesterol concentrations. A metaanalysis of 67 controlled trials suggested that for each gram of dietary fiber, the lowering of total cholesterol concentrations was 70, 37, 28, and 26 mM L−1 plasma g−1 fiber respectively for pectin, oat products, psyllium and guar gum. It has been shown that alginate supplementation of a low-fiber diet at 7.5 g day−1 more than double mean fatty acid excretion in the digesta of a small cohort (n = 6) of human ileostomy patients. Interestingly, while high molecular weight alginate has hypocholesteraemic effects, low molecular weight alginates do not appear to have the same effect. When added into diets with higher total cholesterol and fat contents, sodium alginate can reduce total cholesterol similar to other algal polysaccharides such as sulfated glucuronoxylohamnan, porphyran and furonan (Wei et al., 2009). Fucoidan is also known to be able to reduce blood fat levels by affecting fat absorption, activating the activities of lipid metabolic enzyme and stimulating the expression of LDL-RmRNA, while linolenic acid can regulate fat metabolism through promoting the transformation of LDL to HDL. By lowering the concentration of LDL and raising the concentration of HDL, total blood fat levels can be reduced and atherosclerosis can be prevented. In a study involving pre-school Japanese children, Wada et al. (2011) investigated whether seaweed intake is associated with blood pressure level among Japanese pre-school children. Results showed that the boys with the lowest, middle and highest tertiles of seaweed intake had diastolic blood pressure readings of 62.8, 59.3, and 59.6 mmHg, respectively, while girls with higher seaweed intake had significantly lower systolic blood pressure readings, with girls taking the lowest, middle and highest tertiles of seaweed showing blood pressures of 102.4, 99.2, and 96.9 mmHg respectively. It was concluded that seaweed intake was negatively related to diastolic blood pressure in boys and to systolic blood pressure in girls. This suggests that dietary seaweed may have beneficial effects on blood pressure among children.

29.3.7  Anticoagulant and antithrombotic properties According to investigations by WHO, cardiovascular and cerebrovascular diseases have become the top killer for mankind, with atherosclerosis posing serious risks to public health, especially among elderly people. While there are already many anticoagulant and antithrombotic drugs such as aspirin, heparin, etc., scientists have found that seaweeds are rich in natural anticoagulant and antithrombotic substances, in particular, red and brown seaweeds contain many sulfated polysaccharides with medical functions similar to heparin. Furthermore, seaweed derived polysaccharides can be chemically modified to generate enhanced anticoagulant and antithrombotic properties. For example, alginic acid can be sulfated to produce polysaccharide sulfate (PSS) that have been clinically proven as an effective anticoagulant and antithrombotic agent (Wei et al., 2006).

29.3.8  Anti-inflammatory and anti-allergic properties Inflammatory reaction is a physiological response by the vascular tissue system to foreign body invasion in which macrophages play an important role. Seaweed derived polyphenols are known to have anti-inflammation properties (Le et al., 2009). During the anti-inflammation process, polyphenols function through suppressing the release of inflammatory mediators or blocking their migration to target cells. Fucoidan was found to have similar properties (Fitton et al., 2007). Niu et al. (2003) used methanol to extract 39 species of seaweeds to assess the anti-inflammatory properties. Results showed

460  PART | VII  Algal biomedicine

that 7 types of brown seaweeds and 11 types of red seaweeds showed positive results, while the extract from green seaweeds showed no activity. Among the 18 that showed activity, Chorda filum was found to have the strongest activity.

29.3.9  Anti-bacteria and anti-virus properties Seaweeds contain natural anti-bacteria and anti-virus substances. Shi and Xu (1997) studied nine species of common seaweeds for their antimicrobial properties. Results showed that all nine species had obvious inhibitory effect against Bacillus subtilis. It is proposed that the polyphenol compounds in seaweeds are the main means for seaweeds to resist the invasion of pathogen and has good anti-bacteria and anti-virus properties. More and more studies have shown that seaweeds contain novel antimicrobial substances that can be utilized as new drugs for hospital use (Ma et al., 2006; Hudson et al., 1998). Alginate has the ability to suppress the growth of bacteria in the digestive system. In a study of 21 strains of authentic human intestinal bacteria growing on alginate based media under in vitro conditions, only bacteroides ovatus demonstrated the capability to thrive. In another study where human participants were fed 10 g of alginates per day, results showed an increase in fecal bifidobacterial cultures, and a decrease in both some potentially pathogenic bacterial strains, e.g., Enterobacteriaceae and lecithinase negative Clostridia, and the levels of fecal toxins produced by putrefaction, e.g., ammonia and sulfides. Laboratory studies also showed that incubation of human fecal inocula with alginates did not produce short-chain fatty acids for over 6 h. After 24 h. incubation, between 50% and 80% of the alginate had been degraded under these conditions, suggesting that alginates are slowly fermented by the colonic microflora in humans. In animal studies, the fermentability of dietary alginates increased over time of feeding, suggesting a shift of the colonic microflora to one that was more capable of degrading alginate polysaccharide chains. Literature information suggests that M-rich alginates are less well digested than G-rich ones, and that increasing chain length of alginates results in reduced fermentability. However, owing to the complexity of the human colonic microflora, it is always difficult to predict how changes to specific bacterial species will affect the microflora as a whole, or the knock-on physiological effects these changes may have on the host (Brownlee et al., 2009; Dettmar et al., 2011).

29.3.10  Anti-HIV properties Sulfated polysaccharides have been shown to have inhibitory properties against damages to cell induced by HIV-1 (Zhang et al., 2003). When used together with AZT, it can reduce the toxicity posed by AZT. The anti-HIV properties were generated by regulating the immune system through activating macrophages, reticuloendothelial system, T and B lymphocytes, and by activating complement and promoting the generation of various cytokines. The main mechanism includes disrupting adhesion of HIV-1 to host cells, inhibiting expression of HIV-1 antigen, inhibiting synthesis of syncytium, inhibiting the activities of reverse transcriptase and enhancing immune system functions.

29.3.11  Anti-fatigue properties Liu et al. (2003) studied the anti-fatigue properties of seaweed polysaccharides on rats. Results showed that a diet of seaweed polysaccharides can raise the duration time during a loaded swimming test and also extend the survival time under reduced oxygen pressure. The anti-fatigue property is due to higher hemoglobin, more oxyhemoglobin dissociation, and enhanced release of oxygen.

29.3.12  Anti-aging properties Aging is related to a number of physical and physiological factors. In particular, the healthy function of kidney and spleen play a key role in human health. Seaweeds and seaweeds derived bioactive substances can enhance kidney and spleen function through regulating the nervous system function, promoting immunity, repairing DNA, regulating endocrine function, removing free radicals and promoting healthy metabolism. Recent research found that an extract of Fucus vesiculosus promotes the contraction of fibroblast-populated collagen gels through increased expression of integrin molecules. Topical application of an aqueous extract was found to have positive effect on the thickness and mechanical properties of human skin, whereby after applying the seaweed extract topically to human cheek skin twice daily for 5 weeks, a significant decrease in skin thickness measured by B-mode ultrasound was elicited, as was a significant improvement in elasticity measured with a Cutometer as compared with controls. In cheek skin, the thickness normally increases and the elasticity decreases with age. The positive results suggest that Fucus vesiculosus extract possesses anti-aging activities and may be useful for cosmetic applications (Fujimura et al., 2002).

Health benefits of bioactive seaweed substances Chapter | 29  461



29.3.13  Absorption of heavy metal ions As a polymeric acid, alginate has strong binding to heavy metal ions. In a study on the binding abilities of alginate for divalent metal ions during gel formation of sodium alginate solution, Haug et al. (1967), Smidsrod and Haug (1972) noted that the ability for alginate to bind divalent metal ions is related to the ion exchange coefficient between the divalent metal ion and the sodium ion: K   Metal ion concentration in the gel Sodium ion concentration in the solution 

2

/ Sodium ion concentration in the gel   Metal ion concentration in solution  2

After studying various metal ions, Haug and Smidsrod found that the binding abilities for alginate are in the order of Pb 2   Cu 2   Cd 2   Ba 2   Sr 2   Ca 2   Co 2   Ni 2   Zn 2   Mn 2  The strong binding for lead ions has been successfully utilized to develop formulations for children with high levels of lead in blood.

29.3.14  Suppression of esophageal and esophageal reflux Since alginic acid is insoluble in water, aqueous alginate fluid gels when the water soluble alginate is converted into an alginic acid gel on contact with gastric juice. This gel remains in the stomach for up to 3 h, with the raft floating on top of the gastric juices providing a mechanical barrier against reflux of stomach contents into the esophagus/esophagus. If calcium carbonate and sodium bicarbonate are present in the formulation, a calcium/acid gel is formed, where the acidic environment of the stomach turns the carbonates into carbon dioxide, making the gel buoyant. As the intra-gastric pressure increases, the gel/foam is pushed upward into the lower esophagus/esophagus coating, which can protect the mucous membrane. Once the gel reaches the higher pH intestine fluids, alginic acid is neutralized to its corresponding salt, which forms highly viscous barriers (Brownlee et al., 2009; Dettmar et al., 2011).

29.3.15  Bulking of fecal contents and relief of constipation In a 7 day supplementation of the diet of 5 healthy adult males with sodium alginate at 175 mg kg−1 body weight, it was found that the fecal wet and dry weights significantly increased. Similar results were obtained in porcine studies, where the addition of 5% alginate in the feed increased the volume of colonic luminal contents. The resulting health benefit is that a range of damaging agents which originate from food, microflora and the gastrointestinal tract itself are adsorbed to dietary alginates in a way similar to that of other dietary fibers, with the effect higher for alginate than cellulose, xylan and carrageenan at the same concentration. When alginate is added into the diet, it leads to the bulking of the colonic contents and eventually the passed stools with a dilution of any damaging agents in the colon, thereby effecting a reduced mucosal exposure to these agents (Brownlee et al., 2009; Dettmar et al., 2011).

29.3.16  Slimming properties Chater et al. (2016) studied the effect of three Hebridean brown seaweeds on lipase activity by using a turbidimetric lipase activity assay and an in vitro simulation of the upper digestive tract. In tests involving Ascophyllum nodosum, Fucus vesiculosus, and Pelvetia canaliculata using whole seaweed homogenate, sodium carbonate extract and ethanol extracts, all extracts showed significant inhibition of lipase, suggesting multiple bioactive agents, potentially including alginates, fucoidans and polyphenols. Whole homogenate extract of F. vesiculosus was the most potent inhibitor of lipase, followed by ethanol supernatant while sodium carbonate extract showed relatively weaker inhibition. These inhibitory effects were validated in a model gut system. The results suggest that seaweeds can be used as a potential weight management tool. Aqueous alginate solutions form gel when on contact with either acid or calcium ions. The acidic nature of the stomach means that alginate can be administered in solution form and gel in situ, which is different to other viscous polysaccharides that need to be administered in gel form to be a gel in the stomach. For consumers, high viscosity or gel strength in the mouth is often associated with poor organoleptic acceptability of foods. At the same time, high viscosity in the stomach is linked to increased gastric distension and thereby increased satiety. Due to its unique polymeric structure, alginate can be administered in a low-viscosity form and gel spontaneously in the stomach to give increased satiety. It has been shown that the addition of

462  PART | VII  Algal biomedicine

alginate to a milk based liquid meal replacement resulted in participants reporting an increased feelings of fullness compared to a control. Alginate with high gel strength significantly prolonged a postprandial feeling of hunger. It was also shown that while alginate increased the volume of stomach contents, it did not affect gastric emptying rates. It has been proposed that, following ingestion, ionic gelation of alginate in stomach acid can modulate feeding behavior through slowed gastric clearance, stimulation of gastric stretch receptors and attenuated nutrient uptake (Brownlee et al., 2009; Dettmar et al., 2011). Due to its viscous nature, alginate tends to suppress the bioavailability of certain beneficial dietary components, including β-carotene and minerals such as calcium, iron, chromium and cobalt. In addition, alginate appears to have some inhibitory effects on a range of digestive enzymes in vitro. However, while this property is linked to the slimming effect of alginate containing diets, the inclusion of high levels of alginate in the diets of at-risk individuals, e.g., the elderly, pregnant women and infants, may outweigh any potential health benefits.

29.3.17  Anti-diabetic properties The emergence of type 2 diabetes mellitus (T2DM) as the pre-eminent global non-infectious disease has driven the search for new anti-diabetic strategies including utilizing traditional food and herbs. Seaweeds were found to possess potential anti-diabetic properties. Chin et al. (2015) measured the ability of seaweed extracts to inhibit α-glucosidase and dipeptidylpeptidase-4 (DPP-4) and also the ability to stimulate incretin hormone secretion in vitro. It was found that crude water extracts of Halimeda macroloba, Padina sulcata, Sargassum binderi, and Turbinaria conoides possessed potent inhibitory activities against α-glucosidase and DPP-4. The highest inhibitory activity against α-glucosidase was found in water extracts of the green seaweed species H. macroloba with an IC50 value of 6.388 mg mL−1. Crude water extracts of the brown seaweeds P. sulcata, S. binderi and T. conoides exhibited potent DPP-4 inhibition compared with the green seaweed H. macroloba. The brown seaweed also stimulates secretion of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) from pGIP neo STC-1 cells in  vitro. These results suggest seaweeds extracts possess certain anti-diabetic properties.

29.3.18  Deodorant properties A study has shown that seaweed extract had deodorizing activity against methyl mercaptan, the active compound being phlorotannins. It was shown that seaweed derived substance had stronger deodorizing activity than other natural substances such as chlorophyll and sodium copper chlorophyll (Tokita et al., 1984).

29.3.19  Anti-acne properties Acne vulgaris is a common skin disease that usually begins after puberty between adolescence ages of 11 and 30 years. It can persist for years and results in permanent scars and disfigurement and has an adverse effect on physiological development. Extensive application of antibiotics to treat acne since the 1960s has led to the widespread occurrence of resistance in cutaneous Propionibacterium acnes. Kok et al. (2016) studied the brown seaweed Sargassum polycystum as a potential agent against acne vulgaris. The antioxidant and in vitro antibacterial properties of four methanol fractions (F1-F4) obtained through column chromatography were studied. F1 showed the highest activity against Propionibacterium acnes with minimal inhibitory and bactericidal concentrations of 0.25 and 0.50 mg mL−1, respectively. Additionally, treatment of P. acnes lipase with F1 resulted in the highest lipase inhibition at 71.90%. Another fraction F2 showed the highest scavenging activity. The mass spectrometry profile of F1 and F2 revealed chlorophyll a and fucoxanthin as the major anti-acne constituents. These findings suggest that extracts of S. polycystum could be developed for topical applications against acne vulgaris.

29.3.20  Anti-depression properties Miyake et al. (2014) carried out a study to investigate the association between seaweed consumption and depressive symptoms during pregnancy in Japan, where the study subjects were 1745 pregnant women. Depressive symptoms were defined as present when subjects had a Center for Epidemiologic Studies Depression Scale score of 16 or higher. Dietary consumption during the preceding month was assessed using a self-administered diet history questionnaire. Adjustment was made for age, gestation, region of residence, number of children, family structure, history of depression, family history of depression, smoking, and secondhand smoke exposure at home and at work, job type, household income, education, body mass index, and intake of fish and yogurt. The prevalence of depressive symptoms during pregnancy was 19.3%. After adjustment



Health benefits of bioactive seaweed substances Chapter | 29  463

for possible dietary and non-dietary confounding factors, higher seaweed consumption was independently associated with a lower prevalence of depressive symptoms during pregnancy. It was found that the adjusted odds ratios (95% confidence intervals) for depressive symptoms during pregnancy in the first, second, third, and fourth quartiles of seaweed consumption were 1 (reference), 0.72 (0.51–1.004), 0.71 (0.50–1.01), and 0.68 (0.47–0.96), respectively (P for trend = 0.03). The results suggest that seaweed consumption may be inversely associated with the prevalence of depressive symptoms during pregnancy in Japanese women.

29.3.21  Protection against radiation Seaweeds grow up in a harsh marine environment constantly exposed to sun light and UV. In order to survive in these conditions, seaweeds generate many structurally unique chemical compounds to protect them against radiation induced damages. Sodium alginate is found to be able to absorb radioactive elements in the body. It can inhibit the absorption of 90Sr in the digestive track and promote its excretion. The mannuroic acid and guluronic acid monomers can bind 89Sr and 90Sr to form insoluble gel and promote their removal from the body. Sodium alginate can also promote the removal of radioactive elements 220Ra and 140Ba (Brownlee et al., 2009; Dettmar et al., 2011). Over exposure to ultra-violet is the main cause of skin cancer and skin aging. Fucoxanthin and phlorotannin extracted from seaweeds have shown to have protective effect against ultra-violet to avoid UV induced cell damage (Heo and Jeon, 2009; Connan et al., 2007; Heo et al., 2010).

29.3.22  Inhibition of matrix metalloproteinase Skin and tissue functions are a complex and highly coordinated process where several different cell types and molecules, such as growth factors and extracellular matrix (ECM) components, play an important role. The metalloproteinase (MMP) family is among the many proteins that are essential for tissue integrity. MMPs can act on ECM and non-ECM components affecting degradation and modulation of the ECM, growth-factor activation, and cell-cell and cell-matrix signaling, hence they play an important role in skin health (Fujii et al., 2008; Scharffetter-Kochanek et al., 2000). As people age, the activities of the matrix metalloproteinase, in particular MMP-1, usually grow stronger, causing a deterioration of the collagen synthesis process and a drop in skin elasticity. Hence a major function of cosmetic product is to control the collagen metabolism process. Since seaweed extracts such as phlorotannins can inhibit MMP activities, they are highly valued as cosmetic ingredients (Kim et al., 2006).

29.3.23  Skin whitening effect Seaweed derived substances have inhibitory activities against tyrosinase, which blocks the conversion of tyrosine to melanin (Gao et al., 2008). Many natural compounds have the ability to inhibit tyrosinase activities and have skin whitening effects (Sima et al., 2010), while the activities of seaweed extracts have been proven in an animal study (Cha et al., 2011). The inhibitory activity of phlorotannins have been proven in an animal study (Yoon et al., 2009), showing skin whitening effect better than some commercial whitening agents.

29.4 Summary Seaweeds represent a huge resource for mankind and as demands grow for special drugs, nutraceuticals, cosmetic products and functional foods, there is a strong momentum for the exploration of marine biological resources in general, and seaweeds in particular for novel compounds with health benefits. Seaweeds are arguably the largest biomass in the ocean and hence are one of the biggest providers of marine bioactive substances that can be used to develop functional foods in addition to drugs, cosmetics and other novel health related products. Because of their diverse range of structural features and novel bioactivities, these bioactive seaweed substances will find more and more applications in the health related industries. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c,d, e,f,g,h,i,j,k,l,m,n,o).

Acknowledgments This chapter was sponsored by National Key R&D Program of China, Project No. 2018YFC1105600.

464  PART | VII  Algal biomedicine

References Ahn, G.N., Kim, K.N., Cha, S.H., Song, C.B., Lee, J., Heo, M.S., et al., 2007. Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage. Eur. Food Res. Technol. 226 (1–2), 71–79. Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H., Prinsep, M.R., 2013. Marine natural products. Nat. Prod. Rep. 30 (2), 237–323. Brownlee, I.A., Seal, C.J., Wilcox, M., Dettmar, P.W., Pearson, J.P., 2009. Applications of alginates in food. In: Rehm, B.H.A. (Ed.), Alginates: Biology and Applications. Springer-Verlag, Berlin, Heidelberg, pp. 211–228. Cha, S.H., Ko, S.C., Kim, D., Jeon, Y.J., 2011. Screening of marine algae for potential tyrosinase inhibitor: those inhibitors reduced tyrosinase activity and melanin synthesis in zebrafish. J. Dermatol. 38 (4), 354–363. Chandini, S.K., Ganesa, P., Bhaskar, G.N., 2008. In vitro antioxidant activities of three selected brown seaweeds of India. Food Chem. 107 (2), 707–713. Chater, P.I., Wilcox, M., Cherry, P., Herford, A., Mustar, S., Wheater, H., et al., 2016. Inhibitory activity of extracts of Hebridean brown seaweeds on lipase activity. J. Appl. Phycol. 28 (2), 1303–1313. Chin, Y.X., Lim, P.E., Maggs, C.A., Phang, S.M., Sharifuddin, Y., Brian, B.D., 2015. Anti-diabetic potential of selected Malaysian seaweeds. J. Appl. Phycol. 27 (5), 2137–2148. Conde, E., Moure, A., Domínguez, H., 2015. Supercritical CO2 extraction of fatty acids, phenolics and fucoxanthin from freeze-dried Sargassum muticum. J. Appl. Phycol. 27 (2), 957–964. Connan, S., Deslandes, E., Gall, E.A., 2007. Influence of day–night and tidal cycles on phenol content and antioxidant capacity in three temperate intertidal brown seaweeds. J. Exp. Mar. Biol. Ecol. 349 (2), 359–369. Das, D. (Ed.), 2015. Algal Biorefinery: An Integrated Approach. Springer, New York. Dettmar, P.W., Strugala, V., Richardson, J.C., 2011. The key role alginates play in health. Food Hydrocoll. 25 (2), 263–266. Fitton, J.H., Irhimeh, M., Falk, N., 2007. Macroalgal fucoidan extracts: a new opportunity for marine cosmetics. Cosmet. Toiletries 122 (8), 55–64. Fujii, T., Wakaizumi, M., Ikami, T., Saito, M., 2008. Amla (Emblica officinalis Gaertn.) extract promotes procollagen production and inhibits matrix metalloproteinase-1 in human skin fibroblasts. J. Ethnopharmacol. 119 (1), 53–57. Fujimura, T., Tsukahara, K., Moriwaki, S., Kitahara, T., Sano, T., Takema, Y., 2002. Treatment of human skin with an extract of Fucus vesiculosus changes its thickness and mechanical properties. J. Cosmet. Sci. 53 (1), 1–9. Gao, X.H., Zhang, L., Wei, H., Chen, H.D., 2008. Efficacy and safety of innovative cosmeceuticals. Clin. Dermatol. 26 (4), 367–374. Haug, A., Myklestad, S., Larsen, B., Smidsrod, O., 1967. Correlation between chemical structure and physical properties of alginates. Acta Chem. Scand. 21, 768–778. Heo, S.J., Jeon, Y.J., 2009. Protective effect of fucoxanthin isolated from Sargassum siliquastrum on UV-B induced cell damage. J. Photochem. Photobiol. B 95 (2), 101–107. Heo, S.J., Ko, S.C., Kang, S.M., Kang, H.S., Kim, J.P., Kim, S.H., et al., 2008. Cytoprotective effect of fucoxanthin isolated from brown algae Sargassum siliquastrum against H2O2-induced cell damage. Eur. Food Res. Technol. 228 (1), 145–151. Heo, S.J., Ko, S.C., Kang, S.M., Cha, S.H., Lee, S.H., Kang, D.H., et al., 2010. Inhibitory effect of diphlorethohydroxycarmalol on melanogenesis and its protective effect against UV-B radiation-induced cell damage. Food Chem. Toxicol. 48 (5), 1355–1361. Hill, R.A., 2012. Marine natural products. Annu. Rep. Prog. Chem. B 108, 131–146. Hu, Y., Chen, J., Hu, G., Yu, J., Zhu, X., Lin, Y., et al., 2015. Statistical research on the bioactivity of new marine natural products discovered during the 28 years from 1985 to 2012. Mar. Drugs 13 (1), 202–221. Hudson, J.B., Kim, J.H., Lee, M.K., DeWreede, R.E., Hong, Y.K., 1998. Antiviral compounds in extracts of Korean seaweeds: evidence for multiple activities. J. Appl. Phycol. 10, 427–434. Itoh, H., Noda, H., Amano, H., Zhuaug, C., Mizuno, T., Ito, H., 1993. Antitumor activity and immunological properties of marine algal polysaccharides, especially fucoidan, prepared from Sargassum thunbergii of Phaeophyceae. Anticancer Res. 13 (6A), 2045–2052. Januar, H.I., Dewi, A.S., Marraskuranto, E., Wikanta, T., 2012. In silico study of fucoxanthin as a tumor cytotoxic agent. J. Pharm. Bioallied Sci. 4 (1), 56–59. Jimenez-Escrig, A., Gomez-Ordonez, E., Ruperez, P.J., 2012. Brown and red seaweeds as potential sources of antioxidant nutraceuticals. J. Appl. Phycol. 24 (5), 1123–1132. Kim, M.M., Ta, Q.V., Mendis, E., Rajapakse, N., Jung, W.K., Byun, H.G., et al., 2006. Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity. Life Sci. 79 (15), 1436–1443. Kok, J.M.L., Jee, J.M., Chew, L.Y., Wong, C.L., 2016. The potential of the brown seaweed Sargassum polycystum against acne vulgaris. J. Appl. Phycol. 28 (5), 3127–3133. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.



Health benefits of bioactive seaweed substances Chapter | 29  465

Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Koyanagi, S., Tanigawa, N., Nakagawa, H., Soeda, S., Shimeno, H., 2003. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochem. Pharmacol. 65 (2), 173–179. Le, Q.T., Li, Y., Qian, Z.J., Kim, M.M., Kim, S.K., 2009. Inhibitory effects of polyphenols isolated from marine alga Ecklonia cava on histamine release. Process Biochem. 44 (2), 168–176. Li, Y.X., Kim, S.K., 2011. Utilization of seaweed derived ingredients as potential antioxidants and functional ingredients in the food industry: an overview. Food Sci. Biotechnol. 20 (6), 1461–1466. Li, H.J., Yang, F.M., 2005. Review on extraction technology and biological activity of phycobiliprotein. Food Sci. 26 (7), 243–246. Li, Y., Quian, Z.J., Ryu, B., Lee, S.H., Kim, M.M., Kim, S.K., 2009. Chemical components and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia cava. Bioorg. Med. Chem. 17 (5), 1963–1973. Liu, F., Li, Z., Pang, Y., Luo, Q., Yan, J., 2003. Influence of Laminaria japonica polysaccharides on blood gas of hypoxic mice and its anti-fatigue effect. Chin. J. Public Health 19 (12), 1462–1463. Ma, H., Mei, X., Xu, S., 2006. Overseas research progress in marine antimicrobial substances. Chin. J. Mar. Drugs 25 (3), 46–49. Maruyama, H., Tamauchi, H., Hashimoto, M., Nakano, T., 2003. Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida. In Vivo 17 (3), 245–249. Masaki, H., 2010. Role of antioxidants in the skin: anti-aging effects. J. Dermatol. Sci. 58 (2), 85–90. Miyake, Y., Tanaka, K., Okubo, H., Sasaki, S., Arakawa, M., 2014. Seaweed consumption and prevalence of depressive symptoms during pregnancy in Japan: baseline data from the Kyushu Okinawa Maternal and Child Health Study. BMC Pregnancy Childbirth 14, 301–307. Murphy, C., Hotchkiss, S., Worthington, J., McKeown, S.R., 2014. The potential of seaweed as a source of drugs for use in cancer chemotherapy. J. Appl. Phycol. 26 (5), 2211–2264. Niu, R., Fan, X., Han, L., 2003. A screening for the anti-inflammatory effect of algal extracts. Oceanol. Limnol. Sin 34 (2), 150–154. Nomura, M., Kamogawa, H., Susanto, E., Kawagoe, C., Yasui, H., Saga, N., et al., 2013. Seasonal variations of total lipids, fatty acid composition, and fucoxanthin contents of Sargassum horneri (Turner) and Cystoseira hakodatensis (Yendo) from the northern seashore of Japan. J. Appl. Phycol. 25 (4), 1159–1169. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Queguineur, B., Hanniffy, D., et al., 2011. In vitro and cellular antioxidant activities of seaweed extracts prepared from five brown seaweeds harvested in spring from the west coast of Ireland. Food Chem. 126 (3), 1064–1070. Qin, Y., 2005. The ion exchange properties of alginate fibers. Text. Res. J. 75 (2), 165–168. Qin, Y., 2006. The characterization of alginate wound dressings with different fiber and textile structures. J. Appl. Polym. Sci. 100 (3), 2516–2520. Qin, Y., 2008. The gel swelling properties of alginate fibers and their application in wound management. Polym. Adv. Technol. 19 (1), 6–14. Qin, Y., 2016. Medical Textile Materials. Woodhead Publishing, Cambridge. Qin, Y. (Ed.), 2018. Bioactive Seaweeds for Food Applications: Natural Ingredients for Healthy Diets. Academic Press, San Diego. Scharffetter-Kochanek, K., Brenneisen, P., Wenk, J., Herrmanni, G., Ma, W., Kuhr, L., et al., 2000. Photoaging of the skin from phenotype to mechanisms. Exp. Gerontol. 35 (3), 307–316. Shi, R., Xu, Z., 1997. Research on the antimicrobial properties of lipids and phenols in nine types of seaweeds around the coast of Qingdao. Chin. J. Mar. Drugs 4, 16–19. Sima, V.H., Patris, S., Aydogmus, Z., Sarakbi, A., Sandulescu, R., Kauffmann, J.M., 2010. Tyrosinase immobilized magnetic nanobeads for the amperometric assay of enzyme inhibitors: application to the skin whitening agents. Talanta 83 (3), 980–987. Smidsrod, O., Haug, A., 1972. Dependence upon the gel-sol state of the ion-exchange properties of alginates. Acta Chem. Scand. 26 (5), 2063–2074. Teruya, T., Konishi, T., Uechi, S., Tamaki, H., Tako, M., 2007. Anti-proliferative activity of oversulfated fucoidan from commercially cultured Cladosiphon okamuranus TOKIDA in U937 cells. Int. J. Biol. Marcromol. 41 (3), 221–226. Tokita, F., Ishikawa, M., Shibuya, K., Koshimizu, M., Abe, R., 1984. Deodorizing activity of some plant extracts against methyl mercaptan. Nippon Nogeik. Kaishi 58 (6), 585–589.

466  PART | VII  Algal biomedicine

Wada, K., Nakamura, K., Tamai, Y., Tsuji, M., Sahashi, Y., Watanabe, K., et al., 2011. Seaweed intake and blood pressure levels in healthy pre-school Japanese children. Nutr. J. 10, 83–90. Wei, Y., Li, J., Zhao, A., Zhang, X., Hu, Y., 2006. Research advances in anti-coagulant activity of substances from marine creatures. Chin. J. Mar. Drugs 25 (5), 47–50. Wei, Y., Xu, C., Zhao, A., Liu, L., Ai, G., 2009. Research advances in antilipemic activity of substances from marine creatures. Chin. J. Biochem. Pharm. 30 (5), 356–358. Xia, B., Abbott, I.A., 1987. Edible seaweeds of China and their place in the Chinese diet. Econ. Bot. 41 (3), 341–353. Xu, N., Fan, X., Yan, X., Tseng, C.K., 2004. Screening marine algae from China for their antitumor activities. J. Appl. Phycol. 16 (6), 451–456. Yamamoto, I., Takahashi, M., Suzuki, T., Seino, H., Mori, H., 1984. Antitumor effect of seaweeds. IV. Enhancement of antitumor activity by sulfation of a crude fucoidan fraction from Sargassum kjellmanianum. Jpn. J. Exp. Med. 54 (4), 143–151. Yoon, N.Y., Eom, T.K., Kim, M.M., Kim, S.K., 2009. Inhibitory effect of phlorotannins isolated from Ecklonia cava on mushroom tyrosinase activity and melanin formation in mouse B16F10 melanoma cells. J. Agric. Food Chem. 57 (10), 4124–4129. Zemke-White, W.L., Ohno, M., 1999. World seaweed utilization: an end-of-century summary. J. Appl. Phycol. 11 (4), 369–376. Zhang, S., Li, W., Cai, M., 2003. Anti-HIV-1 activity of sulfated polysaccharides from seaweeds. Mar. Sci. 27 (8), 16–18.

Chapter 30

The pioneering research on the wound care by alginates Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

30.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 indexed-papers published since 1980 (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a,b,c,d,e,f,g,h, 2017a,b,c,d,e,f, 2019, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). The field of algal biomedicine (Konur, 2015a, 2017a, 2020i) has been among the most-prolific research fronts over time as evidenced with over 30,500 papers, comprising over 20% of the algal research as a whole, published during the same study period, corresponding to the public concerns about the development of sustainable and environment-friendly bioproducts and bioprocesses at large. The applications of alginates (Lee and Mooney, 2012; Augst et  al., 2006, Konur, 2017a) in drug delivery (George and Abraham, 2006; Gombotz and Wee, 1998), tissue engineering (Kuo and Ma, 2001; Li et al., 2005), cell engineering (Rowley et al., 1999; Smidsrod and Skjak-Braek, 1990), and wound care (Balakrishnan et al., 2005; Choi et al., 1999) have been among the strategic applications of algal biomedicine in recent years. This book chapter covers the 42 pioneering research papers on the wound care by alginates with at least 45 citations each providing the ample data for the primary stakeholders about the contents of these papers to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate. It also provides the strategic information on the research landscape for these pioneering papers.

30.2  Materials and methodology The search for the literature on the wound care by alginates was carried out in April 2019 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI) and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been developed by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in the Appendix. These keyword sets have been devised in two major parts: the keywords related to wound care and keywords related to the alginates. The papers with at least 45 citations each were selected for this study. First, the strategic information on the research landscape for these papers were presented in summary to put these papers in the context (Konur, 2020a,b,c,d,e,f,g,h,i,j,k ,l,m,n). Next, the concise information about each paper was presented under the three topical headings of wound care by alginates only, by alginates and chitosan, and alginates and other biomaterials. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00030-9 © 2020 Elsevier Inc. All rights reserved.

467

468  PART | VII  Algal biomedicine

30.3 Results 30.3.1  The research landscape There have been 25 prolific authors with 2 or more pioneering papers researching the wound care by alginates. Fifteen, six, three, and one of them have been from South Korea, Japan, India, and China. These are ‘Han-Gon Choi’, ‘Jung-Ae Kim’, ‘Dong Xun Li’, ‘Won Seok Lyoo’, ‘Jung Kil Park’, and ‘Jong Soo Woo’, of Yeungnam University; ‘Sung Giu Jin’, ‘Jeong Hoon Kim’, ‘Jong Oh Kim’, and ‘Jun Yong Choi’ of Dong-A Pharm. Co.; and ‘Young Seon Choi’, ‘Sung Ran Hong’, ‘Young Moo Lee’, ‘Kang Won Song’, and ‘Moon Hyang Park’ of Hanyang University from South Korea. The others are ‘Yoshimi Kakimaru’ of Kuraray Medical Inc.; ‘Kyoko Suzuki’, ‘Yoshihisa Suzuki’, ‘Masaho Tanihara’, ‘Yoshihiko Nishimura’, and ‘Yasuhiko Shimizu’ of Kyoto University from Japan; ‘Biji Bakarishan’, ‘Athipettah Jayakrishnan’, and ‘Mira Mohanty’ of Sree Chitra Tirunal Institute for Medical Sciences and Technology from India; and ‘Xingyi Li’ of Wenzhou Medical College from China. In total, 212 authors have contributed to these papers. There have been 13 prolific institutions from with 2 or more pioneering papers researching the wound care by alginates. Five, three, two, two, and one institutions have been from South Korea, Japan, India, United Kingdom, and China, respectively. These are Seoul National University, Dong A Pharm Co. Ltd., Hanyang University, Inje University, and Yeungnam University from South Korea; Kyoto University, Nara Institute of Science Technology, and Kuraray Co. Ltd. from Japan; Sree Chitra Tirunal Institute for Medical Sciences Technology and Department of Science and Technology of India from India; Cardiff University and University of Birmingham from the United Kingdom; and Sichuan University from China. In total, 80 institutions have contributed to these papers. There have been seven journals publishing two or more pioneering papers in this field. These are ‘Biomaterials’, ‘International Journal of Pharmaceutics’, ‘Journal of Biomedical Materials Research’, ‘British Journal of Plastic Surgery’, ‘International Journal of Biological Macromolecules’, ‘Journal of Materials Science Materials in Medicine’, and ‘Carbohydrate Polymers’. The first three journals have published 18 papers. In total, 20 journals published these papers. There have been eight countries publishing two or more pioneering papers in this field. These are the United Kingdom, South Korea, China, Japan, India, Germany, Sweden, and the United States. The first 4 countries have published 30 papers. In total, 21 countries have published these papers. These papers have been primarily published in the 2000s and 2010s with 16 and 15 papers, respectively. There have also been three and eight papers published in the 1980s and 1990s, respectively. The most-prolific years have been 2008, 2010, and 2011 with four papers each. There have been six prolific subject categories indexing these papers. These are ‘Materials Science Biomaterials’, ‘Engineering Biomedical’, ‘Polymer Science’, ‘Pharmacology Pharmacy’, ‘Surgery’, and ‘Chemistry Applied’ with 19, 18, 7, 6, 6, and 5 papers, respectively. In total, there have been 18 subject categories indexing these papers. There have been 4216 citations to these papers, in total where the average number of citations per paper has been 100 and H-index has been 42.

30.3.2  The pioneering research on the wound care by alginates only There have been 18 papers focusing on the wound care by alginates only (Table 30.1). Qin (2008) discusses the production processes and applications in wound management of alginate fibers in a review paper with 144 citations. He notes that the ion-exchange and gel-forming abilities are particularly useful for the treatment of exuding wounds and many improvements have been made in recent years to enhance the absorption and gel-forming capabilities and the antimicrobial properties of these fibers. In addition, alginate fibers have been used as a carrier to deliver beneficial ingredients for wound healing. He focuses on the production of various fibers from alginate, production processes for the variants of alginates such as calcium alginate. Thomas et al. (2000) study the alginate-based wound dressings to determine whether alginates may interact with wound macrophages in a paper with 112 citations. Alginate fibers taken from four commercially available dressings were cocultured with the human histiocytic lymphoma cell line U937. They find that ‘Seasorb’ and ‘Tegagen’ had a minimal effect while ‘Sorbsan’ and ‘Kaltostat’ induced 302 pg/mL and 839 pg/mL TNFα, respectively. They conclude that some alginate dressings have the potential to activate macrophages within the chronic wound bed. Hashimoto et al. (2004) study the development of alginate wound dressings linked with hybrid peptides derived from laminin and elastin in a paper with 106 citations. They design SIRVXVXPG (X: A or G) of SIKVAV and VGVAPG. They examine the effectiveness of these hybrid peptides for cell attachment and proliferation using normal human dermal fibroblasts (NHDF) in vitro. They find that SIRVXVXPG promoted attachment of NHDF, unlike Ac-KSIKVAV and AcKVGVAPG. They examine the effectiveness of alginates with these hybrid peptides in wound healing using a rabbit ear skin defect model in vivo. Nine days after operation, they find that ears with the alginate dressings linked with the hybrid

The pioneering research on the wound care by alginates Chapter | 30  469



TABLE 30.1  The pioneering research on the wound care by alginates only. Alginates

Form

Focus

Cits.

Refs.

1

Alginate

Fibers

Production processes and applications

144

Qin (2008)

2

Alginate

Fibers

Healing, macrophage activation

112

Thomas et al. (2000)

3

Alginate

Epithelialization

106

Hashimoto et al. (2004)

4

Alginate

Hydrogel

Cytotoxicity, foreign-body reaction

105

Suzuki et al. (1998)

5

Alginate

Films

Antimicrobial

102

Liakos et al. (2014)

6

Alginate

Bilayer films

Slow-release healing

95

Thu et al. (2012)

7

Calcium alginate

Clinical efficacy and safety

94

Jude et al. (2007)

8

Calcium alginate

Healing

85

Attwood (1989)

9

Alginate, silver alginate, nanosilver alginate

Cytotoxicity, antimicrobial activity, and biocompatibility

84

Wiegand et al. (2009)

10

Alginate

Hydrogel

Healing, scar formation

83

Rabbany et al. (2010)

11

Alginate

Fiber

Healing

77

Gilchrist and Martin (1983)

12

Alginate

Healing, reepithelialization

66

Suzuki et al. (1999)

13

Calcium alginate

Healing

64

Doyle et al. (1996)

14

Calcium alginate

Healing

64

Barnett and Varley (1987)

15

Alginate

Healing, cytotoxicity

57

Sikareepaisan et al. (2011)

16

Calcium alginate

Healing, epithelialization

49

Agren (1996)

17

Alginate

Reepithelialization

45

Porter (1991)

18

Alginate

Healing, antimicrobial

45

Hegge et al. (2011)

Films

Foams

peptides showed significantly greater epithelialization and a larger volume of regenerated tissue compared to those treated with SIVAV-linked, VGVAPG-linked and unlinked alginate dressings. Suzuki et al. (1998) evaluate an alginate gel dressing focusing on the cytotoxicity to fibroblasts in vitro and foreignbody reaction in pig skin in vivo in a paper with 105 citations. They use a freeze-dried alginate gel dressing (AGA-100) low in calcium ions. Cytotoxicity testing on L929 cells showed the cytocompatibility of AGA-100 extracts, while extracts from ‘Kaltostat’ induced cytopathic effects. AGA-100-treated wounds showed rapid wound closure compared to control wounds on day 15. Foreign-body reaction was marked in ‘Kaltostat’ and gauze-treated wounds. They conclude that AGA-100 could reduce the cytotoxicity to fibroblasts and foreign-body reaction. Liakos et al. (2014) study the composite wound dressing films of essential oils (EOs) encapsulated in sodium alginate with antimicrobial properties in a paper with 102 citations. Elicriso italic, chamomile blue, cinnamon, lavender, tea tree, peppermint, eucalyptus, lemongrass and lemon oils were encapsulated in the films as potential active substances. They note that such diverse types of essential oil-fortified alginate films can find many applications mainly as disposable wound dressings but also in medical device protection and disinfection. Thu et al. (2012) study the alginate based bilayer hydrocolloid films as potential slow-release wound healing vehicle in a paper with 95 citations. The bilayer is composed of an upper layer impregnated with model drug and a drug-free lower layer. They find that bilayer had superior mechanical and rheological properties than the single layer films. The bilayers also showed low moisture vapor transmission rate, slower hydration rate and lower drug flux in vitro compared to single layer. The bilayers also provided a significant higher healing rate in vivo, with well-formed epidermis with faster granulation tissue formation compared to the controls. They conclude that bilayers are suitable for treating low suppurating wounds and slow release application on wound surfaces. Jude et  al. (2007) study the clinical efficacy and safety of ‘HydrofiberR’ dressing containing ionic silver (SI) and ‘AlgosterilR’ calcium alginate (CA) dressings in non-ischaemic diabetic foot ulcers (DFU) in a paper with 94 citations.

470  PART | VII  Algal biomedicine

They find that all ulcer healing outcomes improved in both groups. Silver dressings were associated with favorable clinical outcomes compared with CA dressings, specifically in ulcer depth reduction and in infected ulcers requiring antibiotic treatment. Attwood (1989) studies calcium alginate (CA) dressing to accelerate split skin-graft (SSG) donor site healing in a paper with 85 citations. They find that in 107 patients with 130 SSG donor operations, there was a statistically highly significant decrease in average time to complete healing (from 10 down to 7 days), and also significantly better patient comfort. They conclude that these dressings were easy to use and the quality of the new skin was significantly better compared to paraffin gauze dressings. Wiegand et al. (2009) carry out the in vitro study on cytotoxicity, antimicrobial activity, and binding capacity for pathophysiological factors in chronic wounds of alginate (A) and silver (S)-containing alginate in a paper with 84 citations. They test A alone, A containing ionic S, and A with nanocrystalline S. They find that A binded considerable amounts of elastase, reduced the concentration of proinflammatory cytokines and inhibited the formation of free radicals. Furthermore, A showed antibacterial activity and high biocompatibility. Incorporation of S into A fibers increased antimicrobial activity and improved the binding affinity for elastase, matrix metalloproteases-2, and the proinflammatory cytokines. Rabbany et al. (2010) study the continuous delivery of ‘Stromal Cell-Derived Factor-1’ (SDF-1) from alginate scaffolds for wound healing in a paper with 83 citations. They find that although SDF-1 plasmid- and protein-loaded patches were able to release therapeutic product over hours to days, SDF-1 protein was released faster than SDF-1 plasmid. Wounds treated with SDF-1 protein and plasmid loaded patches healed faster than sham or control. At day 9, SDF-1-treated wounds significantly accelerated wound closure compared to nontreated controls. They conclude that patch-mediated SDF-1 delivery provide a novel therapy for accelerating healing and reducing scarring in clinical wounds. Gilchrist and Martin (1983) study the wound treatment with an alginate fiber dressing in a paper with 77 citations. They find that the high absorption of exudate is achieved via strong hydrophilic gel formation, controlling wound secretion levels and minimizing bacterial contamination. Furthermore, alginate fibers trapped in a wound are readily biodegraded and do not have to be removed thus avoiding disturbance of granulation tissue formation. Suzuki et al. (1999) study the in vivo evaluation of an alginate dressing, ‘AGA-100’ in a paper with 66 citations. They find that the closure rate of full-thickness wounds treated with ‘AGA-100’ was significantly higher on day 15 compared with that with ‘Kaltostat’ and ‘Sorbsan’. Reepithelization rate of partial-thickness wounds treated with ‘Sorbsan’ was statistically significantly lower on day 3 than those with the other two dressings. A large amount of foreign debris was noted in all the full-thickness wounds treated with ‘Kaltostat’ or ‘Sorbsan’, while only about one-third of wounds treated with ‘AGA-100’ showed a little dressing debris. They conclude that ‘AGA-100’ possesses superior properties compared with commercial alginate dressings. Doyle et al. (1996) study the effect of calcium alginate (CA) on cellular wound healing processes modeled in vitro in a paper with 64 citations. They use human dermal fibroblast, microvascular endothelial cell (HMEC), and keratinocyte cultures. They find that the CA increased the proliferation of fibroblasts but decreased the proliferation of HMEC and keratinocytes. In contrast, the CA decreased fibroblast motility but had no effect on keratinocyte motility. There was no significant effect of CA on the formation of capillary-like structures by HMEC. They conclude that CA improve some cellular aspects of normal wound healing, but not others. Barnett and Varley (1987) study the effects of calcium alginate (CA) on wound healing in a paper with 64 citations. They find that it is an effective hemostat, generally well tolerated by body tissues. Good epidermal healing was seen on all wounds although cellular reactions could be provoked in full thickness wounds without occlusion, if there was an insufficient volume of wound exudate to completely wet the alginate fibers. Sikareepaisan et al. (2011) study the preparation and characterization of asiaticoside (PAC)-loaded alginate films and their potential for use as effectual wound dressings in a paper with 57 citations. The release of PAC from the PAC-loaded alginate immersed films was achieved by both the swelling and the erosion of the alginate matrix in the phosphate buffer solution (PBS). They find that these materials were non-toxic to the skin cells. Agren (1996) studies four calcium alginate (CA) dressings (‘Algosteril’, ‘Comfeel alginate’, ‘Kaltostat’ and ‘Sorbsan’) in the treatment of partial thickness wounds in a paper with 49 citations. They compare these dressings using a standardized partial-thickness wound model in domestic pigs. They find that wound fluid spread laterally onto surrounding normal skin by about 40% more with ‘Sorbsan’ than, with the other alginate dressings after 24 h of treatment. The corresponding figure after 66 h of treatment was 20%. ‘Algosteril’ adhered significantly more to the wounds than ‘Comfeel Alginate’. ‘Kaltostat’ left significantly more dressing residues on the wound surface at dressing removal than the ‘Comfeel Alginate’ dressing. These dressings showed significant differences in important handling characteristics and did not differ significantly in their effect on epithelialization.



The pioneering research on the wound care by alginates Chapter | 30  471

Porter (1991) studies reepithelization of split skin-graft (SKG) donor areas after application of hydrocolloid and alginate dressings in a paper with 45 citations. They find that at the time of the first dressing change 87% of the donor areas dressed with the hydrocolloid and 86% of the donor areas dressed with the alginate were more than 90% healed. The mean time from operation to the observation of complete healing was 10.0 days for the donor areas dressed with the hydrocolloid and 15.5 days for the donor areas dressed with the alginate. They conclude that alginate dressings were more suitable for outpatients, as they are simpler to use. Hegge et al. (2011) study the formulation and bacterial phototoxicity of curcumin loaded alginate foams for wound treatment applications in a paper with 45 citations. They find that both curcumin loaded and unloaded foams hydrated within 1 min and absorbed from 12 to 16 times their dry weight of a model physiological fluid. Exposure to the prepared foams in combination with visible light irradiation resulted in more than 6 log reduction of Enterococcus faecalis cells. An 81% reduction in viable E. coli cells was detected after treatment with the foam containing PEG 400 as the only solubilizer of curcumin combined with visible light irradiation.

30.3.3  The pioneering research on the wound care by alginates and chitosan There have been 11 papers focusing on the wound care by alginates and chitosan (Table 30.2). Murakami et al. (2010) study the hydrogel blends of chitin/chitosan, fucoidan and alginate (ACF-HS) as healing-impaired wound dressings in a paper with 204 citations. They develop a hydrogel sheet composed of a blended powder of alginate, ACF-HS (60:20:2:4 w/w) as a functional wound dressing. They find that ACF-HS effectively interacted with and protected the wound in rats, providing a good moist healing environment with exudates. Although normal rat wound repair was not stimulated by the application of ACF-HS, healing-impaired wound repair was significantly stimulated. There was significantly advanced granulation tissue and capillary formation in the healing-impaired wounds treated with ACF-HS on day 7, as compared to those treated with calcium alginate fiber and those left untreated. Wang et al. (2002) study the chitosan-alginate polyelectrolyte complex (PEC) membrane as a wound dressing with a focus on the incisional wound healing in a paper with 164 citations. They find that these membranes and their aqueous extracts were nontoxic toward mouse and human fibroblast cells. Cell growth was also not hindered by co-incubation with the membranes. Compared to conventional gauze dressing, these membranes caused an accelerated healing of incision wounds in a rat model. Wounds closed at 14 days postoperatively, and there was mature epidermal architecture with keratinized surface of normal thickness and a subsided inflammation in the dermis. They conclude that closure rate and appearance of PEC membrane-treated wounds were comparable with ‘OpsiteR’-treated wounds. Knill et al. (2004) study the alginate fibers modified with unhydrolyzed and hydrolyzed chitosans for wound dressings in a paper with 137 citations. They find that modification of fibers with unhydrolyzed chitosans resulted in a significant reduction in tenacity. Reduction of chitosan molecular weight had a positive effect on its ability to penetrate the alginate fibers compared with unhydrolyzed chitosan/alginate fibers. Hydrolyzed chitosan/alginate fibers demonstrated an antibacterial effect with initial use, and had the ability to provide a slow release/leaching of antibacterially active components. Kim et al. (1999) study the polyelectrolyte complex (PEC) sponge composed of chitosan and sodium alginate (SA) for wound dressing application in a paper with 86 citations. They find that the release of AgSD from AgSD-impregnated PEC wound dressing in PBS buffer was dependent on the number of repeated in situ complex formations for the wound dressing. The PEC wound dressing containing antimicrobial agents could protect the wound surfaces from bacterial invasion and effectively suppress bacterial proliferation. Cellular damage was reduced by the controlled released of AgSD from the sponge matrix of AgSD-medicated wound dressing. Granulation tissue formation and wound contraction for the AgSD plus dihydroepiandrosterone (DHEA) impregnated PEC wound dressing were faster than any other groups. Li et  al. (2012) study the in situ injectable nanocomposite hydrogel composed of nanocurcumin (NC), ‘N,O-carboxymethyl chitosan and oxidized alginate’ (CCS-OA) for wound healing in a paper with 84 citations. They find that the encapsulated nanocurcumin was slowly released from CCS-OA hydrogel with the diffusion-controllable manner at initial phase followed by the corrosion manner of hydrogel at terminal phase. Application of NC/CCS-OA hydrogel could significantly enhance the reepithelialization of epidermis and collagen deposition in the wound tissue. They conclude that the nanocomposite hydrogel could significantly accelerate the process of wound healing. Meng et al. (2010) study the characteristics and drug release properties of chitosan and alginate polyelectrolyte complex (PEC) membranes in a paper with 79 citations. They find that these membranes showed pH- and ionic strength-dependent water uptake properties and had the water vapor transmission rate range from 442 to 618 g/m2/day. The maximum value of the dry membrane of breaking strength was 52.16 MPa and the maximum value of the wet membrane breaking elongation was 46.28%. The silver sulfadiazine release rate was the fastest when the alginate content was 50%.

472  PART | VII  Algal biomedicine

TABLE 30.2  The pioneering research on the wound care by alginates and chitosan. Alginates

Form

Focus

Cits.

Refs.

1

Alginate

Hydrogel

Healing, capillary formation

204

Murakami et al. (2010)

2

Alginate

Polyelectrolyte complex (PEC)

Cytotoxicity, healing

164

Wang et al. (2002)

3

Sodium alginate

Fibers

Antibacterial

137

Knill et al. (2004)

4

Sodium alginate

Polyelectrolyte complex (PEC) sponge

Cytotoxicity, antibacterial

86

Kim et al. (1999)

5

Oxidized alginate

Nano-composite hydrogel

Epithelialization

84

Li et al. (2012)

6

Alginate

Polyelectrolyte complex membrane

Properties

79

Meng et al. (2010)

7

Sodium alginate

Sponge

Healing

56

Dai et al. (2009)

8

Sodium alginate

Polyelectrolyte complex (PEC)

Healing

54

Hong et al. (2008)

9

Alginate dialdehyde

Membranes

Healing, biocompatibility

49

Gu et al. (2013)

10

Alginate

Hydrogels

Healing, antibacterial

47

Chen et al. (2017)

11

Sodium alginate

Films

Healing, epithelization

47

Dantas et al. (2011)

Dai et  al. (2009) study the chitosan-‘sodium alginate’ (SA) sponge focusing on the preparation and application in curcumin delivery for dermal wound healing in rat in a paper with 56 citations. As the chitosan content in the sponge decreased, the swelling ability decreased. All types of the sponges exhibited biodegradable properties. They find that the release of curcumin from the sponges could be controlled by the crosslinking degree. Curcumin could be released from the sponges in an extended period for up to 20 days. An in vivo animal test using SD rat showed that sponge had better effect than cotton gauze, and adding curcumin into the sponge enhanced the therapeutic healing effect. Hong et  al. (2008) study the accelerated wound healing by smad3 ‘antisense oligonucleotides’ (ASO)-impregnated chitosan/sodium alginate (SA) polyelectrolyte complex (PEC) in a paper with 54 citations. They find that zeta potentials and bioadhesive strengths of ASOs-PEC were increased as the chitosan ratio in PEC. In smad3-PEC, the healing process was faster than other groups because collagen contents increased and level of TGF-β1 decreased. They conclude that the smad3-PEC composed of chitosan and sodium alginate could be applied for accelerated wound healing. Gu et al. (2013) study the chitosan/silk fibroin (CS/SF) blending membrane fixed with ‘alginate dialdehyde’ (ADA) for wound dressing to evaluate the physical properties and biocompatibility of the membranes in a paper with 49 citations. They find that the stability, water absorption and water vapor permeability of the ADA fixed CS/SF membranes could meet the needs of wound dressing. The membranes promoted the cell attachment and proliferation. They conclude that ADA fixed CS/SF blending membranes with a suitable ratio could be a promising candidate for wound healing applications. Chen et al. (2017) study the covalently antibacterial oxidized alginate (OA)-‘carboxymethyl chitosan’ (CMCS) hydrogel dressing integrated gelatin microspheres containing tetracycline hydrochloride (TH) for wound healing in a paper with 47 citations. They find that with increasing ratios of microspheres from 10 to 40 mg/mL, the composite dressing showed shorter gelation time and lower swelling ratios, as well as higher mechanical strength. The gel dressing with 30 mg/mL microspheres showed more suitable stabilities and mechanical properties for wound healing. The loaded TH could be sustained release from the composite gel dressing. With the powerful bacteria growth inhibition effects against Escherichia coli and Staphylococcus aureus they conclude that the composite gel dressing has a promising future in treatment of bacterial infection. Dantas et al. (2011) study the dermal burn healing by combining sodium alginate (SA)/chitosan (C)-based films and low level laser therapy in a paper with 47 citations. They find that combined laser therapy and both dressings improved epithelization, blood vessels formation and collagenization, promoted rapid replacement of type III for type I collagen and favored the better arrangement of the newly formed collagen fibers. They conclude that the combination of laser therapy and sodium alginate/chitosan-based dressing improves burn healing, apparently by modulating the epithelization, blood vessels formation and collagenization processes.



The pioneering research on the wound care by alginates Chapter | 30  473

30.3.4  The pioneering research on the wound care by alginates and other biomaterials There have been 13 papers focusing on the wound care by alginates and other biomaterials (Table 30.3). There have been four, four, three, and two papers on the wound care by alginates and gelatin, polyvinyl alcohol (PVA), cellulose, and silk, respectively. Balakrishnan et al. (2005) evaluate an in situ forming hydrogel wound dressing based on oxidized alginate, gelatin, and borax in a paper with 447 citations. The composite matrix has the hemostatic effect of gelatin, the wound healingpromoting feature of alginate and the antiseptic property of borax to make it a potential wound dressing material. They find that the hydrogel had a fluid uptake of 90% of its weight which would prevent the wound bed from accumulation of exudates. The hydrogel can maintain a moist environment over wound bed in moderate to heavily exuding wound which would enhance epithelial cell migration during the healing process. In a rat model, within 2 weeks, the wound covered with gel was completely filled with new epithelium without any significant adverse reactions. Choi et al. (1999) study the gelatin-alginate sponge in a paper with 283 citations. They find that as the alginate content in the sponge increased, the porosity increased, resulting in an enhanced water uptake ability. Sponges loaded with silver sulfadiazine or gentamicin sulfate slowly released drugs for up to 4 days. The crosslinked sponge resisted in vitro collagenase digestion for up to 3 days. An in vivo animal test using witar rat showed rather good wound healing effect of this sponge containing AgSD than vaseline gauze in our full-thickness skin defect model. Shalumon et al. (2011) study the sodium alginate (SA)/polyvinyl alcohol (PVA)/nano ZnO composite nanofibers for antibacterial wound dressings in a paper with 179 citations. They find that those with 0.5 and 1% ZnO concentrations were less toxic where as those with higher concentrations of ZnO were toxic in nature. These mats showed antibacterial activity due to the presence of ZnO. Kim et al. (2008b) study the development of polyvinyl alcohol (PVA)‑sodium alginate (SA) gel-matrix-based wound dressing system containing nitrofurazone (NFZ) in a paper with 139 citations. They find that increased SA concentration decreased the gelation %, maximum strength and break elongation, but it resulted into an increment in the swelling ability, elasticity and thermal stability of hydrogel film. However, SA had insignificant effect on the release of NFZ. The amounts of proteins adsorbed on hydrogel were increased with increasing SA ratio, indicating the reduced blood compatibility. In vivo experiments showed that this hydrogel improved the healing rate of artificial wounds in rats. Roh et al. (2006) study wound healing effect of silk fibroin (SF)/alginate (AA)-blended sponge in full thickness skin defect of rat in a paper with 106 citations. They find that half healing time (HT50) of the sponge was dramatically reduced. Furthermore, sponge significantly increased the size of reepithelialization and the number of proliferating cell nuclear antigen positive cells. They conclude that the wound healing effect of sponge was the best among other treatments including SF and AA, and this synergic effect was mediated by reepithelialization via rapid proliferation of epithelial cell. Pereira et al. (2013) develop alginate-aloe vera (AV) based hydrogel films for wound healing applications in a paper with 102 citations. They find that AV improved the transparency of the films, as well their thermal stability. The developed films present adequate mechanical properties for skin applications, while the solubility studies demonstrated the insolubility of the films after 24 h of immersion in distilled water. The water absorption and swelling behavior of these films were greatly improved by the increase in AV proportion. Choi et al. (2001) study cross-linked gelatin/alginate (GA), gelatin/hyaluronate (GH) and chitosan/hyaluronate (CH) sponges and their application as a wound dressing in full-thickness skin defect of rat in a paper with 90 citations. The AgSD-impregnated sponges showed good wound healing performances on the whole. However, there were meaningful differences of wound healing between the gelatin-based sponges (GH, GA) and the CH. Walker et al. (2003) study the bacterial immobilization in a carboxymethyl cellulose (CMC) and alginate dressings in a paper with 81 citations. They show that following hydration of the wound dressing, the subsequent formation of a cohesive gel was effective in encapsulating large populations of potentially pathogenic bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus under the gelled surface, as well as being immobilized within the swollen fibers. They conclude that the unique absorbent gelling properties of the CMC dressing provide an ideal environment for immobilizing bacteria. Balakrishnan et al. (2006) study the ‘dibutyryl cyclic adenosine monophosphate’ (cAMP) in an in situ-forming hydrogel wound dressing based on oxidized alginate and gelatin in a paper with 71 citations. They find that in vitro release of DBcAMP from the matrix into phosphate buffered saline was slow and increased with time. Only 50–60% of the compound was released into the medium over a period of 2 days suggestive of a sustained release into the wound bed over a period of few days. Dressing promoted wound healing lead to complete reepithelialization of wounds within 10 days, whereas control wounds took 15 days for complete reepithelialization. They conclude that the presence of DBcAMP accelerated healing and re-epithelialization of full-thickness wounds.

Alginates

Form

Co-matrices

Focus

Cits.

Refs.

1

Oxidized alginate

Hydrogel

Gelatin, borax

Healing, antiseptic, epithelial cell migration

447

Balakrishnan et al. (2005)

2

Alginate

Sponge

Gelatin

Healing

283

Choi et al. (1999)

3

Sodium alginate

Nanofibers

Polyvinyl alcohol/nano zno

Cytotoxicity, antibacterial

179

Shalumon et al. (2011)

4

Sodium alginate

Hydrogels

Polyvinyl alcohol

Compatibility, healing

139

Kim et al. (2008a)

5

Alginate

Sponge

Silk fibroin

Epithelialization, healing

106

Roh et al. (2006)

6

Alginate

Hydrogel

Aloe vera

Water absorption and swelling

102

Pereira et al. (2013)

7

Alginate

Sponge

Gelatin

Healing

90

Choi et al. (2001)

8

Alginate

Carboxymethyl cellulose

Bacterial immobilization

81

Walker et al. (2003)

9

Oxidized alginate

Hydrogel

Gelatin

Healing, epithelialization

71

Balakrishnan et al. (2006)

10

Sodium alginate

Hydrogel membranes

Polyvinyl alcohol

Healing

65

Kamoun et al. (2015)

11

Sodium calcium alginate

Cellulose

Foreign body reaction, healing

65

Matthew et al. (1995)

12

Sodium alginate

Hydrogel films

Polyvinyl alcohol

Healing

47

Kim et al. (2008b)

13

Silver alginate

Fibers

Carboxymethylcellulose

Healing, antimicrobial

45

Beele et al. (2010)

474  PART | VII  Algal biomedicine

TABLE 30.3  The pioneering research on the wound care by alginates and other biomaterials.



The pioneering research on the wound care by alginates Chapter | 30  475

Kamoun et al. (2015) study the polyvinyl alcohol (PVA)‑sodium alginate (SA) hydrogel membranes for wound dressing applications in a paper with 65 citations. They find that increased SA content with PVA decreased gel fraction, elasticity, and elongation to break of PVA-SA membranes. However, it resulted in an increase in swelling degree, protein adsorption, and roughness of membrane surface. The hydrolytic degradation of PVA-SA membranes has prominently increased with increasing SA content. Furthermore, hemolysis (%) and in vitro inhibition (%) for both Gram positive and negative bacteria have been sharply affected by addition of SA into PVA, indicating the improved blood hemocompatibility. They conclude that this membrane based wound dressing system containing ampicillin could be a good candidate in wound care. Matthew et al. (1995) study the subperiosteal behavior of sodium calcium alginate (SCA) and cellulose (C) wound dressing materials in a paper with 65 citations. They find that both biomaterials caused a foreign body reaction, persisting up to 12 weeks after surgery. New bone formation occurred along the surface of the mandible in some specimens, but was not apparently related to the implants. The implantation of these biomaterials between bone and periosteum in the jaws caused a delay in wound healing, and had no effect on bone induction. Kim et al. (2008a) develop clindamycin-loaded wound dressing with polyvinyl alcohol (PVA) and sodium alginate (SA) in a paper with 47 citations. They find that increased SA concentration decreased the gelation %, maximum strength and break elongation, but it resulted into an increment in the swelling ability, elasticity and thermal stability of hydrogel film. This hydrogel improved the healing rate of artificial wounds in rats. Beele et al. (2010) evaluate the potential of a new silver alginate (SIA)/carboxymethyl cellulose (CMC) antimicrobial wound dressing to promote wound healing in a paper with 45 citations. The dressing showed a statistically significant improvement to healing as indicated by a reduction in the surface area of the wound, over the 4-week study period, compared with calcium alginate (CA) controls. This dressing showed a greater ability to prevent wounds progressing to infection when compared with the CA control dressing. There was an improvement in wound healing for this dressing compared with a non‑silver dressing.

30.4 Discussion 30.4.1  The research landscape The section on the research landscape highlights the most-prolific authors, institutions, journals, countries, publication years, and subject categories putting the pioneering research in a context. The authors and institutions from South Korea, Japan, and India have primarily contributed to the pioneering research in this field. ‘Biomaterials’, ‘International Journal of Pharmaceutics’, and ‘Journal of Biomedical Materials Research’ have been the primary research outlets. The United Kingdom, South Korea, China, and Japan have published 30 papers. The data on the publication years show that this research field has primarily developed in recent years. It is notable that 37 of these papers have been indexed by the subject categories of ‘Materials Science Biomaterials’ and ‘Engineering Biomedical’ highlighting the materials science emphasis of the research. The citation impact of these papers has been relatively significant with 4216 citations in total, 100 citations per paper and H-index of 42. There have been 18, 11, and 13 pioneering papers related to the wound care by alginates only, by alginates and chitosan, and alginates and other biomaterials, respectively.

30.4.2  The pioneering research on the wound care by alginates only The research on the wound care by alginates only has formed a significant part of the research in this field with 18 papers (Table 30.1). Qin (2008) has been the only review paper focusing on the production processes and applications of alginates in the wound care. The research in this field has primarily focused on the healing of the deep wounds (Agren, 1996; Attwood, 1989; Barnett and Varley, 1987; Doyle et al., 1996; Gilchrist and Martin, 1983; Hegge et al., 2011; Rabbany et al., 2010; Sikareepaisan et al., 2011; Suzuki et al., 1999; Thomas et al., 2000; Thu et al., 2012). All these studies have confirmed the healing effect of the alginate wound dressings in the wound care. Some studies have focused on the reepithelization in the wound healing process (Agren, 1996; Hashimoto et al., 2004; Porter, 1991; Suzuki et al., 1999). All these studies have confirmed the healing effect of the alginate wound dressings in the wound care through reepithelization process. As the cytotoxicity of the wound dressings is an important public issue, Jude et al. (2007), Sikareepaisan et al. (2011), Suzuki et al. (1998), and Wiegand et al. (2009) have focused on this issue. All these studies have confirmed the non-toxicity of the alginate wound dressings in the wound care.

476  PART | VII  Algal biomedicine

Some studies have focused on the antimicrobial effects of the alginate wound dressings (Hegge et al., 2011; Liakos et al., 2014; Wiegand et al., 2009). All these studies have confirmed the antimicrobial effect of the alginate wound dressings in the wound care. Although most of these studies have primarily studied the alginates in general, some studies have focused on calcium alginate (Agren, 1996; Attwood, 1989; Barnett and Varley, 1987; Doyle et  al., 1996; Jude et  al., 2007) and silver and nanosilver alginate (Wiegand et al., 2009). Although most of the studies have studied alginate dressings in general some studies have focused on the alginate fibers (Gilchrist and Martin, 1983; Qin, 2008; Thomas et al., 2000), films (Liakos et al., 2014; Sikareepaisan et al., 2011; Thu et al., 2012), foams (Hegge et al., 2011), and hydrogels (Suzuki et al., 1998). Thus, the pioneering research on the wound care by alginates only has provided ample evidence for the value of alginate dressings in the wound care. The optimization of the wound care emerges as a strategic point in these studies.

30.4.3  The pioneering research on the wound care by alginates and chitosan The research on the wound care by alginates and chitosan has formed a significant part of the research in this field with 11 papers (Table 30.2). The research in this field has primarily focused on the healing of the deep wounds (Chen et al., 2017; Dai et al., 2009; Dantas et al., 2011; Gu et al., 2013; Hong et al., 2008; Murakami et al., 2010; Wang et al., 2002). All these studies have confirmed the healing effect of the alginate and chitosan wound dressings in the wound care. Some studies have focused on the reepithelization in the wound healing process (Dantas et al., 2011; Li et al., 2012). All these studies have confirmed the healing effect of the alginate and chitosan wound dressings in the wound care through reepithelization process. As the cytotoxicity of the wound dressings is an important public issue, Kim et al. (1999) and Wang et al. (2002) have focused on this issue. All these studies have confirmed the non-toxicity of the alginate and chitosan wound dressings in the wound care. Some studies have focused on the antimicrobial effects of the alginate and chitosan wound dressings (Chen et al., 2017; Knill et al., 2004). All these studies have confirmed the antimicrobial effect of the alginate and chitosan wound dressings in the wound care. Although most of these studies have primarily studied the alginates in general, some studies have focused on sodium alginate (Dai et al., 2009; Dantas et al., 2011; Hong et al., 2008; Kim et al., 1999; Knill et al., 2004), oxidized alginate (Li et al., 2012), and alginate dialdehyde (Gu et al., 2013). All these studies have studied some form of alginate dressings: hydrogels (Chen et al., 2017; Li et al., 2012; Murakami et al., 2010); polyelectrolyte complexes (Hong et al., 2008; Kim et al., 1999; Meng et al., 2010; Wang et al., 2002), sponge (Dai et al., 2009), fibers (Knill et al., 2004), membranes (Gu et al., 2013), and films (Dantas et al., 2011). Thus, the pioneering research on the wound care by alginates and chitosan has provided ample evidence for the value of alginate and chitosan dressings in the wound care. The optimization of the wound care emerges as a strategic point in these studies.

30.4.4  The pioneering research on the wound care by alginates and other biomaterials The research on the wound care by alginates and other biomaterials has formed a significant part of the pioneering research in this field with 13 papers (Table 30.3). The research in this field has primarily focused on the healing of the deep wounds (Balakrishnan et al., 2005; Choi et al., 1999, 2001; Kim et al., 2008a,b; Roh et al., 2006; Kamoun et al., 2015; Matthew et al., 1995; Beele et al., 2010). All these studies have confirmed the healing effect of the alginate and other biomaterial wound dressings in the wound care. Some studies have focused on the reepithelization in the wound healing process (Balakrishnan et al., 2006; Roh et al., 2006). All these studies have confirmed the healing effect of these dressings in the wound care through reepithelization process. As the cytotoxicity of the wound dressings is an important public issue, Shalumon et al. (2011) have focused on this issue. This study has confirmed the non-toxicity of these wound dressings in the wound care. Some studies have focused on the antimicrobial effect of these wound dressings (Balakrishnan et al., 2005, 2006, Beele et al., 2010; Shalumon et al., 2011; Walker et al., 2003). All these studies have confirmed the antimicrobial effect of these wound dressings in the wound care.



The pioneering research on the wound care by alginates Chapter | 30  477

Although most of these studies have primarily studied the alginates in general, some studies have focused on sodium alginate (Kamoun et al., 2015; Kim et al., 2008a,b; Shalumon et al., 2011), sodium calcium alginate (Matthew et al., 1995), silver alginate (Beele et al., 2010), and oxidized alginate (Balakrishnan et al., 2005, 2006). Most of these studies have studied some form of alginate dressings: hydrogels (Balakrishnan et al., 2005, 2006; Kamoun et al., 2015; Kim et al., 2008a,b; Pereira et al., 2013), nanofibers (Shalumon et al., 2011), sponge (Choi et al., 2001), and fibers (Beele et al., 2010). Thus, the pioneering research on the wound care by these wound dressings has provided ample evidence for the value of alginate and other biomaterial dressings in the wound care. The optimization of the wound care emerges as a strategic point in these studies.

30.5 Conclusion This study of the pioneering research on the wound care by alginates at the global scale covering the whole range of research fronts as well as all types of alginates has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. The research in this field have progressed in three subfields: wound care by alginates only, by alginates and chitosan, and by alginates and other biomaterials with 18, 11, and 13 papers, respectively (Tables 30.1–30.3). There has been a research focus on the healing of wounds by alginates in general as well as specific issues such as antimicrobial effects, cytotoxicity, biocompatibility, reepithelialization, and foreign body reaction. Although some studies studied alginates in general, there have also been some specific studies of sodium alginate, calcium alginate, silver alginate, and oxidized alginate. Similarly, some studies have covered alginates in general whereas other studies have focused on alginate hydrogels, films, sponges, membranes, fibers, nanofibers, and polyelectrolyte complexes. It has been found that the detailed keyword set provided in the appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. The citation impact of these pioneering studies has been significant, contributing to the wider research fields of chitosan, regenerative medicine, alginates, tissue engineering, drug delivery, and hydrogels. It is notable that the applications of nanotechnology in this field has not been significant (Li et al., 2012; Shalumon et al., 2011; Wiegand et al., 2009). It is expected the applications of nanotechnology in the wound care by alginates would accelerate to further optimize the healing processes in the wound care (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e,f). It appears that the structure-processing-property relationships form the basis of the research in this field as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002). These pioneering studies with at least 45 citations each in this field have formed the basis for the rapid expansion of this research field with the promising results for the effective wound care through the optimization of alginate types, other biomaterials, and forms of alginates among others. It is recommended that the corresponding studies should be carried out in other related fields such as applications of alginates in drug delivery, tissue engineering, and cell engineering. It is further recommended that a full scientometric study of these fields should be carried.

Appendix. The keyword sets A.1  Alginate-related keywords TI = (algin or alginic* or alginate*).

A.2  Wound-related keywords (TI = (wound* or ‘pressure ulcer*’ or ‘foot ulcer*’ or dressing* or ‘leg ulcer*’ or skin or burn or sorbsan or kaltostat or kaltogel or tegagen or algosteril or algisite or comfeel) or SO = (‘Advances in Skin & Wound*’ or ‘Advances in Wound*’

478  PART | VII  Algal biomedicine

or ‘Chronic Wound*’ or ‘International Journal of Lower Extremity Wounds’ or ‘International Wound*’ or ‘Journal of Wound*’ or ‘Ostomy Wound*’ or Wound*)) NOT TI = (Pseudomonas or food* or release or tissue*).

References Agren, M.S., 1996. Four alginate dressings in the treatment of partial thickness wounds: a comparative experimental study. Br. J. Plast. Surg. 49 (2), 129–134. Attwood, A.I., 1989. Calcium alginate dressing accelerates split skin-graft donor site healing. Br. J. Plast. Surg. 42 (4), 373–379. Augst, A.D., Kong, H.J., Mooney, D.J., 2006. Alginate hydrogels as biomaterials. Macromol. Biosci. 6 (8), 623–633. Balakrishnan, B., Mohanty, M., Umashankar, P.R., Jayakrishnan, A., 2005. Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials 26 (32), 6335–6342. Balakrishnan, B., Mohanty, M., Fernandez, A.C., Mohanan, P.V., Jayakrishnan, A., 2006. Evaluation of the effect of incorporation of dibutyryl cyclic adenosine monophosphate in an in situ-forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials 27 (8), 1355–1361. Barnett, S.E., Varley, S.J., 1987. The effects of calcium alginate on wound-healing. Ann. R. Coll. Surg. 69 (4), 153–155. Beele, H., Meuleneire, F., Nahuys, M., Percival, S.L., 2010. A prospective randomised open label study to evaluate the potential of a new silver alginate/ carboxymethylcellulose antimicrobial wound dressing to promote wound healing. Int. Wound J. 7 (4), 262–270. Brennan, L., Owende, P., 2010. Biofuels from microalgae-a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14 (2), 557–577. Chen, H.N., Xing, X.D., Tan, H.P., Jia, Y., Zhou, T.L., Chen, Y., et al., 2017. Covalently antibacterial alginate-chitosan hydrogel dressing integrated gelatin microspheres containing tetracycline hydrochloride for wound healing. Mater. Sci. Eng. C Mater. Biol. Appl. 70, 287–295. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Choi, Y.S., Hong, S.R., Lee, Y.M., Song, K.W., Park, M.H., Nam, Y.S., 1999. Study on gelatin-containing artificial skin: I. Preparation and characteristics of novel gelatin-alginate sponge. Biomaterials 20 (5), 409–417. Choi, Y.S., Lee, S.B., Hong, S.R., Lee, Y.M., Song, K.W., Park, M.H., 2001. Studies on gelatin-based sponges. Part III: a comparative study of cross-linked gelatin/alginate, gelatin/hyaluronate and chitosan/hyaluronate sponges and their application as a wound dressing in full-thickness skin defect of rat. J. Mater. Sci. Mater. Med. 12 (1), 67–73. Dai, M., Zheng, X.L., Xu, X., Kong, X.Y., Li, X.Y., Guo, G., et al., 2009. Chitosan-alginate sponge: preparation and application in curcumin delivery for dermal wound healing in rat. J. Biomed. Biotechnol. 2009, 595126. Dantas, M.D.M., Cavalcante, D.R.R., Araujo, F.E.N., Barretto, S.R., Aciole, G.T.S., Pinheiro, A.L.B., et al., 2011. Improvement of dermal burn healing by combining sodium alginate/chitosan-based films and low level laser therapy. J. Photochem. Photobiol. B 105 (1), 51–59. Doyle, J.W., Roth, T.P., Smith, R.M., Li, Y.Q., Dunn, R.M., 1996. Effect of calcium alginate on cellular wound healing processes modeled in vitro. J. Biomed. Mater. Res. 32 (4), 561–568. George, M., Abraham, T.E., 2006. Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review. J. Control. Release 114 (1), 1–14. Gilchrist, T., Martin, A.M., 1983. Wound treatment with sorbsan—an alginate fiber dressing. Biomaterials 4 (4), 317–320. Gombotz, W.R., Wee, S.F., 1998. Protein release from alginate matrices. Adv. Drug Deliv. Rev. 31 (3), 267–285. Gu, Z.P., Xie, H.X., Huang, C.C., Li, L., Yu, X.X., 2013. Preparation of chitosan/silk fibroin blending membrane fixed with alginate dialdehyde for wound dressing. Int. J. Biol. Macromol. 58, 121–126. Hashimoto, T., Suzuki, Y., Tanihara, M., Kakimaru, Y., Suzuki, K., 2004. Development of alginate wound dressings linked with hybrid peptides derived from laminin and elastin. Biomaterials 25 (7–8), 1407–1414. Hegge, A.B., Andersen, T., Melvik, J.E., Bruzell, E., Kristensen, S., Tonnesen, H.H., 2011. Formulation and bacterial phototoxicity of curcumin loaded alginate foams for wound treatment applications: studies on curcumin and curcuminoides XLII. J. Pharm. Sci. 100 (1), 174–185. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol. Adv. 29 (2), 189–198. Hong, H.J., Jin, S.E., Park, J.S., Ahn, W.S., Kim, C.K., 2008. Accelerated wound healing by smad3 antisense oligonucleotides-impregnated chitosan/ alginate polyelectrolyte complex. Biomaterials 29 (36), 4831–4837. Jude, E.B., Apelqvist, J., Spraul, M., Martini, J., Silver Dressing Study Group, 2007. Prospective randomized controlled study of HydrofiberR dressing containing ionic silver or calcium alginate dressings in non-ischaemic diabetic foot ulcers. Diabet. Med. 24 (3), 280–288. Kamoun, E.A., Kenawy, E.R.S., Tamer, T.M., El-Meligy, M.A., Eldin, M.S.M., 2015. Poly (vinyl alcohol)-alginate physically crosslinked hydrogel membranes for wound dressing applications: characterization and bio-evaluation. Arab. J. Chem. 8 (1), 38–47. Kim, H.J., Lee, H.C., Oh, J.S., Shin, B.A., Oh, C.S., Park, R.D., et al., 1999. Polyelectrolyte complex composed of chitosan and sodium alginate for wound dressing application. J. Biomater. Sci. Polym. Ed. 10 (5), 543–556. Kim, J.O., Choi, J.Y., Park, J.K., Kim, J.H., Jin, S.G., Chang, S.W., et al., 2008a. Development of clindamycin-loaded wound dressing with polyvinyl alcohol and sodium alginate. Biol. Pharm. Bull. 31 (12), 2277–2282. Kim, J.O., Park, J.K., Kim, J.H., Jin, S.G., Yong, C.S., Li, D.X., et al., 2008b. Development of polyvinyl alcohol-sodium alginate gel-matrix-based wound dressing system containing nitrofurazone. Int. J. Pharm. 359 (1–2), 79–86.



The pioneering research on the wound care by alginates Chapter | 30  479

Knill, C.J., Kennedy, J.F., Mistry, J., Miraftab, M., Smart, G., Groocock, M.R., et al., 2004. Alginate fibres modified with unhydrolysed and hydrolysed chitosans for wound dressings. Carbohydr. Polym. 55 (1), 65–76. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2019. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

480  PART | VII  Algal biomedicine

Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Kuo, C.K., Ma, P.X., 2001. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. structure, gelation rate and mechanical properties. Biomaterials 22 (6), 511–521. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Li, Z.S., Ramay, H.R., Hauch, K.D., Xiao, D.M., Zhang, M.Q., 2005. Chitosan-alginate hybrid scaffolds for bone tissue engineering. Biomaterials 26 (18), 3919–3928. Li, X.Y., Chen, S., Zhang, B.J., Li, M., Diao, K., Zhang, Z.L., et al., 2012. In situ injectable nano-composite hydrogel composed of curcumin, N,Ocarboxymethyl chitosan and oxidized alginate for wound healing application. Int. J. Pharm. 437 (1–2), 110–119. Liakos, I., Rizzello, L., Scurr, D.J., Pompa, P.P., Bayer, I.S., Athanassiou, A., 2014. All-natural composite wound dressing films of essential oils encapsulated in sodium alginate with antimicrobial properties. Int. J. Pharm. 463 (2), 137–145. Matthew, I.R., Browne, R.M., Frame, J.W., Millar, B.G., 1995. Subperiosteal behavior of alginate and cellulose wound dressing materials. Biomaterials 16 (4), 275–278. Meng, X., Tian, F., Yang, J., He, C.N., Xing, N., Li, F., 2010. Chitosan and alginate polyelectrolyte complex membranes and their properties for wound dressing application. J. Mater. Sci. Mater. Med. 21 (5), 1751–1759. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal‐free organic dyes for dye‐sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Murakami, K., Aoki, H., Nakamura, S., Nakamura, S., Takikawa, M., Hanzawa, M., et al., 2010. Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-impaired wound dressings. Biomaterials 31 (1), 83–90. Pereira, R., Carvalho, A., Vaz, D.C., Gil, M.H., Mendes, A., Bartolo, P., 2013. Development of novel alginate based hydrogel films for wound healing applications. Int. J. Biol. Macromol. 52, 221–230. Porter, J.M., 1991. A comparative investigation of reepithilialization of split skin-graft donor areas after application of hydrocolloid and alginate dressings. Br. J. Plast. Surg. 44 (5), 333–337. Qin, Y.M., 2008. Alginate fibres: an overview of the production processes and applications in wound management. Polym. Int. 57 (2), 171–180. Rabbany, S.Y., Pastore, J., Yamamoto, M., Miller, T., Rafii, S., Aras, R., et al., 2010. Continuous delivery of stromal cell-derived factor-1 from alginate scaffolds accelerates wound healing. Cell Transplant. 19 (4), 399–408. Roh, D.H., Kang, S.Y., Kim, J.Y., Kwon, Y.B., Kweon, H.Y., Lee, K.G., et al., 2006. Wound healing effect of silk fibroin/alginate-blended sponge in full thickness skin defect of rat. J. Mater. Sci. Mater. Med. 17 (6), 547–552. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes-towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Shalumon, K.T., Anulekha, K.H., Nair, S.V., Nair, S.V., Chennazhi, K.P., Jayakumar, R., 2011. Sodium alginate/poly(vinyl alcohol)/nano ZnO composite nanofibers for antibacterial wound dressings. Int. J. Biol. Macromol. 49 (3), 247–254. Sikareepaisan, P., Ruktanonchai, U., Supaphol, P., 2011. Preparation and characterization of asiaticoside-loaded alginate films and their potential for use as effectual wound dressings. Carbohydr. Polym. 83 (4), 1457–1469. Smidsrod, O., Skjak-Braek, G., 1990. Alginate as immobilization matrix for cells. Trends Biotechnol. 8 (3), 71–78. Suzuki, Y., Nishimura, Y., Tanihara, M., Suzuki, K., Nakamura, T., Shimizu, Y., et al., 1998. Evaluation of a novel alginate gel dressing: cytotoxicity to fibroblasts in vitro and foreign-body reaction in pig skin in vivo. J. Biomed. Mater. Res. 39 (2), 317–322.



The pioneering research on the wound care by alginates Chapter | 30  481

Suzuki, Y., Tanihara, M., Nishimura, Y., Suzuki, K., Yamawaki, Y., Kudo, H., et al., 1999. In vivo evaluation of a novel alginate dressing. J. Biomed. Mater. Res. 48 (4), 522–527. Thomas, A., Harding, K.G., Moore, K., 2000. Alginates from wound dressings activate human macrophages to secrete tumour necrosis factor-α. Biomaterials 21 (17), 1797–1802. Thu, H.E., Zulfakar, M.H., Ng, S.F., 2012. Alginate based bilayer hydrocolloid films as potential slow-release modern wound dressing. Int. J. Pharm. 434 (1–2), 375–383. Walker, M., Hobot, J.A., Newman, G.R., Bowler, P.G., 2003. Scanning electron microscopic examination of bacterial immobilisation in a carboxymethyl cellulose (AQUACELR) and alginate dressings. Biomaterials 24 (5), 883–890. Wang, L.H., Khor, E., Wee, A., Lim, L.Y., 2002. Chitosan-alginate PEC membrane as a wound dressing: assessment of incisional wound healing. J. Biomed. Mater. Res. 63 (5), 610–618. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718.

Chapter 31

The scientometric analysis of the research on the algal foods Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

31.1 Introduction The algae have increasingly gained public importance as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et  al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environmentfriendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 papers published since 1980. The algal foods have been among the most-prolific research fronts over time as evidenced with nearly 15,000 papers published during the same study period, corresponding to the public concerns about the development of sustainable and environment-friendly bioproducts and bioprocesses in food science and technology. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate, the stakeholders should have timely and thorough access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b, 2012a,b). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal foods and the underlying research areas as in fields of the algal research (Konur, 2011a, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a,b,c, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012c,d,e,f,g,h,i,j,k,l, 2018a,b,c), energy and fuels (Konur, 2012m,n,o, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e, 2019d), biomedicine (Konur, 2016i, 2018d), and social sciences (Konur, 2011b, 2012p,q,r,s,t,u,v,x,y,z, 2013a,b,c,d,e,f,g,h,i,j,k,l). Although there have been over 550 literature reviews on the algal foods, there has been no published scientometric studies in the journal literature. This is contrast to the many published scientometric studies on food science and technology in the journal literature (Blazquez‐Ruiz et al., 2016; Guerrero‐Bote and Moya‐Anegon, 2015; Yeung et al., 2018). Therefore, this paper presents the first-ever scientometric study of the research in algal foods covering the whole range of research fronts as well as whole range of algae at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field with a relatively low funding rate.

31.2  Materials and methodology The search for the scientometric analysis of the literature on the algal foods was carried out in January 2019 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI) and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in the Appendix. These keyword sets have been devised in three major parts: the keywords related to foods and keywords related to the algae as well as the cross-subject keywords related to algal foods. There have been two distinct keyword sets for the first part: the set of core subject categories related to the foods and keywords related to the foods.

Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00031-0 © 2020 Elsevier Inc. All rights reserved.

485

486  PART | VIII  Algal foods

On the other hand, the second part consists of the keywords related to the algae in general, dinoflagellates and coccolithophores, microalgae, macroalgae, diatoms, cyanobacteria, and journal titles related to the algae. A detailed set of cross-subject keywords related to the both foods and algae has formed the third keyword set. The papers located through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subject to the quality control exercise to ensure that these references have been primarily related to the algal foods. This refined list of papers has formed the core sample for the scientometric and content overview of the literature on the algal foods. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. Additionally, the search has been carried out for the papers with at least 100 citations and the relevant scientometric data has been collected. These papers have been termed as ‘influential papers’. Furthermore, the data on the scientometric analysis and brief content overview of 20 most-cited papers have also been provided focusing on the determination of the key research fronts for these citation classics, complementing sections on the keywords and research fronts. The presented results have also been discussed through an interdisciplinary perspective. Additionally, a number of conclusions have been drawn and a number of recommendations for the further research have been made.

31.3 Results 31.3.1  Documents and indexes The search has resulted in 17,044 papers where there have been 14,152 articles, 1587 meeting abstracts, 583 reviews, 335 notes, 122 letters, 99 corrections, 73 editorial materials, and 69 news items. In the first instance, the papers excluding meeting abstracts, news items, and corrections have been selected resulting in 15,265 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 14,789 papers in English. The other major languages have been Japanese, German, French, Spanish, and Russian. This set of 14,789 papers has formed the core sample for the scientometric analysis of the literature on the algal foods. The articles have formed 93.0% of the final sample while reviews, notes, letters, and editorial matters have formed 3.7%, 2.0%, 0.8%, and 0.4% of this sample, respectively. Additionally, 2.4% of these papers have been ‘proceedings papers’ and there have been four ‘retracted papers’. On the other hand, 98.0% of these papers have been indexed by the SCI-E while only 0.3% of the papers have been indexed by the SSCI and there have been no papers indexed by the A&HCI. Additionally, 2.0% of the papers have been indexed by the ESCI.

31.3.2 Keywords The most-prolific keywords used in algal foods have been determined based on the influential papers to locate the hot topics and the primary research fronts in the algal foods. There have been a number of most-prolific keywords for the first set of keywords for the foods: ‘edible, *food*, nutrition*, dietary, anti*, inhibit*, bioactiv*, *cancer, cytotoxic* or immuno*, pharm*’. On the other hand, there have been a number of prolific journals related to algal foods: ‘Marine Drugs’, ‘Journal of Natural Products’, ‘Journal of Agricultural and Food Chemistry’, ‘Food Chemistry’, ‘Carbohydrate Polymers’, ‘Food Hydrocolloids’, ‘Phytotherapy Research’, Virology or ‘Journal of the Science of Food and Agriculture’. Similarly, the most-prolific subject categories related to the algal foods have been ‘Food Science Technology’, ‘Pharmacology Pharmacy’, ‘Biochemistry Molecular Biology’, ‘Biotechnology Applied Microbiology’, ‘Chemistry Medicinal’, ‘Plant Sciences’, and ‘Chemistry Applied’. Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, carrageenan*, macroalga*, rhodophyt*, seaweed*, bacillariophycea*, diatom, diatoms, and cyanobacter*’. The other prolific keywords for the algae have been ‘coccolith*, dinophycea*, Alexandrium, chlorophycea*, chlorophyt*, “green alga*”, microalga*, “micro-alga*”, Chlamydomonas, *Chlorella, Dunaliella, Euglena, Scenedesmus, “brown alga*”, phaeophycea*, kelp*, phaeophyt*, “red alga*”, Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, Ulva, bacillariophyt*, “blue green alga*”, “blue-green alga*”, *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, Synechocystis’.



Algal food research Chapter | 31  487

On the other hand, the most-prolific cross-subject keywords have been ‘phlorotannin*, dieckol, ulvan, porphyran, laminaran’.

31.3.3 Authors There have been 39,209 authors contributing to the research on the algal foods in total. The information on the most-­ prolific and influential 20 authors is provided in Table  31.1: Authors’ names, gender, institutions, countries, primary research fronts, number of papers produced by these authors, the number of influential papers with at least 100 citations received (I-100), and the percentage of the number of influential papers with relative to the number of all the papers published (I-100%). The data on these authors shows that the most-prolific author with the highest citation impact has been ‘Richard E Moore’ of the University of Hawaii at Manoa, working primarily on the ‘cryptophycins’ from cyanobacteria, with 74 papers. His citation impact is highest with 18 influential papers. The other most-prolific authors with the high citation impact have been ‘Gregory ML Patterson’, ‘Michael R Boyd’, ‘William H Gerwick’, ‘Quanbin Zhang’, ‘You-Jin Jeong’, ‘Se-Kwon Kim’, ‘Anatolii I Usov’, ‘Zhien Li’ with six or more influential papers each. Of them, ‘Se-Kwon Kim’ has been a ‘highly cited researcher’ in 2018 (Basu, 2006). The United States has been the most-prolific country for these authors with seven authors while Japan and Spain have been the other prolific countries with three authors each. Additionally, China, Russia, and South Korea have had two authors each. On the other hand, Europe has had only three authors as a whole. There has been a significant gender deficit among these top prolific and influential authors as only four of them are females: ‘Pilar Ruperez’, ‘Kyoko Hayashi’, ‘Elena Ibanez’, and ‘Elsa B Damonte’. Similarly, the most-prolific institution has been ‘Spanish National Research Council’ (CSIC) with three authors. The other prolific institutions have been ‘Chinese Academy of Sciences’, ‘National Cancer Institute’, ‘Russian Academy of Sciences’, ‘Toyoma University’, and ‘University of Hawaii Manoa’ with two authors each. The most-prolific research fronts have been the ‘fucoidans’ and other ‘sulfated polysaccharides’ with five authors each. The other prolific research fronts have been ‘cryptophycins’ and ‘foods’ with three authors each and ‘curacins’ and ‘cyanovirins’ with three authors each. Similarly, the most prolific types of algae studied by these top authors have been ‘macroalgae’ and ‘cyanobacteria’ with 11 and 7 authors, respectively. Additionally, two authors focused on ‘microalgae’. There have been no focus on ‘algae in general’, ‘diatoms’, and ‘dinoflagellates and coccolithophores’. The number of papers published by these authors have ranged from 12 to 125. These most-prolific authors have also contributed to nearly 6.1% and 31.1% of all the papers and influential papers, respectively. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the influential papers with relative to the number of all the papers published for each author in the last column (I-100%). These data shows that ‘Richard E Moore’ and ‘Gregory ML Patterson’ have been the top influential authors with 41.7% and 40.0% ratios. The other most-influential authors have been ‘Pilar Ruperez’, ‘Michael R Boyd’, ‘William H Gerwick’, and ‘Kyoko Hayashi’ with over 20% ratio each.

31.3.4 Countries Nearly 99.7% of the papers have had country information in their abstract pages and 134 countries and territories have contributed to these papers overall. Table 31.2 provides the information about the most-prolific and influential 20 countries. These 20 most-prolific countries have produced 96.0% and 115.1% of all the papers and influential papers, respectively, as a whole. The most-prolific and influential country has been the US producing 16.1% and 30.7% of all the papers and influential papers, respectively. The second-top country has been Japan with 12.8% and 15.8% of all the papers and influential papers, respectively. Additionally, Spain, France, China, United Kingdom, and Germany have emerged as the other most-prolific and influential countries following the top-two countries producing at least 5.6% of the influential papers each. These countries have also produced at least 4.0% of all the papers each. The European countries have been dominant in the top-20 country list as they have produced 26.7% and 38.7% of all the papers and influential papers, respectively, as a whole, surpassing significantly the United States, Japan, and China. Similarly, the Asian countries in this top-20 list, have produced 43.5% and 35.5% of all the papers and influential papers, respectively, as a whole.

Author

Gender

Institution

Country

Research fronts

Algae

1

Richard E Moore

M

Univ. Hawaii Manoa

United States

Cryptophycins

Cyanobacteria

2

Gregory ML Patterson

M

Univ. Hawaii Manoa

United States

Cryptophycins

3

Michael R Boyd

M

Natl. Cancer Inst.

United States

4

William H Gerwick

M

Univ. Calif. San Diego

5

Quanbin Zhang

M

6

You-Jin Jeong

7

I-0

I-100

I-100%

74

18

41.7

Cyanobacteria

41

14

40.0

Cyanovirins

Cyanobacteria

29

11

31.3

United States

Curacins

Cyanobacteria

125

8

29.4

Chinese Acad. Sci.

China

Polysaccharides

Macroalgae

62

7

16.0

M

Cheju Natl. Univ.

S. Korea

Polysaccharides

Macroalgae

124

6

15.4

Se-Kwon Kim

M

Pukyong Natl. Univ.

S. Korea

Polysaccharides

Macroalgae

70

6

37.9

8

Anatolii I Usov

M

Russian Acad. Sci.

Russia

Polysaccharides

Macroalgae

30

6

20.0

9

Zhien Li

M

Chinese Acad. Sci.

China

Polysaccharides

Macroalgae

15

6

15.2

10

Barry R O’Keefe

M

Natl. Cancer Inst.

United States

Cyanovirins

Cyanobacteria

37

5

12.1

11

Elena Ibanez

F

CSIC

Spain

Foods

Microalgae

33

5

12.1

12

Charles D Smith

M

Fox Chase Cancer Ctr.

United States

Cryptophycins

Cyanobacteria

17

5

13.5

13

Takahisa Nakano

M

Riken Vitamin

Japan

Fucoidans

Macroalgae

16

5

10.3

14

Pilar Ruperez

F

CSIC

Spain

Foods

Macroalgae

12

5

34.1

15

Hendrik Luesch

M

Univ. Florida

United States

Dispeptides

Cyanobacteria

58

4

6.9

16

Toshimitsu Hayashi

M

Toyoma Univ.

Japan

Fucoidans

Macroalgae

39

4

11.3

17

Elsa B Damonte

F

Univ. Buenos Aires

Argentina

Fucoidans

Macroalgae

33

4

8.6

18

Kyoko Hayashi

F

Toyoma Univ.

Japan

Fucoidans

Macroalgae

33

4

24.3

19

Alejandro Cifuentes

M

CSIC

Spain

Foods

Microalgae

26

4

4.8

20

Nikolay E Nifantiev

M

Russian Acad. Sci.

Russia

Fucoidans

Macroalgae

25

4

6.4

Average

45.0

6.6

Total %

6.1

31.1

19.6

M, Male; F, Female; I-0, no. papers, the number of papers for at least 12 papers; I-100, the number of influential papers with at least 100 citations for at least 4 papers; I-100%, the percentage of the number of influential papers with relative to the number of all the papers published.

488  PART | VIII  Algal foods

TABLE 31.1  The most-prolific and influential authors in algal foods.

Algal food research Chapter | 31  489



TABLE 31.2  The most-prolific and influential countries in algal foods. Country

I-0

I-0%

I-100

Europe

3945

26.7

159

38.7

Asia

6438

43.5

145

35.3

1

United States

2383

16.1

126

30.7

2

Japan

1908

12.8

65

15.8

3

Spain

648

4.4

31

7.5

4

France

670

4.5

28

6.8

5

China

1719

11.6

28

6.8

6

United Kingdom

593

4.0

27

6.6

7

Germany

890

6.0

23

5.6

8

S. Korea

1217

8.2

20

4.9

9

India

1028

7.0

16

3.9

11

Canada

428

3.9

15

3.7

10

Portugal

258

1.7

13

3.2

12

Italy

436

2.9

11

2.7

13

Russia

311

2.1

11

2.7

14

Australia

358

2.4

10

2.4

15

Brazil

559

3.8

10

2.4

16

Denmark

104

0.7

9

2.2

17

Ireland

191

1.3

9

2.2

18

Netherlands

155

1.0

8

2.0

19

Argentina

132

0.9

7

1.7

20

Malaysia

208

1.4

6

1.5

14,196

96.0

473

115.1

Total

I-100%

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Europe and Asia values are for only the top-20 countries.

31.3.5 Institutions Over 99.6% of the papers have had their institutions listed in their abstract pages. For these papers, 6851 institutions have contributed to the research on the algal foods in total. The information about the 20 most-prolific and influential institutions is given in Table 31.3. The most-prolific and influential institution has been the ‘Scientific Applications International Company’ (SAIC) of the US publishing 0.5% and 4.4% of the all and influential papers, respectively. ‘Spanish National Research Council’ (CSIC), ‘National Cancer Institute’ (NCI), ‘Chinese Academy of Sciences’, ‘University of Hawaii’ at Manoa, ‘French National Research Council’ (CNRS), and ‘Russian Academy of Sciences’ have been the other influential institutions as they have produced 4.1%, 3.7%, 3.4%, 2.9%, 2.7%, and 2.4% of the influential papers, respectively. The most-prolific countries for these institutions have been the United States, France, and Japan with four, three, and three institutions, respectively. On the other hand, Europe has had six institutions as a whole where these European institutions produced 5.6% and 12.9% of the all the papers and influential papers, respectively. Similarly, the Asian institutions have produced 9.9% and 13.4% the all the papers and influential papers, respectively while the US institutions have produced 1.6% and 12.4% of all the papers and influential papers, respectively.

490  PART | VIII  Algal foods

TABLE 31.3  The most-prolific and influential institutions in algal foods. Institutions

Country

I-0

I-0%

I-100

I-100%

United States

237

1.6

51

12.4

Europe

805

5.6

53

12.9

1466

9.9

55

13.4

72

0.5

18

4.4

210

1.4

17

4.1

83

0.6

15

3.7

394

2.7

14

3.4

64

0.4

12

2.9

Asia 1

Sci. Apps. Int. Corp.-SAIC

United States

2

Spanish Natl. Res. Counc.-CSIC

Spain

3

Natl. Cancer Inst.-NCI

United States

4

Chin. Acad. Sci.

China

5

Univ. Hawaii Manoa

United States

6

French Natl. Res. Counc.-CNRS

France

291

2.0

11

2.7

7

Russ. Acad. Sci.

Russia

220

1.5

10

2.4

8

Pukyong Natl. Univ.

S. Korea

287

1.9

8

1.9

9

Hokkaido Univ.

Japan

177

1.2

8

1.9

10

Counc. Sci. Ind. Res.-CSIR

India

189

1.3

7

1.7

11

Univ. Buenos Aires

Argentina

82

0.6

7

1.7

12

Mac Planck Soc.

Germany

57

0.4

7

1.7

13

Jeju Natl. Univ.

S. Korea

209

1.4

6

1.5

14

Univ. Tokyo

Japan

133

0.9

6

1.5

15

Univ. Porto

Portugal

104

0.7

6

1.5

16

Ilichev Pacific Ocan. Inst.

Russia

86

0.6

6

1.5

17

Tohoku Univ.

Japan

77

0.5

6

1.5

18

Natl. Inst. Agron. Res.-INRA

France

72

0.5

6

1.5

19

French Res. Inst. Exploit. Sea-IFREMER

France

71

0.5

6

1.5

20

Zelinsky Inst. Org. Chem.

Russia

38

0.4

6

1.5

2916

19.7

182

44.3

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Europe and Asia values are for only the top-20 institutions.

The contribution of these institutions has ranged from 0.4% to 2.7% for all the papers and from 1.5% to 4.4% for the influential papers. Overall, these 20 institutions have contributed to 19.7% and 44.3% of all the papers and influential papers, respectively.

31.3.6  Research funding bodies Only 49.0% of these papers have had declared any research funding in their abstract pages and overall, 8042 funding bodies have funded these papers. The corresponding funding rate for the influential papers has been 33.8%. The most-prolific funding bodies have been the ‘National Natural Science Foundation of China’, ‘National Institute of General Medical Sciences’ of the United States, and ‘National Cancer Institute’ of the US funding 3.8%, 1.2%, and 1.00.9% of the papers, respectively. The other prolific funding bodies have been the ‘National Council for Scientific and Technological Development’(CNPQ) of Brazil, ‘Coordination of Improvement for Higher Level Personnel’ (CAPES) of Brazil, ‘Fundamental Research Funds for the Central Universities’ of China, ‘National Science Foundation’ of the United States, and ‘Natural Science Foundation of China’ with at least 0.4% of the papers each.

Algal food research Chapter | 31  491



1400

Number of papers

1200 1000 800 600 400 200 0 Publication years FIG. 31.1  The number of publications in the algal foods between 1980 and 2018.

31.3.7  Publication years The Fig. 31.1 shows the number of papers on the algal foods, published between 1980 and 2018 as of January 2019. The data in this figure shows that the number of papers has risen from 102 papers in 1980 to 1327 papers in 2018. The most prolific decade has been the 2010s with 55.1% of the papers. Additionally, 8.4%, 14.2%, and 22.3% of the papers have been published in the 1980s, 1990s, and 2000s, respectively. Thus, the figure shows that there has been a steadily increasing trend between 1980 and January 2019.

31.3.8  Source titles Overall, these papers have been published in 2136 journals. The Table 31.4 provides the information on the 20 most-prolific and influential journals. These 20 journals have published 21.4% and 43.1% of all the papers and influential papers, respectively, in total. The most-prolific and influential journal has been ‘Food Chemistry’ publishing 1.7% and 5.1% of all the papers and influential papers, respectively. ‘Journal of the American Chemical Society’, ‘Journal of Applied Phycology’, ‘Journal of Natural Products’, and ‘Carbohydrate Research’ have followed the top journal as the other most prolific and influential journals with at least 2.7% of the influential papers each. The most-prolific subject categories for these journals have been ‘Chemistry Applied’ and ‘Food Science and Technology’ with six journals each, followed by ‘Chemistry Organic’ and ‘Biochemistry Molecular Biology’ with four journals each. The other prolific subjects have been ‘Polymer Science’ and ‘Chemistry Medicine’ with two journals each and ‘Plant Sciences’ and ‘Biotechnology Applied Microbiology’ with two papers each.

31.3.9  Subject categories These papers have been indexed by 152 subject categories. The information about the 10 most-prolific and influential subject categories are given in Table 31.5. As expected, the most-prolific and influential subject category has been ‘Food Science Technology’ indexing 19.3% and 21.3% all the papers and influential papers, respectively. The other prolific and influential subjects have been ‘Chemistry Applied’, ‘Biochemistry Molecular Biology’, and ‘Pharmacology Pharmacy’ with at least 15.0% of the influential papers each. Thus, the first-four categories have been the key pillars of the research in algal foods, indexing together 59.6% and 72.0% of all the papers and influential papers, respectively.

Journals

Abbr.

Subject

I-0

I-0%

I-100

I-100%

Food Sci. Technol.

643

4.3

44

10.7

Algae

641

4.3

24

5.8

255

1.7

21

5.1

55

0.4

17

4.1

1

Food Chemistry

Food Chem.

Chem. Appl., Food Sci. Technol., Nutr. Diet.

2

Journal of the American Chemical Society

J. Am. Chem. Soc.

Chem. Mult.

3

Journal of Applied Phycology

J. Appl. Phycol.

Biot. Appl. Microb., Mar. Fresh. Biol.

478

3.2

14

3.4

4

Journal of Natural Products

J. Nat. Prod.

Plant Sci., Chem. Med., Phar. Phar.

448

3.0

12

2.9

5

Carbohydrate Research

Carbohyd. Res.

Bioch. Mol. Biol., Chem. Appl., Chem. Org.

74

0.5

11

2.7

6

Journal of Agricultural and Food Chemistry

J. Agr. Food Chem.

Agr. Mult., Chem. Appl., Food Sci. Technol.

275

1.9

10

2.4

7

International Journal of Biological Macromolecules

Int. J. Biol. Macromol.

Bioch. Mol. Biol., Chem. Appl., Polym. Sci.

226

1.5

10

2.4

8

Journal of Phycology

J. Phycol.

Plant Sci., Mar. Fresh. Biol.

163

1.1

10

2.4

9

Proceedings of the National Academy of Sciences of the United States of America

P. Natl. Acad. Sci. USA

Mult. Sci.

24

0.2

8

1.9

10

Marine Drugs

Mar. Drugs

Chem. Med.

526

3.6

7

1.7

11

Journal of Organic Chemistry

J. Org. Chem.

Chem. Org.

68

0.5

7

1.7

12

Journal of Biological Chemistry

J. Biol. Chem.

Bioch. Mol. Biol.

56

0.4

7

1.7

13

Trends in Food Science Technology

Trends Food Sci. Tech.

Food Sci. Technol.

16

0.1

7

14

Food Hydrocolloids

Food Hydrocolloid.

Chem. Appl., Food Sci. Technol.

97

0.7

6

1.5

15

Tetrahedron Letters

Tetrahedron Lett.

Chem. Org.

77

0.5

6

1.5

16

Marine Biology

Mar. Biol.

Mar. Fresh. Biol.

61

0.4

6

1.5

17

Carbohydrate Polymers

Carbohyd. Polym.

Chem. Appl., Chem. Org., Polym. Sci.

214

1.4

5

1.2

18

Natural Product Reports

Nat. Prod. Rep.

Bioch. Mol. Biol., Chem. Med., Chem. Org.

14

0.1

5

1.2

19

Applied Microbiology and Biotechnology

Appl. Microbiol. Biot.

Biot. Appl. Microb.

34

0.2

4

1.0

20

Antimicrobial Agents and Chemotherapy

Antimicrob. Agents Ch.

Microbiol., Phar. Phar.

11

1.0

4

1.0

3172

21.4

177

43.1

Total

17.

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Values for biomedical sciences are only for the top-20 journals.

492  PART | VIII  Algal foods

TABLE 31.4  The most-prolific and influential journals in algal foods.

Algal food research Chapter | 31  493



TABLE 31.5  The most-prolific and influential subject categories in algal foods. Subject categories

I-0 no. papers

I-0% papers

I-100 no. papers

I-100% papers

Food and Nutrition

3638

24.6

125

30.4

1

Food Science Technology

2850

19.3

89

21.7

2

Chemistry Applied

1739

11.8

75

18.2

3

Biochemistry Molecular Biology

2056

13.9

70

17.0

4

Pharmacology Pharmacy

2166

14.6

62

15.1

5

Biotechnology Applied Microbiology

2051

13.9

45

11.0

6

Chemistry Organic

834

5.6

41

10.0

7

Marine Freshwater Biology

1293

8.7

41

10.0

8

Plant Sciences

1795

12.1

40

9.7

9

Nutrition Dietetics

788

5.3

36

8.8

10

Chemistry Medicinal

2039

13.8

35

8.5

17,611

119.1

534

129.9

Total

I-0, the number of all the papers; I-0%, the percentage of the number of all the papers; I-100, the number of influential papers with at least 100 citations; I-100%, the percentage of the number of influential papers. Values for biomedical sciences are only for the top-10 subject categories.

31.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers with at least 100 citations, 2.8% of the research sample of 14,789 papers, Table 31.6. The records in this dataset has been refined from 411 papers to 353 papers to focus on the core papers for the field of algal foods. The data shows that the field of ‘macroalgal foods’ and ‘cyanobacterial foods’ have been the most prolific research fronts with 91 and 90 papers, respectively. The other key research fronts have been ‘sulfated polysaccharides in general’, ‘fucoidans’, ‘alginates’, and ‘microalgal foods in general’ with 12.4%, 8.7%, 10.4%, and 7.3% of the influential papers, respectively. The most-studied type of algae has been ‘macroalgae’ with 62.8% of the influential papers. Additionally, the papers on the ‘cyanobacteria’ and ‘microalgae’ have formed 25.4% and 7.3% of these papers, respectively.

TABLE 31.6  The most-prolific research fronts in algal foods. Research fronts

Algae

26

Cyanobacteria 90

Diatoms

Dinoflagellates

Macroalgae

Total

0

4

91

221 (62.3%)

1

General

2

Alginates

37

37 (10.4%)

3

Carrageenans

20

20 (5.6%)

Fucoidans

31

31 (8.7%)

Sulfated polysaccharides

44

44 (12.4%)

223 (62.8%)

353 (100%)

4

Total

10

Microalgae

10 (2.81%)

26 (7.3%)

90 (25.4%)

0

4 (1.1%)

Numbers, the number of influential papers for each research front and type of algae; %, the percentage of influential papers for each research front and type of algae; the number in bold, the most-prolific research fronts with at least 50 influential papers; dinoflagellates, it includes coccolithophores.

494  PART | VIII  Algal foods

The subresearch fronts in ‘cyanobacterial foods’ have been ‘cyanovirins’, ‘phycocyanins’, ‘hapalosins’, ‘cryptophycins’, ‘patellamides’, ‘largazole’, ‘curacins’, ‘cyanobactins’, ‘spirulan’, and ‘welwitindolinones’. Similarly, the subresearch fronts in ‘macroalgal foods’ have been ‘fucoxanthins’, ‘griffithsins’, ‘phlorotannins’, ‘kahalilides’ and other ‘specific cyanobacterial peptides’.

31.3.11  Citation classics This section provides the information on both the scientometric analysis and brief content overview of the most-cited 20 papers in algal foods. The information on these papers is given in Table 31.7: authors’ names, publication years, document type, number of authors per paper, lead authors’ names, gender, and paper numbers (lead authors were determined based on the number of papers produced in this field with at least 12 papers in general and 3 influential papers), journals, subject areas, research fronts, types of algae, academic focus of the papers, number of citations received, and the number of citations per year.

31.3.11.1  Scientometric overview of the citation classics These papers have been published between 1983 and 2011. The most-prolific decade has been the 2000s with 12 papers. Additionally, there have been one, four, and three papers published in the 1980s, 1990s, and 2010s, respectively. The reviews have been over-represented in these classical papers as there have been 8 articles and 12 reviews. The number of the authors of these papers has ranged from 1 to 18 while the mean number of authors has been 4.5. There have been 17 authors with at least 12 papers and 3 influential papers as the lead authors of the citation classics. There has been a significant gender deficit among the lead authors of these classical papers as only three authors are female: ‘Elena Ibanez’, ‘Maria I Bilan’, and ‘Pilar Ruperez’. The most-prolific and influential lead authors have been ‘Quanbin Zhang’ and ‘Zhien Li’ with two citation classics each working primarily on the fucoidans and other sulfated polysaccharides. In total, these citation classics have been published by only 17 journals. The most-prolific journals have been ‘Food Chemistry’, ‘International Journal of Biological Macromolecules’ and ‘Journal of Applied Phycology’ with two papers each. In total, these papers have been indexed by 14 subject categories. The most-prolific category has been ‘Biochemistry and Molecular Biology’ with seven papers, closely followed by ‘Chemistry Applied’, ‘Food Science Technology’, and ‘Polymer Science’ with six, five, and four papers, respectively. Additionally there have been three papers each indexed by ‘Biotechnology and Applied Microbiology’ and ‘Microbiology’. In total, there have been six research fronts. The most-prolific research front has been ‘sulfated polysaccharides’ with nine papers. The other prolific research fronts have been ‘fucoidans’ and ‘algal foods’ with four papers each. There have also been one paper each for ‘alginates’, ‘astaxanthin’, and ‘cyanovirins’. There have been three types of algae covered by these classical papers. The most prolific type of algae has been ‘macroalgae’ with 15 papers. The other prolific type of algae has been ‘microalgae’ and ‘cyanobacteria’ with two and three papers, respectively. There have been no papers related to ‘diatoms’, ‘dinoflagellates and coccolithophores’, and ‘algae in general’. The most-studied topic has been the ‘bioactivity’ of algae and their derivatives with 13 papers. The other prolific topics have been ‘proteins’, ‘cyanovirins’, ‘food ingredients’, ‘bioactive compounds’, ‘bacteria encapsulation’, and ‘applications of microalgae’. These papers have received between 279 and 649 citations each, with a mean value of 379 citations per paper. On the other hand, the number of citations per year has ranged from 9 to 61 with a mean value of 31 citations per year. The paper by Holdt and Kraan (2011) on the ‘macroalgal foods’ with 429 total citations and 61 citations per year has been the mostcited paper.

31.3.11.2  Brief overview of the content of the citation classics There have been four major classes of papers: ‘sulfated polysaccharides’, ‘fucoidans’, ‘algal foods’, and ‘other research fronts’ with nine, four, four, and three papers, respectively. Sulfated polysaccharides Jiao et al. (2011) review the progress in the research on the chemical structures and bioactivities of ‘sulfated polysaccharides’ (SPs) in a review paper with 365 citations. They focus first on the structural chemistry of the major macroalgal SPs

Guerin et al.

Herrero et al.

Li et al.

Cumashi et al.

Holdt and Kraan

Boyd et al.

Sultana et al.

Jiao et al.

Wijesekara et al.

Smit

Fleurence

Patankar et al.

1

2

3

4

5

6

7

8

9

10

11

12

Authors

1993

1999

2004

2011

2011

2000

1997

2011

2007

2008

2006

2003

Year

A

R

R

R

R

A

A

R

A

R

R

R

Doc.

5

1

1

3

4

6

17

2

18

4

3

3

N auths.

TABLE 31.7  The citation classics in algal foods.

J Fleurence-3

SK Kim-6

MR Boyd11; BR O’Keefe-5; JB McMahon-4

AI Usov6; NE Nifantiev-4; MI Bilan-4; AA Grachev-4

A Cifuentes-4; M Herrero-4; E Ibanez-5

Lead authors

J. Biol. Chem.

Trends Food Sci. Tech.

J. Appl. Phycol.

Carbohydr. Polym.

Mar. Drugs

Int. J. Food Microbiol.

Antimicrob. Agents Ch.

J. Appl. Phycol.

Glycobiology

Molecules

Food Chem.

Trends Biotechnol.

Journal

Bioch. Mol. Biol.

Food Sci. Technol.

Biot. Appl. Microb.; Mar. Fresh. Biol.

Chem. Appl., Chem. Org.; Polym. Sci.

Chem. Med.

Food Sci. Technol., Microbiol.

Microbiol., Phar. Phar.

Biot. Appl. Microb.; Mar. Fresh. Biol.

Bioch. Mol. Biol.

Bioch. Mol. Biol.; Chem. Mult.

Chem. Appl., Food Sci. Tech., Nutr. Diet.

Biot. Appl. Microb.

Subject area

Fucoidans

Foods

Sulf. polysacch.

Sulf. polysacch.

Sulf. polysacch.

Alginates

Cyanovirins

Foods

Fucoidans

Fucoidans

Foods

Astaxanthin

Res. fronts

Macroalgae

Macroalgae

Macroalgae

Macroalgae

Macroalgae

Macroalgae

Cyanobacteria

Macroalgae

Macroalgae

Macroalgae

Microalgae

Microalgae

Algae

Bioactivity

Proteins

Bioactivity

Bioactivity

Bioactivity

Bacteria encapsulation

Cyanovirins

Bioactive compounds

Bioactivity

Bioactivity

Ingredients

Applications

Topic

14

19

25

51

52

22

20

61

41

46

44

43

Av. cits

Continued

354

354

355

357

365

393

410

429

450

456

524

649

Cits.

2006

2003

Ruperez et al.

Tan

Witvrouw and De Clercq

Qi et al.

Lahaye and Robic

Duan et al.

Average

15

16

17

18

19

20

A

R

A

R

R

A

R

A

Doc.

5

4

2

7

2

1

3

1

4

N auths.

M Lahaye-3

HM Qi-3; QB Zhang-7; Z Li-6

P Ruperez-5

QB Zhang-7; Z Li-6

Lead authors

Food Chem.

Biomacromolecules

Int. J. Biol. Macromol.

Gen. Pharmacol.Vasc. S.

Phytochemistry

J. Agr. Food Chem.

Microbiol. Rev.

Int. J. Biol. Macromol.

Journal

Chem. Appl., Food Sci. Tech.; Nutr. Diet.

Bioch. Mol. Biol.; Chem. Org., Polym. Sci.

Bioch. Mol. Biol.; Chem. Appl.; Polym. Sci.

Phar. Phar.

Bioch. Mol. Biol.; Plant Sci.

Agr. Mult., Chem. Appl.; Food Sci. Tech.

Microbiol.

Bioch. Mol. Biol., Chem. Appl.; Polym. Sci.

Subject area

Sulf. polysacch.

Sulf. polysacch.

Sulf. polysacch.

Sulf. polysacch.

Sulf. polysacch.

Sulf. polysacch.

Foods

Fucoidans

Res. fronts

Macroalgae

Macroalgae

Macroalgae

Macroalgae

cyanobacteria

Macroalgae

Cyanobacteria

Macroalgae

Algae

Bioactivity

Bioactivity

Bioactivity

Bioactivity

Bioactivity

Bioactivity

Proteins

Bioactivity

Topic

379

279

299

302

303

304

317

331

347

Cits.

31

23

27

23

14

28

20

9

35

Av. cits

Doc., document; A, article; R, review; Gender, gender of lead authors—female authors in italic. N paper, for the authors with at least 12 papers with 0 citations and with at least 3 influential papers—number after the author names. Subject, Web of Science subjects. Topic, primary topic of the papers; Algae, type of algae studied; Res. fronts, primary research fronts studied; Cits., number of citations received in total; Av. Cits., number of citations per year.

2007

2005

1997

2007

2002

1983

Ciferri

14

2008

Wang et al.

Year

13

Authors

TABLE 31.7  The citation classics in algal foods—cont’d



Algal food research Chapter | 31  497

such as agarans, carrageenans, ulvans, and fucans. They then focus on the anticoagulant/antithrombotic, antiviral, immunoinflammatory, antilipidemic, and antioxidant activities of these SPs. Wijesekara et al. (2011) discuss the biological activities and health benefits of SPs in a review paper with 357 citations. They focus on the fucoidans in brown algae, carrageenans in red algae, and ulvans in green algae. These SPs show many beneficial biological activities such as anticoagulant, antiviral, antioxidative, anticancer, and immunomodulating activities. Smit (2004) discusses medicinal and pharmaceutical applications of macroalgal natural products in a review paper with 355 citations. He notes that these natural products have included SPs as antiviral substances, halogenated furanones as antifouling compounds, and kahalalide F as a possible treatment of lung cancer, tumors, and AIDS. He further notes that macroalgal lectins, fucoidans, kainoids, and aplysiatoxins are routinely used in biomedical research and many other substances have known biological activities. Ruperez et al. (2002) study the antioxidant capacity of SPs from the Fucus vesiculosus in a paper with 317 citations. They determine the contents of the algae as neutral sugars, uronic acids, sulfate, proteins, and polyphenols. The main neutral sugars were fucose, glucose, galactose, and xylose. They find that fucose showed the highest potential to be antioxidant. Tan (2007) discusses the cyanobacterial bioactive natural products for drug discovery in a review paper with 304 citations. He reviews the research on the biosynthesis and biological activities of 128 new natural products with a focus on the bioactive compounds for the development of anticancer agents. Witvrouw and De Clercq (1997) discusses the macroalgal SPs as potential anti-HIV drugs in a review paper with 303 citations. They note that SPs not only inhibit the cytopathic effect of HIV, but also prevent HIV-induced syncytium (giant cell) formation and antiviral activity increases with increasing molecular weight and degree of sulfation. They argue that PSs exert their anti-HIV activity by shielding off the positively charged sites in the V3 loop of the viral envelope glycoprotein (gp120). They further argue that SPs could be used for not only treatment of patients with HIV, but also prophylaxis and protection from HIV. Qi et al. (2005) study the antioxidant activity of sulfated ulvans in vitro in a paper with 302 citations. They focus on the scavenging activity of superoxide and hydroxyl radicals, reducing power and metal chelating ability of the sulfated ulvans. They find that ulvans with high sulfate content had more effective scavenging activity on hydroxyl radical than natural ulvan, exhibited stronger reducing power, and showed more pronounced chelating ability on ferrous ion at high concentration than natural ulvans. Lahaye and Robic (2007) discuss the structure and functional properties of ulvan in a review paper with 299 citations. They note that the physicochemical, rheological, and biological properties sulfated ulvans are suitable for novel biomedical applications. Duan et al. (2006) evaluate antioxidant properties of macroalgal extract and fractions from Polysiphonia urceolata in relation to ‘butylated hydroxytoluene’ (BHT), ‘gallic acid’ (GA), and ‘ascorbic acid’ (AscA) in a paper with 279 citations. They focus on the antioxidant activity (AA), total phenolic content, and reducing power of the crude extract, fractions, and subfractions. They find that the crude extract and the ethyl acetate-soluble fraction exhibited higher ‘antioxidant activity (AA) than BHT in the DPPH assay model. They show significant association between the antioxidant potency and total phenolic content as well as between the antioxidant potency and reducing power. Fucoidans Li et al. (2008) discuss structure and bioactivity of fucoidans in a review paper with 456 citations. They review the research on the structure and bioactivity of fucoidan as well as the relationships between structure and bioactivity. Cumashi et  al. (2007) study the bioactivity of fucoidans for the development of potential drugs for thrombosis, inflammation, and tumor progression using 9 macroalgal species in a paper with 450 citations. They focus on the anti-­ inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans to examine the effect of fucoidan origin and composition on their biological activities. They find that all fucoidans inhibited leucocyte recruitment in an inflammation model in rats, most fucoidans could serve as P-selectin inhibitors, exhibited anticoagulant activity, displayed strong antithrombin activity, and strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets. Patankar et al. (1993) study the structure-bioactivity relationships of fucoidans in a paper with 354 citations. They report the revised average structure for fucoidan. They find that the core region of the fucan is composed primarily of a polymer of alpha 1-3-linked fucose with sulfate groups substituted at the 4 position on some of the fucose residues. Fucose is also attached to this polymer to form branch points, one for every 2-3 fucose residues within the chain. Wang et al. (2008) study the antioxidant activity of fucoidans in a paper with 347 citations. They focus on the superoxide and hydroxyl radical scavenging activity, chelating ability, and reducing power. They find that all fractions possessed considerable antioxidant activity and there was a positive correlation between the sulfate content and scavenging superoxide radical ability. They further find that the ratio of sulfate content/fucose was an effective indicator to antioxidant activity of the fucoidans.

498  PART | VIII  Algal foods

Algal foods Herrero et al. (2006) discuss the clean extraction of functional ingredients from plants and microalgae in a review paper with 524 citations. They focus on the supercritical fluid extraction and subcritical water extraction of compounds with antibacterial, antiviral, antifungal, or antioxidant activity. Holdt and Kraan (2011) discuss bioactive compounds in seaweed with a focus on the functional food applications and legislation in a review paper with 429 citations. They review the research on bioactive compounds from 10 macroalgal species. They present data in tables with species, effect and test organism (if present) with examples of uses to enhance comparisons. They also review EU, United States, and Japanese legislation on functional foods. Fleurence (1999) discusses biochemical and nutritional aspects of macroalgal proteins in a review paper with 354 citations. He also provides some perspectives on the potential uses of these proteins for the development of new foods or additives for human or animal consumption. Ciferri (1983) discuss the cyanobacterial proteins in an early review paper with 331 citations. He focuses on the structure and nutritional properties of Spirulina. Other research fronts Guerin et al. (2003) discuss the applications of microalgal astaxanthin for human health and nutrition in a review paper with 649 citations. He focuses on the antioxidant, UV-light protection, anti-inflammatory, and other biomedical properties of astaxanthin and its possible role in many human health problems. He argues that protecting body tissues from oxidative damage with daily ingestion of natural astaxanthin might be a practical and beneficial strategy in health management. Boyd et al. (1997) study the development of ‘cyanovirin-N’ (CV-N) from Nostoc ellipsosporum in a paper with 420 citations. They find that CV-N irreversibly inactivate diverse laboratory strains and primary isolates of ‘human immunodeficiency virus’ (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. They argue that the antiviral activity of CV-N is due to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. Sultana et al. (2000) study the microencapsulation of probiotic bacteria with alginate-starch in a paper with 393 citations. They additionally evaluate the effects of microencapsulation on the survival of Lactobacillus acidophilus and Bifidobacterium spp. in yoghurt over a period of 8 weeks. They find that the survival of encapsulated cultures of bacteria showed a decline in viable count of about 0.5 log while there was a decline of about 1 log in cultures which were incorporated as free cells in yoghurt.

31.4 Discussion As there have been nearly 15,000 core papers related to the algal foods, comprising more than 10% of the papers on the algae as a whole, it can be argued that this field has gained the public importance in recent years, corresponding to the increasing public concerns about the sustainable and environment-friendly bioproducts (Konur, 2011a, 2012c,d,e,f,g,h,i,j,k,l, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a,b,c,d,e,f,g,h,i, 2017a,b,c,d,e, 2018a,b,c, 2019a,b,c,d, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). The primary mode of scientific communication has been articles while reviews have formed 3.7% of the sample. The primary index has been SCI-E indexing more than 98.0% of the papers while only 0.3% of the papers have been indexed by the SSCI focusing on the societal aspects of algal foods. There have been no papers indexed by the A&HCI. These findings suggest that there is substantial room for the research in social and humanitarian aspects such as policy-related studies as well as scientometric and consumer studies in this field. The most-prolific keywords related to the algal foods have been determined through the detailed examination of the 353 influential papers with at least 100 citations. A detailed keyword set has been devised for the search (given in the Appendix) and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the most-prolific keywords for the biomedicine have been ‘edible, *food*, nutrition*, dietary, anti*, inhibit*, bioactiv*, *cancer, cytotoxic* or immuno*, pharm*’. On the other hand, there have been a number of prolific journals related to algal foods: ‘Marine Drugs’, ‘Journal of Natural Products’, ‘Journal of Agricultural and Food Chemistry’, ‘Food Chemistry’, ‘Carbohydrate Polymers’, ‘Food Hydrocolloids’, ‘Phytotherapy Research’, Virology, or ‘Journal of the Science of Food and Agriculture’. Similarly, the most-prolific subject categories related to the algal foods have been ‘Food Science Technology’, ‘Pharmacology Pharmacy’, ‘Biochemistry Molecular Biology’, ‘Biotechnology Applied Microbiology’, ‘Chemistry Medicinal’, ‘Plant Sciences’, and ‘Chemistry Applied’.



Algal food research Chapter | 31  499

Similarly, the most-prolific keywords for the algae have been ‘alga, algae, algal, dinoflagellate*, carrageenan*, macroalga*, rhodophyt*, seaweed*, bacillariophycea*, diatom, diatoms, and cyanobacter*’. The other prolific keywords for the algae have been ‘coccolith*, dinophycea*, Alexandrium, chlorophycea*, chlorophyt*, “green alga*”, microalga*, “micro-alga*”, Chlamydomonas, *Chlorella, Dunaliella, Euglena, Scenedesmus, “brown alga*”, phaeophycea*, kelp*, phaeophyt*, “red alga*”, Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, Ulva, bacillariophyt*, “blue green alga*”, “blue-green alga*”, *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, Synechocystis’. These keywords have formed the primary research fronts for the algal foods. On the other hand, the most-prolific cross-subject keywords have been ‘phlorotannin*, dieckol, ulvan, porphyran, laminaran’. The findings show that although over 39,000 authors have contributed to the research, 20 most-prolific and influential authors have shaped the literature on the algal foods publishing 6.1% and 31.1% of all the papers and the influential papers, respectively (Table 31.1). The success of these authors, their institutions and countries could be explained by the ‘firstmover advantage’ paradigm (Lieberman and Montgomery, 1988). The data provides the evidence for the presence of the significant gender deficit among both the lead authors (Table 31.1) and the lead authors of the citation classics as only four and three these top authors are female, respectively (Table 31.7) (Bordons et al., 2003). The data on the papers by the most-prolific and influential authors highlight the primary research fronts as the ‘fucoidans’ and ‘other sulfated polysaccharides’. The other minor research fronts have been ‘cryptophycins’, ‘algal foods’, ‘curacins’, and ‘cyanovirins’. The data in Table 31.1 provides information on the most-prolific and influential authors, institutions, countries, journals, topics, and their citation impact in terms of the I-100 and I-100% by these authors. It has been found during the search process that the author names with two or more forenames or surnames have been spelt differently by the databases with significant implications for the recovery of their papers. Similar difficulties have also been observed for the common names such as ‘Wang Y’ or ‘Li Y’ for the recovery of their papers and the related analysis. The data shows that although over 130 countries and territories have contributed to the research in algal foods, mostprolific 20 countries contributed to 96.0% and 115.1% of all the papers and the influential papers, respectively (Table 31.2). The major producers of the research have been the United States, Japan, China, and Europe as these countries have had the ‘first-mover advantage’ over the other countries. It is notable that the citation impact of China has been relatively small in relation to other top producers as China has produced 11.6% and 6.8% of all the papers and influential papers, respectively (Guan and Ma, 2007). As in the case of countries, although nearly 7000 institutions have contributed to the research in algal foods, the 20 most-prolific institutions mainly from the United States and Europe, having the first-mover advantages, have published more than 19.7% of all the papers and 44.3% of the influential papers, respectively (Table 31.3). As only 49% and 39% of all the papers and influential have declared a research funding, respectively, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). It is notable that there has been significant research funding opportunities in China and Latin America in relation to the United States and Europe. Thus, there is ample room to extend the research funding opportunities to support the research and development in algal foods. The steady rise in the number of the publications in this field in the 2000s and 2010s (as of January 2019) provides the strong evidence for the increasing public importance of the algal foods in recent years (Fig. 31.1). The annual number of publications have risen to over 1300 papers and it is expected that the number of papers would continue to rise in the next decade with at least another 15,000 papers, provided that the opportunities for research funding would increase significantly, corresponding to the increasing importance of the algal foods to the global society at large. Although over 2100 journals have contributed to the research in algal foods, the 20 most-prolific journals, having the first-mover advantages, have published over 21.4% and 43.1% of all the papers and influential papers, respectively (Table 31.4). This finding has been most relevant for the top journals. The data on the Web of Science subject categories suggests that the first-four categories have been the key pillars of the research in algal foods, indexing together 59.6% and 72.0% of all the papers and influential papers, respectively, forming the scientific basis of the research in this field: ‘Food Science Technology’, ‘Chemistry Applied’, ‘Biochemistry Molecular Biology’, and ‘Pharmacology Pharmacy’ (Table 31.5). As the journals related to algae and foods in the top 20 journal list have published only 4.3% both all the papers and influential papers each, the broad search strategy, covering all subject categories and journals, developed for this study, has been justified.

500  PART | VIII  Algal foods

The data on the research fronts have confirmed that the major research fronts have been ‘macroalgal foods’ and ‘cyanobacterial foods’ (Table 31.6). The other key research fronts have been ‘sulfated polysaccharides in general’, ‘fucoidans’, ‘alginates’, and ‘microalgal foods in general’. The most-studied the type of algae has been ‘macroalgae’ with 62.8% of the papers. The other prolific types of algae have been ‘microalgae’ and ‘cyanobacteria’. The extensive data on the 20 citation classics largely confirm the findings of the earlier sections based on nearly 15,000 papers (Table 31.7). There has been a significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely over-represented in the citation classic sample as there have been 12 reviews. Similarly, the most-prolific research fronts have been ‘sulfated polysaccharides’, ‘fucoidans’, and ‘algal foods’ with nine, four, and four papers, respectively. There have been also one paper each for ‘alginates’, ‘astaxanthin’, and ‘cyanovirins’. The most prolific type of algae have been ‘macroalgae’ with 15 papers. The other prolific type of algae has been ‘microalgae’ and ‘cyanobacteria’ with two and three papers, respectively. The most-studied topics have been ‘bioactivity’ of algae and their derivatives with 13 papers. The other prolific topics have been ‘proteins’, ‘cyanovirins’, ‘food ingredients’, ‘bioactive compounds’, ‘bacteria encapsulation’, and ‘applications of microalgae’. It appears that the structure-processing-property relationships form the basis of the research in algal foods as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

31.5 Conclusion This analytical study of the research in algal foods at the global scale covering the whole range of research fronts as well as all types of algae has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the adequate development of the research and practice in this field. Thus, it emerges that the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as bioenergy and biofuels (Konur, 2012c,d,e,f,g,h,i,j,k,l, 2018a,b,c), energy and fuels (Konur, 2012m,n,o, 2015l,m), nanobiomaterials (Konur, 2016b,c,d,e,f,g,h, 2017b,c,d,e, 2019d), biomedicine (Konur, 2016i, 2018d), complementing over 550 literature reviews. The data has shown that the annual number of papers in this field has risen to over 1300 papers while there have been nearly 15,000 papers over the study period from 1980 to 2018. It is further expected that the size of the research output would continue to increase in the incoming years and decades, with at least another 15,000 papers in the next decade, corresponding to the increasing public importance of the algal foods to the global society at large. The provision of the adequate research funding is essential for the further development of the research and practice in this field as only 49.0% and 39% of all the papers and influential papers have declared a research funding, respectively. The key research fronts have been ‘macroalgal foods’, ‘cyanobacterial foods’, ‘sulfated polysaccharides in general’, ‘fucoidans’, ‘alginates’, ‘microalgal foods in general’. The most-studied types of algae has been ‘macroalgae’ with 62.8% of the papers. The other prolific types of algae have been ‘microalgae’ and ‘cyanobacteria’. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies (Konur, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n). It has been found that the detailed keyword set provided in the Appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts.

Appendix. The keyword sets A.1  Algae and foods separate keywords A.1.1 Foods WC = (food* or nutr* or pharm* or ‘chemistry med*’ or med* or immun* or oncol* or ‘integrative comp*’ or ‘materials science biom*’ or hemat* or virol* or endoc* or infect* or ‘engineering biomed*’ or ‘tropical med*’ or gastro* or cardiac*



Algal food research Chapter | 31  501

or surgery or path* or obstet* or urol* or respir* or allergy or opht* or geriat* or pediatr* or otorhin* or dent* or periph* or neurosci* or ‘clinical neuro*’) OR (TI = (edible or *food* or nutrition* or nutraceut* or frankfurter* or sausage* or dietary or poultry or ingredient* or anti* or ‘biological* activ*’ or ‘radical scaveng*’ or yoghurt* or melanoma or apoptosis or inhibit* or diabet*or hepat* or bioactiv* or *cancer or carcin* or skin or *cholesterol* or cardio* or meat* or ‘alphaglucosidase’ or ‘alpha-amylase’ or pancrea* or insulin or angiotensin or cytotoxic* or immuno* or probiot* or prebiot* or hypotens* or hypertens* or pasta or ‘health benefit*’ or pharm* or hiv or therap* or antigen* or *drug* or *herpes or dengue or *medic* or *obes* or vaccine* or bactericidal or arthritis or heart) NOT TI = (‘food and drug’ or ‘food chain*’ or ‘food web*’ or foodweb*)).

A.1.2  Keywords related to the algae Algal general (TI = (alga or algae or algal or phycolog*) OR SO = (Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘European Journal of Phycology’ or Fottea* or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Research’)) NOT TI = (‘shewanella alga*’). Dinoflagellates and coccolithophores TI = (Amphidinolid* or chrysophycea* or chlorococcales or chrysophyt* or *coccolith* or dinocyst* or dinoflagell* or dinophycea* or dinophyt* or haptophyt* or peridiniales or prymnesiophycea* or raphidophycea* or raphidophyt* or zooxanthella* or Akashiwo or Amphidinium or Aureococcus or *Ceratium or *Chattonella or Cochlodinium or Crypthecodinium or Dinophysis or Emiliania or Gambierdiscus or *Gonyaulax or *Gymnodinium or Gyrodinium or Hematodinium or Heterocapsa or Heterosigma or Isochrysis or Karenia* or Karlodinium or Mallomonas or Noctiluca or Ochromonas or Ostreopsis or Oxyrrhis or Peridinium or Pfiesteria or Phaeocystis or Prorocentrum or Prymnesium or Scrippsiella or Symbiodinium or Vaucheria). Microalgae TI = (chlorophycea* or chlorophyt* or cryptomonad* or cryptophycea* or cryptophyt* or euglen* or eustigmatophycea* or ‘green alga*’ or microalga* or ‘micro-alga*’ or ‘micro alga*’ or prasinophycea* or streptophyt* or trebouxiophycea* or volvocales or Acetabularia or Ankistrodesmus or Asteromonas or Aurantiochytrium or Botryococcus or Chaetoceros or Chlamydomonas or *Chlorella or *Chlorococcum or Coccomyxa or Cyanidioschyzon or Cyanidium or Desmodesmus or Dunaliella or Euglena or Galdieria or Haematococcus or Micrasterias or Micromonas or Monoraphidium or Nannochloropsis or Neochloris or Ostreococcus or Phormidium or Platymonas or Polytomella or Porphyridium or Prototheca or Pseudokirchneriella or Pyramimonas or Scenedesmus or Schizochytrium or Selenastrum or Tetraselmis or Trebouxia or Volvox). Macroalgae TI = (‘brown alga*’ or ‘macro-alga*’ or ‘macro alga*’ or ‘red alga*’ or agaran* or agarophyt* or ascophyllan or carrageenan* or characea* or charophyt* or cladophorales or cryptonemiales or dictyotales or dieckol or dulse or florideophycea* or fucale* or fucoidan* or fucoidin* or fucosterol or fucoxanthin* or furcellaran or gelidiales or gigartinale* or gracilariales or griffithsin or kelp* or laminariale* or laminarin* or laminaran* or macroalga* or mekabu or phaeophycea* or phlorotannin* or phaeophyt* or phlorofucofuroeckol or phycocolloid* or porphyran or rhodophyce* or rhodophyt* or seaweed* or ulvale* or ulvan or ulvophycea* or Wakame or zygnematophycea* ‘Chara vulgaris’ or Alaria or Ascophyllum or Asparagopsis or Asparagopsis or Bangia* or Bostrychia or Bryopsis or Catenella or Caulerpa or Ceramium or Chaetomorpha or Chondrus or Cladophora or Codium or Corallina or Cystoseira or Ecklonia or Ectocarpus or ‘Eisenia bicyclis’ or Enteromorpha or Eucheuma or Fucus or Gelidium or Gigartina* or Gracilaria or Gracilariopsis or Grateloupia or Halimeda or Halocynthia or Hizikia or Hypnea or Kappaphycus or Laminaria or Laurencia* or Lessonia or Lomentaria or Macrocystis or Monostroma or Mougeotia or Nitella or Nitellopsis or Oedogonium or Padina or Palmaria or Pelvetia or Plocamium or Polysiphonia or Porphyra or Pyropia or Saccharina or Sargassum or Scytosiphon or Solieria or Spirogyra or Turbinaria or Ulva or Undaria). Diatoms (TI = (bacillariophycea* or bacillariophyt* or diatom or diatoms or Asterionella or Chaetoceros or Coscinodiscus or Cyclotella or Cylindrotheca or Didymosphenia or Navicula or Nitzschia or Phaeodactylum or Skeletonema or Stephanodiscus

502  PART | VIII  Algal foods

or Thalassiosira) OR SO = (‘Diatom Research’)) NOT (TI = (diatomic* or atom* or *molecule*or amphorae or diatomyid* or dissociation or ‘rare gas*’ or *silica) OR SO = (‘Journal of Chemical Physics’)). Cyanobacteria TI = (‘blue green alga*’ or ‘blue-green alga*’ or calothrixin* or cryptophycin* or cyanelle or *cyanobacter* or cyanobactin* or cyanophycin* or cyanophyt* or cyanophycea* or cyanovirin* or cyanoviridin* or curacin or dendroamide* or fischerindole* or hapalindole* or hapalosin or hormothamnione or largamide* or largazole or lyngbyabellin or majusculamide* or microcolin*or microginin* or microvirin* or nostocales or nostocine* or oscillatoriales or patellamide* or *phycocyanobilin* or *phycocyanin* or prochlorophyt* or scytovirin or scytophycin* or spirulan* or symplostatin or tasiamide or venturamide* or welwitindolinone* or Acaryochloris or *Anabaena or Anacystis or Aphanizomenon or Aphanothece or Arthrospira or Calothrix or Cyanophora or Cyanothece or *Lyngbya* or Mastigocladus or Microcoleus or Moorea or Nostoc or Oscillatoria or Planktothrix or Plectonema or Prochlorococcus or Prochloron or Prochlorothrix or Scytonema or Spirulina or *Synechococcus or Synechocystis or ThermoSynechococcus or Tolypothrix or Trichodesmium). Journals SO = (Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘Cryptogamie Algologie’ or ‘Diatom Research’ or ‘European Journal of Phycology’ or Fottea* or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Research’).

A.2  Algal foods- cross-subject keywords TI = (cyanobactin* or fucoidan* or fucosterol* or furcellaran* or spirulan* or dieckol or phlorotannin* or welwitindolinone* or curacin* or cryptophycin* or cyanovirin* or griffithsin* or calothrixin* or microginin or hapalosin* or microcolin or majusculamide or dendroamide* or *ulvan or laminarin or laminaran or fucoidin or phlorofucofuroeckol or porphyran or nostocine or ascophyllan or hapalindole* or agaran*). TI = (alginate* or carrageenan*) and TI = (edible or *food* or nutrition* or nutraceut* or frankfurter* or sausage* or dietary or poultry or ingredient* or anti* or ‘biological* activ*’ or ‘radical scaveng*’ or yoghurt* or melanoma or apoptosis or diabet*or hepat* or bioactiv* or *cancer or carcin* or skin or *cholesterol* or cardio* or meat* or ‘alpha-glucosidase’ or ‘alpha-amylase’ or pancrea* or insulin or angiotensin or cytotoxic* or immuno* or probiot* or prebiot* or hypotens* or hypertens* or pasta or ‘health benefit*’ or antigen* or *herpes or dengue or *obes* or vaccine* or bactericidal or arthritis or heart). NOT (WC = (ecol* or ener* or env* or toxic* or ocean* or fish* or biophys* or ‘engineering env*’ or limnol* or water* or veterin* or zool* or geo* or physiol*) OR TI = (*toxin* or microcystin* or lipid* or delivery or tissue* or *fuel* or ecol* or optogen* or *foul*)).

Acknowledgments The significant contribution of the authors of the pioneering studies in algal foods to the development of the research in in this field have been gratefully acknowledged. The authors listed as the ‘most-prolific and influential authors’ in Table 31.1 have published at least 12 papers and 4 influential papers with at least 100 citations and authors listed as the ‘lead authors’ in Table 31.7 have published at least 12 papers and 3 influential papers in algal foods.

References Abramo, G., D’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Basu, A., 2006. Using ISI's' Highly Cited Researchers’ to obtain a country level indicator of citation excellence. Scientometrics 68 (3), 361–375. Blazquez‐Ruiz, J., Guerrero‐Bote, V.P., Moya‐Anegon, F., 2016. New scientometric‐based knowledge map of food science research (2003 to 2014). Compr. Rev. Food Sci. Food Saf. 15 (6), 1040–1055. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Boyd, M.R., Gustafson, K.R., McMahon, J.B., Shoemaker, R.H., OKeefe, B.R., Mori, T., et al., 1997. Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob. Agents Chemother. 41 (7), 1521–1530. Brennan, L., Owende, P., 2010. Biofuels from microalgae-a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14 (2), 557–577.



Algal food research Chapter | 31  503

Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Ciferri, O., 1983. Spirulina, the edible microorganism. Microbiol. Rev. 47 (4), 551–578. Cumashi, A., Ushakova, N.A., Preobrazhenskaya, M.E., D’Incecco, A., Piccoli, A., Totani, L., et al., 2007. A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology 17 (5), 541–552. Duan, X.J., Zhang, W.W., Li, X.M., Wang, B.G., 2006. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem. 95 (1), 37–43. Fleurence, J., 1999. Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Sci. Technol. 10 (1), 25–28. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Guan, J., Ma, N., 2007. China’s emerging presence in nanoscience and nanotechnology: a comparative bibliometric study of several nanoscience ‘giants’. Res. Policy 36 (6), 880–886. Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21 (5), 210–216. Guerrero‐Bote, V.P., Moya‐Anegon, F., 2015. Analysis of scientific production in food science from 2003 to 2013. J. Food Sci. 80 (12), R2619–R2626. Herrero, M., Cifuentes, A., Ibanez, E., 2006. Sub- and supercritical fluid extraction of functional ingredients from different natural sources: plants, foodby-products, algae and microalgae—a review. Food Chem. 98 (1), 136–148. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol. Adv. 29 (2), 189–198. Holdt, S.L., Kraan, S., 2011. Bioactive compounds in seaweed: functional food applications and legislation. J. Appl. Phycol. 23 (3), 543–597. Jiao, G.L., Yu, G.L., Zhang, J.Z., Ewart, H.S., 2011. Chemical structures and bioactivities of sulfated polysaccharides from marine algae. Mar. Drugs 9 (2), 196–223. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374. Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books Ltd., Stoke on Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011a. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2011b. The scientometric evaluation of the research on the students with disabilities in higher education. Soc. Polit. Econ. Cult. Res. 3 (2), 81–148. Konur, O., 2012a. The gradual improvement of disability rights for the disabled tenants in the UK: the promising road is still ahead. Soc. Polit. Econ. Cult. Res. 4 (3), 71–112. Konur, O., 2012b. The policies and practices for the academic assessment of blind students in higher education and professions. Energy Educ. Sci. Technol. B 4 (si1), 240–244. Konur, O., 2012c. Prof. Dr. Ayhan Demirbas’ scientometric biography. Energy Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012d. The evaluation of the biogas research: a scientometric approach. Energy Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012e. The evaluation of the bio-oil research: a scientometric approach. Energy Educ. Sci. Technol. A 30 (si1), 379–392. Konur, O., 2012f. The evaluation of the biorefinery research: a scientometric approach. Energy Educ. Sci. Technol. A 30 (si1), 347–358. Konur, O., 2012g. The evaluation of the research on the biodiesel: a scientometric approach. Energy Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012h. The evaluation of the research on the bioethanol: a scientometric approach. Energy Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012i. The evaluation of the research on the biofuels: a scientometric approach. Energy Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012j. The evaluation of the research on the biohydrogen: a scientometric approach. Energy Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012k. The evaluation of the research on the microbial fuel cells: a scientometric approach. Energy Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012l. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012m. The evaluation of the global energy and fuels research: a scientometric approach. Energy Educ. Sci. Technol. A 30 (1), 613–628. Konur, O., 2012n. 100 citation classics in energy and fuels. Energy Educ. Sci. Technol. A 30 (si1), 319–332. Konur, O., 2012o. What have we learned from the citation classics in Energy and Fuels: a mixed study. Energy Educ. Sci. Technol. A 30 (si1), 255–268. Konur, O., 2012p. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Energy Educ. Sci. Technol. B 4 (4), 1893–1908. Konur, O., 2012q. The evaluation of the research on the Arts and Humanities in Turkey: a scientometric approach. Energy Educ. Sci. Technol. B 4 (3), 1603–1618. Konur, O., 2012r. The evaluation of the educational research: a scientometric approach. Energy Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012s. The scientometric evaluation of the research on the deaf students in higher education. Energy Educ. Sci. Technol. B 4 (3), 1573–1588.

504  PART | VIII  Algal foods

Konur, O., 2012t. The scientometric evaluation of the research on the students with ADHD in higher education. Energy Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2012u. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Energy Educ. Sci. Technol. B 4 (si1), 250–257. Konur, O., 2012v. The evaluation of the research on the doctoral education: a scientometric approach. Energy Educ. Sci. Technol. B 4 (si1), 593–600. Konur, O., 2012x. The scientometric evaluation of the institutional research: the Karadeniz Universities-Part 3. Energy Educ. Sci. Technol. B 4 (si1), 850–856. Konur, O., 2012y. The scientometric evaluation of the institutional research: the Karadeniz Universities-Part 2. Energy Educ. Sci. Technol. B 4 (si1), 844–849. Konur, O., 2012z. The scientometric evaluation of the institutional research: the Karadeniz Universities-Part 1. Energy Educ. Sci. Technol. B 4 (si1), 836–843. Konur, O., 2013a. The scientometric evaluation of the institutional research. The Marmara Universities-part  4. Energy Educ. Sci. Technol. B 5 (2), 365–380. Konur, O., 2013b. The scientometric evaluation of the institutional research. The Marmara Universities-part  3. Energy Educ. Sci. Technol. B 5 (2), 349–364. Konur, O., 2013c. The scientometric evaluation of the institutional research. The Marmara Universities-part  2. Energy Educ. Sci. Technol. B 5 (2), 333–348. Konur, O., 2013d. The scientometric evaluation of the institutional research. The Marmara Universities-part  1. Energy Educ. Sci. Technol. B 5 (2), 317–332. Konur, O., 2013e. The scientometric evaluation of the institutional research. The Ege Universities-part 3. Energy Educ. Sci. Technol. B 5 (1), 83–98. Konur, O., 2013f. The scientometric evaluation of the institutional research. The Ege Universities-part 2. Energy Educ. Sci. Technol. B 5 (1), 67–82. Konur, O., 2013g. The scientometric evaluation of the institutional research. The Ege Universities-part 1. Energy Educ. Sci. Technol. B 5 (1), 51–66. Konur, O., 2013h. The scientometric evaluation of the institutional research. The Akdeniz Universities-part 3. Energy Educ. Sci. Technol. B 5 (1), 151–166. Konur, O., 2013i. The scientometric evaluation of the institutional research. The Akdeniz Universities-part 2. Energy Educ. Sci. Technol. B 5 (1), 135–150. Konur, O., 2013j. The scientometric evaluation of the institutional research. The Akdeniz Universities-part 1. Energy Educ. Sci. Technol. B 5 (1), 119–134. Konur, O., 2013k. The scientometric evaluation of the institutional research. The Inner Anatolian Universities-part 4. Energy Educ. Sci. Technol. B 5 (2), 267–282. Konur, O., 2013l. The scientometric evaluation of the institutional research. The Inner Anatolian Universities-part 3. Energy Educ. Sci. Technol. B 5 (2), 251–266. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489. Konur, O., 2016a. Algal Omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729.



Algal food research Chapter | 31  505

Konur, O., 2016c. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016d. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016f. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453. Konur, O., 2016g. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016h. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016i. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Algal drugs: the state of the research. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019b. Algal genomics. In: Kim, S.K. (Ed.), Encyclopedia of Marine Biotechnology. Wiley-Blackwell, Oxford. Konur, O., 2019c. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019d. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

506  PART | VIII  Algal foods

Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Lahaye, M., Robic, A., 2007. Structure and functional properties of ulvan, a polysaccharide from green seaweeds. Biomacromolecules 8 (6), 1765–1774. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Li, B., Lu, F., Wei, X.J., Zhao, R.X., 2008. Fucoidan: structure and bioactivity. Molecules 13 (8), 1671–1695. Lieberman, M.B., Montgomery, D.B., 1988. First‐mover advantages. Strateg. Manag. J. 9 (S1), 41–58. Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal‐free organic dyes for dye‐sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. Patankar, M.S., Oehninger, S., Barnett, T., Williams, R.L., Clark, G.F., 1993. A revised structure for fucoidan may explain some of its biological activities. J. Biol. Chem. 268 (29), 21770–21776. Qi, H.M., Zhang, Q.B., Zhao, T.T., Chen, R., Zhang, H., Niu, X.Z., et al., 2005. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (chlorophyta) in vitro. Int. J. Biol. Macromol. 37 (4), 195–199. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Ruperez, P., Ahrazem, O., Leal, J.A., 2002. Potential antioxidant capacity of sulfated polysaccharides from the edible marine brown seaweed Fucus vesiculosus. J. Agric. Food Chem. 50 (4), 840–845. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes-towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Smit, A.J., 2004. Medicinal and pharmaceutical uses of seaweed natural products: a review. J. Appl. Phycol. 16 (4), 245–262. Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., Kailasapathy, K., 2000. Encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt. Int. J. Food Microbiol. 62 (1–2), 47–55. Tan, L.T., 2007. Bioactive natural products from marine cyanobacteria for drug discovery. Phytochemistry 68 (7), 954–979. Wang, J., Zhang, Q.B., Zhang, Z.S., Li, Z., 2008. Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica. Int. J. Biol. Macromol. 42 (2), 127–132. Wijesekara, I., Pangestuti, R., Kim, S.K., 2011. Biological activities and potential health benefits of sulfated polysaccharides derived from marine algae. Carbohydr. Polym. 84 (1), 14–21. Witvrouw, M., De Clercq, E., 1997. Sulfated polysaccharides extracted from sea algae as potential antiviral drugs. Gen. Pharmacol. 29 (4), 497–511. Yeung, A.W.K., Mocan, A., Atanasov, A.G., 2018. Let food be thy medicine and medicine be thy food: a bibliometric analysis of the most cited papers focusing on nutraceuticals and functional foods. Food Chem. 269, 455–465.

Chapter 32

Microalgae: A new and promising source of food Eliane Collaa, Anne Luize Lupatini Menegottob, Daneysa Lahis Kalschnea, Rosana Aparecida da Silva-Buzanelloa, Cristiane Canana, Deisy Alessandra Drunklera a

Federal Technological University of Parana, Medianeira, Brazil, bUniversity Regional Integrated High Uruguay and Missions (URI), Erechim, Brazil

32.1 Introduction Microalgae have been consumed for several centuries, including species such as Spirulina platensis, Nostoc spp., and Aphanizomenon spp., which were consumed as food by indigenous tribes (Stanic-Vucinic et al., 2018; Demir and Tukel, 2010; Spolaore et al., 2006). In the 1960s, microalgae were once again highlighted as a “future source of food” because of their nutritional quality (Habib et al., 2008) and as a source of “single-cell protein” capable of feeding the exponentially growing world population (Barka and Blecker, 2016). In the 1970s, the “Intergovernmental Institution for the Use of Microalgae Spirulina against Malnutrition” (IIMSAM) was inaugurated considering the excellent nutritional properties of Spirulina and its potential to act against hunger and malnutrition worldwide (Deng and Chow, 2010; Garcia et al., 2017). Currently, microalgae are considered an excellent alternative source of protein (Lupatini et al., 2017a; Roy and Pal, 2015) that can treat nutritional insufficiency through diversifying and increasing the protein sources in the diet (Barka and Blecker, 2016). Furthermore, microalgae present potential for commodity-scale production and high biomass production and provide several advantages over plant and animal food sources, such as requiring a small cultivation area and being independent of growing seasons (Degnechew and Buzayehu, 2018). The size of the market for products developed from microalgae, including whole microalgae, is still smaller compared to that for cereals or other commodity crops. However, as a result of scientific and technological developments, the microalgae market has grown impressively and will likely continue to grow in the next decade (Vigani et al., 2015). In addition to their protein content, microalgae are a promising source of nutrients such as amino acids, fatty acids, vitamins, and minerals (El-Tantawy, 2015). For example, S. platensis is one of the highest sources of B12 (Kumudha and Sarada, 2015). Different nutraceutical properties have also been attributed to microalgae, such as their capacity to improve the state of health or reduce the risk of disease. Studies have shown that bioactive compounds in microalgae may present antioxidant activity (Konickova et al., 2014; Su et al., 2014), which can be associated with some of their therapeutic properties, including their anticancer (E Silva et al., 2017) and anti-inflammatory properties (Xia et al., 2016). Moreover, the addition of microalgae to different foods may improve their techno-functional properties and, consequently, facilitate the processing of these foods (Medina et al., 2015). To demonstrate that microalgae can improve food systems, including both microalgae as a whole and their separate compounds, we carried out a literature review of the main characteristics and properties of microalgae as a source of food. At following, we outline the potential use of microalgae as a food source and present their composition, biochemical characteristics, and properties, including their nutritional, nutraceutical, and techno-functional. In addition, we conducted a small review on the use of microalgae in animal feed, which has been highlighted as a promising area and has already been applied in first world countries at a large scale.

32.2  Microalgae composition Microalgae have been highlighted as a food or a potential ingredient that can enrich the nutritional content of conventional foods because of their chemical composition (Table 32.1) (Becker, 2007; Zhu et al., 2014). Protein is the major organic component of microalgae followed by carbohydrates or fiber, and lipids (Matos, 2017). Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00032-2 © 2020 Elsevier Inc. All rights reserved.

507

508  PART | VIII  Algal foods

TABLE 32.1  General composition of different microalgae (% of dry matter). Microalgae

Proteins

Carbohydrates

Lipids

Chlorella vulgaris

51–58

12–17

14–22

Chlorella pyrenoidosa

57

26

2

Dunaliella salina

57

32

6

Scenedesmus obliquus

50–56

10–17

12–14

Scenedesmus quadricauda

47



1.9

Spirulina (Arthrospira) maxima

60–71

13–16

6–7

Spirulina (Arthrospira) platensis

46–63

8–14

4–9

Adapted from Becker, E.W., 2007. Some promising microalgal species for commercial applications: a review. Energy Procedia 110, 510–517 and Zhu, L.D., Hiltunen, E., Antila, E., Zhong, J.J., Yuan, Z.H., Wang, Z.M., 2014. Microalgal biofuels: flexible bioenergies for sustainable development. Renew. Sust. Energ. Rev. 30, 1035–1046.

Most notably, several microalgae can synthesize large quantities of proteins, and this outstanding capacity is one of the main reasons why these organisms are considered as an alternative source of food (Matos, 2017). In particular, the high protein content of Spirulina stands out in relation to other microalgal species (Table 32.1). On the other hand, the carbohydrate composition varies among microalgae. Carbohydrates are generally present in the form of cellulose, β-1-3 glucan, glucose, and monosaccharides (Safi et al., 2014; Spolaore et al., 2006). Some microalgae in the genera Spirulina, Chlorella, and Dunaliella also stand out for their high concentrations (6.0, with the maximum at pH 12. The EC and FC of the protein extract were maintained over 50% as well as the ES at 24 h, presenting values of 63%, 92.3%, and 82.9%, respectively. On the other hand, the protein hydrolysate did not show EC; small peptides are responsible for increasing the solubility and decreasing the EC.

Medina et al. (2015)

Chlorella pyrenoidosa

Protein concentrate obtained by threephase partitioning

WAC, OAC, and FC

The WAC was 3.1% (w/w), and the OAC was 2.02%. The microalgal protein concentrate presented a 95% FC, a higher value than some vegetal protein concentrates. After 180 min of storage, the FS was about 97%.

Waghmare et al. (2016)

Haematococcus pluvialis

Water-soluble matrix extracted under high pressure at native (5.7) and neutral pH (7.0)

EC, emulsification activity index (EAI), and ES

The EC was similar between sample types, corresponding with 558 and 534 mL oil g−1 protein at pH 5.7 and 7.0, respectively. After 24 h, the supernatant obtained at neutral pH had greater stability (94%) than that extracted at native pH (84%). The EAI values are associated with the ES due to the large interfacial area, which probably maintained the emulsion stable, corresponding with values of 55 m2 g−1 at pH 5.7 and 80 m2 g−1 at pH 7.0.

Ba et al. (2016)

Spirulina platensis

Biomass and protein concentrate and isolate obtained by solubilization (pH 11) and precipitation (pH 4.2) with the aid of a high-speed homogenizer

Solubility, FC, and FS

The solubility of the protein concentrate and isolate (~85%) increased compared to the biomass (~65%); the highest values occurred at pH 10–11. The protein extraction process may increase protein solubility due to cell wall rupture and separation of soluble proteins at alkaline pH. The protein concentrate (~55%) and isolate (45%) presented lower FC than the biomass (~75%); in contrast, the protein concentrate and isolate presented higher FS after 60 min (75% and 90%, respectively) compared to the biomass (35%). The authors related that the ash content may affect the foam properties and that the presence of salt can reduce the electrostatic repulsion among the protein molecules, increasing the FC.

Pereira et al. (2018)

514  PART | VIII  Algal foods

32.4  Microalgae as feed The demand for animal protein has increased as a result of population growth. Given this panorama, the increased production of protein and the improvement of protein sources are required. The main sources of nutrients for animal feed are soy and corn obtained from agriculture, which demand a large portion of arable land for cultivation and are dependent upon input prices, uncertainty, and availability as well as climatic conditions (Altmann et al., 2019; Alqaisi et al., 2019). Therefore, microalgae have been highlighted as an alternative source or ingredient of animal feed because of their high quality and quantity of nutrients that can improve the nutritional properties (Holman and Malau-Aduli, 2012). Spirulina stands out as a potential feed source or supplement due to its nutritional properties, especially its protein, fatty acid, and mineral contents, which can be directly incorporated in feed in the form of whole biomass or extracts (Madeira et al., 2017). Notably, the use of microalgae in animal feed is not recent; in fact, microalgae been used in feed since the 1950s (Lum et al., 2013). Several studies have been reported and demonstrated the positive effects of adding microalgae to feed, as summarized in Table 32.5. The use of microalgae as a substitute for conventional ingredients in aquiculture was also previously reported (Yaakob et al., 2014). Some microalgae, such as Isochrysis galbana, Pavlova lutheri, Chaetoceros calcitrans, and Thalassiosira pseudonana, have been used in the culture of bivalve mollusks, crustacean larvae, and zooplankton in view of their good fatty acid profiles. According to the presence of the carotenoid astaxanthin, additional applications of microalgae in aquaculture can include coloring the flesh of salmonids and inducing nutraceutical properties, promoting the animal health (Makkar, 2012). In regard to monogastric organisms, research on the use of Spirulina as feed has mostly focused on poultry. The impact of microalgal dietary inclusion on chicken growth and growth rate depends on the feed type previously used and the microalgal ratio. However, Spirulina supplementation ranging from 50 to 100 g kg−1 of feed was shown to maintain typical growth rates. On the other hand, Spirulina dietary inclusion was associated with greater cost efficiency in poultry production (Holman and Malau-Aduli, 2012).

TABLE 32.5  Use of microalgae in animal feed and main effects on growth performance and/or meat quality. Animals

Microalgae

Main results

References

Pigs

Laminaria digitata

10% enhancement of growth and potential production of iodine-rich pork for human consumption

He et al. (2002)

C. vulgaris

Increase in average daily intake

Yan et al. (2012)

S. platensis; C. vulgaris

No effect on average daily intake, average daily gain, and gain:feed ratio

Furbeyre et al. (2017)

S. platensis

Increase in γ-linolenic acid compared to the control

Altmann et al. (2019)

S. platensis

Higher live weights and body conformation measurements

Holman and Malau-Aduli (2012)

S. platensis

Improved final live body weight, daily live weight gain, feed intake, and feed conversion ratio; Higher hemoglobin and total white blood cell counts, serum globulin, and vitamin A and lower glutathione compared to the control; Lower aspartate amino transferase, alanine amino transferase, cholesterol, glucose, and serum malondialdehyde levels compared to the control

El-Sabagh et al. (2014)

Chlorella spp.

Increase in average daily gain

Kang et al. (2013)

S. platensis

Increase in average daily gain and decrease in feed conversion ratio

Shanmugapriya et al. (2015)

C. vulgaris

Increase in average daily gain and feed intake; No effect on gain:feed ratio

Oh et al. (2015)

S. platensis

No effect on growth performance and carcass yield Improvement in the fatty acid profile, especially γlinolenic acid

Zotte et al. (2013) and Peiretti and Meineri (2011)

Lambs

Poultry

Rabbits



Microalgal foods Chapter | 32  515

Finally, microalgae are a great source of carbohydrates, which constitute the highest proportion of macronutrients in livestock diets. Microalgae can contribute to the energy supply and microorganism activity in the rumen in ruminants, keeping the gastrointestinal tract healthy. Additionally, microalgae provide dietary fiber that is also beneficial for animals’ gut health (Madeira et al., 2017).

32.5  Final considerations The potential use of microalgae as source of food was highlighted in the present chapter, including its benefits and properties. The use of microalgae in food remains in the development stage, and the body of scientific research on this topic is continuing to grow. Similarly, the techno-functional properties of microalgae are continuously being explored. Protein deficiency and malnutrition have prompted the search for alternative sources of food or protein such as microalgae. At the same time, the demand for bioactive foods has increased. In this regard, microalgae can represent an alternative source of protein yet also provide numerous health benefits, including nutraceutical benefits that extend beyond their nutritional value. Also, microalgae are also a potential source of animal feed, and studies have associated microalgal supplementation with improvements in animal growth, fertility, and nutritional quality. Developed countries, such as the United States, for example, have already begun to use microalgae in feed. However, further studies are needed to clarify the potential applications of microalgae, including surveys of the active ingredients and associated biological pathways. Greater knowledge of microalgae would support their future applications in the food system and in animal production. Several studies have highlighted the techno-functional properties of microalgae, which represent important additional qualities that can determine the potential applications of microalgae in the food industry. Currently, the food industry uses different synthetic additives during food processing, whereas the application of microalgae might improve some important food characteristics, such as the emulsifying and foaming properties, thereby decreasing the need for other artificial ingredients. However, further research on the techno-functional properties of microalgae is necessary. Studies on the applications of microalgae in food systems and the knowledge of these systems are necessary, so that microalgae can be effectively used in food industry in the future. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c, d,e,f,g,h,i,j,k,l,m,n,o).

References Ahmed, F., Fanning, K., Netzel, M., Turner, W., Li, Y., Schenk, P.M., 2014. Profiling of carotenoids and antioxidant capacity of microalgae from subtropical coastal and brackish waters. Food Chem. 165, 300–306. Alqaisi, O., Moraes, L.E., Ndambi, O.A., Williams, R.B., 2019. Optimal dairy feed input selection under alternative feeds availability and relative prices. Inform. Process. Agric. https://doi.org/10.1016/j.inpa.2019.03.004. Altmann, B.A., Neumann, C., Rothstein, S., Liebert, F., Morlein, D., 2019. Do dietary soy alternatives lead to pork quality improvements or drawbacks? A look into micro-alga and insect protein in swine diets. Meat Sci. 153, 26–34. Ba, F., Ursu, A.V., Laroche, C., Djelveh, G., 2016. Haematococcus pluvialis proteins: extraction, characterization, concentration/fractionation and emulsifying properties. Bioresour. Technol. 200, 147–152. Barba, F.J., Grimi, N., Vorobiev, E., 2014. New approaches for the use of non-conventional cell disruption technologies to extract potential food additives and nutraceuticals from microalgae. Food Eng. Rev. 7 (1), 45–62. Barka, A., Blecker, C., 2016. Microalgae as a potential source of single-cell proteins. A review. BASE 20, 427–436. Becker, E.W., 2007. Some promising microalgal species for commercial applications: a review. Energy Procedia 110, 510–517. Benelhadj, S., Gharsallaoui, A., Degraeve, P., Attia, H., Ghorbel, D., 2016. Effect of pH on the functional properties of Arthrospira (Spirulina) platensis protein isolate. Food Chem. 194, 1056–1063. Bishop, W.M., Zubeck, H.M., 2012. Evaluation of microalgae for use as nutraceuticals and nutritional supplements. J. Nutr. Food Sci. 2 (5), 1–6. Borowitzka, M.A., 2013. High-value products from microalgae-their development and commercialisation. J. Appl. Phycol. 25 (3), 743–756. Colla, L.M., Reinehr, C.O., Reichert, C., Costa, J.A.V., 2007. Production of biomass and nutraceutical compounds by Spirulina platensis under different temperature and nitrogen regimes. Bioresour. Technol. 98 (7), 1489–1493. da Silva Gorgonio, C.M., Aranda, D.A.G., Couri, S., 2013. Morphological and chemical aspects of Chlorella pyrenoidosa, Dunaliella tertiolecta, Isochrysis galbana and Tetraselmis gracilis microalgae. Nat. Sci. 5 (7), 783–791. Damodaran, S., 1996. Amino acids, peptides, and proteins. In: Fennema, O.R. (Ed.), Food Chemistry, third ed. Marcel Dekker Inc., New York, pp. 321–429. de Bhowmick, G., Sarmah, A.K., Sen, K., 2019. Zero-waste algal biorefinery for bioenergy and biochar: a green leap towards achieving energy and environmental sustainability. Sci. Total Environ. 650, 2467–2482. de Morais, M.G., da Silva Vaz, B., de Morais, E.G., Costa, J.A.V., 2015. Biologically active metabolites synthesized by microalgae. Biomed. Res. Int. 2015, 835761.

516  PART | VIII  Algal foods

Degnechew, G.D., Buzayehu, D.B., 2018. Applications of Arthrospira platensis as an alternative source of food, maintaining nutritional security and awareness creation; there by reducing problems of malnutrition in the society. World News Nat. Sci. 19, 1–8. Demir, B.S., Tukel, S.S., 2010. Purification and characterization of lipase from Spirulina platensis. J. Mol. Catal., B Enzym. 64 (3–4), 123–128. Deng, R.T., Chow, T.J., 2010. Hypolipidemic, antioxidant, and antiinflammatory activities of microalgae spirulina. Cardiovasc. Ther. 28 (4), 33–45. Diprat, A.B., Menegol, T., Boelter, J.F., Zmozinski, A., Vale, M.G.R., Rodrigues, E., et al., 2017. Chemical composition of microalgae Heterochlorella luteoviridis and Dunaliella tertiolecta with emphasis on carotenoids. J. Sci. Food Agric. 97 (10), 3463–3468. E Silva, E.F., da Silva Figueira, F., Lettnin, A.P., Carrett-Dias, M., Filgueira, D.M.V.B., Kalil, S., et al., 2017. C-Phycocyanin: cellular targets, mechanisms of action and multi drug resistance in cancer. Pharmacol. Rep. 70 (1), 75–80. Ejike, C.E.C.C., Collins, S.A., Balasuriya, N., Swanson, A.K., Mason, B., Udenigwe, C.C., 2017. Prospects of microalgae proteins in producing peptidebased functional foods for promoting cardiovascular health. Trends Food Sci. Technol. 59, 30–36. El-Baky, H.H.A., El-Baz, F.K., El-Baroty, G.S., 2002. Spirulina species as a source of carotenoids and a-tocoferol and its anticarcinoma factors. Biotechnology 2 (3), 222–240. El-Sabagh, M.R., Eldaim, M.A., Mahboub, D., Abdel-Daim, M., 2014. Effects of Spirulina platensis algae on growth performance, antioxidative status and blood metabolites in fattening lambs. J. Agric. Sci. 6 (3), 92–98. El-Tantawy, W.H., 2015. Antioxidant effects of Spirulina supplement against lead acetate-induced hepatic injury in rats. J. Tradit. Complement. Med. 6 (4), 327–331. Fakhry, E.M., El Maghraby, D.M., 2013. Fatty acids composition and biodiesel characterization of Dunaliella salina. J. Water Resour. Prot. 5, 894–899. Fassett, R.G., Coombes, J.S., 2011. Astaxanthin: a potential therapeutic agent in cardiovascular disease. Mar. Drugs 9 (3), 447–465. Furbeyre, H., van Milgen, J., Mener, T., Gloaguen, M., Labussiere, E., 2017. Effects of dietary supplementation with freshwater microalgae on growth performance, nutrient digestibility and gut health in weaned piglets. Animal 11 (2), 183–192. Garcia, J.L., Vicente, M., Galan, B., 2017. Microalgae, old sustainable food and fashion nutraceuticals. Microb. Biotechnol. 10 (5), 1017–1024. Gouda, K.G.M., Kavitha, M.D., Sarada, R., 2015. Antihyperglycemic, antioxidant and antimicrobial activities of the butanol extract from Spirulina platensis. J. Food Biochem. 39 (5), 594–602. Guil-Guerrero, J.L., Navarro-Juarez, R., Lopez-Martinez, J.C., Campra-Madrid, P., Rebolloso-Fuentes, M.M., 2004. Functional properties of the biomass of three microalgal species. J. Food Eng. 65 (4), 511–517. Habib, M.A.B., Parvin, M., Huntington, T.C., Hasan, M.R., 2008. A Review on Culture, Production and Use of Spirulina as Food for Humans and Feeds for Domestic Animals and Fish. Food and Agricultural Organization, Roma. Harun, R., Singh, M., Forde, G.M., Danquah, M.K., 2010. Bioprocess engineering of microalgae to produce a variety of consumer products. Renew. Sust. Energ. Rev. 14 (3), 1037–1047. He, M.L., Hollwich, W., Rambeck, W.A., 2002. Supplementation of algae to the diet of pigs: a new possibility to improve the iodine content in the meat. J. Anim. Physiol. Anim. Nutr. (Berl) 86 (3–4), 97–104. Holman, B.W.B., Malau-Aduli, A.E.O., 2012. Spirulina as a livestock supplement and animal feed. J. Anim. Physiol. Anim. Nutr. (Berl) 97 (4), 615–623. Kang, H.K., Salim, H.M., Akter, N., Kim, D.W., Kim, J.H., Bang, H.T., et al., 2013. Effect of various forms of dietary Chlorella supplementation on growth performance, immune characteristics, and intestinal microflora population of broiler chickens. J. Appl. Poult. Res. 22 (1), 100–108. Kitada, K., Machmudah, S., Sasaki, M., Goto, M., Nakashima, Y., Kumamoto, S., et al., 2009. Antioxidant and antibacterial activity of nutraceutical compounds from Chlorella vulgaris extracted in hydrothermal condition. Sep. Sci. Technol. 44 (5), 1228–1239. Koller, M., Muhr, A., Braunegg, G., 2014. Microalgae as versatile cellular factories for valued products. Algal Res. 6 (A), 52–63. Konickova, R., Vankova, K., Vanikova, J., Vancova, K., Muchova, L., Subhanova, I., et al., 2014. Natural source of bilirubin-like tetrapyrrolic compounds. Ann. Hepatol. 13 (2), 273–283. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.



Microalgal foods Chapter | 32  517

Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Koyande, A.K., Chew, K.W., Rambabu, K., Tao, Y., Chu, D.T., Show, P.L., 2019. Microalgae: a potential alternative to health supplementation for humans. Food Sci. Human Wellness. https://doi.org/10.1016/j.fshw.2019.03.001. Kulkarni, S., Nikolov, Z., 2018. Process for selective extraction of pigments and functional proteins from Chlorella vulgaris. Algal Res. 35, 185–193. Kumudha, A., Sarada, R., 2015. Effect of different extraction methods on vitamin B12 from blue green algae, Spirulina platensis. Pharm. Anal. Acta 6 (2), https://doi.org/10.4172/2153-2435.1000337. Liguori, I., Russo, G., Curcio, F., Bulli, G., Aran, L., Della-Morte, D., et al., 2018. Oxidative stress, aging, and diseases. Clin. Interv. Aging 13, 757–772. Lum, K.K., Kim, J., Lei, X.G., 2013. Dual potential of microalgae as a sustainable biofuel feedstock and animal feed. J. Anim. Sci. Biotechnol. 4 (1), 53. Lupatini, A.L., de Oliveira Bispo, L., Colla, L.M., Costa, J.A.V., Canan, C., Colla, E., 2017a. Protein and carbohydrate extraction from S. platensis biomass by ultrasound and mechanical agitation. Food Res. Int. 99 (3), 1028–1035. Lupatini, A.L., Colla, L.M., Canan, C., Colla, E., 2017b. Potential application of microalgae Spirulina platensis as a protein source. J. Sci. Food Agric. 97 (3), 724–732. Madeira, M.S., Cardoso, C., Lopes, P.A., Coelho, D., Afonso, C., Bandarra, N.M., et al., 2017. Microalgae as feed ingredients for livestock production and meat quality: a review. Livest. Sci. 205, 111–121. Makkar, H., 2012. Biofuel Co-Products as Livestock Feed. Opportunities and Challenges. Food and Agricultural Organization, Rome. Matos, A.P., 2017. The impact of microalgae in food science and technology. J. Am. Oil Chem. Soc. 94 (11), 1333–1350. Matsudo, M.C., Bezerra, R.P., Sato, S., Perego, P., Converti, A., Carvalho, J.C.M., 2009. Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source. Biochem. Eng. J. 43 (1), 52–57. Medina, C., Rubilar, M., Shene, C., Torres, S., Verdugo, M., 2015. Protein fractions with techno-functional and antioxidante properties from Nannochloropsis gaditana microalgal biomass. J. Biobased Mater. Bioenergy 9 (4), 417–425. Mobin, S., Alam, F., 2017. Some promising microalgal species for commercial applications: a review. Energy Procedia 110, 510–517. Nakos, M., Pepelanova, I., Beutel, S., Krings, U., Berger, R.G., Scheper, T., 2017. Isolation and analysis of vitamin B12 from plant samples. Food Chem. 216, 301–308. Oh, S.T., Zheng, L., Kwon, H.J., Choo, Y.K., Lee, K.W., Kang, C.W., et al., 2015. Effects of dietary fermented Chlorella vulgaris (CBT®) on growth performance, relative organ weights, cecal microflora, tibia bone characteristics, and meat qualities in Pekin ducks. Asian-Australas J. Anim. Sci. 28 (1), 95–101. Packer, M.A., Harris, G.C., Adams, S.L., 2016. Food and feed applications of algae. In: Bux, F., Chisti, Y. (Eds.), Algae Biotechnology: Products and Process. Springer, Cham, pp. 217–247. Peiretti, P.G., Meineri, G., 2011. Effects of diets with increasing levels of Spirulina platensis on the carcass characteristics, meat quality and fatty acid composition of growing rabbits. Livest. Sci. 140 (1–3), 218–224. Pereira, A.M., Lisboa, C.R., Costa, J.A.V., 2018. High protein ingredients of microalgal origin: obtainment and functional properties. Innov. Food Sci. Emerg. Technol. 47, 187–194. Plaza, M., Herrero, M., Cifuentes, A., Ibanez, E., 2009. Innovative natural functional ingredients from microalgae. J. Agric. Food Chem. 57 (16), 7159–7170. Rodriguez-Sanchez, R., Ortiz-Butron, R., Blas-Valdivia, V., Hernandez-Garcia, A., Cano-Europa, E., 2012. Phycobiliproteins or C-phycocyanin of Arthrospira (Spirulina) maxima protect against HgCl2-caused oxidative stress and renal damage. Food Chem. 135 (4), 2359–2365. Roy, S.S., Pal, R., 2015. Microalgae in aquaculture: a review with special references to nutritional value and fish dietetics. Proc. Zool. Soc. 68 (1), 1–8. Safi, C., Zebib, B., Merah, O., Pontalier, P.Y., Vaca-Garcia, C., 2014. Morphology, composition, production, processing and applications of Chlorella vulgaris: a review. Renew. Sustain. Energy Rev. 35, 265–278. Salla, A.C.V., Marguerites, A.C., Seibel, F.I., Holz, L.C., Briao, V.B., Bertolin, E.E., et al., 2016. Increase in the carbohydrate content of the microalgae Spirulina in culture by nutrient starvation and the addition of residues of whey protein concentrate. Bioresour. Technol. 209, 133–141. Sathasivam, R., Radhakrishnan, R., Hashem, A., Abd-Allah, E.F., 2017. Microalgae metabolites: a rich source for food and medicine. Saudi J. Biol. Sci. https://doi.org/10.1016/j.sjbs.2017.11.003. Schwenzfeir, A., Lech, F., Wierenga, P.A., Eppink, M.H.M., Gruppen, H., 2013. Foam properties of algae soluble protein isolate: effect of pH and ionic strength. Food Hydrocoll. 33, 111–117. Shanmugapriya, B., Babu, S.S., Hariharan, T., Sivaneswaran, S., Anusha, M.B., College, C.N., et al., 2015. Research article dietary administration of Spirulina platensis as probiotics on growth performance and histopathology in broiler chicks. Int. J. Recent Sci. Res. 6 (2), 2650–2653. Shibata, S., Natori, Y., Nishihara, T., Tomisaka, K., Matsumoto, K., Sansawa, H., et al., 2003. Antioxidant and anti-cataract effects of Chlorella on rats with streptozotocin-induced diabetes. J. Nutr. Sci. Vitaminol. 49 (5), 334–339.

518  PART | VIII  Algal foods

Sinha, S., Patro, N., Patro, I.K., 2018. Maternal protein malnutrition: current and future perspectives of Spirulina supplementation in neuroprotection. Front. Neurosci. 12, 966. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101 (2), 87–96. Stanic-Vucinic, D., Minic, S., Nikolic, M.R., Velickovic, T.C., 2018. Spirulina phycobiliproteins as food components and complements. In: Jacob-Lopez, E. (Ed.), Microalgal Biotechnology. IntechOpen, Santa Maria, pp. 129–149. Su, C.H., Liu, C.S., Yang, P.C., Syu, K.S., Chiuh, C.C., 2014. Solid-liquid extraction of phycocyanin from Spirulina platensis: kinetic modeling of influencial factors. Sep. Purif. Technol. 123, 64–68. Tafreshi, A.H., Shariati, M., 2009. Dunaliella biotechnology: methods and applications. J. Appl. Microbiol. 107 (1), 14–35. Ursu, A.V., Marcatti, A., Sayd, T., Sante-Lhoutellier, V., Djelveh, G., Michaud, P., 2014. Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris. Bioresour. Technol. 157, 134–139. Vanthoor-Koopmans, M., Wijffels, R.H., Barbosa, M.J., Eppink, M.H.M., 2013. Biorefinery of microalgae for food and fuel. Bioresour. Technol. 135, 142–149. Verdasco-Martin, C.M., Diaz-Lozano, A., Otero, C., 2019. Advantageous enzyme selective extraction process of essential Spirulina oil. Catal. Today. https://doi.org/10.1016/j.cattod.2019.02.066. Vigani, M., Parisi, C., Rodriguez-Cerezo, E., Barbosa, M.J., Sijtsma, L., Ploeg, M., et al., 2015. Food and feed products from micro-algae: market opportunities and challenges for the EU. Trends Food Sci. Technol. 42 (1), 81–92. Vo, T.S., Ngo, D.H., Kim, S.K., 2015. Nutritional and pharmaceutical properties of microalgal Spirulina. In: Kim, S.K. (Ed.), Handbook of Marine Microalgae. Academic Press, San Diego, pp. 299–308. Waghmare, A.G., Salve, M.K., LeBlanc, J.G., Arya, S.S., 2016. Concentration and characterization of microalgae proteins from Chlorella pyrenoidosa. Bioresour. Bioprocess. 3, 16. Xia, D., Liu, B., Xin, W.Y., Liu, T.S., Sun, J.Y., Liu, N., et al., 2016. Protective effects of C-phycocyanin on alcohol-induced subacute liver injury in mice. J. Appl. Phycol. 28 (2), 765–772. Yaakob, Z., Ali, E., Zainal, A., Mohamad, M., Takriff, M.S., 2014. An overview: biomolecules from microalgae for animal feed and aquaculture. J. Biol. Res. 21 (1), 6. Yan, L., Lim, S.U., Kim, I.H., 2012. Effect of fermented Chlorella supplementation on growth performance, nutrient digestibility, blood characteristics, fecal microbial and fecal noxious gas content in growing pigs. Asian-Australas J. Anim. Sci. 25 (12), 1742–1747. Yeh, K.L., Chang, J.S., 2012. Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31. Bioresour. Technol. 105, 120–127. Yousefi, R., Saidpour, A., Mottaghi, A., 2019. The effects of Spirulina supplementation on metabolic syndrome components, its liver manifestation and related inflammatory markers: a systematic review. Complement. Ther. Med. 42, 137–144. Zhang, W.G., Zhang, P.L., Sun, H., Chen, M.Z., Lu, S., Li, P.F., 2014. Effects of various organic carbon sources on the growth and biochemical composition of Chlorella pyrenoidosa. Bioresour. Technol. 173, 52–55. Zhu, L.D., Hiltunen, E., Antila, E., Zhong, J.J., Yuan, Z.H., Wang, Z.M., 2014. Microalgal biofuels: flexible bioenergies for sustainable development. Renew. Sust. Energ. Rev. 30, 1035–1046. Zotte, A.D., Sartori, A., Bohatir, P., Remignon, H., Ricci, R., 2013. Effect of dietary supplementation of Spirulina (Arthrospira platensis) and Thyme (Thymus vulgaris) on growth performance, apparent digestibility and health status of companion dwarf rabbits. Livest. Sci. 152 (2–3), 182–191.

Chapter 33

The scientometric analysis of the research on the algal toxicology Ozcan Konur Formerly, Ankara Yildirim Beyazit University, Ankara, Turkey

33.1 Introduction The algae has increasingly gained public importance for the benefit of the global society at large as a sustainable resource for the biomedicine (Lee and Mooney, 2012; Rowley et al., 1999) and bioenergy and biofuels (Brennan and Owende, 2010; Chisti, 2007) in an environment-friendly way (Ho et al., 2011; Wang et al., 2008) as evidenced with over 150,000 papers published since 1980. The toxicological aspects of the algae have similarly been among the most-researched subjects over time as evidenced with over 18,000 papers during the same study period, corresponding to the public concerns about the toxic effects of the algae on both the humans and animals. In line with the teachings of North’s New Institutional Theory (North, 1991, 1994), for devising efficient incentive structures for the optimal development of the research and practice in this field, the stakeholders should have timely access to the information on the relevant research (Konur, 2000, 2002a,b,c, 2004, 2006a,b, 2007a,b, 2012a). In this respect, the scientometric studies (Garfield, 1972, 2006) have had a lot to offer to enable the key stakeholders to inform themselves about algal toxicology and the underlying research areas as in other fields of the algal research (Konur, 2011, 2015a,b,c,d,e,f,g,h,i,j,k, 2016a, 2017a, 2019a, 2020a,b,c,d,e,f,g,h,i,j,k,l,m,n), bioenergy and biofuels (Konur, 2012b,c,d,e,f,g,h,i,j,k, 2018a,b,c), nanobiomaterials (Konur, 2016b,c,d,e,f,g, 2017b,c,d,e,f, 2019b), biomedicine (Konur, 2016h, 2018d), and social sciences (Konur, 2012o,p,q,r,s,t). There have been two scientometric studies in the algal toxicology focusing on the microcystin research in China (Wang et al., 2015) and themes and geography of the cyanotoxin research (Merel et al., 2013). However, there have been no scientometric study covering the whole field of algal toxicology at the global scale as of September 2018 as in other research fields. This paper presents the first-ever scientometric study of the research in algal toxicology covering the whole range of toxins and harmful algal blooms at the global scale and provides the ample data for the primary stakeholders to devise the efficient set of incentive structures for the optimal development of the research and practice in this field.

33.2  Materials and methodology The search for the scientometric analysis of the literature on the algal toxicology was carried out in September 2018 using four databases of the Web of Science: Science Citation Index-Expanded (SCI-E), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (A&HCI) and Emerging Sources Citation Index (ESCI). The keyword set used for the search has been constituted by taking the relevant words from the titles, abstracts, author keywords, and journal titles of the searched papers. The full keyword set is given in the Appendix. These keyword sets have been devised in two major parts: the direct algal toxicology-related keywords and the combined keyword sets from the fields of the toxicology and the algae. There have been four distinct keyword sets for the first part: toxins related to the algae in general, toxins related to dinoflagellates, toxins related to cyanobacteria, and toxins related to diatoms. On the other hand, there have been two distinct sub-parts for the second part: keywords related to toxicology and keywords related to the algae. Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00033-4 © 2020 Elsevier Inc. All rights reserved.

521

522  PART | IX  Algal toxicology

The first sub-part of the second part consists of the keywords for the titles of the papers and the keywords related to the core set of journals related to the toxicology. The second sub-part consists of the keywords related to the algae in general, phytoplankton, dinoflagellates, and coccolithophores, microalgae, macroalgae, diatoms, cyanobacteria, and journals related to the algae. The papers found through the use of this detailed keyword set have been refined for the document types (articles, reviews, notes, letters, and editorial materials) and language (English). The found references have also been subject to the quality control exercise to ensure that these references have been primarily related to the algal toxicology. This refined list of papers has formed the sample for the scientometric analysis and content overview of the literature on the algal toxicology. The data on the document types and indexes, keywords, authors, institutions, funding bodies, countries and territories, journal titles, Web of Science subject categories, and the research fronts have been collected during the study. The results on these aspects have been presented in the text and through a number of figures and tables where necessary. The data on the scientometric analysis and content analysis of 20 most-cited papers have also been provided focusing on the determination of the key research streams for these citation classics, complementing sections on the keywords and research fronts. The presented results have also been discussed through an interdisciplinary perspective and a number of recommendations have been made.

33.3 Results 33.3.1  Documents and indexes The search has resulted in 20,647 papers where there have been 17,327 articles, 1681 meeting abstracts, 677 reviews, 350 notes, 206 editorial materials, 170 letters, and 133 corrections. In the first instance, the papers excluding meeting abstracts and corrections have been selected resulting in 18,730 papers in total. In the next step, these papers have been further refined on the language basis. On this basis, there have been 18,238 papers in English. The other major languages have been Japanese, Chinese, Spanish, and German. This set of 18,238 papers has formed the sample for the scientometric analysis of the literature on the algal toxicology. The articles have formed 92.8% of the final sample while reviews, notes, editorial matters, and letters have formed 3.5%, 1.9%, 1.0%, and 0.9% of this sample, respectively. Additionally, 3.6% of these papers have been ‘proceedings papers’ and two of them have been ‘retracted papers’. On the other hand, 99.5% of these papers have been indexed by the SCI-E while only 0.5% of them have been indexed by the other indices, SSCI, ESCI, and A&HCI.

33.3.2 Keywords The most-prolific keywords used in algal toxicology have been determined to assess the hot topics and the primary research fronts in the algal toxicology. There have been three most-prolific keywords for the first set of keywords for the algal toxicology: ‘microcystin*’, ‘okadaic acid’, and ‘red tide’. The other prolific keywords for the algal toxicology have been ‘phycotoxin* and “harmful algal bloom*”’ for the algae in general; ‘azaspiracid, brevetoxin*, ciguatera, ciquatoxin*, “harmful dinoflagellate*”, maitotoxin*, palytoxin*, pectenotoxin*, saitotoxin*, “shellfish poison*”, “shellfish toxin*”, and yessotoxin*’ for the dinoflagellates; ‘anatoxin*, “β-methylamino-l-alanine”, BMAA, cyanotoxin*, cylindrospermopsin*, “harmful cyanobacteria”, nodularin, saitotoxin, and teleocidin’ for the cyanobacteria; and “domoic acid” for the diatoms. There have been 12 most-prolific keywords for the algae: ‘alga, algae, and algal’ for the algae; ‘dinoflagellate* and phytoplankton’ for the dinoflagellates and phytoplankton; ‘macroalga*, rhodophyt*, and seaweed*’ for the macroalgae; ‘bacillariophycea*, diatom, and diatoms’ for the diatoms; and ‘cyanobacter*’ for the cyanobacteria. The other prolific keywords for the algae have been ‘periphyton*’ for the algae; ‘coccolith*, dinophycea*, and Alexandrium’ for the dinoflagellates; ‘chlorophycea*, chlorophyt*, green alga*, microalga*, micro-alga*, Chlamydomonas, *Chlorella, Dunaliella, Euglena, and Scenedesmus’ for the microalgae; ‘brown alga*, carrageenan*, fucoid*, phaeophycea*, kelp*, phaeophyt*, red alga*, Fucus, Gracilaria, Laminaria, Porphyra, Sargassum, and Ulva’ for the macrolgae; ‘bacillariophyt*’ for the diatoms; ‘“blue green alga*”, “blue-green alga*”, *Anabaena, *Microcystis, *Nostoc, Spirulina, *Synechococcus, and Synechocystis’ for the cyanobacteria.



The scientometric analysis of the research on the algal toxicology Chapter | 33  523

On the other hand the most-prolific journals related solely to the algal research have been ‘Algal Research*, European Journal of Phycology, Harmful Algae, Journal of Applied Phycology, Journal of Phycology, and Phycologia’.

33.3.3 Authors There have been 36,496 authors contributing to the research on the algal toxicology in total. The information on the mostprolific 20 authors is provided in Table  33.1: Authors’ names, gender, research fields, institutions, countries, primary research topics, number of papers produced, the number of papers with at least 100 citations received (I-100), and the percentage of the number of papers with at least 100 citations received with relative to the total number of papers published (I-100%). The data on the authors shows that the most-prolific author has been ‘Takeshi Yasumoto’ of the Japan Food Research Laboratories, working primarily on the toxins of dinoflagellates, with 274 papers. His citation impact is highest with 46 influential papers with at least 100 citations. It is notable that only three of these authors are female. The academic disciplines of these authors have ranged from ecology to pharmacology. Japan has been the most-prolific country for these authors with five authors while the United States followed Japan with four authors. The other prolific countries have been China, Finland, and Spain. On the other hand, Europe has had six authors as a whole. The most-prolific research fields have been the ‘toxins of dinoflagellates and cyanobacteria’ with 12 and 7 papers, respectively. The number of papers published by these authors have ranged from 77 to 274. These most-prolific authors have contributed to nearly 13.5% of the papers. The data on the I-100 of these most-prolific authors shows that ‘Wayne W Carmichael’ of Wright State University, working primarily on the ‘cyanobacterial toxins’, has followed the top author, ‘Takeshi Yasumoto’ with 42 influential papers. The other most-prolific authors with significant citation impact have been ‘Geoffrey A Codd’ of University of Dundee working primarily on the ‘cyanobacterial toxins’ with 27 papers, ‘Kaarina Sivonen’ of University of Helsinki working primarily on the ‘cyanobacterial toxins’ with 23 papers, ‘Donald M Anderson’ of Woods Hole Oceanographic Institute primarily working on the ‘harmful algal blooms’ with 18 papers, and ‘Masayuki Stake’ of University of Tokyo primarily working on the ‘toxins of dinoflagellates’ with 18 papers. Additionally, the citation impact of these prolific authors has been examined in terms of the percentage of the I-100 papers with relative to the total number of papers published for each author in the last column (I-100%). These data shows that ‘Wayne W Carmichael’ has been the top influential author with 34%. The other most-influential authors have been ‘Masayuki Stake’, ‘Kaarina Sivonen’, ‘Geoffrey A Codd’, ‘Takeshi Yasumoto’, and ‘Brett A Neilan’ with 21%, 20%, 19%, 19%, and 17%, respectively.

33.3.4 Countries Nearly 99.5% of the papers have had country information in their abstract pages and 147 countries and territories have contributed to these papers overall. Table 33.2 provides information about the most-prolific 10 countries. These 10 mostprolific countries have produced 82.9% of the papers as a whole. The most-prolific country has been the US producing 27.2% of the papers. Additionally, China and Japan have emerged as the prolific countries following the United States with 12.4% and 12.2% of the papers, respectively. The European countries have been dominant in the top 10 country list as they have produced 48.4% of the papers as a whole, surpassing both the United States and China.

33.3.5 Institutions Only 99.5% of the papers have had their institutions listed in their abstract pages. For these papers, 6813 institutions have contributed to the research on the algal toxicology in total. The information about the 10 most-prolific institutions is given in Table 33.3. The most-prolific institution has been the ‘Chinese Academy of Sciences’ publishing 3.6% of the papers. The ‘National Oceanic Atmospheric Administration’, ‘Centre National de la Recherche Scientifique’, ‘Tohoku University’, and ‘Universite Cote D’Azur’ have followed the top institution with 2.7%, 2.4%, 2.4%, and 2.3% of the papers, respectively. The most-prolific country for these institutions has been France with three institutions. On the other hand, Japan and the United States have had two papers each and Europe has had four papers as a whole.

Author

Gender

Res. field

Institute

Country

Topics

No. papers

I-100

I-100%

1

Takeshi Yasumoto

M

Marine toxins

Japan Food Research Lab.

Japan

Toxins of dinoflagellates

274

46

17

2

Luis M Botana

M

Pharmacology

Univ. Santiago de Compostela

Spain

Toxins of dinoflagellates

145

0

0

3

Geoffrey A. Codd

M

Microbiology

Univ. Dundee

Scotland

Cyanotoxins

144

27

19

4

Donald M. Anderson

M

Biology

Woods Hole Ocean. Inst.

United States

Harmful algal blooms

126

18

14

5

Wayne W. Carmichael

M

Biology

Wright State Univ.

United States

Cyanotoxins

125

42

34

6

Ping Xie

M

Ecology

Chin. Acad. Sci.

China

Cyanotoxins

119

4

3

7

Hirota Fujiki

M

Pharmacy

Saga Univ.

Japan

Toxins of dinoflagellates

117

14

12

8

Kaarina Sivonen

F

Microbiology

Univ. Helsinki

Finland

Cyanotoxins

113

23

20

9

Vitor Vasconcelos

M

Biology

Univ. Porto

Portugal

Cyanotoxins

109

4

4

10

Michael A. Quilliam

M

Chemistry

Natl. Res. Counc. Canada

Canada

Toxins of dinoflagellates

107

16

15

11

Mercedes R. Vieytes

F

Marine toxins

Univ. Santiago de Compostela

Spain

Toxins of dinoflagellates

105

0

0

12

Yuji Oshima

M

Environment

Kyushu Univ.

Japan

Toxins of dinoflagellates

104

13

13

13

Toshiyuki Suzuki

M

Fisheries

Nat. Res. Inst. Fisher.

Japan

Toxins of dinoflagellates

96

1

1

14

Brett A. Neilan

M

Microbiology

Univ. Newcastle

Australia

Cyanotoxins

95

16

17

15

Daniel G. Baden

M

Chemistry

Univ. N Carolina Wilmington

United States

Toxins of dinoflagellates

91

11

12

16

John S. Ramsdell

M

Physiology

Natl. Ocean. Atmosph. Admn.

United States

Toxins of dinoflagellates

91

0

0

17

Jun Chen

F

Ecology

Chin. Acad. Sci.

China

Cyanotoxins

88

2

2

18

Masayuki Stake

M

Chemistry

Univ. Tokyo

Japan

Toxins of dinoflagellates

85

18

21

19

Stephan Pflugmacher

M

Ecology

Univ. Helsinki

Finland

Cyanotoxins

78

9

12

20

Philip Hess

M

Toxicology

French Res. Inst. Exploit. Sea

France

Toxins of dinoflagellates

77

2

3

M, male; F, female; Res. field, academic specialty of the authors; I-100, the number of papers with at least 100 citations received; I-100%, the percentage of the number of papers with at least 100 citations received with relative to the total number of papers published.

524  PART | IX  Algal toxicology

TABLE 33.1  The most-prolific authors in algal toxicology.

The scientometric analysis of the research on the algal toxicology Chapter | 33  525



TABLE 33.2  The most-prolific countries in algal toxicology. Country

No. papers

% Papers

1

United States

4961

27.2

2

China

2259

12.4

3

Japan

2216

12.2

4

Spain

1023

5.6

5

Germany

1010

5.5

6

France

975

5.4

7

Australia

931

5.1

8

Italy

611

3.4

9

India

575

3.1

10

South Korea

561

3.1

TABLE 33.3  The most-prolific institutions in algal toxicology. Institutions

Country

No. papers

% Papers

1

Chinese Academy of Sciences

China

664

3.6

2

National Oceanic Atmospheric Administration

United States

489

2.7

3

Centre National de la Recherche Scientifique

France

434

2.4

4

Tohoku University

Japan

434

2.4

5

Universite Cote D’Azur

France

424

2.3

6

University of Tokyo

Japan

280

1.5

7

University of North Carolina

United States

272

1.5

8

Consejo Superior de Investigaciones Cientificas

Spain

239

1.3

9

National Research Council Canada

Canada

229

1.3

10

Institut Français de Recherche pour L'exploitation de la Mer

France

220

1.2

The contribution of these institutions has ranged from 1.2% to 3.6% of the papers. Overall, these 10 institutions have contributed to 20.2% of the papers.

33.3.6  Research funding bodies Only 51.5% of these papers have had declared any research funding in their abstract pages. Overall, 9498 funding bodies funded these papers. The most-prolific funding body has been the ‘National Natural Science Foundation of China’ funding 4.5% of the papers. The other prolific funding bodies have been the ‘National Institutes of Health’ and ‘National Science Foundation’ of the United States, ‘Fundamental Research Funds for the Central Universities of China’ and ‘National Basic Research Program of China’, ‘Australian Research Council’, and ‘European Union’ funding between 70 and 168 papers each.

526  PART | IX  Algal toxicology

33.3.7  Publication years Fig. 33.1 shows the number of papers on the algal toxicology, published between 1980 and 2018 as of September 2018. The data in this figure shows that the number of papers has risen from 86 papers in 1980 to 964 papers in 2016. The most prolific publication decade has been the 2010s with 43.3% of the papers. On the other hand, 8.1%, 17.9%, and 30.7% of the papers have been published in the 1980s, 1990s, and 2000s, respectively. Thus, it is notable that 74% of the papers have been published between 2000 and 2018 as of September 2018. There has been a continuous increasing trend since 1980.

33.3.8  Source titles Overall, these papers have been published in 2011 journals. Table 33.4 provides the information on the 10 most-prolific journals. These 10 journals have published 29% of all the papers in total.

1200

Number of papers

1000 800 600 400 200 0 Publication years

FIG. 33.1  The number of publications in the algal toxicology between 1980 and 2018.

TABLE 33.4  The most-prolific journals in algal toxicology. Institutions

Subject

No. papers

% Papers

1

Harmful Algae

Marine & Freshwater Biology

1438

7.9

2

Toxicon

Pharmacology & Pharmacy; Toxicology

1240

6.8

3

Aquatic Toxicology

Marine & Freshwater Biology; Toxicology

522

2.9

4

Bulletin of Environmental Contamination and Toxicology

Environmental Sciences; Toxicology

431

2.4

5

Ecotoxicology and Environmental Safety

Environmental Sciences; Toxicology

418

2.3

6

Environmental Toxicology and Chemistry

Environmental Sciences; Toxicology

310

1.7

7

Environmental Toxicology

Environmental Sciences; Toxicology; Water Resources

275

1.5

8

Tetrahedron Letters

Chemistry, Organic

229

1.3

9

Chemosphere

Environmental Sciences

225

1.2

10

Water Research

Engineering, Environmental; Environmental Sciences; Water Resources

204

1.1

The scientometric analysis of the research on the algal toxicology Chapter | 33  527



The most-prolific journal has been ‘Harmful Algae’ publishing 7.9% of the papers, primarily focusing on harmful algae. ‘Toxicon’, ‘Aquatic Toxicology’, ‘Bulletin of Environmental Contamination and Toxicology’, and ‘Ecotoxicology and Environmental Safety’ have followed this top journal with 6.8%, 2.9%, 2.4%, and 2.3% of the papers, respectively. The most-prolific subject category for these journals have been ‘Toxicology’ and ‘Environmental Sciences’ with six journals each. It is notable that ‘Harmful Algae’ has been the only journal related to the algae in this top-10 list. However, there have been four other journals in the top 100 journal list, related to algae publishing 2.3% of the papers in total: ‘Journal of Phycology’, ‘Journal of Applied Phycology’, ‘Phycologia’, and ‘European Journal of Phycology’. Thus, these five algaerelated journals have published 10.2% of the papers, in total.

33.3.9  Subject categories The information about the 10 most-prolific subject categories are given in Table 33.5. As expected, the most-prolific subject category has been ‘Toxicology’ indexing 27% of the papers. On the other hand, 21.7% and 21.5% of the papers have been indexed by the subject categories of ‘Environmental Sciences’ and ‘Marine Freshwater Biology’, respectively. Thus, these three categories have been the key pillars of the research in algal toxicology. It is notable that 11.1% of the papers has been indexed by the category of ‘Pharmacology and Pharmacy’. On the other hand, four categories related to basic sciences have taken place in the top 10 category list: ‘Marine and Freshwater Biology’, ‘Biochemistry and Molecular Biology’, ‘Analytical Chemistry’, and ‘Organic Chemistry’. Similarly, three categories related to technology have taken place in this list as well: ‘Water Resources’, ‘Biotechnology and Applied Microbiology’, and ‘Environmental Engineering’. Thus, the research in algal toxicology shows an interdisciplinary character covering science, technology, and medicine.

33.3.10  Research fronts The most-prolific research fronts have been determined by collecting the data on the influential papers with at least 100 citations, 4.3% of the research sample of 18,235 papers, Table 33.6. The data shows that there have been two major research fronts: ‘algal toxins’ and ‘harmful algal blooms’ with 91% and 9% of the sample papers, respectively. There have been three sub-research fronts for the algal toxins: ‘cyanobacterial toxins’ (cyanotoxins), ‘dinoflagellate toxins’ (dinotoxins) and ‘diatom toxins’ with 53%, 32%, and 5% of the sample, respectively.

TABLE 33.5  The most-prolific subject categories in algal toxicology. Subject categories

No. papers

% Papers

1

Toxicology

4926

27.0

2

Environmental Sciences

3958

21.7

3

Marine and Freshwater Biology

3912

21.5

4

Pharmacology and Pharmacy

2019

11.1

5

Biochemistry and Molecular Biology

1128

6.2

6

Chemistry, Analytical

937

5.1

7

Water Resources

789

4.3

8

Chemistry, Organic

766

4.2

9

Biotechnology and Applied Microbiology

713

3.9

10

Engineering, Environmental

695

3.8

528  PART | IX  Algal toxicology

TABLE 33.6  The most-prolific research fronts in algal toxicology. Toxins, blooms

I-100 no. papers

I-100% papers

1

Algal toxins

680

91

1.1.

Cyanobacterial toxins

399

53

1.1.1.

Cyanotoxins in general

69

9

1.1.2.

Other cyanotoxins

15

2

1.1.3.

Microcystins

219

29

1.1.4.

Cylindrospermopsins

38

5

1.1.5.

BMAA-beta-N-methylamino-l-alanine

15

2

1.1.6.

Lyngbyatoxins

12

2

1.1.7.

Nodularin

6

1

1.1.8.

Saxitoxin

13

2

1.1.9.

Anatoxins

12

2

1.2.

Dinoflagellate toxins

240

32

1.2.1.

Dinoflagellate toxins in general

28

4

1.2.2.

Other dinoflagellate toxins

36

5

1.2.3.

Okadaic acid

49

7

1.2.4.

Ciquatoxins

26

3

1.2.5.

Brevetoxins

30

4

1.2.6.

Palytoxins

30

4

1.2.7.

Maitotoxins

8

1

1.2.8.

Saitotoxins

9

1

1.2.9.

Azaspiracid

9

1

1.2.10

Yessotoxin

8

1

1.2.11.

Pfisteria toxins

7

1

1.3.

Diatom toxins

41

5

1.3.1.

Domoic acid

41

5

2.

Harmful algal blooms

69

9

2.1.

Algae

32

4

2.2.

Cyanobacteria

21

3

2.3.

Dinoflagellates

17

2

749

100

Total

I-100 no. papers, the number of papers with at least 100 citations received for each research front; I-100% papers, the percentage of the number of papers with at least 100 citations received with relative to the total number of papers published.

Similarly, there have been three sub-research fronts for the ‘harmful algal blooms’ based on the type of algae: ‘harmful algal blooms’ in general, ‘harmful cyanobacterial blooms’, and ‘harmful dinoflagellate blooms’ with 4%, 3%, and 2% of the sample, respectively. The data shows that the most prolific research front on the basis of the type of toxins has been the research on the ‘microcystins’ with 29% of the sample. The other prolific research fronts have been ‘cyanotoxins’ in general, ‘okadaic acid’, ‘cylindrospermopsins’, other ‘dinoflagellate toxins’, and ‘domoic acid’ with 9%, 7%, 5%, 5%, and 5% of the sample, respectively.



The scientometric analysis of the research on the algal toxicology Chapter | 33  529

33.3.11  Citation classics This section provides the information on both the scientometric analysis and content overview of the most-cited 20 papers in algal toxicology. The information on these papers are given in Table 33.7: authors’ names, publication years, number of authors per paper, lead authors’ names, gender, institutions, and countries (lead authors were determined based on the number of papers produced in this field), number of papers published by the lead authors, subject and topics of the papers (toxins or harmful algae), type of toxins studied, academic focus of the papers, number of citations received, and the number of citations per year.

33.3.11.1  Scientometric overview of the citation classics These papers have been published between 1985 and 2012. The most-prolific decade has been the 1990s with nine papers while there have been five papers each in the 1980s and 2000s. There has been only one paper in the 2010s. The reviews have been over-represented in these classical papers as there have been 10 reviews and 10 articles. The number of the authors of these papers has ranged from 1 to 15 while the mean number of authors has been 5. The most-prolific lead author has been ‘Wayne W Carmichael’ with four papers. The other prolific lead authors have been ‘Donald M Anderson’, ‘JoAnn M Burkholder’, ‘Geoffrey A Codd’, ‘Hirota Fujiki’, ‘Patricia M Glibert’, ‘Christopher J Gobler’, and ‘Masami Suganuma’ with two papers each. There has been a significant gender deficit among the lead authors of these classical papers as only seven papers have been authored in part by female authors while one paper has been published solely by a female author. Similarly, only seven of these prolific lead authors have been females. The most-prolific institutions have been the ‘Wright State University’ and ‘University of Dundee’ with four and three papers, respectively. The other prolific institutions have been the ‘National Cancer Center Research Institute’, ‘North Carolina State University’, ‘University of Maryland’, and ‘Woods Hole Oceanographic Institute’ with two papers each. In total, 20 institutions have contributed to these classical papers. In total, eight countries contributed to these classical papers. The most-prolific country has been the US contributing to nine papers. The other prolific countries have been Australia, Japan, Scotland, and Germany with four, three, three, and two papers, respectively. In total, these papers have been published by 19 journals where only ‘Harmful Algae’ have had 2 papers. In total, these papers have been indexed by 17 subject categories. The most-prolific categories have been ‘Biochemistry and Molecular Biology’, ‘Marine and Freshwater Biology’, and ‘Multidisciplinary Sciences’ with 5, 4, and 3 papers, respectively. The other prolific categories have been ‘Biophysics’, ‘Pharmacology and Pharmacy’, and ‘Toxicology’ with 2 papers each. There have been two broad topical areas of ‘harmful algal blooms’ and ‘algal toxins’ with 6 and 14 papers, respectively. The most-prolific types of algae have been ‘cyanobacteria’ and ‘dinoflagellates’ with 10 and 7 papers, respectively. Additionally, 3 papers have covered algae in general. As 14 papers have covered algal toxins, six and five papers have covered microcystins and okadaic acid, respectively. The focus of these papers has been mostly on the toxicology and ecology with 13 and 4 papers, respectively. Additionally, two and one papers have focused on the medicine and biochemistry, respectively. These papers have received between 585 and 1567 citations each, totaling in 16,979 citations with a mean value of 849 citations. On the other hand, the number of citations per year has ranged from 19 to 112 with a mean value of 17 citations per year.

33.3.11.2  Brief overview of the content of the citation classics There have been two major classes of papers: algal toxins and harmful algal blooms. In the first section, there have been five, six, and three papers on the ‘okadaic acid’ from dinoflagellates, ‘microcystins’, and ‘cyanotoxins’ in general from cyanobacteria. In the second part, there have been six papers on the ‘harmful algal blooms’. Algal toxins Dinoflagellates-okadaic acid Bialojan and Takai (1988) study the inhibitory effect of okadaic acid on protein phosphatases in a most-cited paper with 1567 citations. They find that okadaic acid had a relatively high specificity for type 2A, type 1 and polycation-modulated phosphatases and okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes. Cohen et al. (1990) review okadaic acid in a paper with 1411 citations. They note that the okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A and this toxin is extremely useful for identifying biological processes that are controlled through the reversible phosphorylation of proteins.

530  PART | IX  Algal toxicology

TABLE 33.7  The citation classics in algal toxicology. Authors

Year

Doc.

N auths.

Lead authors

Gender

Inst.

Count.

N paper

1

Bialojan and Takai

1988

A

2

Akira Takai

M

Univ. Heidelberg

Germany

18

2

Hallegraeff

1993

R

1

Gustaaf M. Hallegraeff

M

Univ. Tasmania

Australia

55

3

Cohen et al.

1990

R

3

Charles F.B. Holmes

M

Natl. Res. Counc. Can.

Canada

27

4

Mackinstosh et al.

1990

A

5

Geoffrey A. Codd; Kenneth A. Beattie

M; M

Univ. Dundee

Scotland

144; 38

5

Deng et al.

2008

A

5

Donyuan Zhao

M

Fudan Univ.

China



6

Anderson et al.

2002

R

3

Donald M. Anderson; Patricia M. Glibert; JoAnn M. Burkholder

M; F; F

Woods Hole Ocean. Inst.; Univ. Maryland; North Carolina St. Univ.

United States

126; 33; 52

7

Ishihara et al.

1989

A

11

Daisuke Uemura

M

Shizuoka Univ.

Japan

31

8

Carmichael

1992

R

1

Wayne W. Carmichael

M

Wright St. Univ.

United States

125

9

Haystead et al.

1989

A

7

Philip Cohen

M

Univ. Dundee

Scotland



10

Jochimsen et al.

1998

A

12

Wayne W. Carmichael; Sandra M.F.O. Azevedo

M; F

Wright St. Univ.; Univ. Fed. Rio de Janeiro

United States; Brazil

125; 40

11

Smayda

1997

R

1

Theodore J. Smayda

M

Univ. Rhode Isl.

United States



12

Heisler et al.

2008

A

15

Donald M. Anderson; Patricia M. Glibert; JoAnn M. Burkholder; William P. Cochlan; Christopher J. Gobler

M; F; F; M; M

Woods Hole Ocean. Inst.; Univ. Maryland; North Carolina St. Univ.; San Francisco St. Univ.

United States

126; 33;52; 17; 66

13

Codd et al.

2005

R

3

Geoffrey A. Codd; James S. Metcalf

M; M

Univ. Dundee

Scotland

144; 62

The scientometric analysis of the research on the algal toxicology Chapter | 33  531



Journal

Subject area

Topic

Algae

Toxin

Subject

Cits.

Av. cits

Biochem. J.

Biochem. Mol. Biol.

Toxins

Dinoflagellates

Okadaic acid

Toxicology

1567

52

Phycologia

Plant Sci.; Mar. Fresh. Biol.

Harmful blooms

Dinoflagellates



Ecology, toxicology

1430

57

Trends Biochem. Sci.

Biochem. Mol. Biol.

Toxins

Dinoflagellates

Okadaic acid

Toxicology

1411

50

FEBS Lett.

Biochem. Mol. Biol.; Biophys., Cell Biol.

Toxins

Cyanobacteria

Microcystin-LR

Toxicology

1174

42

J. Am. Chem. Soc.

Chem. Mult.

Toxins

Cyanobacteria

Microcystin-LR

Toxicology

1122

112

Estuaries

Env. Sci.; Mar. Fresh. Biol.

Harmful blooms

Algae



Ecology

988

62

Biochem. Biophys. Res. Commun.

Bioch. Mol. Biol.; Biophys.

Toxins

Dinoflagellates

Okadaic acid

Toxicology

960

34

J. Appl. Bacteriol.

Biot. Appl. Microb.; Microb.

Toxins

Cyanobacteria



Toxicology

909

35

Nature

Mult. Sci.

Toxins

Dinoflagellates

Okadaic acid

Toxicology

839

29

N. Engl. J. Med.

Med. Gen. Int.

Toxins

Cyanobacteria

Microcystins

Medicine

711

36

Limnol. Oceanogr.

Limnol.; Ocean.

Harmful blooms

Dinoflagellates



Ecology

648

31

Harmful Algae

Mar. Fresh. Biol.

Harmful blooms

Algae



Ecology

617

62

Toxicol. Appl. Pharmacol.

Pharm. Pharm.; Toxic.

Toxins

Cyanobacteria



Toxicology

612

19

Continued

532  PART | IX  Algal toxicology

TABLE 33.7  The citation classics in algal toxicology—cont’d Authors

Year

Doc.

N auths.

Lead authors

Gender

Inst.

Count.

N paper

14

Landsberg

2002

R

1

Jan H. Landsberg

F

Florida Mar. Res. Inst.

United States

18

15

Suganuma et al.

1988

A

9

Masami Suganuma; Hirota Fujiki; Seiji Yoshizawa; Takashi Sugimura

F; M; M; M

Natl. Cancer Ct. Res. Inst.

Japan

58; 117 ; 20; 62

16

Carmichael

1994

R

1

Wayne W. Carmichael

M

Wright St. Univ

United States

125

17

NishiwakiMatsushima et al.

1992

A

8

Wayne W. Carmichael; Masami Suganuma; Hirota Fujiki;

M; F; M

Wright St. Univ.; Natl. Cancer Ct. Res. Inst.

United States; Japan

125; 58; 117

18

Dawson

1998

R

1

Raymond M. Dawson

M

Def. Sci. Tech. Org.

Australia



19

O’Neill et al.

2012

R

4

Timothy W Davis; Michele A Burford; Christopher J. Gobler

M; F; M

Bowling Green St. Univ.; Griffith Univ.; Stony Brook Univ.

United States; Australia; United States

22; 21; 66

20

Tillett et al.

2000

A

6

Elke Dittmann; Thomas Borner; Brett A. Neilan

F; M; M

Univ. Postdam; Humboldt Univ.; Univ. Newcastle

Germany; Germany; Australia

38; 24; 95

Doc., document; A, article; R, review; Gender, gender of lead authors; M, male; F, female; Inst., Institute of the lead authors; Count., country of lead authors; N paper, number of papers published by the lead authors with at least 17 papers; Subject, Web of Science subjects; Topic, primary topic of the papers, type of algae studied; Toxins, type of toxins studied; Cits., number of citations received in total; Av. cits., number of citations per year.

The scientometric analysis of the research on the algal toxicology Chapter | 33  533



Journal

Subject area

Topic

Algae

Toxin

Subject

Cits.

Av. cits

Rev. Fish. Sci.

Fisher.

Harmful blooms

Algae



Ecology

609

38

Proc. Natl. Acad. Sci. U.S.A.

Mult. Sci.

Toxins

Dinoflagellates

Okadaic acid

Toxicology

609

20

Sci. Am.

Mult. Sci.

Toxins

Cyanobacteria



Toxicology

583

24

J. Cancer Res. Clin. Oncol.

Oncol.

Toxins

Cyanobacteria

Microcystin-LR

Toxicology

565

22

Toxicon

Pharm. Pharm.; Toxic.

Toxins

Cyanobacteria

Microcystin-LR

Medicine

553

28

Harmful Algae

Mar. Fresh. Biol.

Harmful blooms

Cyanobacteria



Ecology

549

92

Chem. Biol.

Biochem. Mol. Biol.

Toxins

Cyanobacteria

Microcystis

Biochemistry

523

29

534  PART | IX  Algal toxicology

Ishihara et al. (1989) study the inhibitory effect of okadaic acid on protein phosphatases in a paper with 960 citations. They find that okadaic acid is inhibitor of type 2A, type 1 phosphatases as well as endogenous phosphatase of smooth muscle myosin B. Haystead et al. (1989) study the effects of the okadaic acid on intracellular ‘protein-phosphorylation’ and metabolism in a paper with 839 citations. They find that okadaic acid behaves like a specific protein phosphatase inhibitor in a number of metabolic processes and PP1 and PP2A are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo. Suganuma et al. (1988) study the tumor promotion by okadaic acid in a paper with 609 citations. They find that okadaic acid is a potent additional tumor promoter in mice treated with ‘dimethylbenz[a]anthracene’ (DMBA) and okadaic acid. They conclude that okadaic acid is a non-phorbol ‘12-tetradecanoate 13-acetate’ (TPA) type tumor promoter in mouse skin carcinogenesis. Cyanobacteria-microcystins MacKintosh et al. (1990) study the microcystins in a paper with 1174 citations. They note that ‘microcystin-LR’ inhibits ‘protein phosphatases’ 1 (PP1) and 2A (PP2A). It inhibits protein phosphatase 2B less potently, while it does not inhibit other phosphatases and protein kinases. They establish that okadaic acid prevents the binding of microcystin-LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PP1. They speculate that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin-LR. Deng et al. (2008) study the removal of microcystins in a paper with 1122 citations. They synthesize superparamagnetic microspheres with a Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell with accessible large pores and excellent magnetic property and achieve a fast removal of microcystins with high efficiency. Jochimsen et al. (1998) report a case study of the exposure to microcystins at a hemodialysis center in Brazil where 50 patients died of acute liver failure in 1996 in a paper with 711 citations. They find that the water supplied to this hemodialysis center was not treated, filtered, and chlorinated. Microcystins were detected in water from the reservoir and from dialysis center as well as in serum and liver tissue of the patients. Nishiwaki-Matsushima et al. (1992) study the liver tumor promotion in animals by microcystins (Microcystin-LR) in a paper with 565 citations. They show that microcystin-LR is a liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. Dawson (1998) reviews toxicology of microcystins (Microcystin-LR) in a paper with 553 citations. He notes that this toxin causes massive hepatic hemorrhage in the liver through the inhibition of protein phosphatases 1 and 2A. Tillett et al. (2000) outline the structural organization of microcystin biosynthesis in Microcystis based on the characterization of biosynthetic gene cluster of this toxin from Microcystis in a paper with 523 citations. They conclude that the enzymatic organization of this toxin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a β-amino-pentaketide precursor and the formation of β- and γ-carboxyl-peptide bonds, respectively. Cyanobacterial toxins in general Carmichael (1992) reviews cyanotoxins with a focus on microcystins and nodularin in a paper with 909 citations. Codd et al. (2005) review the epidemiology and general properties of cyanobacterial toxins with a focus on the human health risk management in a paper with 612 citations. Finally, Carmichael (1994) reviews cyanotoxins in general in a paper with 583 citations. Harmful algal blooms Hallegraeff (1993) reviews harmful algal blooms in a paper with 1430 citations. He notes that algal blooms can have detrimental consequences to aquaculture, fisheries and tourism, with major economic, environmental and human health problems. For example, they discolor the sea and produce toxins that can pass to humans through the digestion of fish and shellfish. Anderson et al. (2002) review harmful algal blooms and eutrophication of coastal waters focusing on sources of nutrients, effects of nutrient loading and reduction in a paper with 988 citations. They note the strong correlations between total phosphorus inputs and phytoplankton production in freshwaters, and between total nitrogen input and phytoplankton production in estuarine and marine waters. They confirm that there is a linear relationship between nutrient loading and the development of large biomass blooms. Although eutrophication is one of several mechanisms by which harmful algae grow, it is not the only explanation for harmful blooms or toxic outbreaks. Smayda (1997) reviews harmful algal blooms in a paper with 648 citations. He notes that harmful dinoflagellates show significant ecophysiological differences such as greater biophysical vulnerability to turbulence, greater bloom dependence



The scientometric analysis of the research on the algal toxicology Chapter | 33  535

on water-mass stratification, greater nutritional diversity involving mixotrophic tendencies, greater potential use of allelochemical mechanisms in interspecific competition and antipredation defenses, and unique behavioral consequences of their motility compared to diatoms. Heisler et al. (2008) present a consensus statement on the relationship between eutrophication and harmful algal blooms in a paper with 617 citations. They note that the degraded water quality from increased nutrient pollution promotes the development and persistence of many harmful algal blooms and is one of the reasons for their expansion. Landsberg (2002) reviews the impact of harmful algal blooms on aquatic organisms in a paper with 609 citations. O'Neil et  al. (2012) review harmful cyanobacterial blooms with a focus on their determinants, eutrophication, and climate change, in a paper with 553 citations. They examine the relationships between eutrophication, climate change and these blooms and marine ecosystems. They conclude that while the interactive effects of eutrophication and climate change on these blooms are complex, these processes are likely to enhance the magnitude and frequency of these blooms.

33.4 Discussion As there have been over 18,000 papers related to the algal toxicology, comprising more than 10% of the papers on the algae as a whole, it can be argued that this field has gained the public importance in recent years corresponding to the increasing public concerns about the toxicity of the algae to both humans and animals over time. The primary mode of scientific communication has been articles while reviews have formed 3.5% of the sample. The primary index has been SCI-E indexing more than 99.5% of the papers. The most-prolific keywords related to the algal toxicology have been determined through the detailed examination of the papers with at least 100 citations. A detailed keyword set has been devised for the search and the hit rate of this keyword set has been significant with a minimum level of the unrelated records. It has been found that the first keyword set directly related to the algal toxicology has secured nearly two-thirds of the papers. The second keyword set has additionally secured the remaining one-third of the papers. It has been found that the most-prolific keywords have been ‘microcystin*, “okadaic acid”, red tide, phycotoxin*, “harmful algal bloom*”, azaspiracid, brevetoxin*, ciguatera, ciquatoxin*, “harmful dinoflagellate*”, maitotoxin*, palytoxin*, pectenotoxin*, saitotoxin*, “shellfish poison*”, “shellfish toxin*”, and yessotoxin*, anatoxin*, BMAA, cyanotoxin*, cylindrospermopsin*, “harmful cyanobacteria”, “β-methylamino-l-alanine”, nodularin, saitotoxin, teleocidin, and “domoic acid”’. These keywords have formed the primary research fronts for the algal toxicology. The findings show that although over 36,000 authors have contributed to the research, 20 most-prolific authors have shaped the literature on the algal toxicology publishing 12.6% of the papers (Table 33.1). The success of these authors and their institutions and countries could be explained by the ‘first-mover advantage’ paradigm (Lieberman and Montgomery, 1988). The data provides the evidence for the presence of the significant gender deficit among both the lead authors and the authors of the citation classics (Bordons et al., 2003). The data on the papers by the most-prolific authors highlight the primary research areas as ‘cyanotoxins, dinotoxins, and harmful algal blooms’. The data in Table 33.1 provides information on the most-prolific authors, institutions, countries, journals, topics, and their citation impact in terms of the I-100 and I-100% by these authors. The data shows that although 147 countries contributed to the research in algal toxicology, most-prolific 10 countries contributed to 83% of the papers (Table 33.2). The major producers of the research have been the United States, China, Japan, and Europe as these countries have had the ‘first-mover advantage’ over the other countries. As in the case of countries, although over 6800 institutions have contributed to the research in algal toxicology, the 10 most-prolific institutions from the United States, China, Japan, and Europe, having the first-mover advantages, have published more than 20% of the papers (Table 33.3). As over 51% of the papers have declared a research funding, the role of the incentive structures for the development of the research in this field has been significant (Abramo et al., 2009). The steep rise in the number of the publications in this field over the study period provides the strong evidence for the increasing public importance of the algal toxicology over time (Fig. 33.1). The annual number of publications have risen to nearly 1000 papers and it is expected that it would continue to rise in the incoming years and decades corresponding to the increasing importance of the algae to the global society at large. As the algae-related journals have published only over 10% of the papers, the broad search strategy, covering all the journals indexed by the databases, developed for this study, has been justified (Table 33.4). The data on the Web of Science subject categories suggest that the first four subjects, covering over 80% of the papers, have formed the scientific basis of the research in this field: ‘Toxicology, Environmental Sciences, Marine and Freshwater Biology, and Pharmacology and Pharmacy’ (Table 33.5).

536  PART | IX  Algal toxicology

These data also show the lack of the emphasis on the societal and humanitarian aspects of the research on the algal toxicology as 99.5% of the papers have been indexed the SCI-E. It is expected that the research in these interdisciplinary areas would be conducted in the coming years and decades as in the case of research on the nanoscience and nanotechnology and other emerging technologies. The data on the research fronts have confirmed that the major research fronts have been ‘cyanobacterial toxins, dinoflagellate toxins, diatom toxins, and harmful algal blooms’ (Table 33.6). Similarly, on the basis of toxins, ‘microcystins’ have been the most-prolific toxins with 29% of the papers. The other prolific research fronts have been ‘cyanotoxins in general, okadaic acid, cylindrospermopsins, other dinoflagellate toxins, and domoic acid’. The extensive data on the 20 citation classics largely confirm the findings of the earlier sections based on the 18,235 papers (Table 33.7). There has been significant overlap between these two samples with regard to the prolific authors, institutions, keywords, research fronts, institutions, countries, and subject categories. However, it is noted that the reviews have been largely over-represented in the citation classic sample (50% vs 4%). The brief overview of the content of these classical papers have provided important clues on the primary scientific issues in this field over time between 1980 and 2018. The primary focus of these papers has been the toxicity of the algae to the humans and animals. It appears that the structure-processing-property relationships form the basis of the research in algal toxicology as in other hard sciences but most specifically in materials science and engineering (Konur and Matthews, 1989; Mishra et al., 2009; Scherf and List, 2002).

33.5 Conclusion This first-ever analytical study of the research in algal toxicology at the global scale covering the whole range of toxins and harmful algal blooms has provided the ample data for the primary stakeholders for devising efficient set of incentive structures for the development of the research and practice in this field. Thus, the scientometric analysis as an analytical tool has a great potential to gain valuable insights into the evolution of the research the in this field as in the case of new emerging technologies and processes such as nanoscience and nanotechnology, complementing literature reviews. The data have shown that the annual number of papers in this field has risen to nearly 1000 papers while there have been over 18,000 papers over the study period from 1980 to 2018. It is further expected that the size of the research output would continue to increase in the incoming years and decades, corresponding to the increasing public importance of the algae to the global society at large. The key research fronts have been the ‘cyanobacterial toxins (especially microcystins), dinoflagellate toxins (especially okadaic acid), diatom toxins (especially domoic acid), and harmful algal blooms’ producing these toxins. The focus of the research has been on the impact of these toxins and blooms on the humans and animals. As in the case of the most academic fields such as bioenergy or nanoscience and nanotechnology, limited number of authors and their institutions and countries, having the first-mover advantage, have effectively shaped the literature in this field through the significant citation impact of their studies. It has been found that the detailed keyword set provided in the appendix to the paper has been effective to locate the core literature in this field with a relatively significant hit rate. It is recommended that the further scientometric studies should be carried in this field focusing on major research fronts such as ‘cyanobacterial toxins, dinoflagellate toxins, diatoms toxins, and harmful algal blooms’.

Appendix. The keyword sets A.1  Algal toxicology direct keywords Algal toxins in general: TI=(phycotoxin* or ‘harmful algal bloom*’) OR SO=(‘Harmful Algae’). Dinoflagellate toxins: TI=(azaspiracid* or brevetoxicosis or brevetoxin* or *ciguatera or *ciguatoxin* or cooliatoxin* or dinophysistoxin* or dinophytoxin* or gambiertoxin* or gambierol or gonyautoxin* or gymnodimine or ‘harmful dinoflagellate*’ or karlotoxin* or maitotoxin* or ‘okadaic acid*’ or ostreotoxin* or palytoxin* or *pectenotoxin* or prymnesin* or ‘red tide’ or saxitoxin* or ‘shellfish poison*’ or ‘shellfish toxin*’ or *yessotoxin* or zooxanthellatoxin). Cyanotoxins: TI=(antillatoxin* or *anatoxin* or *aplysiatoxin* or apratoxin* or aeruginosin* or ‘β-methylamino-lalanine*’ or bmaa or cyanoginosin* or cyanohab* or cyanotoxin* or *cylindrospermopsin* or ‘harmful cyanobacteria*’ or ‘l-arginyl-poly-l-aspartate*’ or lyngbyabellin* or lyngbyatoxin* or *microcystin* or microviridin or nodularin* or *oscillatoxin* or *saxitoxin* or *teleocidin* or tolytoxin*).

The scientometric analysis of the research on the algal toxicology Chapter | 33  537



Diatom toxins: TI=(‘domoic acid*’ or domoate).

A.2  Combined keyword sets A.2.1 Toxicology TI=(*toxin* or *toxic*) OR SO=(‘aquatic toxicology’ or ‘archives of environmental contamination and toxicology’ or ‘archives of toxicology’ or ‘bulletin of environmental contamination and toxicology’ or ‘cardiovascular toxicology’ or ‘chemical research in toxicology’ or ‘clinical toxicology’ or ‘critical reviews in toxicology’ or ‘cutaneous and ocular toxicology’ or ecotoxicology* or ‘environmental toxicology*’ or ‘forensic toxicology’ or ‘human & experimental toxicology’ or ‘inhalation toxicology’ or ‘international journal of toxicology’ or ‘journal of analytical toxicology’ or ‘journal of applied toxicology’ or ‘journal of biochemical and molecular toxicology’ or ‘journal of environmental science and health part cenvironmental carcinogenesis & ecotoxicology reviews’ or ‘journal of immunotoxicology’ or ‘journal of toxicolog*’ or ‘molecular & cellular toxicology’ or nanotoxicology or neurotoxicology* or ‘particle and fiber toxicology’ or ‘reviews of environmental contamination and toxicology’ or toxicolog* or toxicon or toxin*).

A.2.2 Algae A.2.2.1  Algal general TI=(alga or algae or algal or phycolog* or algicid* or periphyton*) OR SO=(Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘Cryptogamie Algologie’ or ‘European Journal of Phycology’ or Fottea* or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or ‘Phycologia’ or ‘Phycological Research’). A.2.2.2  Phytoplankton, dinoflagellates, coccolithophores TI=(chrysophycea* or chlorococcales or chrysophyt* or *coccolith* or dinocyst* or dinoflagell* or dinophycea* or dinophyt* or haptophyt* or peridiniales or *phytoplankton* or prymnesiophycea* or raphidophycea* or raphidophyt* or zooxanthella* or Acropora or Akashiwo or Alexandrium or Amphidinium or Aureococcus or *Ceratium or *Chattonella or Cochlodinium or Crypthecodinium or Dinophysis or Emiliania or Gambierdiscus or *Gonyaulax or *Gymnodinium or Gyrodinium or Hematodinium or Heterocapsa or Heterosigma or Isochrysis or Karenia* or Karlodinium or Mallomonas or Noctiluca or Ochromonas or Ostreopsis or Oxyrrhis or Peridinium or Pfiesteria or Phaeocystis or Prorocentrum or Prymnesium or Scrippsiella or Symbiodinium or Vaucheria). A.2.2.3 Microalgae TI=(chlorophycea* or chlorophyt* or cryptomonad* or cryptophycea* or euglenophycea* or eustigmatophycea* or ‘green alga*’ or microalga* or ‘micro-alga*’ or ‘micro alga*’ or prasinophycea* or streptophyt* or trebouxiophycea* or volvocales or Acetabularia or Ankistrodesmus or Asteromonas or Aurantiochytrium or Botryococcus or Chaetoceros or Chlamydomonas or *Chlorella or *Chlorococcum or Coccomyxa or Cyanidioschyzon or Cyanidium or Desmodesmus or Dunaliella or Euglena or Galdieria or Haematococcus or Micrasterias or Micromonas or Monoraphidium or Nannochloropsis or Neochloris or Ostreococcus or Pediastrum or Phormidium or Platymonas or Polytomella or Porphyridium or Prototheca or Pseudokirchneriella or Pyramimonas or Scenedesmus or Schizochytrium or Selenastrum or Tetraselmis or Trebouxia or Volvox). A.2.2.4 Macroalgae TI=(agarophyt* or ‘brown alga*’ or carrageenan* or characea* or charophyt* or cladophorales or cryptonemiales or dictyotales or florideophycea* or fucale* or fucoid* or gelidiales or gigartinale* or gracilariales or kelp* or laminariale* or macroalga* or ‘macro-alga*’ or ‘macro alga*’ or phaeophycea* or phaeophyt* or ‘red alga*’ or rhodophycea* or rhodophyt* or seaweed* or ‘sulfated polysaccharide*’ or ulvale* or ulvan* or ulvophycea* or zygnematophycea* or ‘Chara Vulgaris’ or Alaria or Ascophyllum or Asparagopsis or Asparagopsis or Bangia* or Bostrychia or Bryopsis or Catenella or Caulerpa or Ceramium or Chaetomorpha or Chondrus or Cladophora or Codium or Corallina or Cystoseira or Ecklonia or Ectocarpus or ‘Eisenia bicyclis’ or Enteromorpha or Eucheuma or Fucus or Gelidium or Gigartina* or Gracilaria or Gracilariopsis or Grateloupia or Halimeda or Halocynthia or Hizikia or Hypnea or Kappaphycus or Laminaria or Laurencia* or Lessonia or Lomentaria or Macrocystis or Monostroma or Mougeotia or Nitella or Nitellopsis or Oedogonium or Padina or Palmaria or

538  PART | IX  Algal toxicology

Pelvetia or Plocamium or Polysiphonia or Porphyra or Pyropia or Saccharina or Sargassum or Scytosiphon or Solieria or Spirogyra or Turbinaria or Ulva or Undaria). A.2.2.5 Diatoms TI=(bacillariophycea* or bacillariophyt* or diatom or diatoms or Asterionella or Chaetoceros or Coscinodiscus or Cyclotella or Cylindrotheca or Didymosphenia or Navicula or *Nitzschia or Phaeodactylum or Skeletonema or Stephanodiscus or Thalassiosira) OR SO=(‘Diatom Research’). A.2.2.6 Cyanobacteria TI=(‘blue green alga*’ or ‘blue-green alga*’ or *cyanobacter* or cyanophage* or cyanophyt* or cyanophycea* or nostocales or oscillatoriales or Acaryochloris or *Anabaena or Anacystis or Aphanizomenon or Aphanothece or Arthrospira or Calothrix or Cyanophora or Cyanothece or Cylindrospermopsis or *Lyngbya* or Mastigocladus or Microcoleus or *Microcystis or Moorea or Nodularia or *Nostoc or Oscillatoria or Planktothrix or Plectonema or Prochlorococcus or Prochloron or Prochlorothrix or Scytonema or Spirulina or *Synechococcus or Synechocystis or Tolypothrix or Trichodesmium). A.2.2.7 Journals SO=(Algae or ‘Algal Research*’ or ‘British Phycological Journal’ or ‘Cryptogamie Algologie’ or ‘Diatom Research’ or ‘European Journal of Phycology’ or Fottea* or ‘Harmful Algae’ or ‘Journal of Applied Phycology’ or ‘Journal of Phycology’ or Phycologia or ‘Phycological Research’).

References Abramo, G., D’Angelo, C.A., Caprasecca, A., 2009. Allocative efficiency in public research funding: can bibliometrics help? Res. Policy 38 (1), 206–215. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25 (4B), 704–726. Bialojan, C., Takai, A., 1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases—specificity and kinetics. Biochem. J. 256 (1), 283–290. Bordons, M., Morillo, F., Fernandez, M.T., Gomez, I., 2003. One step further in the production of bibliometric indicators at the micro level: differences by gender and professional category of scientists. Scientometrics 57 (2), 159–173. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and coproducts. Renew. Sustain. Energy Rev. 14 (2), 557–577. Carmichael, W.W., 1992. Cyanobacteria secondary metabolites—the cyanotoxins. J. Appl. Bacteriol. 72 (6), 445–459. Carmichael, W.W., 1994. The toxins of cyanobacteria. Sci. Am. 270 (1), 78–86. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Codd, G.A., Morrison, L.F., Metcalf, J.S., 2005. Cyanobacterial toxins: risk management for health protection. Toxicol. Appl. Pharmacol. 203 (3), 264–272. Cohen, P., Holmes, C.F.B., Tsukitani, Y., 1990. Okadaic acid: a new probe for the study of cellular-regulation. Trends Biochem. Sci. 15 (3), 98–102. Dawson, R.M., 1998. The toxicology of microcystins. Toxicon 36 (7), 953–962. Deng, Y.H., Qi, D.W., Deng, C.H., Zhang, X.M., Zhao, D.Y., 2008. Superparamagnetic high-magnetization microspheres with an Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell for removal of microcystins. J. Am. Chem. Soc. 130 (1), 28–29. Garfield, E., 1972. Citation analysis as a tool in journal evaluation. Science 178 (4060), 471–479. Garfield, E., 2006. Citation indexes for science. A new dimension in documentation through association of ideas. Int. J. Epidemiol. 35 (5), 1123–1127. Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32 (2), 79–99. Haystead, T.A.J., Sim, A.T.R., Carling, D., Honnor, R.C., Tsukitani, Y., Cohen, P., et al., 1989. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature 337 (6202), 78–81. Heisler, J., Glibert, P.M., Burkholder, J.M., Anderson, D.M., Cochlan, W., Dennison, W.C., et al., 2008. Eutrophication and harmful algal blooms: a scientific consensus. Harmful Algae 8 (1), 3–13. Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S., 2011. Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnol. Adv. 29 (2), 189–198. Ishihara, H., Martin, B.L., Brautigan, D.L., Karaki, H., Ozaki, H., Kato, Y., et al., 1989. Calyculin-A and okadaic acid: inhibitors of protein phosphataseactivity. Biochem. Biophys. Res. Commun. 159 (3), 871–877. Jochimsen, E.M., Carmichael, W.W., An, J.S., Cardo, D.M., Cookson, S.T., Holmes, C.E.M., et al., 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N. Engl. J. Med. 338 (13), 873–878. Konur, O., 2000. Creating enforceable civil rights for disabled students in higher education: an institutional theory perspective. Disabil. Soc. 15 (7), 1041–1063. Konur, O., 2002a. Access to employment by disabled people in the UK: is the disability discrimination act working? Int. J. Discrim. Law 5 (4), 247–279. Konur, O., 2002b. Access to nursing education by disabled students: rights and duties of nursing programs. Nurse Educ. Today 22 (5), 364–374.



The scientometric analysis of the research on the algal toxicology Chapter | 33  539

Konur, O., 2002c. Assessment of disabled students in higher education: current public policy issues. Assess. Eval. High. Educ. 27 (2), 131–152. Konur, O., 2004. Disability and racial discrimination in employment in higher education. In: Law, I., Phillips, D., Turney, L. (Eds.), Institutional Racism in Higher Education. Trentham Books, Stoke on Trent, pp. 83–92. Konur, O., 2006a. Participation of children with dyslexia in compulsory education: current public policy issues. Dyslexia 12 (1), 51–67. Konur, O., 2006b. Teaching disabled students in higher education. Teach. High. Educ. 11 (3), 351–363. Konur, O., 2007a. A judicial outcome analysis of the disability discrimination act: a windfall for the employers? Disabil. Soc. 22 (2), 187–204. Konur, O., 2007b. Computer-assisted teaching and assessment of disabled students in higher education: the interface between academic standards and disability rights. J. Comput. Assist. Learn. 23 (3), 207–219. Konur, O., 2011. The scientometric evaluation of the research on the algae and bio-energy. Appl. Energy 88 (10), 3532–3540. Konur, O., 2012a. The policies and practices for the academic assessment of blind students in higher education and professions. Energ. Educ. Sci. Technol. B 4 (si1), 240–244. Konur, O., 2012b. Prof. Dr. Ayhan Demirbas’ scientometric biography. Energ. Educ. Sci. Technol. A 28 (2), 727–738. Konur, O., 2012c. The evaluation of the biogas research: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (2), 1277–1292. Konur, O., 2012d. The evaluation of the bio-oil research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 379–392. Konur, O., 2012e. The evaluation of the biorefinery research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (si1), 347–358. Konur, O., 2012f. The evaluation of the research on the biodiesel: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1003–1014. Konur, O., 2012g. The evaluation of the research on the bioethanol: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 1051–1064. Konur, O., 2012h. The evaluation of the research on the biofuels: a scientometric approach. Energ. Educ. Sci. Technol. A 28 (2), 903–916. Konur, O., 2012i. The evaluation of the research on the biohydrogen: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 323–338. Konur, O., 2012j. The evaluation of the research on the microbial fuel cells: a scientometric approach. Energ. Educ. Sci. Technol. A 29 (1), 309–322. Konur, O., 2012k. The scientometric evaluation of the research on the production of bioenergy from biomass. Biomass Bioenergy 47, 504–515. Konur, O., 2012o. Evaluation of the research on the social sciences in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1893–1908. Konur, O., 2012p. The evaluation of the research on the arts and humanities in Turkey: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (3), 1603–1618. Konur, O., 2012q. The evaluation of the educational research: a scientometric approach. Energ. Educ. Sci. Technol. B 4 (4), 1935–1948. Konur, O., 2012r. The scientometric evaluation of the research on the deaf students in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1573–1588. Konur, O., 2012s. The scientometric evaluation of the research on the students with ADHD in higher education. Energ. Educ. Sci. Technol. B 4 (3), 1547–1562. Konur, O., 2012t. The research on the attitudes toward disabled people in the educational settings: a scientometric evaluation. Energ. Educ. Sci. Technol. B 4 (si1), 250–257. Konur, O., 2015a. Algal biosorption of heavy metals from wastes. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 597–625. Konur, O., 2015b. Algal economics and optimization. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 691–716. Konur, O., 2015c. Algal high-value consumer products. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 653–681. Konur, O., 2015d. Algal photobioreactors. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 81–107. Konur, O., 2015e. Algal photosynthesis, biosorption, biotechnology, and biofuels. In: Kim, S.K. (Ed.), Springer Handbook of Marine Biotechnology. Springer, New York, NY, pp. 1131–1161. Konur, O., 2015f. Current state of research on algal biodiesel. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 487–512. Konur, O., 2015g. Current state of research on algal bioelectricity and algal microbial fuel cells. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 527–555. Konur, O., 2015h. Current state of research on algal bioethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 217–244. Konur, O., 2015i. Current state of research on algal biohydrogen. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 393–421. Konur, O., 2015j. Current state of research on algal biomethane. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 273–301. Konur, O., 2015k. Current state of research on algal biomethanol. In: Kim, S.K., Lee, C.G. (Eds.), Marine Bioenergy: Trends and Developments. CRC Press, Boca Raton, FL, pp. 327–369. Konur, O., 2016a. Algal omics: the most-cited papers. In: Kim, S.K. (Ed.), Marine Omics: Principles and Applications. CRC Press, Boca Raton, FL, pp. 9–34. Konur, O., 2016b. Scientometric overview in nanobiodrugs. In: Holban, A.M., Grumezescu, A.M. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 405–428. Konur, O., 2016c. Scientometric overview regarding nanoemulsions used in the food industry. In: Grumezescu, A.M. (Ed.), Emulsions. Elsevier, Amsterdam, pp. 689–711. Konur, O., 2016d. Scientometric overview regarding the nanobiomaterials in antimicrobial therapy. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Antimicrobial Therapy. Elsevier, Amsterdam, pp. 511–535. Konur, O., 2016e. Scientometric overview regarding the nanobiomaterials in dentistry. In: Grumezescu, A.M. (Ed.), Nanobiomaterials in Dentistry. Elsevier, Amsterdam, pp. 425–453.

540  PART | IX  Algal toxicology

Konur, O., 2016f. Scientometric overview regarding the surface chemistry of nanobiomaterials. In: Grumezescu, A.M. (Ed.), Surface Chemistry of Nanobiomaterials. Elsevier, Amsterdam, pp. 463–486. Konur, O., 2016g. The scientometric overview in cancer targeting. In: Holban, A.M., Grumezescu, A. (Eds.), Nanoarchitectonics for Smart Delivery and Drug Targeting. Elsevier, Amsterdam, pp. 871–895. Konur, O., 2016h. Glycoscience: the current state of the research. In: Kim, S.K. (Ed.), Marine Glycobiology: Principles and Applications. CRC Press, Boca Raton, FL, pp. 7–21. Konur, O., 2017a. The top citation classics in alginates for biomedicine. In: Venkatesan, J., Anil, S., Kim, S.K. (Eds.), Seaweed Polysaccharides: Isolation, Biological and Biomedical Applications. Elsevier, Amsterdam, pp. 223–249. Konur, O., 2017b. Recent citation classics in antimicrobial nanobiomaterials. In: Ficai, A., Grumezescu, A.M. (Eds.), Nanostructures for Antimicrobial Therapy. Elsevier, Amsterdam, pp. 669–685. Konur, O., 2017c. Scientometric overview in nanopesticides. In: Grumezescu, A.M. (Ed.), New Pesticides and Soil Sensors. Elsevier, Amsterdam, pp. 719–744. Konur, O., 2017d. Scientometric overview regarding oral cancer nanomedicine. In: Andronescu, E., Grumezescu, A.M. (Eds.), Nanostructures for Oral Medicine. Elsevier, Amsterdam, pp. 939–962. Konur, O., 2017e. Scientometric overview regarding water nanopurification. In: Grumezescu, A.M. (Ed.), Water Purification. Elsevier, Amsterdam, pp. 693–716. Konur, O., 2017f. Scientometric overview in food nanopreservation. In: Grumezescu, A.M. (Ed.), Food Preservation. Elsevier, Amsterdam, pp. 703–729. Konur, O., 2018a. Bioenergy and biofuels science and technology: scientometric overview and citation classics. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. 3–63. Konur, O. (Ed.), 2018b. Bioenergy and Biofuels. CRC Press, Boca Raton, FL. Konur, O., 2018c. Preface. In: Konur, O. (Ed.), Bioenergy and Biofuels. CRC Press, Boca Raton, FL, pp. ix–xx. Konur, O., 2018d. Scientometric evaluation of the global research in spine: an update on the pioneering study by Wei et al. Eur. Spine J. 27 (3), 525–529. Konur, O., 2019a. Cyanobacterial bioenergy and biofuels science and technology: a scientometric overview. In: Mishra, A.K., Tiwari, D.N., Rai, A.N. (Eds.), Cyanobacteria: From Basic Science to Applications. Elsevier, Amsterdam, pp. 419–442. Konur, O., 2019b. Nanotechnology applications in food: a scientometric overview. In: Pudake, R.N., Chauhan, N., Kole, C. (Eds.), Nanoscience for Sustainable Agriculture. Springer International Publishing, Cham. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., Matthews, F.L., 1989. Effect of the properties of the constituents on the fatigue performance of composites: a review. Composites 20 (4), 317–328. Landsberg, J.H., 2002. The effects of harmful algal blooms on aquatic organisms. Rev. Fish. Sci. 10 (2), 113–390. Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37 (1), 106–126. Lieberman, M.B., Montgomery, D.B., 1988. First‐mover advantages. Strateg. Manag. J. 9 (S1), 41–58. MacKintosh, C., Beattie, K.A., Klumpp, S., Cohen, P., Codd, G.A., 1990. Cyanobacterial Microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 264 (2), 187–192. Merel, S., Villarin, M.C., Chung, K., Snyder, S., 2013. Spatial and thematic distribution of research on cyanotoxins. Toxicon 76, 118–131.



The scientometric analysis of the research on the algal toxicology Chapter | 33  541

Mishra, A., Fischer, M.K., Bauerle, P., 2009. Metal-free organic dyes for dye-sensitized solar cells: from structure: property relationships to design rules. Angew. Chem. Int. Ed. 48 (14), 2474–2499. Nishiwaki-Matsushima, R., Ohta, T., Nishiwaki, S., Suganuma, M., Kohyama, K., Ishikawa, T., et al., 1992. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res. Clin. Oncol. 118 (6), 420–424. North, D.C., 1991. Institutions. J. Econ. Perspect. 5 (1), 97–112. North, D.C., 1994. Economic performance through time. Am. Econ. Rev. 84 (3), 359–368. O'Neil, J.M., Davis, T.W., Burford, M.A., Gobler, C.J., 2012. The rise of harmful cyanobacteria blooms: the potential roles of eutrophication and climate change. Harmful Algae 14, 313–334. Rowley, J.A., Madlambayan, G., Mooney, D.J., 1999. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20 (1), 45–53. Scherf, U., List, E.J., 2002. Semiconducting polyfluorenes—towards reliable structure-property relationships. Adv. Mater. 14 (7), 477–487. Smayda, T.J., 1997. Harmful algal blooms: their ecophysiology and general relevance to phytoplankton blooms in the sea. Limnol. Oceanogr. 42 (5), 1137–1153. Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., et al., 1988. Okadaic acid: an additional non-phorbol-12-tetradecanoate13-acetate-type tumor promoter. Proc. Natl. Acad. Sci. USA 85 (6), 1768–1771. Tillett, D., Dittmann, E., Erhard, M., von Dohren, H., Borner, T., Neilan, B.A., 2000. Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chem. Biol. 7 (10), 753–764. Wang, B., Li, Y.Q., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Wang, Y., Hou, S.W., Ke, F., Gao, H., 2015. Bibliometric analysis of research on microcystins in China and worldwide from 1991 to 2011. Desalin. Water Treat. 53 (1), 272–283.

Further reading Konur, O., 2012l. The evaluation of the global energy and fuels research: a scientometric approach. Energ. Educ. Sci. Technol. A 30 (1), 613–628. Konur, O., 2012m. 100 citation classics in energy and fuels. Energ. Educ. Sci. Technol. A 30 (si1), 319–332. Konur, O., 2012n. What have we learned from the citation classics in energy and fuels: a mixed study. Energ. Educ. Sci. Technol. A 30 (si1), 255–268. Konur, O., 2015l. The review of citation classics on the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 490–526. Konur, O., 2015m. The scientometric study of the global energy research. In: Prasad, R., Sivakumar, S., Sharma, U.C. (Eds.), Energy Science and Technology. V. 1. Opportunities and Challenges. Studium Press LLC, Houston, TX, pp. 475–489.

Chapter 34

Toxic effects of harmful algal blooms on finfish and shellfish Yukihiko Matsuyamaa, Tatsuya Odab a

Fisheries Research and Education Agency, Nagasaki, Japan, bNagasaki University, Nagasaki, Japan

34.1 Introduction Increased incidence of ‘harmful algal blooms’ (HABs) and the associated death of life and toxins contamination have hampered the development of finfish and shellfish aquaculture last half century (Hallegraeff, 1993; Anderson, 1994; Honjo, 1994; Shumway, 1990; Matsuyama and Shumway, 2009). The bloom of certain algal species occasionally attains concentrations of several million cells per liter, and may form visible patches on the sea surface commonly referred to as ‘red tides’ or harmful algal blooms (HABs). Some algal species produce natural toxins (here after terms ‘phycotoxins’) which pose a threats to humans or are detrimental to aquatic organisms. While the term ‘red tide’ had been used historically and generally to describe these phenomena, recent studies now refer to the phenomena as ‘harmful algal blooms’ (HABs) which have been associated with various types of incidence of large-scale mortality of aquatic organisms (Shumway, 1990; Landsberg, 2002; Matsuyama and Shumway, 2009) and shellfish poisonings (Hallegraeff, 1993; Smayda and Villareal, 1989; Shumway, 1990; Matsuyama and Shumway, 2009; Landsberg, 2002; Montes et al., 2018). Physical damage directly caused by HABs and phycotoxin production have become a marked increasing social and industrial concern for marine products during the last quarter century (Anderson, 1994; Hallegraeff, 1993; Okaichi, 2004; Smayda and Villareal, 1989; Shumway, 1990). Some attribute the apparent increased incidence to an increased awareness, number of observers, or prompt dissemination of information worldwide, but almost all scientists now agree that there is a very real increase in the frequency, intensity, duration, and geographic spreading of the blooms (Matsuyama and Shumway, 2009). The increased frequency has resulted in public health hazards, economical losses, and destruction of aquatic ecosystem globally. Numerous factors have been suggested as causes of these increases in HABs and are briefly discussed below—this is not meant to be an all-inclusive review, but rather a primer for the finfish and shellfish aquaculturists. While all of these factors have merit for some HAB species, there is no one cause to which we can attribute either the individual HABs or their global proliferation. Almost all toxic microalgal species are autotrophic, unicellular microbes, and their growth is affected by numerous factors including water temperature, salinity, light intensity, coastal current and stratification, fresh water runoff, concentration and/or ratio of nutrients, grazing by zooplankters, competition with other phytoplankton species, and presence or absence of algicidal microbe. Drastic changes in these factors driven by the global warming (Hallegraeff, 1993), El Nino (LeonMunoz et al., 2018), eutrophication as a result of increased utilization of coastal areas (Anderson et al., 2002; Okaichi, 2004), dispersal of toxic algal species by ships’ ballast water (Hallegraeff, 1998), dams (Humborg et al., 1997), and acid rain (Graneli and Haraldsson, 1993), all impact HAB species. Urbanization and concurrent increase of sewage effluents, industrial and agricultural waste can stimulate toxic algal blooms (Hallegraeff, 1993; Okaichi, 2004; Smayda and Villareal, 1989; Matsuyama and Shumway, 2009). In the last half century, finfish and shellfish cultivations have expanded in many coastal areas, and these may have contributed to an increased awareness of toxic algal species (Rensel and Whyte, 2004) that were commonly recognized as hidden flora in indigenous phytoplankton communities (Smayda and Villareal, 1989). There is increasing evidence that toxic algal species are being transported to new areas via ships’ ballast water or through infected shellfish species (Hallegraeff, 1998) and with shellfish transportation (Scholin et al., 1995; Honjo et al., 1998; Hegaret et al., 2007; Matsuyama et al., 2010) or large-amount unfiltered seawater due to live fish transportation (Matsuyama et  al., 2010). Thus, the HAB impacts are related to the development of aquaculture industry, and the effects on fish and shellfish will continue in future. There is an excellent review on the ecophysiological impacts of HABs Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00034-6 © 2020 Elsevier Inc. All rights reserved.

543

544  PART | IX  Algal toxicology

­ entioned above. In this paper, we summarize the impact of HABs with special emphasis on death of finfish and shellfish m life, and discuss the mechanism of toxic occurrences in their physiology.

34.2  Impact of HAB species on finfish and shellfish industries Blooms of toxic algal species are common occurrences in finfish and shellfish culture areas worldwide, and pose various threats to public health and aquaculture damages. HABs showed adverse effects not only on cultivated fish and bivalves, but also on higher trophic organisms such as birds and marine mammals (Landsberg, 2002), various invertebrates, their predators such as zooplankton and protozoan (Tillmann et al., 2007), the same phytoplankters (competitor, e.g., Uchida et al., 1999). HABs have wide and varied effects on marine ecosystems. In general, due to great concern for commercially important cultured species, this paper will focus mainly on the effects on fish and shellfish (bivalves and gastropods).

34.2.1  Fish-killing species and its mechanism of detrimental effects In the past half-century, HABs has adversely affected various aquatic animal life. Among them, finfish are one of the most affected marine organisms. Since the 1970’s, the development of fish farming industry has especially been remarkable, and the production of fish aquaculture is increasing all over the world (FAO, 2016). In 2014, harvested amounts from finfish aquaculture accounted for 49.8 million tons (FAO, 2016). Large-scale incidences of cultured fish death due to highly ichthyotoxic HABs occurs mainly in intensive farming locations (Canada, Chili, Japan, Korea, Norway, USA, etc.). Among them, the mass mortality in yellowtail (14 millions dying) by raphidophyte Chattonella antiqua occurred in the Seto Inland Sea in 1971 (Okaichi, 1989), massive fish kills in the Pamlico and Neuse Estuaries of the southeastern United States (Burkholder et al., 1992), and in cultured salmon (27 million dying) by Peudochattonella cf. verruculosa and Alexandrium catenella in Patagonia, Chile in 2016 (Montes et al., 2018) are recorded as historical and catastrophic episodes. Overall characteristic and mitigation technique for fish-killing HABs are reviewed in Rensel and Whyte (2004). Here, we outline the main HABs species ultimately toxic to fish, and discuss the mechanisms of fish-killing. Fig. 34.1 shows the fish death mechanism exposed to ichthyotoxic HABs arranged as a schematic diagram based on the previous efforts. Regarding the death mechanism of fish by HABs, though various findings have been presented, it has not yet been fully understood. Observation on dying fish affected by HABs showed that adverse effect appears in a short-term period (from several tens of minutes to several hours), that there is less noticeable trauma except for the gills. These results showed that target organs by HABs is mainly gills tissue, and good agreement with damaged gill lost gasexchange function subsequently resulting in suffocation due to rapid decline of blood oxygen concentration (e.g., Ishimatsu et al., 1990). Field observation also showed that slight decrease in environmental oxygen concentration accelerates rapid death of fish exposed to HABs, and it was also found that HABs caused critical gill damage to finfish (Okaichi, 1989). Further, HABs directly injure the gill tissue (Fig. 34.1) and the death of fish is closely related to the cell density of HABs. Fig. 34.2 shows the schematic flow for death of finfish life exposed to harmful algal species. Previous efforts showed various factors causing cytotoxicity to gill tissue: ‘reactive oxygen species’ (ROS) (Oda et  al., 1992), free fatty acid (Okaichi, 1989; Marshall et al., 2003), phycotoxins (Onoue et al., 1990), mucus (Matsusato and Kobayashi, 1974), mechanical irritation and clogging, are presented for the mechanism by which HAB injures the gill of fish. Nevertheless, fine mechanism causing detrimental effect to gill tissue due to HABs remain unclear except for mechanical injure by giant diatoms Chaetoceros concavicornis and C. convoltus (Albright et al., 1993). The adverse effect of HABs on finfish is species-specific, therefore, quantifying of toxicity is complicated. Furthermore, toxic effects of HABs also vary depending on physiological conditions of the fish. Here we outline the portent toxic HABs to finfish.

34.2.1.1 Chattonella Genus Chattonella (C. antiqua, C. marina, C. ovate) is a group of Raphidophyte, described as highly toxic to fish (Honjo, 1994). Cells are ‘naked’ but covered with thin glycoprotein complex (Glycocalyx, see Yokote and Honjo, 1985) and cell lengths are relatively longer (50–100 μm) than other species. Chattonella formed bloom in summer in coastal and enclosed bays, caused mass mortality of wild fish and cultured fish (Honjo, 1994; Edvardsen and Imai, 2006; Aoki et al., 2015). In general, fish death will occur when the cell density of Chattonella exceeds 105–106 cells/L (e.g., Hiroishi et al., 2005; Aoki et al., 2015). Photomicrographs of gills of dead fish (juvenile yellowtails) including secondary lamellae of fish gill exposed to C. antiqua are shown in Fig. 34.1C The discharge of epithelial cells is remarkable, and disappearance of chloride and mucus



Toxicity of harmful algal blooms Chapter | 34  545

FIG.  34.1  Photomicrographs showing representative histopathological alterations in yellowtail Seriola quinqueradiata (juvenile, three month old, body length 73–94 mm) exposed to culture isolates of three fish-killing harmful algae (unpublished data). (A) Negative control, (B) dying individuals due to simple hypoxia (105 cells/ mL) bloom have occurred in the eastern Long Island (Gobler et al., 2008), Chesapeake Bay (Mulholland et al., 2009) in the United States, and in the Arabian Gulf and Gulf of Oman (Richlen et al., 2010) associated with massive damage to natural fish and cultured fish. As shown in Fig 34.1E, the large amount of mucoid-like substances to the rim of secondary gill lamellae of the dead fish is observed, and discharging of epithelial cells is characteristic. Some studies implied ROS production in C. polykrikoides (Kim et al., 1999). However, several works suggested that massive mucus production is responsible for fish mortality (Kim et al., 2000c, 2002; Gobler et al., 2008) although Onoue and Nozawa (1989) found hemolytic and neurotoxic phycotoxin from C. polykrikoides. This species has a large amount of mucous substances in the cell surface layer, and release of mucocyst is also recognized by cell irritation. To date, toxic factors in this species are unclear, but a causal relationship between large amount of mucus production and fish toxicity is greatly suspected.

34.2.1.4 Heterosigma Raphidophyte Heterosigma akashiwo is small (cell size of 10–15 μm), frequent bloom-forming species in the inner bay and near brackish water area (see Edvardsen and Imai, 2006). Although this species frequently forms red tides in the inner bay, the incidence of fishery damage occasionally occurs compared with Chattonella, Karenia, and Cochlodinium. It appears that the adverse effects of this species will vary with geographical localities. The agents causing fish toxicity remain unclear. H. akashiwo is known to be producer of ROS (Kim et al., 2000b; Twiner et al., 2001) that is related to the death of rainbow trout (Yang et al., 1995), while H2O2 produced by this species is not related to fish toxicity (Twiner et al., 2001).

34.2.1.5  Other species In addition to Chattonella, Karenia, Cochlodinium, there are several genera causing fish-kill during the bloom periods. However, it is emphasized that large-scale fish-killing blooms due to the species mentioned below do not occur frequently in worldwide. However, major salmon-growing areas in Chili record catastrophic mortality of cultured salmon (27 million dying) in early 2016 (Garcia-Mendoza et  al., 2018). Causative species is Peudochattonella verruculosa (Montes et al., 2018), and co-existed sub-lethal species Alexandrium catenalla affected farmed salmon. Karenia degitata (Yang et al., 2001) and Karlodinium micrum (Deeds et al., 2006) are also occasionally kill wild and cultured finfish. Chrysochromulina spp., an ichthyotoxic flagellate, bloomed along another European coast caused fish farm mortalities (Graneli et al., 1993).

Toxicity of harmful algal blooms Chapter | 34  549



34.2.2  Effects of fish species and physiological conditions on survival rate Fish reared in farming cages is generally affected more than wild fish that can avoid from bloom water (Rensel and Whyte, 2004). In addition, the adverse effect of HABs on fish is usually recognized as species-specific. Fig. 34.6 shows schematic diagram of relationships between fish specific susceptibility and HABs. The large-ocean-dwelling fish such as tuna (Thunnus spp.) and yellowtail (Seriola quinqueradiata) are highly susceptible to HAB exposure (Munday and Hallegraeff, 1998; Aoki et al., 2015) and coastal and benthic fish tend to be tolerance to HABs toxicity (e.g., Kim et al., 2000c). Also, even for the same fish species, for example in the case of yellowtail, young (less than 12 month) are more resistant to lethal level than adult fish (Fisheries Research Agency, 2012). This phenomenon may be related to the different characteristic of oxygen demand and the swimming activity. Further, structures of the gills (e.g., gill surface area against body weight) also greatly differ among fish species. It is also well recognized that stop-feeding therapy in an aquaculture can reduce susceptibility of fish exposed to HABs. Fig. 34.7 shows the results of exposure tests using juvenile yellowtail. Test fish reared in three conditions (full feeding, half feeding, non-feeding) were proceeded to exposure experiment. Survival time of non-feeding juvenile was significantly prolonging as compared with other conditions (full feeding and half feeding). Unfortunately, present result dose not reveal the scientific mechanism, but it is clear that the change of physiological state of the fish caused by non-feeding therapy results in life-prolongation effect on toxic algal blooms.

34.2.3  Mass mortality and detrimental effects on molluscan shellfish Shellfish mariculture industries usually are hampered by phycotoxin contamination in products (Shumway, 1990; Shumway and Cembella, 1993). The toxic algal blooms responsible for the shellfish poisoning were reviewed in elsewhere (Anderson, 1994; Shumway, 1990; Bricelj and Shumway, 1998). Toxic HABs-derived phycotoxins usually do not cause shellfish dieoff, thus, shellfish harvesting can resume once the phycotoxins have depurated (e.g., Matsuyama and Shumway, 2009). In some instances, toxic algal species have had an adverse effect on shellfish physiology (Shumway, 1990; Landsberg, 2002). Intense blooms have devastated the cultures, i.e., caused a decrease in growth rate and declines in yield through inhibition of byssus production and filtration rates, increase of mortality rate and suppression of reproduction due to recruitment failure and seed production failure due to contaminated seawater (Bricelj and Lonsdale, 1997; Bricelj et al., 2001; Gainey and Shumway, 1988; Gallager et al., 1989; Gobler et al., 2008; Ho and Zubkoff, 1979; Landsberg, 2002; Luckenbach et al., 1993; Lesser and Shumway, 1993; Matsuyama et al., 2001a; Nielsen and Stromgren, 1991; Smolowitz and Shumway, 1997; Tracey, 1988; Wikfors and Smolowitz, 1995). The economic losses of local shellfish industries resulting from the direct impact of toxic algae on shellfish cultivation and natural stock are of great concerns. Though some phycotoxins are shown to have weak effects on shellfish physiology (Gainey and Shumway, 1988), other phycotoxins that remain uncharacterized have potent adverse effects (Bricelj et al., 2001; Landsberg, 2002; Matsuyama et  al., 1997; Leverone et  al., 2007; Matsuyama and Shumway, 2009). While these uncharacterized toxins are detrimental to shellfish physiology, they are not toxic to fish and mammals and are therefore, do not pose a public health hazard. However, loss of shellfish due to sublethal algal species is of great concern for aquaculture industry and it should be clarified the outline of some detrimental species.

Flounder Black sea bream File fish

Survival time

Juvenile yellowtail

Puffer Red sea bream

Trevally salmon Adult yellowtail Tuna

LC50 of HABs cell density FIG. 34.6  Scheme of finfish susceptibility among species for HABs exposure.

550  PART | IX  Algal toxicology

1

Survival rate

0.8 0.6 0.4 0.2 0

0

2

4

6

8

10

12

14

16

18

20

Time after exposure (hr) FIG. 34.7  Changes in the survival rate of juvenile yellowtail exposed to a cultured toxic isolate Chattonella antiqua at lethal cell density. Each six individuals were used for C. antiqua exposure, the cell density was 1800 cells/mL, the dissolved oxygen concentration was kept 5–6 mg/L during the experiment. Black line: Food-satiated breeding individual (daily 7% feed against body weight), dot line: half breeding individuals (daily 3.5% feed against body weight), gray line: 4-day starved breeding individual.

In addition, gamete of wild molluscan larvae disperses off the coastal water at the initial stage of life, thus, there is a risk of being directly exposed to toxic bloom waters. Though almost all marine animals are susceptible to environmental stressors (suspended matter, oxygen deficient, heavy metal, contaminated chemicals, etc.) in their early life stage, while molluscan larvae are adversely affected by harmful bloom, they had have a greatly concerned cause ecosystem collapse by sudden and/or prolonged natural resources population responsible for key species dismiss (e.g., Southgate et al., 1984) and massive recruitment failure. Therefore, we especially should be aware of impact of HABs on early larva via result of laboratory rearing experiment. Effects of harmful algal species on several molluscan species are summarize in Table 34.1 referred to previous laboratory experiments. Here we outline the portent toxic HABs to shellfish.

34.2.3.1  Karenia mikimotoi The dinoflagellate Karenia mikimotoi (formerly Gyrodinium aureolum, Gymnodinium cf.nagasakiense, Gymnodinium mikimotoi) is widely known as a both finfish and shellfish-killing species (Tangen, 1977; Mahoney et al., 1990; Gentien, 1998; Landsberg, 2002; Matsuyama et al., 1999; Shumway, 1990; Smolowitz and Shumway, 1997; Landsberg, 2002; Matsuyama and Shumway, 2009). The oldest report of a K. mikimotoi bloom and associated shellfish death was mentioned in Nishikawa (1903) in detail. He found that red-tide due to K. mikimotoi killed a number of cultured pearl oysters (Pinctada fucata martensii) from Ago Bay. Half of the individuals died within 44 h of exposure to K. mikimotoi bloom water indoor tanks. Since the 1960s, this species has formed massive blooms around European and East Asian coastal waters (Tangen, 1977; Takayama and Matsuoka, 1991). Previous efforts revealed that the mussel Mytilus edulis or M. galloprovincialis reduces filtration rate when exposed to more than 5 × 102 cells/mL of K. mikimotoi, and death occurs at concentrations greater than 5 × 103 cells/mL (Widdows et al., 1979; Matsuyama et al., 1998, 1999). K. brevis, which is closely related to K. mikimotoi, has been shown to have inhibitory effects on bivalves mollusks (Leverone et al., 2006; Table 34.1). The recent increase in the duration and extent of K. mikimotoi blooms is obvious, suggesting that shellfish industries should be aware of potential risks associated with K. mikimotoi.

34.2.3.2  Aureococcus anophagefferen It was recognized as a prolonged discoloration of the water commonly referred to as ‘brown tide’ (Cosper et al., 1987). The occurrence of brown tide severely impacts the commercially important bay scallop industry, affecting more than 80% of the harvest (Cosper et al., 1987). Further, brown tide affected other commercially important shellfish industries (Gallager et al., 1989; Shumway, 1990; Tracey, 1988; Gobler et al., 2008). Similar bloom events occurred concurrently in Narragansett Bay, RI, Barnegat Bay, and the Texas coast associated with near elimination of mussel population. These events were associated with massive die-offs of marine life (Bricelj and Lonsdale, 1997; Gallager et al., 1989; Shumway, 1990; Tracey, 1988). Laboratory experiments also revealed that exposure of mussel to A. anophagefferen culture isolates resulted in significant inhibitory effects (Bricelj and Lonsdale, 1997; Gainey and Shumway, 1991). However, toxic effects of A. anophagefferen magnitude varied among isolates, toxic mechanism responsible for shellfish mortality remain unclear (Padilla et al., 2006).



TABLE 34.1  Comparison of toxicity of harmful algal species to bivalve larvae. HAB species (class)

Concentrations (cells/ml) 7

Molluscan larvae species

Larval age (d)

Chattonella antiqua (Raphidophyceae)

1 × 10 cells/mL

Crassostrea gigas

0–1

Heterosigma akashiwo (Raphidophyceae)

1 × 105 cells/mL

Crassostrea gigas

0–1

Heterosigma akashiwo (Raphidophyceae)

104 cells/mL

Aureococcus anophagefferens (Pelagomonadaceae)

Exposure time (d)

Mortality (%) or adverse effect

References

None

Matsuyama et al. (2001a,b)

None

Matsuyama et al. (2001a,b)

Agropecten irradiance

Significant arrese of embryos development and eye-spot larvae mortality

Wang et al. (2006)

Mercenaria mercenaria

slight growth inhibiton

Padilla et al. (2006)

0.42

Alexandrium catenalla (Dinophyceae)

1 × 103 cells/mL

Pinctada fucata martensii

21

3

40

Basti et al. (2015)

Alexandrium affine (Dinophyceae)

1 × 103 cells/mL

Pinctada fucata martensii

21

3

60

Basti et al. (2015)

Alexandrium monilatum (Dinophyceae)

5.5 × 102 cells/mL

Crassostrea virginica

10–14

2 h

10

May et al. (2010)

2

5.5 × 10 cells/mL

Mercenaria mercenaria

10–14

2 h

62

May et al. (2010)

Alexandrium tamarense (Dinophyceae)

1 × 104 cells/mL

Crassostrea gigas

0–1

0.42

96

Matsuyama et al. (2001a,b)

Alexandrium tamarense (Dinophyceae)

1 × 104 cells/mL

Chlamys farreri

3.00

10

Minimal

Wikfors and Smolowitz (1995)

Pfiesteria shumwayae (Dinophyceae)

>3 × 103 cells/mL

Crassostrea virginica

1

60

Shumway et al. (2006)

Pfiesteria piscicida (Dinophyceae)

5 × 103 cells/mL

Agropecten irradiance

0.04

100

Springer et al. (2002)



Modifed from Tang, Y.Z., Gobler, J.C., 2009. Cochlodinium polykrikoides blooms and clonal isolates from the northwest Atlantic coast cause rapid mortality in larvae of multiple bivalve species. Mar. Biol. 156 (12), 2601–2611.

Toxicity of harmful algal blooms Chapter | 34  553

554  PART | IX  Algal toxicology

34.2.3.3  Cochlodinium polykrikoides Fish-killing dinoflagellate Cochlodinium polykrikoides also interfere molluscan shellfish physiology. Previous studies have reported that C. polykrikoides exhibits negative effects on oyster (Crassostrea gigas) larvae such as slow down of the metamorphosis during blooms (Matsuyama et al., 2001b), and mortality of larvae of the American oyster, C. verginica, is elevated concomitant with calcium uptake by C. polykrikoides exposure (Ho and Zubkoff, 1979). Similar adverse or sub-­lethal effect of C. polykrikoides on bay scallop and American oyster has been observed in a bloom site of Long Island (Gobler et al., 2008). Likewise fish-killing, biochemical matrix (thick polysaccharide surrounding their cell surface) may coincide with gill inflammation associated with valve closure, and subsequent reduction of gas exchange within scallop (Gobler et al., 2008). Furthermore, laboratory experiments (Tang and Gobler, 2009) revealed that culture isolates of C. polykrikoides showed potent sublethal effect to molluscan shellfish (C. verginica, A. irradiance, M. mercenaria, Table 34.1) unlike Matsuyama et al. (2001a). Recent finding indicates that toxicity in HAB species magnitude varied among isolates (e.g., Zou et al., 2010). We should be aware that the toxicity of the culture isolate may also greatly depend on culture conditions and/ or established localities. Peconic Estuary in nearly a decade, a major brown tide has not occurred, but the scallop population has not recovered, appears to demonstrate that the failure of this population to recover could be due, in part, to the recent outbreaks of C. polykrikoides blooms (Gobler et al., 2008).

34.2.3.4  Heterocapsa circularisquama The novel dinoflagellate Heterocapsa circularisquama is the causal species of red tide on the Japanese coast and has destroyed the shellfish aquaculture industries around the western part of Japan (Matsuyama et  al., 1992, 2001a; Nagai et  al., 1996; Matsuyama, 1999). This dinoflagellate exhibits potent and specific detrimental effects on bivalve and gastropod molluscs (Matsuyama et al., 1999; Matsuyama, 2003). Novel and persistent blooms of this species in Japanese coastal waters have adversely affected shellfish cultivation of the Pacific oyster (C. gigas), manila clam (Ruditapes philippinarum), pearl oyster (Pinctada fucata martensii) and abalone Haliotis discus (Matsuyama, 1999). Since 1998, H. circularisquama was identified in water samples from Hong Kong Bay, China (Iwataki et al., 2002), and Bizerte Lac, Tunisia (Turki, 2004). In Japan, economic losses of shellfish aquaculture industry resulting from the direct mortality of marketable products were estimated at a minimum of 10 billion-yen in the last three decades. Interestingly, there is no evidence that this species poses a threat, either by fish-kills or public health hazards coincident with red tide. Several hemolytic toxins are characterized as the possible agent of shellfish mortality (Oda et al., 2001; Sato et al., 2001), however, the principal toxin (labile glycoprotein like substances) that causes a potent detrimental effect on shellfish species remains unclear (Matsuyama et al., 1997; Matsuyama, 2003).

34.2.3.5  Other potentially toxic species to shellfish Alexandrium, responsible for PSP, also has an adverse effect on shellfish physiology (Ford et  al., 2008; Gainey and Shumway, 1988; Matsuyama et al., 2001b; Yan et al., 2001; Yan et al., 2003; Leverone et al., 2006). In these cases, shellfish species are affected by PST or uncharacterized toxins because some Alexandrium showed PST-independent cytotoxic effect on shellfish physiology and function (Ford et al., 2008; Matsuyama et al., 2001b; May et al., 2010) since some sublethal species (A. taylori, A. affine) does not produces any PSTs (Matsuyama et al., 2001b; Basti et al., 2015). Recent studies imply that Alexandrium produces a cytotoxic substance different from PST that may have deleterious effects on shellfish species (Emura et al., 2004; Katsuo et al., 2007; Ford et al., 2008). The effect of Alexandrium on shellfish species is speciesspecific, and its toxicity varies significantly among the isolates (Ford et al., 2008; Hegaret et al., 2008). The 1988 toxic bloom of Chrysochromulina polylepis extended over most of the Skagerrak and the entire Kattegat Seas, an area of about 75,000 km2, and lasted four weeks, from May to early June (Graneli et al., 1993). Although less significant shellfish mortality is seen in C. polylepis bloom, laboratory bioassay indicated that this species inhibited the fertilization and embryonic development (Table 34.1; Granmo et al., 1988). The ichthyotoxic raphidophyte Heterosigma akashiwo is a widespread species that remains a potential threat to not only fish farming, but also shellfish cultivation. H. akashiwo blooms have disrupted cultivation and the harvesting of wild stocks of shellfish in Canada, Portugal and New Zealand (Hegaret et al., 2007). Exposure of commercially important shellfish to H. akashiwo under laboratory conditions shows sublethal effects on American oyster (Keppler et al., 2005; Wang et al., 2006; Hegaret and Wikfors, 2007).

34.2.4  Adverse effects on gastropod species Southgate et al. (1984) observed the selective mortalities of key animal species notably grazing gastropods and subsequent a marked increase in fucoid algae in rocky shore in following a bloom of the toxic dinoflagellate, Gyrodinium aureolum



Toxicity of harmful algal blooms Chapter | 34  555

in 1979, S.W. Ireland. Gastropods are also victims exposed to HABs in marine ecosystem. Matsuyama (2003) also noticed several incidence of mass gastropod mortalities in Turbo (Batillus) cornutus, Haliotis discus, Sulculus diversicolor, Glossaulax didyma during Karenia mikimotoi and Heterocapsa circularisquama blooms. Harding et al. (2009) also report mortality of the Rapana venosa during Alexandrium monilatum bloom occurred in York River, United Sates. Abalone is also an aquaculture target species and some species are an economically important organisms. For this reason, the potential influence of red tide is of great concerned. Matsuyama et al. (1998) demonstrated the clarified effect of three harmful algae (K. mikimotoi, H. circularisquama, Heterosigma akashiwo) on two abalone species by a laboratory bioassay. In K. mikimotoi and H. circularisquama exposure, unusual locomotion and death in gastropods were found within 48 h, suggested potent cytotoxicity to abalone life. Botes et al. (2003) screening 17 species of potentially toxic dinoflagellates to commercially important abalone (Haliotis midae). Of the 17 dinoflagellate species tested, A. sanguinea, K. cristata, K. brevis and K. mikimotoi were found to cause the largest mortalities among abalone larvae or spat, and thus may pose a significant threat to the abalone mariculture industry if blooms were to enter farms at concentrations tested (Botes et al., 2003).

34.3 Conclusions The impact of a toxic algal species is serious and major barrier in the development of finfish and shellfish aquaculture. However, recent efforts have been contributed to scientific understanding in this field. Although direct control of a harmful algal bloom appears to be difficult, scientific clarification of mechanism responsible for finfish and shellfish mortalities will present key solutions to the threat of toxic algal species (e.g., stop feeding therapy to finfish). Estuarine ecosystems have provided various types of services to mariculture industries in the last half century. For the sustained and environmentally acceptable aquaculture systems in the world, we need to learn techniques to suppress environmental key factors underlying HABs outbreak and develop new aquaculture techniques that minimize adverse effects of HABs to aquatic organisms in future. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c, d,e,f,g,h,i,j,k,l,m,n,o).

Acknowledgements We thank the members of the Fisheries Research Agency (FRA) and Nagasaki University for helping us with valuable studies and Dr. Ishimatsu of Nagasaki University for kindly providing us with the result of blood oxygen measure experiments. Thanks are also due to Professor Tsuneo Honjo of Kyushu University for comments on the draft. This work was supported in part by a grant from the Fisheries Agency of Japan and Ministry of Education of Japan.

References Albright, L.J., Yang, C.Z., Johnson, S., 1993. Sub-lethal concentrations of the harmful diatoms, Chaetoceros concavicornis and C. convolutus, increase mortality rates of penned Pacific salmon. Aquaculture 117 (3–4), 215–225. Anderson, D.M., 1994. Red tide. Sci. Am. 271 (2), 52–58. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25 (4), 704–726. Aoki, K., Onitsuka, M., Shimizu, M., Kuroda, H., Matsuo, H., Kitadai, Y., 2015. Interregional differences in mortality of aquacultured yellowtail Seriola quinqueradiata in relation to a Chattonella bloom in the Yatsushiro Sea, Japan, in 2010. Fish. Sci. 81 (3), 525–532. Baden, D.G., Mende, T.J., 1982. Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis. Toxicon 20 (2), 457–461. Basti, L., Go, J., Higuchi, K., Nagai, K., Segawa, S., 2011. Effects of the toxic dinoflagellate Heterocapsa circularisquama on larvae of the pearl oyster Pinctada fucata martensii (Dunker, 1873). J. Shellfish Res. 30 (1), 177–186. Basti, L., Nagai, S., Go, J., Okano, S., Nagai, K., Watanabe, R., et al., 2015. Differential inimical effects of Alexandrium spp. and Karenia spp. on cleavage, hatching, and two larval stages of Japanese pearl oyster Pinctada fucata martensii. Harmful Algae 43, 1–12. Botes, L., Smit, A.J., Cook, P.A., 2003. The potential threat of algal blooms to the abalone (Haliotis midae) mariculture industry situated around the South African coast. Harmful Algae 2 (4), 247–259. Brand, L.E., Campbell, L., Bresnan, E., 2012. Karenia: the biology and ecology of a toxic genus. Harmful Algae 14, 156–178. Bricelj, V.M., Lonsdale, D.J., 1997. Aureococcus anophagefferens: causes and ecological consequences of brown tides in U.S. mid-Atlantic coastal waters. Limnol. Oceanogr. 42 (5), 1023–1038. Bricelj, V., MacQuarrie, S., Schaffner, R., 2001. Differential effects of Aureococcus anophagefferens isolates (“brown tide”) in unialgal and mixed suspensions on bivalve feeding. Mar. Biol. 139 (4), 605–616. Bricelj, V.M., Shumway, S.E., 1998. Paralytic shellfish toxins in bivalve molluscs: occurrences, transfer kinetics, and biotransformation. Rev. Fish. Sci. 6 (4), 315–383.

556  PART | IX  Algal toxicology

Burkholder, J.M., Noga, E.J., Hobbs, C.W., Glasgow, H.B., Smith, S.A., 1992. New ‘phantom’ dinoflagellate is the causative agent of major estuarine fish kills. Nature 358 (6385), 407–410. Cosper, E.M., Dennison, W.C., Carpenter, E.J., Bricelj, V.M., Mitchell, J.G., Kuenstner, S.H., et al., 1987. Recurrent and persistent brown tide blooms perturb coastal marine ecosystem. Estuaries 10 (4), 284–290. Deeds, J.R., Reimschuessel, R., Place, A.R., 2006. Histopathological effects in fish exposed to the toxins from Karlodinium micrum. J. Aquat. Anim. Health 18 (2), 136–148. Edvardsen, I., Imai, I., 2006. The ecology of harmful flagellates within prymnesiophyceae and raphidophyceae. In: Granéli, E., Turner, J.T. (Eds.), Ecology of Harmful Algae, Ecological Studies, vol. 189. Springer-Verlag, Berlin, Heidelberg, pp. 67–79. Emura, A., Matsuyama, Y., Oda, T., 2004. Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori. Harmful Algae 3 (1), 29–37. FAO, 2016. The state of World Fisheries and Aquaculture 2016: Contributing to Food Security and Nutrition for All. Food and Agriculture Organization, Rome. Fisheries Research Agency, 2012. Red tide due to the genus Chattonella occurred in Yatsushiro Bay, 2010 and associated massive killing of the farming yellowtail Seriola quinqueradiata in Kagoshima area: Report of factor affecting fish-killing by statically analysis method of a hearing survey for aquaculturists. Seikai National Fisheries Research Institute, Fisheries Research Agency. Nagasaki, Japan p. 52. Ford, S.E., Bricelj, V.M., Lambert, C., Paillard, C., 2008. Deleterious effects of a non PST bioactive compounds from Alexandrium tamarense on bivalve hemocytes. Mar. Biol. 154 (2), 241–253. Gainey, L.F., Shumway, S.E., 1988. A compendium of the responses of bivalve molluscs to toxic dinoflagellates. J. Shellfish Res. 7, 623–628. Gainey, L.F., Shumway, S.E., 1991. The physiological effect of Aureococcus anophagefferens (“brown tide”) on the lateral cilia of bivalve mollusks. Biol. Bull. 181 (2), 298–306. Gallager, M.S., Stoecker, K.D., Bricelj, M.V., 1989. Effects of the brown tide alga on growth, feeding physiology and locomotory behavior of scallop larvae (Argopecten irradians). In: Cosper, E.M., Bricelj, M.V., Carpenter, E.J. (Eds.), Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Springer, Berlin, pp. 511–541. Garcia-Mendoza, E., Caceres-Martinez, J., Rivas, D., Fimbres-Martinez, M., Sanchez-Bravo, Y., Vasquez-Yeomans, R., et al., 2018. Mass mortality of cultivated Northern bluefin tuna Thunnus thynnus orientalis associated with Chattonella species in Baja California. Mexico. Front. Mar. Sci. 5 (454), 1–16. Gentien, P., 1998. Bloom dynamics and ecophysiology of the Gymnodinium mikimotoi complex. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer, Berlin, pp. 155–173. Gobler, C.J., Berry, D.L., Anderson, O.R., Burson, A., Koch, F., Rodgers, B.S., et al., 2008. Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA. Harmful Algae 7 (3), 293–307. Graneli, E., Haraldsson, C., 1993. Can increased leaching of trace metals from acidified areas influence phytoplankton growth in coastal waters? Ambio 22 (5), 308–311. Graneli, E., Paasche, E., Maestrini, S.Y., 1993. Three years after the Chrysochromulina polylepis bloom in Scandinavian waters in 1988: some conclusions of recent research and monitoring. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in the Sea. Elsevier, Amsterdam, pp. 23–32. Granmo, A., Havenhand, J., Magnusson, K., Svane, I., 1988. Effects of the planktonic flagellate Chrysochromulina polylepis Manton et Park on fertilization and early development of the ascidian Ciona intestinalis (L.) and the blue mussel Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 124 (1), 65–71. Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32 (2), 79–99. Hallegraeff, G.M., 1998. Transport of toxic dinoflagellates via ships’ ballast water: bioeconomic risk assessment and efficacy of possible ballast water management strategies. Mar. Ecol. Prog. Ser. 168, 297–309. Harding, J.M., Mann, R., Moeller, P.D.R., Hsia, M.S., 2009. Mortality of the veined rapa whelk, Rapana venosa, in relation to a bloom of Alexandrium monilatum in the York River, United States. J. Shellfish Res. 28 (2), 363–367. Hegaret, H., Shumway, S.E., Wikfors, G.H., Pate, S., Burkholder, J.M., 2008. Potential transport of harmful algae through relocation of bivalve mollusks. Mar. Ecol. Prog. Ser. 361, 169–179. Hegaret, H., Wikfors, G.H., 2007. Time–dependent changes in hemocytes of eastern oysters, Crassostrea virginica, and northern bay scallops, Argopecten irradians irradians, exposed to a cultured strain of Prorocentrum minimum. Harmful Algae 4 (2), 187–199. Hegaret, H., Wikfors, G.H., Shumway, S.E., 2007. Diverse feeding responses of five species of bivalve mollusc when exposed to three species of harmful algae. J. Shellfish Res. 26 (2), 549–559. Hiroishi, S., Okada, H., Imai, I., Yoshida, T., 2005. High toxicity of the novel bloom–forming species Chattonella ovata (raphidophyceae) to cultured fish. Harmful Algae 4 (4), 783–787. Ho, M.S., Zubkoff, P.L., 1979. The effects of a Cochlodinium heterolobatum bloom on the survival and calcium uptake by larvae of the American oyster, Crassostrea virginica. In: Taylor, D.L., Selinger, H.H.Y. (Eds.), Toxic Dinoflagellate Blooms. Elsevier, New York, pp. 409–412. Honjo, T., 1994. The biology and prediction of representative red tides associated with fish kills in Japan. Rev. Fish. Sci. 2 (3), 225–253. Honjo, T., Imada, N., Ohshima, Y., Maema, Y., Nagai, K., Matsuyama, Y., et al., 1998. Potential transfer of Heterocapsa circularisquama with pearl oyster consignments. In: Reguera, B., Blanco, J., Fernandez, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Paris, pp. 224–226. Humborg, C., Ittekkot, V., Cociasu, A., Bodungen, B.V., 1997. Effect of Danube River dam on Black Sea biogeochemistry and ecosystem structure. Nature 386 (6623), 385–388. Ishimatsu, A., Maruta, H., Tsuchiyama, T., Ozaki, M., 1990. Respiratory, ionoregulatory and cardiovascular responses of the yellowtail Seriola quinqueradiata to exposure to the red tide plankton Chattonella. Nippon Suisan Gakkaishi 56 (2), 189–199.



Toxicity of harmful algal blooms Chapter | 34  557

Ishimatsu, A., Oda, T., Yoshida, M., Ozaki, M., 1996b. Oxygen radicals are probably involved in the mortality of yellowtail by Chattonella marina. Fish. Sci. 62 (5), 836–837. Ishimatsu, A., Sameshima, M., Tamura, A., Oda, T., 1996a. Histological analysis of the mechanisms of Chattonella-induced hypoxemia in yellowtail. Fish. Sci. 62 (1), 50–58. Iwataki, M., Wong, M.W., Fukuyo, Y., 2002. New record of Heterocapsa circularisquama (Dinophyceae) from Hong Kong. Fish. Sci. 68 (5), 1161–1163. Katsuo, D., Kim, D., Yamaguchi, K., Matsuyama, Y., Oda, T., 2007. A new simple screening method for the detection of cytotoxic substances produced by harmful red tide phytoplankton. Harmful Algae 6 (6), 790–798. Keppler, C.J., Hoguet, J., Smith, K., Ringwood, A.H., Lewitus, A.J., 2005. Sublethal effects of the toxic alga Heterosigma akashiwo on the southeastern oyster (Crassostrea virginica). Harmful Algae 4 (2), 275–285. Kim, H.G., 1998. Cochlodinium polykrikoides blooms in Korean coastal waters and their mitigation. In: Reguera, B., Blanco, J., Fernandez, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Paris, pp. 227. Kim, C.S., Lee, S.G., Kim, H.G., 2000c. Biochemical response of fish exposed to a harmful dinoflagellate Cochlodinium polykrikoides. J. Exp. Mar. Biol. Ecol. 254 (2), 131–141. Kim, C.S., Lee, S.G., Kim, H.G., Jung, J., 1999. Reactive oxygen species as causative agents in the ichthyotoxicity of red tide dinoflagellate Cochlodinium polykrikoides. J. Plankton Res. 21 (11), 2105–2155. Kim, D., Nakamura, A., Okamoto, T., Komatsu, N., Oda, T., Lida, T., et al., 2000a. Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase. Biochim. Biophys. Acta 1524 (2-3), 220–227. Kim, D., Okamoto, T., Oda, T., Tachibana, K., Lee, K.S., Ishimatsu, A., et al., 2001. Possible involvement of the glycocalyx in the ichthyotoxicity of Chattonella marina (Raphidophyceae): immunological approach using antiserum against cell surface structures of the flagellate. Mar. Biol. 139 (4), 625–632. Kim, D., Nakashima, T., Matsuyama, Y., Niwano, Y., Yamaguchi, K., Oda, T., 2007. Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata. J. Plankton Res. 29 (3), 241–247. Kim, D., Oda, T., 2010. Possible factors responsible for the fish-killing mechanisms of the red tide phytoplankton, Chattonella marina and Cochlodinium polykrikoides. In: Ishimatsu, A., Lie, H.J. (Eds.), Coastal Environmental and Ecosystem Issues of the East China Sea. TERRAPUB and Nagasaki University, Nagasaki, pp. 245–268. Kim, D., Oda, T., Ishimatsu, A., Muramatsu, T., 2000b. Galacturonic-acid-induced increase of superoxide production in red tide phytoplankton Chattonella marina and Heterosigma akashiwo. Biosci. Biotechnol. Biochem. 64 (4), 911–914. Kim, D., Oda, T., Muramatsu, T., Kim, D., Matsuyama, Y., Honjo, T., 2002. Possible factors responsible for the toxicity of Cochlodinium polykrikoides, a red tide phytoplankton. Comp. Biochem. Physiol. C 132 (4), 415–423. Koizumi, Y., Uchida, T., Honjo, T., 1996. Diurnal vertical migration of Gymnodinium mikimotoi during a red tide in Hoketsu Bay, Japan. J. Plankton Res. 18 (2), 289–294. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

558  PART | IX  Algal toxicology

Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Kudela, R.M., Gobler, C.J., 2012. Harmful dinoflagellate blooms caused by Cochlodinium sp: global expansion and ecological strategies facilitating bloom formation. Harmful Algae 14, 71–86. Landsberg, J.H., 2002. The effects of harmful algal blooms on aquatic organisms. Rev. Fish. Sci. 10 (2), 113–390. Leon-Munoz, J., Urbina, M.A., Garreaud, R., Iriarte, J.L., 2018. Hydroclimatic conditions trigger record harmful algal bloom in western Patagonia (summer 2016). Sci. Rep. UK 8 (1), 1330. Lesser, M.P., Shumway, S.E., 1993. Effects of toxic dinoflagellates on clearance rates and survival in juvenile bivalve mollusks. J. Shellfish Res. 12 (2), 377–381. Leverone, J.R., Blake, N.J., Pierce, R.H., Shumway, S.E., 2006. Effects of the dinoflagellate Karenia brevis on larval development in three species of bivalve mollusc from Florida. Toxicon 48 (1), 75–84. Leverone, J.R., Shumway, S.E., Blake, N.J., 2007. Comparative effects of the toxic dinoflagellate Karenia brevis on clearance rates in juveniles of four bivalve molluscs from Florida, USA. Toxicon 49 (5), 634–645. Lin, Y.Y., Risk, M., Ray, S.M., Engen, D.V., Clardy, J., Golik, J., et al., 1981. Isolation and structure of brevetoxin B from the “red tide” dinoflagellate Ptychodiscus brevis (Gymnodinium breve). J. Am. Chem. Soc. 103 (22), 6773–6775. Luckenbach, M.W., Sellner, K.G., Shumway, S.E., Greene, K., 1993. Effects of two bloom–forming dinoflagellates, Prorocentrum minimum and Gyrodinium uncatenum, on the growth and survival of the Eastern oyster, Crassostrea virginica (Gmelin 1791). J. Shellfish Res. 12, 411–415. Mahoney, J.B., Olsen, P., Cohn, M., 1990. Blooms of a dinoflagellate Gyrodinium cf. aureolum in New Jersey coastal waters and their occurrence and effects worldwide. J. Coastal Res. 6 (1), 121–135. Marshall, J.A., Nichols, P.D., Hamilton, B., Lewis, R.J., Hallegraeff, G.M., 2003. Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids. Harmful Algae 2 (4), 273–281. Matsuoka, K., Iwataki, M., 2004. Present status in study on a harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef. Bull. Plankton Soc. Jpn. 51 (1), 38–45. Matsusato, T., Kobayashi, H., 1974. Studies on death of fish caused by red tide. Bull. Nansei Reg. Fish. Res. Lab. 7, 43–67. Matsuyama, Y., 1999. Harmful effect of dinoflagellate Heterocapsa circularisquama on shellfish aquaculture in Japan. JARQ-Jpn. Agr Res. Q. 33, 283–293. Matsuyama, Y., 2003. Physiological and ecological studies on harmful dinoflagellate Heterocapsa circularisquama: II. Clarification on toxicity of H. circularisquama and its mechanisms causing shellfish kills. Bull. Fish. Res. Agency 9, 13–117. Matsuyama, Y., 2008. Red tide due to the dinoflagellate Karenia mikimotoi in Hiroshima Bay 2002: environmental features during the red tide and associated fisheries damages to finfish and shellfish aquaculture. In: Moestrup, O., et al. (Eds.), Proceedings of the 12th International Conference on Harmful Algae. UNESCO, Copenhagen. pp. 209–211. Matsuyama, Y., Nagai, K., Mizuguchi, T., Fujiwara, M., Ishimura, M., Yamaguchi, M., et al., 1992. Ecological features and mass mortality of pearl oysters during red tides of Heterocapsa sp. in Ago Bay in 1992. Nippon Suisan Gakk. 61 (1), 35–41. Matsuyama, Y., Nishitani, G., Nagai, S., 2010. Direct detection of harmful algae from the oyster spat and live fish transporting trailers. In: Ho, K.C., Zhou, M.J., Qi, Z.Y. (Eds.), Proceedings of XIII International Conference on Harmful Algae. Hong Kong. pp. 185–189. Matsuyama, Y., Shumway, S.E., 2009. Impacts of harmful algal blooms on shellfisheries aquaculture. In: Burnell, G., Allan, G. (Eds.), New technologies in Aquaculture. Woodhead Publishing, Sawston, Cambridge, pp. 580–609. Matsuyama, Y., Uchida, T., Honjo, T., 1997. Toxic effects of the dinoflagellate Heterocapsa circularisquama on clearance rate of the blue mussel, Mytilus galloprovincialis. Mar. Ecol. Prog. Ser. 146 (1-3), 73–80. Matsuyama, Y., Uchida, T., Honjo, T., 1998. The harmful Effect of red tide dinoflagellates, Heterocapsa circularisquama and Gymnodinium mikimotoi on the clearance rate and survival of the blue mussel, Mytilus galloprovincialis. In: Reguera, B., Blanco, J., Fernandez, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Paris, pp. 422–424. Matsuyama, Y., Uchida, T., Honjo, T., 1999. Effects of harmful dinoflagellates, gymnodinium mikimotoi and heterocapsa circularisquama, red-tide on filtering rate of bivalve molluscs. Fish. Sci. 65 (2), 248–253. Matsuyama, Y., Uchida, T., Honjo, T., Shumway, S.E., 2001a. Impacts of the harmful dinoflagellate, Heterocapsa circularisquama, on shellfish aquaculture in Japan. J. Shellfish Res. 20 (3), 1269–1272. Matsuyama, Y., Usuki, H., Uchida, T., Kotani, Y., 2001b. Effects of harmful algae on the early planktonic larvae of the oyster, Crassostrea gigas. In: Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J., Lewis, R.J. (Eds.), Harmful Algal Blooms 2000. UNESCO, Paris, pp. 411–414. May, S.P., Burkholder, J.M., Shumway, S.E., Hegaret, H., Wikfors, G.H., Frank, D., 2010. Effects of the toxic dinoflagellate Alexandrium monilatum on survival, grazing and behavioral response of three ecologically important bivalve molluscs. Harmful Algae 9 (3), 281–293. Montes, R.M., Rojas, X., Artacho, P., Tello, A., Quinones, R.A., 2018. Quantifying harmful algal bloom thresholds for farmed salmon in southern Chile. Harmful Algae 77, 55–65. Mulholland, M.R., Morse, R.E., Boneillo, G.E., Bernhardt, P.W., Filippino, K.C., Procise, L.A., et al., 2009. Understanding causes and impacts of the dinoflagellate, Cochlodinium polykrikoides blooms in the Chesapeake Bay. Estuar. Coasts 32 (4), 734–747. Munday, B.L., Hallegraeff, G.M., 1998. Mass mortality of captive southern bluefin tuna (Thunnus maccoyii) in April/May 1996 in Boston Bay, South Australia: a complex diagnostic problem. Fish Pathol. 33 (4), 343–350. Munday, R., Towers, N.R., Mackenzie, L., Beuzenberg, V., Holland, P.T., Miles, C.O., 2004. Acute toxicity of gymnodimine to mice. Toxicon 44 (2), 173–178.



Toxicity of harmful algal blooms Chapter | 34  559

Nagai, K., Matsuyama, Y., Uchida, T., Yamaguchi, M., Ishimura, M., Nishimura, A., et al., 1996. Toxicity and LD50 levels of the red tide dinoflagellate Heterocapsa circularisquama on juvenile pearl oysters. Aquaculture 144 (1-3), 149–154. Nakamura, A., Okamoto, T., Komatsu, N., Ooka, S., Oda, T., Ishimatsu, A., et al., 1998. Fish mucus stimulates the generation of superoxide anion by Chattonella marina and Heterosigma akashiwo. Fish. Sci. 64 (6), 866–869. Nielsen, M.V., Stromgren, T., 1991. Shell growth response of mussels (Mytilus edulis) exposed to toxic microalgae. Mar. Biol. 108 (2), 263–267. Nishikawa, T., 1903. Futatabi akashiwo ni tsuite [On a red tide, again]. Zool. Mag. 15, 147–153. Oda, T., Ishimatsu, A., Shimada, M., Takeshita, S., Muramatsu, T., 1992. Oxygen-radical-mediated toxic effects of the red tide flagellate Chattonella marina on Vibrio alginolyticus. Mar. Biol. 112 (3), 505–509. Oda, T., Sato, Y., Kim, D., Muramatsu, T., Matsuyama, Y., Honjo, T., 2001. Hemolytic activity of Heterocapsa circularisquama (Dinophyceae) and its possible involvement in shellfish toxicity. J. Phycol. 37 (4), 509–516. Okaichi, T., 1989. Red tide problems in the Seto Inland Sea, Japan. In: Okaichi, T., Anderson, D.M., Nemoto, T. (Eds.), Red Tides, Biology, Environmental Science, and Toxicology. Elsevier, Amsterdam, pp. 137–142. Okaichi, T., 2004. Red-tide phenomena. In: Okaichi, T. (Ed.), Red Tides. Springer, Dordrecht, pp. 7–60. Onoue, Y., Haq, M.S., Nozawa, K., 1990. Separation of neurotoxins from Chattonella marina. Nippon Suisan Gakkaishi 56 (4), 695. Onoue, Y., Nozawa, K., 1989. Zinc–bound PSP toxins separated from Cochlodinium red tide. In: Natori, S., Hashimoto, K., Ueno, Y. (Eds.), Mycotoxins and Phycotoxins 88. Elsevier, Amsterdam, pp. 359–366. Padilla, D.K., Doall, M.H., Gobler, C.J., Hartson, A., O’Boyle, K., 2006. Brown tide alga, Aureococcus anophagefferens, can affect growth but not survivorship of Mercenaria mercenaria larvae. Harmful Algae 5 (6), 736–748. Raine, R., O’Boyle, S., O’Higgins, T., White, M., Patching, J., Cahill, B., et al., 2001. A satellite and field portrait of a Karenia mikimotoi bloom off the south coast of Ireland, August 1998. Hydrobiologia 465 (1-3), 187–193. Rensel, J.E., Whyte, J.N.C., 2004. Finfish mariculture and harmful algal blooms. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D., Enevoldsen, H.O. (Eds.), Manual on Harmful Marine Microalgae. UNESCO, Paris, pp. 693–722. Richlen, M.L., Morton, S.L., Jamali, E.A., Rajan, A., Anderson, D.M., 2010. The catastrophic 2008–2009 red tide in the Arabian Gulf Region, with observations on the identification and phylogeny of the fish-killing dinoflagellate Cochlodinium polykrikoides. Harmful Algae 9 (2), 163–172. Sato, Y., Oda, T., Muramatsu, T., Matsuyama, Y., Honjo, T., 2001. Photosensitizing hemolytic toxin in Heterocapsa circularisquama, a newly indentified harmful red tide dinoflagellate. Aquat. Toxicol. 56 (3), 191–196. Scholin, C.A., Hallegraeff, G.M., Anderson, D.M., 1995. Molecular evolution of the Alexandrium tamarense ‘species complex’ (Dinophyceae): dispersal in the North American and West Pacific regions. Phycologia 34 (6), 472–485. Shimada, M., Kawamoto, S., Nakatsuka, Y., Watanabe, M., 1993. Localization of superoxide anion in the red tide alga Chattonella antiqua. J. Histochem. Cytochem. 41 (4), 507–511. Shimada, M., Murakami, T.H., Imahayashi, T., Ozaki, H.S., Toyoshima, T., Okaichi, T., 1983. Effects of sea bloom, Chattonella antiqua, on gill primary lamellae of the young yellowtail, Seriola quinqueradiata. Acta Histochem. Cytochem. 16 (3), 232–244. Shumway, S.E., 1990. A review of the effects of algal blooms on shellfish and aquaculture. J. World Aquacult. Soc. 21 (2), 65–104. Shumway, S.E., Burkholder, J.M., Springer, J., 2006. Effects of the estuarine dinoflagellates Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species. Harmful Algae 5 (4), 442–458. Shumway, S.E., Cembella, A.D., 1993. The impact of toxic algae on scallop culture and fisheries. Rev. Fish. Sci. 1 (2), 121–150. Smayda, T.J., Villareal, T., 1989. The 1985 “brown tide” and the open phytoplankton niche in Narragansett Bay during summer. In: Cosper, E.M., Bricelj, V.M., Carpenter, E.J. (Eds.), Novel Phytoplankton Blooms. Springer, Dordrecht, pp. 159–187. Smolowitz, R., Shumway, S.E., 1997. Possible cytotoxic effects of the dinoflagellate, Gyrodinium aureolum, on juvenile bivalve mollusks. Aquac. Int. 5 (4), 291–300. Southgate, T., Wilson, K., Cross, T.F., Myers, A.A., 1984. Recolonization of a rocky shore in S.W. Ireland following a toxic bloom of the dinoflagellate, Gyrodinium aureolum. J. Mar. Biol. Assoc. UK 64 (2), 485–492. Springer, J.J., Shumway, S.E., Burkholder, J.M., Glasgow, H.B., 2002. Interactions between the toxic estuarine dinoflagellate Pfiesteria piscicida and two species of bivalve molluscs. Mar. Ecol. Prog. Ser. 245, 1–10. Stoecker, D.K., Adolf, J.E., Place, A.R., Glibert, P.M., Meritt, D.W., 2008. Effects of the dinoflagellates Karlodinium veneficum and Prorocentrum minimum on early life history stages of the Eastern oyster (Crassostrea virginica). Mar. Biol. 154, 81–90. Takayama, H., Matsuoka, K., 1991. A reassessment of the specific characters of Gymnodinium mikimotoi Miyake et Kominami ex Oda and Gymnodinium nagasakiense Takayama et Adachi. Bull. Plankton Soc. Jpn. 38 (1), 53–68. Tang, Y.Z., Gobler, J.C., 2009. Cochlodinium polykrikoides blooms and clonal isolates from the northwest Atlantic coast cause rapid mortality in larvae of multiple bivalve species. Mar. Biol. 156 (12), 2601–2611. Tangen, K., 1977. Bloom of Gyrodinium aureolum (Dinophyceae) in north European waters, accompanied by mortality in marine organism. Sarsia 63 (2), 123–133. Tester, P.A., Steidinger, K.A., 1997. Gymnodinium breve red tide blooms: initiation, transport, and consequences of surface circulation. Limnol. Oceanogr. 42 (5), 1039–1051. Thain, J.E., Watts, J., 1987. The use of a bioassay to measure changes in water quality associated with a bloom of Gyrodinium aureolum Hulbult. Rap. Process. 187, 103–107. Tillmann, T., John, U., Cembella, A., 2007. On the allelochemical potency of the marine dinoflagellate Alexandrium ostenfeldii against heterotrophic and autotrophic protists. J. Plankton Res. 29 (6), 527–543.

560  PART | IX  Algal toxicology

Tracey, G.A., 1988. Feeding reduction, reproductive failure, and mortality in Mytilus edulis during the 1985 ‘brown tide’ in Narragansett Bay, Rhode Island. Mar. Ecol. Prog. Ser. 50, 73–81. Turki, S., 2004. Suivi des microalgues planktoniques toxiques dans les zones de production, de levage des mollusques bivalves et d’exploitation des oursins du Nord de la Tunisie. Bull. Inst. Nat. Sci. Tech. Mer Salammbo 31, 83–96. Twiner, M.J., Dixon, S.J., Trick, C.G., 2001. Toxic effects of Heterosigma akashiwo do not appear to be mediated by hydrogen peroxcide. Limnol. Oceanogr. 46 (6), 1400–1405. Uchida, T., Toda, S., Matsuyama, Y., Yamaguchi, M., Kotani, Y., Honjo, T., 1999. Interaction between the red tide dinoflagellates Heterocapsa circularisquama and Gymnodinium mikimotoi in laboratory culture. J. Exp. Mar. Biol. Ecol. 241 (2), 285–299. Wang, L., Yan, T., Zhou, M., 2006. Impacts of HAB species Heterosigma akashiwo on early development of the scallop Argopecten irradians Lamarck. Aquaculture 255, 374–383. Whyte, J.N.C., Haigh, N., Ginthie, N.G., Keddy, L.J., 2001. First record of blooms of Cochlodinium sp. (Gymnodiniales, Dinophyceae) causing mortality to aquacultured salmon on the west coast of Canada. Phycologia 40 (3), 298–304. Widdows, J., Moore, M.N., Lowe, D.M., Salkeld, P.N., 1979. Some effects of a dinoflagellate bloom (Gyrodinium aureolum) on the mussel Mytilus edulis. J. Mar. Biol. Assoc. UK 59 (2), 522–524. Wikfors, G.H., Smolowitz, R.M., 1995. Experimental and histological studies of four life–history stages of the Eastern oyster, Crassostrea virginica, exposed to a cultured strain of the dinoflagellate Prorocentrum minimum. Biol. Bull. 188 (3), 313–328. Yamasaki, Y., Kim, D., Matsuyama, Y., Oda, T., Honjo, T., 2004. Production of superoxide anion and hydrogen peroxide by the red tide dinoflagellate Karenia mikimotoi. J. Biosci. Bioeng. 97 (3), 212–215. Yamatogi, T., Sakamoto, S., Yamaguchi, M., Murata, K., Sakurada, K., Takano, Y., et al., 2010. Geographical distribution and growth characteristics of a harmful unarmored dinoflagellate Cochlodinium sp. type–Kasasa in west Kyushu. Japan. Bull. Jap. Soc. Phycol. 58 (3), 167–172. Yan, T., Zhou, M.J., Fu, M., Wang, Y., Yu, R.C., Li, J., 2001. Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense. Toxicon 39 (8), 1239–1244. Yan, T., Zhou, M.J., Fu, M., Yu, R.C., Wang, Y.F., Li, J., 2003. Effects of the dinoflagellate Alexandrium tamarense on the early development of the scallop Argopecten irradians concentricus. Aquaculture 217 (1-4), 167–178. Yang, C.Z., Albright, L.J., Yousif, A.N., 1995. Oxygen–radical–mediated effects of the toxic phytoplankter Heterosigma carterae on juvenile rainbow trout Oncorhyncus mykiss. Dis. Aquat. Org. 23 (2), 101–108. Yang, Z.B., Takayama, H., Matsuoka, K., Hodgkiss, I.J., 2001. Karenia digitata sp. nov. (Gymnodiniales, Dinophyceae), a new harmful algal bloom species from the coastal waters of west Japan and Hong Kong. Phycologia 39 (6), 463–470. Yokote, M., Honjo, T., 1985. Morphological and histochemical demonstration of a glycocalyx on the cell surface of Chattonella antiqua, a ‘naked’ flagellate. Experientia 41 (9), 1143–1145. Yuki, K., Yoshimatsu, S., 1989. Two fish–killing species of Cochlodinium from Harima Nada, Seto Inland Sea, Japan. In: Okaichi, T., Anderson, D.M., Nemoto, T. (Eds.), Red tides, Biology, Environmental Science and Toxicology. Elsevier, Amsterdam, pp. 451–454. Zou, Y., Yamasaki, Y., Matsuyama, Y., Yamaguchi, K., Honjo, T., Oda, T., 2010. Possible involvement of hemolytic activity in the contact–dependent lethal effects of the dinoflagellate Karenia mikimotoi on the rotifer Brachionus plicatilis. Harmful Algae 9 (4), 367–373.

Chapter 35

Cyanobacterial toxins and their effects on human and animal health N.R. Souza, J.S. Metcalf Brain Chemistry Labs, Jackson, WY, United States

35.1 Introduction Cyanobacteria, ancient photosynthetic prokaryotes, are commonly associated with freshwaters, but are also found in marine systems, deserts environments and hot springs, as examples (Fogg et al., 1973). Their ubiquity may be due, in part, to being among some of the oldest living organisms on the planet, with fossilized specimens reported as being 3.6 billion years old (Schopf, 2000). They are considered to have contributed to the Great Oxidation Event and possibly to the evolution of chloroplasts in higher plants (Knoll, 2003). They are components of food chains providing the basis for many of these food webs, supporting zooplankton and phytoplanktivorous and zooplanktivorous fish. Through photosynthesis, these primary producers are able to fix carbon and produce complex sugars (Fogg et al., 1973). Some cyanobacteria are also capable of fixing atmospheric nitrogen in heterocysts, allowing cyanobacterial growth in waters that are nitrogen limited (Fogg et al., 1973; Smith, 1983). The enzymatically-fixed diazotrophic nitrogen is converted into organic nitrogen sources, which are then available for metabolism (Stewart, 1972, 1980). Other cellular specializations that exist in some species are akinetes, which act as seasonal resting spores for the population, or when environmental conditions are not favorable for growth such as during nutrient depletion (Yamamoto, 1976; Cardemil and Wolk, 1979). When conditions, such as high nutrients and temperatures allow, cyanobacteria can become dominant in marine and freshwaters and proliferate, forming massive populations or blooms (Paerl and Huisman, 2008; Humbert and Fastner, 2017). Such blooms can impact high-resource waters such as drinking water reservoirs, recreational waterbodies or those used for animal-watering and fisheries, becoming a concern due to economic impacts and the risk of adverse health effects. Cyanobacterial blooms are often unsightly and have the potential to produce taste and odor compounds such as geosmin or 2-methylisoborneol (MIB), which have a pungent earthy or musty smell (Metcalf and Codd, 2012). However of greater concern is the capacity of cyanobacteria to produce a range of potent toxins. The effects of these cyanotoxins may well vary depending on toxin type, dosage, exposure route, in addition to the possible effects of size and age of the exposed individuals. Although many known and unknown toxic compounds exist in cyanobacteria and discovery and isolation of these continues, the following outlines some of the more commonly-found toxins produced by cyanobacteria.

35.2  Types of toxins produced by cyanobacteria 35.2.1 Hepatotoxins The hepatotoxins are considered to be among the most common cyanobacterial toxins, being comprised of two classes of cyclic peptides, the microcystins and nodularins. Microcystins (Fig. 35.1) can be produced by several genera of cyanobacteria including Microcystis, Planktothrix, and Dolichospermum, while nodularins (Fig. 35.2) are largely confined to the genus Nodularia (Metcalf and Codd, 2012). Microcystins, although only having seven amino acids in the peptide structure, are known to exist as at least 200 different congeners (Catherine et al., 2017). Variability at certain positions within the structure has provided the nomenclature, as demonstrated by microcystin-LR, one of the most common and toxic variants, that possesses the amino acids l-leucine (L) and l-arginine (R) at positions 2 and 4 of the molecule (Spoof and Catherine, 2017). Nodularins were originally characterized by Rinehart et al. (1988) and showed hepatotoxic effects similar to those of the microcystins, with the difference being that nodularins were comprised of only five amino acids. Nodularins have also Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00035-8 © 2020 Elsevier Inc. All rights reserved.

561

562  PART |IX  Algal toxicology

H

O

CH3

COOH

N

N

N H3C H

OCH3

O

H

H3C N

H3C

H CH2

O

H

H

H

CH3

CH3

X

N

Y H

O

O

COOH

FIG. 35.1  Microcystin generic structure where X and Y represent variable amino acids at positions 2 and 4.

CH3O

N

N H3C

H

H2N

CH3

CH3

N

H

H

COOH

O

H3C

CH3

H

N

O O NH

N

O

CH3

O

H

COOH

C NH

FIG. 35.2  Nodularin.

shown modifications to their structure with 10 naturally occurring variants known (Spoof and Catherine, 2017). These cyclic peptides are capable of inhibiting protein phosphatases and phosphoprotein phosphatases (Ward et al., 1997; Hastie et al., 2005), essential mammalian enzymes, and can also interfere with the liver cytoskeleton, resulting in adverse effects to the liver macrostructure with death possible by hypovolaemic shock (Carmichael, 1994; Metcalf et al., 2001). Research on low dose exposure to these toxins has shown that tumor promotion (microcystins) and carcinogenic (nodularins) effects may be possible (Nishiwaki-Matsushima et al., 1992; Ohta et al., 1994; Ueno et al., 1996; Svircev et al., 2013).

35.2.2 Cytotoxins Cylindrospermopsin (Fig. 35.3), a guanidine alkaloid, was first characterized after investigations of an outbreak of human hepatic enteritis in Australia. The drinking water supply was identified as the source of the illness, and ultimately a toxic strain of the cyanobacterium Cylindrospermopsis raciborskii was isolated from these waters that produced cylindrospermopsin (Hawkins et al., 1985; Terao et al., 1994).

OH –

O3SO

H

H

N

NH

O

HN

NH

H3C H FIG. 35.3  Cylindrospermopsin.

NH

O



Cyanobacterial toxins and their effects on human and animal health Chapter | 35  563

Toxicology has revealed hepatotoxic and genotoxic effects of this alkaloid (Kokocinski et  al., 2017 and references therein) and its interaction with the DNA double helix indicates potential carcinogenic effects (Falconer and Humpage, 2001). Inhibition of protein synthesis in plants and animals has been documented (Metcalf et al., 2004; Terao et al., 1994) with the liver, kidneys, lungs and intestines of mammals being susceptible (Seawright et al., 1999). Since its discovery, other genera of cyanobacteria have been reported to produce cylindrospermopsin worldwide, including Dolichospermum and Chrysosporum (Metcalf and Codd, 2012). In addition further variants of cylindrospermopsin such as 7-epicylindrospermopsin, 7-deoxy-desulfo-cylindrospermopsin, 7-deoxy-desulfo-12-acetylcylindrospermopsin and deoxycylindrospermopsin have been isolated (Banker et al., 1997, 2001; Norris et al., 1999; Wimmer et al., 2014).

35.2.3 Neurotoxins 35.2.3.1 Anatoxin-a Anatoxin-a (Fig. 35.4) was originally isolated from the genus Dolichospermum (formerly Anabaena; Carmichael et al., 1975) and since then several other genera of cyanobacteria (e.g., Phormidium, Cylindrospermum, and Aphanizomenon) have been shown to produce this toxin (Metcalf and Codd, 2012). Three structural variants, homoanatoxin-a, 4-­hydroxyhomoanatoxin-a and 11-carboxyl anatoxin-a, and several breakdown products have been documented (Devlin et al., 1977; Carmichael et al., 1979; Carmichael, 1994; James et al., 1998; Bruno et al., 2017). These neurotoxic alkaloids mimic acetylcholine, binding to nicotinic acetylcholine receptors at the neuromuscular junction causing continuous stimulation (Devlin et al., 1977; Carmichael et al., 1979; Carmichael, 1994). In high doses it can cause death as a result of the paralysis of muscles that control breathing. Currently, it is believed that there are no known long-term adverse health effects from exposure to anatoxin-a (Bruno et al., 2017).

35.2.3.2 Anatoxin-a(S) Anatoxin-a(S) (Fig. 35.5) is a naturally occurring organophosphate, toxicologically similar to synthetic organophosphate pesticides and insecticides (Mahmood and Carmichael, 1986), capable of inhibiting acetylcholine esterases and leading to paralysis and death in sufficiently high doses (Mahmood and Carmichael, 1986, Cook et al., 1989; Carmichael, 1994; Henriksen et al., 1997). As anatoxin-a(S) is an inhibitor of AChE’s, genetic engineering of these enzymes has led to detection systems that are able to distinguish anatoxin-a(S) from synthetic organophosphorous compounds (Devic et al., 2002). Although mainly found in aquatic environments with the genus Dolichospermum being the major producer, the presence of this toxin has also recently been shown in cryptogamic crusts of desert environments, suspected of being produced by the genus Microcoleus (Richer et al., 2012; Metcalf et al., 2012).

35.2.3.3 Saxitoxins Saxitoxins (Fig. 35.6) are a group of potent toxic alkaloids (Schantz et al., 1957; Bordner et al., 1975) with over 20 structural variants reported (Ballot et  al., 2017). Known mainly as a product of dinoflagellates causing the syndrome PSP (Paralytic Shellfish Poisoning) as a result of ingestion of contaminated shellfish, this toxin can cause significant impacts on O +

NH2

CH3

FIG. 35.4  Anatoxin-a.

HN H2N+

FIG. 35.5  Anatoxin-a(S).

CH3

N

CH3

N



O

O

P O

CH3

564  PART |IX  Algal toxicology

R4

CH2

R1

H N

N

N+H2

H2N+

N

N

OH OH R2

R3

FIG. 35.6  Saxitoxin generic structure where R represents variable groups.

O

H3 C NH

OH NH2

FIG. 35.7  β-N-methylamino-l-alanine (BMAA).

marine shellfish fisheries. Cyanobacteria (e.g., Lyngbya, Aphanizomenon, Dolichospermum, and Cylindrospermopsis) are also capable of producing saxitoxins in freshwaters (Ballot et al., 2017; Metcalf and Codd, 2012). These toxic alkaloids are capable of blocking sodium channels resulting in paralysis and respiratory arrest in high doses (Carmichael, 1994; Ballot et al., 2017).

35.2.3.4 BMAA β-N-methylamino-l-alanine (BMAA; Fig. 35.7) is a non-protein amino acid that can act as a glutamate agonist at NMDA receptors (Weiss et al., 1989). In addition to excitotoxic effects, BMAA can also have further effects in mammals, including mis-incorporation into proteins in place of l-serine (Dunlop et al., 2013) and through chiral conversion from l- to d-BMAA in the central nervous system, with toxicity demonstrated at different receptors (Metcalf et al., 2017). Laboratory experiments in animals have shown that BMAA can cause neurofibrillary tangles, amyloid plaques and neurological deficits consistent with its association with human neurodegenerative diseases such as ALS and Alzheimer’s (Cox et al., 2016; Scott and Downing, 2018). BMAA has been identified in aquatic and terrestrial environments (Vega and Bell, 1967), produced by several genera of cyanobacteria worldwide (Cox et al., 2005, 2009; Metcalf et al., 2008).

35.2.4 Dermatotoxins Common cyanotoxins that have demonstrated dermatotoxic effects in mammals include lyngbyatoxins, aplysiatoxin and debromoaplysiatoxin. Lyngbyatoxins A, B, and C, are indole alkaloids produced by genera of benthic cyanobacteria (e.g., Moorea producens) that have been implicated in cases of human dermatitis as well as inflammation of oral and gastrointestinal tissues (Aimi et al., 1990; Cardellina et al., 1979). Aplysiatoxin and debromoaplysiatoxin are both phenolic bislactones that can cause skin irritation, rashes and blistering (Mynderse et al., 1977) and along with lyngbyatoxins are also considered to be skin tumor promoters (Fujiki et al., 1981, 1983).

35.2.4.1 LPS Cyanobacteria, being Gram negative bacteria have cell wall structures that possess lipopolysaccharide (LPS) (Drews and Weckesser, 1982). LPS acts as an endotoxin that can be toxic to mammals with the potential of causing gastroenteritis (Codd et al., 2005; Metcalf and Codd, 2012). LPS from cyanobacteria is not as toxic to mammals as other Gram negative bacterial LPS, such as from Salmonella typhimurium and Escherichia coli (Monteiro et al., 2017). Nevertherless, cyanobacterial blooms, may contain large amounts of cyanobacterial LPS and support other LPS-producing and disease-causing heterotrophic bacteria, such as Vibrio cholerae within the bloom (Islam et al., 1994).

35.3  Routes of exposure and poisoning incidents Although several animal and human illnesses have been attributed and associated with cyanobacterial blooms and/or their toxins, in many cases cyanobacteria may have initially been overlooked as the cause, with analysis occurring r­ etrospectively



Cyanobacterial toxins and their effects on human and animal health Chapter | 35  565

(Metcalf and Codd, 2012; Saker et al., 1999). Direct causation can be difficult as blooms may have died or dissipated, the toxins may have degraded or clinical samples may no longer be available for assessment. Understanding how animals and humans can be exposed to these toxins is crucial for assessing risks associated with cyanobacteria and for managing and implementing mitigation strategies (Codd et al., 2005).

35.3.1 Animals In 1878, Francis conducted an investigation into the deaths of sheep, which, he suspected, was caused by a cyanobacterial bloom (Francis, 1878). He experimentally dosed sheep with scum, identified as Nodularia, reproducing the previously observed poisoning symptoms. This was one of the first scientific experiments documenting the association of cyanobacteria with livestock deaths. Since then, reports of intoxications worldwide, including in the United States, Canada, Norway, and Qatar, have confirmed wild and domestic animal poisonings and/or deaths due to cyanotoxins (Pybus et al., 1986; Handeland and Ostensvik, 2010; Sivonen and Jones, 1999; Codd et al., 1992, 2005; Mez et al., 1997, Saker et al., 1999, Mahmood et al., 1988, Henriksen et al., 1997; Metcalf and Codd, 2012; Edwards et al., 1992; Wood et al., 2007; Chatziefthimiou et al., 2014). Fish kills have been reported and attributed to the direct effects of cyanotoxins (Rodger et al., 1994) and also indirectly due to depletion of oxygen in water (Townsend et al., 1992). When a bloom of cyanobacteria senesces after changes in environmental conditions and/or reduction in nutrients that support the bloom, heterotrophic bacteria undergo respiration during decay of this cyanobacterial organic material. This metabolic process leads to the consumption of oxygen and a decrease in its availability in the water column for other aquatic animals. Deaths of birds implicated with cyanotoxins, based on post-mortem investigations and analysis of bloom material are well documented worldwide (Metcalf and Codd, 2012; Krienitz et al., 2003). Bird deaths attributed to cyanotoxins include Chilean Flamingos, Greater Flamingo chicks, Lesser Flamingos and Dalmatian pelicans (Krienitz et al., 2005; AlonsoAndicoberry et al., 2002; Codd et al., 2003; Papadimitriou et al., 2018). At a Canadian lake in 1985, approximately 1000 bats and 24 ducks were reported to have died after exposure to a bloom of Anabaena. Bat and duck carcasses that had visible scum on their surface were sent for analysis and a toxic cyanobacterial alkaloid was identified (Pybus et al., 1986). Soll and Williams (1985) correlated the deaths of white rhinoceros (Ceratotherium simum) in South Africa to a reservoir located in the Barakologadi Game Reserve that supported a bloom of Microcystis. Analysis of one of the animal’s livers showed severe hepatomegaly indicating possible cyanobacterial intoxication from drinking water. Similarly, Van Halderen et al. (1996) correlated three episodes of sheep deaths to cyanobacteria (Nodularia and Microcystis) with liver analysis indicating necrosis and severe hemorrhage. Thomas et al. (1998) reported the deaths of cattle in north Queensland relating it to a toxic strain of Cylindrospermopsis raciborskii. Histopathological analysis of liver was indicative of hepatotoxin poisoning. Cattle have also succumbed to the action of microcystins as evidenced by deaths at Scottish lochs and in alpine pastures (Lawton et al., 1995; Mez et al., 1997). The odor of geosmin and MIB is distinctive and can be particularly attractive to certain animals, including dogs (Codd et al., 1992). Exposures and intoxications can occur due to ingestion of mats/scums of toxic cyanobacteria (e.g., Phormidium), drinking of water containing a bloom and also by self-cleaning their fur after immersion. Several reports have documented poisonings or deaths of dogs exposed to cyanobacteria (Edwards et al., 1992; Cadel-Six et al., 2007; Wood et al., 2007; Gugger et al., 2005; Puschner et al., 2008; Fastner et al., 2018). Backer et al. (2013) reported 115 poisoning events that occurred in the United States from the late 1920s to mid-2012. Many of the cyanobacterial genera involved were Dolichospermum, Microcystis, Lyngbya, Aphanizomenon and the toxins included anatoxin-a and microcystins. Dermatitis in dogs has also been reported and attributed to cyanobacteria (Puschner et al., 2017). Dog poisoning and deaths due to suspected cyanobacterial neurotoxins have also been reported in desert environments (Cox et al., 2009; Metcalf et al., 2012; Chatziefthimiou et al., 2014). This is largely because cyanobacteria in desert environments such as those in Qatar are present as crusts and can cover up to 87% of the land surface (Richer et al., 2012). When rains occur, then such crusts can flourish and produce toxins in standing pools that the dogs then consume accidentally (Chatziefthimiou et al., 2014).

35.3.2 Humans One of the earliest noticeable reports of human incidents associated with cyanobacteria occurred during the 1930s in Charleston, WV. Gastrointestinal symptoms were reported and could not be attributed to any infectious organism other than

566  PART |IX  Algal toxicology

an association of a heavy bloom of cyanobacteria that was present in a branch of the Ohio River which served as a water source for the city (Miller and Tisdale, 1931; Veldee, 1931). Cases of cyanobacterial poisoning have been reported and can range from skin rashes to fatalities (Aimi et al., 1990; Azevedo et al., 2002) and multiple exposure routes are possible—recreation, aerosols/inhalation, ingestion of water, food and dietary supplements, and medicinal water use (Codd et al., 2005).

35.3.2.1 Water Recreational exposure Cyanobacterial blooms often occur and intensify during summer periods with warmer temperatures and longer hours of sunlight. This is also the time when human outdoor activities increase, often involving water, with long periods of recreation (e.g., sunbathing) and possible unintentional drinking of water (e.g., through swimming, jet skiing). Cases of dermatitis and flu-like symptoms have been reported after recreation in waters containing cyanobacteria (Aimi et al., 1990; Pilotto et al., 1997; Osborne and Shaw, 2008). Such incidents have been reported in Canada, Sweden, Finland and the United States, with symptoms including diarrhea, fever, muscular pain, vomiting, and pneumonia (Annadotter et al., 2001; Giannuzzi et al., 2011). In the United Kingdom soldiers undergoing kayak training in water containing a bloom of Microcystis developed symptoms of sore throat, headaches, diarrhea and vomiting with two of the soldiers developing severe pneumonia and hospitalization (Turner et al., 1990). In Uruguay, Vidal et al. (2017) reported the case of a 20 month-old baby that had severe illness and liver failure after exposure at a lake containing a bloom of Microcystis. Ultimately, a liver transplant was required and the presence of MC-LR and a d-Leucine variant were identified in the clinical materials. Drinking water Cyanotoxins have been reported in drinking water supplies worldwide (e.g., Molica et al., 2005; Mhlanga et al., 2006; Chatziefthimiou et al., 2016; Gaget et al., 2017). Consuming contaminated drinking water presents another exposure route with reported incidents dating back to at least 1931 (Miller and Tisdale, 1931; Veldee, 1931). Although often not fatal, symptoms of gastroenteritis have been associated with the use of drinking water contaminated by cyanobacteria (Teixeira et al., 1993; Cronberg et al., 1995). A high-profile incident occurred in 1979 in Palm Island (Australia) where more than 100 people suffered from bloody diarrhea and vomiting after drinking water from Solomon Dam (Byth, 1980). Later investigations of the waterbody isolated and identified a strain of Cylindrospermopsis raciborskii and its toxic metabolite cylindrospermopsin (Hawkins et al., 1985; Ohtani et al., 1992). In Toledo, Ohio, in 2014, closure of the drinking water supply and restrictions to water use were applied due to a bloom of microcystin-containing Microcystis present in Lake Erie that affected the water treatment facility (Pelley, 2016). In addition to health effects from short-term exposure, long-term exposure to cyanobacteria and cyanotoxins may adversely affect human health. Correlating primary liver cancer with drinking water sources that support frequent blooms of cyanobacteria indicates that waterbodies with cyanobacteria are positively associated with increased risk of the disease, consistent with microcystins being considered tumor promoters (Yu, 1995; Ueno et al., 1996). Aerosol Inhalation of aerosols and airborne particles comprised of, or containing cyanobacteria have been reported and constitute an under-researched route of exposure. Cyanotoxins such as microcystins have been detected at lakeside and in laboratory experiments using air-sampling devices (Cheng et al., 2007; Backer et al., 2008, 2010). During a cyanobacterial bloom event in Florida in 2016, four counties declared a state of emergency due to the severity of the bloom. Residents that came into contact with the aerosol from the bloom complained of flu-like symptoms, headaches, eye irritation, respiratory issues, and even rashes (Lantigua, 2017; Metcalf et al., 2018). Analysis of ALS patients has indicated that exposure to BMAA may result in the development of neurological disease (Murch et al., 2004; Cox et al., 2016). Assessment of ALS patient residences in New Hampshire found clusters of patients living around lakes that supported cyanobacterial blooms, and assessment of air filters from such lakes found the cyanotoxin BMAA and its isomers to be present (Caller et al., 2009; Henegan et al., 2017). Medical procedures Although less frequently encountered, adverse health incidents due to the use of contaminated water in medical procedures have been reported. Hindman et al. (1975) investigated a case in which 49 patients undergoing hemodialysis presented



Cyanobacterial toxins and their effects on human and animal health Chapter | 35  567

pyrogenic reactions. Their findings indicated that these reactions were likely due to endotoxin contamination of the water used for preparing dialysate, possibly caused by an increase in algal numbers and associated bacteria. The need for water to use in such medical procedures emphasizes the importance of having water that is cyanobacteria and/or cyanotoxin free. When waters are not monitored or treated effectively, then human fatalities can result. This was demonstrated in 1996, in Caruaru, Brazil, where 100 hemodialysis patients died of liver failure, and 52 of these patients showed an association with cyanotoxins. The water used for the procedures was taken from a reservoir containing a bloom of cyanobacteria that was ultimately administered to people after insufficient/inappropriate procedures to remove the cyanotoxins (Jochimsen et al., 1998; Pouria et al., 1998; Carmichael et al., 2001; Azevedo et al., 2002).

35.3.2.2 Food Cyanotoxins, including the hepatotoxic, neurotoxic and cytotoxic groups, have been identified in various trophic levels (Codd et al., 1999; Kittler et al., 2012; Berry et al., 2011; Magalhaes et al., 2001; Sipia et al., 2007; Al-Sammak et al., 2014) with many of the organisms being used as food sources (see Testai et al., 2016). Codd et al. (1999) investigated accidentally contaminated lettuce after spray irrigation of the crops and found Microcystis aeruginosa and microcystins in the salad lettuce, making it unfit for human consumption. Similar laboratory findings were reported by Kittler et al. (2012) confirming cylindrospermopsin in the tissues of Brassica crop plants. Marine foods such as fish and molluscs such as mussels can also be contaminated with cyanobacterial filaments and colonies containing cyanotoxins (Sipia et al., 2007; Magalhaes et al., 2001). Dietary supplements are potentially a health concern as they may contain cyanotoxins. Microcystins, anatoxin-a and BMAA have been detected in commercial food supplements (Bruno et al., 2006; Bautista et al., 2015; Glover et al., 2015; Roy-Lachapelle et al., 2017), which have led to the implementation of legislation in certain places, such as in the State of Oregon, that has microcystin guidelines of 1 μg/g in cyanobacterial supplements (Dietrich and Hoeger, 2005).

35.3.2.3 Air The natural phenomenon of dust storms in desert environment can be another possible exposure route for cyanobacteria and their toxins (Chatziefthimiou et al., 2015). Evaluating the amount of microcystins present in desert crust and comparing it with the amount of dust that can potentially be inhaled during a dust storm alongside known toxicity data indicated that there could be a risk (Metcalf et al., 2012). Furthermore, with respect to BMAA, Horner et al. (2003) found that military personnel deployed to the Persian Gulf during Operation Desert Storm had an increased risk of developing ALS than those who underwent the same training but were not deployed, which may have been associated with the presence of BMAA in desert crust material (Cox et al., 2009).

35.4  Bioassays for cyanobacterial toxins As the number of cyanobacterial toxins with differing toxicities in environmental populations of cyanobacteria can be extremely large, such as with the microcystins, then assessment of the toxicity of a cyanobacterial bloom may be necessary. Although there are many ways to determine the presence and concentration of cyanotoxins and the genes related to their production (Metcalf and Codd, 2012), toxicity assessment generally requires some form of bioassay to be applied.

35.4.1  Small animal After the initial work of Francis (1878) where sheep were shown to be susceptible to the action of Nodularia scum, mammals have been used to determine the potential adverse risk that cyanobacteria and their toxins may pose to health. This has largely been accomplished through the use of small animal bioassays. Using mice or rats, extracts of cyanobacterial blooms, strains or purified compounds can be assessed for toxicity (Blaha et al., 2017). Furthermore, based upon careful analysis of organs of these experimentally-dosed animals, toxicological targets have been determined, such as the liver in the case of microcystins, that provide some specificity of the nature of the toxicant in unknown animal poisonings. Other animals used to assess cyanobacterial toxins include pigs (Falconer et al., 1994) and fish to determine what risk cyanobacterial toxins may pose to humans and aquatic organisms that live with cyanobacterial blooms, respectively. Certainly, in the case of the dosing pigs with Microcystis scum (Falconer et al., 1994), along with assessment of other cyanobacterial toxins has led to the derivation and introduction of Guideline Values (GVs) for some cyanobacterial toxins (Ibelings et al., 2014). Although small animals have proven useful for the assessment of cyanobacterial toxins, whether present in extracts or as purified compounds, due, in part to ethical considerations, these are being replaced by alternative bioassays. Such

568  PART |IX  Algal toxicology

b­ ioassays, often use small organisms such as early life stages of crustaceans (e.g., Artemia salina) or fish (e.g., Danio rerio). Their smaller size and the ability to use larger numbers of organisms during toxicity assessment provides greater sensitivity and statistical power to help understand the risk of exposure to these toxic compounds (Blaha et al., 2017). Assessment of the risk will depend upon the information required by the end user. The bioassay may be required for the assessment of chronic versus acute toxicity. Other scenarios may include assessment of aquatic organisms that may be affected by cyanobacterial toxins, whether through mortality or effects on reproduction, as examples.

35.4.2  Enzyme and molecular systems Although small animal bioassays have been useful for the assessment of known and unknown compounds, once the nature of the toxin is understood, then molecular targets can be isolated for adaptation and development as in vitro detection systems. Such systems can be purified or produced as genetically modified systems to detect the target compound, either for increasing specificity and/or sensitivity. Enzyme inhibition systems can be used as a surrogate for bioassays when the specific toxicant is known. Enzyme inhibition systems are known for microcystins (protein phosphatase inhibition assays; PPIA; Ward et al., 1997), cylindrospermopsin (protein translation assays; Froscio et al., 2001) and anatoxin-a(S) (acetylcholine esterase inhibition assays; Metcalf et al., 2012) as examples. Often, radioactive or colorimetric substrates can be used to detect the nature of the toxicant present. Furthermore, where more than one class of compound may inhibit a particular enzyme, then antibodies can be used to separate the various classes of toxins, as evidenced by microcystin antibodies with PPIA where the cyclic hepatotoxins were distinguished from okadaic acid, calyculin A and tautomycin (Metcalf et al., 2001). In addition, the use of such enzyme assays where specific toxicants are known can be applied to the field, along with or in addition to field ELISA kits for known cyanotoxins. When analyzing known cyanotoxins using methods such as mass spectrometry, unknown compounds with toxic potential may also be present alongside co-occurring cyanotoxins. This scenario demonstrates that bioassays still have a considerably important place alongside analytical methods for cyanotoxins.

35.5  Detoxication, metabolism and deposition The need to protect people from cyanobacterial toxins has led to the introduction of Guideline Values (GVs) for bathing, recreational and drinking water by the World Health Organization (Ibelings et al., 2014). Long-term exposure to cyanobacteria and their toxins continues to attract attention, and evidence links exposures to these organisms with diseases such as neurodegeneration and some cancers. In the case of heterotrophic bacteria, metabolism of cyanotoxins is known to occur (e.g., converting microcystins into non-toxic derivatives in a matter of days to weeks; Bourne et al., 1996). In mammalian systems, metabolic systems can degrade microcystins and cylindrospermopsin using glutathione-S-transferases (Metcalf et al., 2000) and cytochrome P450 (Norris et al., 2002), respectively. The products of these detoxication systems result in lower toxicity compounds that can then be removed or excreted from the body. Organisms that have more of a symbiotic relationship with cyanobacteria are capable of carrying out other processes with cyanobacterial toxins. This has been highlighted by species such as the Lesser Flamingo, which although undergo periodic mass mortalities from the action of cyanobacterial toxins in acute doses, are capable of depositing these toxins within keratinous tissues, such as their feathers alongside the carotenoid pigments obtained from these photosynthetic organisms (Metcalf et  al., 2006, 2013). Other bird species which indirectly consume cyanobacterial toxins, such as eider ducks, through nodularin-containing mussels, are also capable of depositing these toxins within their feathers (Sipia et al., 2008). Ultimately, the health status of the organism that is being exposed to cyanobacterial toxins may have a bearing on the ultimate toxicological outcome. When healthy and if in sub-acute doses, many organisms are capable of detoxifying or removing cyanobacterial toxins. The introduction of GVs for cyanobacterial toxins will most likely allow these metabolic pathways to remain active to protect human and animal health.

35.6 Conclusion Eutrophication of water bodies, anthropogenic or naturally occurring, combined with climate change can lead to an increase in algal blooms as well as the expansion of certain sub-tropical species, such as Cylindrospermopsis, to temperate regions (Anderson et al., 2012; Paerl and Huisman, 2008, 2009; Paerl and Paul, 2012, Sinha et al., 2012). As human populations grow, the pressure on water resources will also increase, along with the likely potential of exposure to cyanobacteria. In order to reduce exposure risks, safety and preventive strategies need to be implemented such as: 1. The monitoring and surveillance of waterbodies, especially the ones used as drinking water sources and/or with high recreational activities (Hilborn and Beasley, 2015; Figgatt et al., 2017).



Cyanobacterial toxins and their effects on human and animal health Chapter | 35  569

2. Displaying information and warning signs about blooms. 3. Communication with appropriate authorities and awareness of multidisciplinary fields of professions (e.g., medical field; Vidal et al., 2017; Hilborn and Beasley, 2015; Metcalf et al., 2018). 4. Use of satellite imagery and remote sensing (Wynne and Stumpf, 2015; Recknagel et al., 2018; Hunter et al., 2017). 5. Appropriate water treatment strategies (e.g., activated carbon) and research (Cheung et  al., 2013; He et  al., 2016; Simmons, 1998). 6. Guidelines and legislation for drinking and recreational waters (Metcalf et al., 2018). Ultimately, reducing pollution, eutrophication and the responsible use of water supplies may reduce the frequency and intensity of cyanobacterial blooms to the point where humans and animals can safely enjoy such water resources. This study contributes to the wider research on the science, technology, and medicine of the algae (Konur, 2020a,b,c,d, e,f,g,h,i,j,k,l,m,n,o).

References Aimi, N., Odaka, H., Sakai, S., Fujiki, H., Suganuma, M., Moore, R.E., et al., 1990. Lyngbyatoxins B and C, two new irritants from Lyngbya majuscula. J. Nat. Prod. 53 (6), 1593–1596. Alonso-Andicoberry, C., Garcia-Villada, L., Lopez-Rodas, V., Costas, E., 2002. Catastrophic mortality of flamingos in a Spanish national park caused by cyanobacteria. Vet. Rec. 151 (23), 706–707. Al-Sammak, M.A., Hoagland, K.D., Cassada, D., Snow, D.D., 2014. Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-A in Nebraska reservoirs, fish, and aquatic plants. Toxins 6 (2), 488–508. Anderson, D.M., Cembella, A.D., Hallegraeff, G.M., 2012. Progress in understanding harmful algal blooms: paradigm shifts and new technologies for research, monitoring, and management. Ann. Rev. Mar. Sci. 4, 143–176. Annadotter, H., Cronberg, G., Lawton, L.A., Hansson, H.B., Gothe, U., Skulberg, O.M., 2001. An extensive outbreak of gastroenteritis associated with the toxic cyanobacterium Planktothrix agardhii (oscillatoriales, cyanophyceae) in Scania, South Sweden. In: Chorus, I. (Ed.), Cyanotoxins—Occurrence, Causes, Consequences. Springer, Berlin, pp. 200–208. Azevedo, S.M.F.O., Carmichael, W.W., Jochimsen, E.M., Rinehard, K.L., Lau, S., Shaw, G.R., et al., 2002. Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil. Toxicology 181–182, 441–446. Backer, L.C., Carmichael, W., Kirkpatrick, B., Williams, C., Irvin, M., Zhou, Y., et al., 2008. Recreational exposure to low concentrations of microcystins during an algal bloom in a small lake. Mar. Drugs 6 (2), 389–406. Backer, L.C., McNeel, S.V., Barber, T., Kirkpatrick, B., Williams, C., Irvin, M., et al., 2010. Recreational exposure to microcystins during algal blooms in two California lakes. Toxicon 55 (5), 909–921. Backer, L.C., Landsberg, J.H., Miller, M., Keel, K., Taylor, T.K., 2013. Canine cyanotoxin poisonings in the United States (1920s-2012): review of suspected and confirmed cases from three data sources. Toxins 5 (9), 1597–1628. Ballot, A., Bernard, C., Fastner, J., 2017. Saxitoxins and analogues. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 148–154. Banker, R., Carmeli, S., Hadas, O., Teltsch, B., Porat, R., Sukenik, A., 1997. Identification of cylindrospermopsin in Aphanizomenon ovalisporum (cyanophyceae) isolated from lake Kinneret, Israel. J. Phycol. 33 (4), 613–616. Banker, R., Carmeli, S., Werman, M., Teltsch, B., Porat, R., Sukenik, A., 2001. Uracil moiety is required for toxicity of the cyanobacterial hepatotoxin cylindrospermopsin. J. Toxicol. Environ. Health A 62 (4), 281–288. Bautista, A.C., Moore, C.E., Lin, Y., Cline, M.G., Benitah, N., Puschner, B., 2015. Hepatopathy following consumption of a commercially available bluegreen algae dietary supplement in a dog. BMC Vet. Res. 11, 136. Berry, J.P., Lee, E., Walton, K., Wilson, A.E., Bernal-Brooks, F., 2011. Bioaccumulation of microcystins by fish associated with a persistent cyanobacterial bloom in Lago de Patzcuaro (Michoacan, Mexico). Environ. Toxicol. Chem. 30 (7), 1621–1628. Blaha, L., Camean, A.M., Fessard, V., Gutierrez-Praena, D., Jos, A., Marie, B., et al., 2017. Bioassay use in the field of toxic cyanobacteria. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 272–279. Bordner, J., Thiessen, W.E., Bates, H.A., Rapoport, H., 1975. The structure of a crystalline derivative of saxitoxin. The structure of saxitoxin. J. Am. Chem. Soc. 97 (21), 6008–6012. Bourne, D.G., Jones, G.J., Blakeley, R.L., Jones, A., Negri, A.P., Riddles, P., 1996. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. Appl. Environ. Microbiol. 62 (11), 4086–4094. Bruno, M., Fiori, M., Mattei, D., Melchiorre, S., Messineo, V., Volpi, F., et al., 2006. ELISA and LC-MS/MS methods for determining cyanobacterial toxins in blue-green algae food supplements. Nat. Prod. Res. 20 (9), 827–834. Bruno, M., Ploux, O., Metcalf, J.S., Mejean, A., Pawlik-Skowronska, B., Furey, A., 2017. Anatoxin-A, homoanatoxin-A, and natural analogues. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 138–147. Byth, S., 1980. Palm Island mystery disease. Med. J. Aust. 2 (1), 40–42. Cadel-Six, S., Peyraud-Thomas, C., Brient, L., de Marsac, N.T., Rippka, R., Mejean, A., 2007. Different genotypes of anatoxin-producing cyanobacteria coexist in the Tarn River, France. Appl. Environ. Microbiol. 73 (23), 7605–7614.

570  PART |IX  Algal toxicology

Caller, T.A., Doolin, J.W., Haney, J.F., Murby, A.J., West, K.G., Farrar, H.E., et al., 2009. A cluster of amyotrophic lateral sclerosis in New Hampshire: a possible role for toxic cyanobacteria blooms. Amyotroph. Lateral Scler. 10 (S2), 101–108. Cardellina, J.H., Marner, F.J., Moore, R.E., 1979. Seaweed dermatitis: structure of lyngbyatoxin A. Science 204 (4389), 193–195. Cardemil, L., Wolk, C.P., 1979. The polysaccharides from heterocyst and spore envelopes of a blue-green alga. Structure of the basic repeating unit. J. Biol. Chem. 254 (3), 736–741. Carmichael, W.W., 1994. The toxins of cyanobacteria. Sci. Am. 270 (1), 78–86. Carmichael, W.W., Biggs, D.F., Gorham, P.R., 1975. Toxicology and pharmacological action of Anabaena flos-aquae toxin. Science 187 (4176), 542–544. Carmichael, W.W., Biggs, D.F., Peterson, M.A., 1979. Pharmacology of anatoxin-a, produced by the freshwater cyanophyte Anabaena flos-aquae NRC44-1. Toxicon 17 (3), 229–236. Carmichael, W.W., Azevedo, S.M.F.O., An, J.S., Molica, R.J.R., Jochimsen, E.M., Lau, S., et al., 2001. Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ. Health Perspect. 109 (7), 663–668. Catherine, A., Bernard, C., Spoof, L., Bruno, M., 2017. Microcystins and nodularins. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 109–126. Chatziefthimiou, A.D., Richer, R., Rowles, H., Powell, J.T., Metcalf, J.S., 2014. Cyanotoxins as a potential cause of dog poisonings in desert environments. Vet. Rec. 174 (19), 484–485. Chatziefthimiou, A.D., Metcalf, J.S., Powell, J.T., Glover, W.B., Bannack, S., Cox, P., Richer, R., 2015. One Health: the case of human exposure to cyanobacteria toxins in natural and built environments. In: Qatar Green Building Conference 2015-the Vision, 27-28 April, Qatar. Chatziefthimiou, A.D., Metcalf, J.S., Glover, W.B., Banack, A.A., Dargham, S.R., Richer, R.A., 2016. Cyanobacteria and cyanotoxins are present in drinking water impoundments and groundwater wells in desert environments. Toxicon 114, 75–84. Cheng, Y.S., Zhou, Y., Irvin, C.M., Kirkpatrick, B., Backer, L.C., 2007. Characterization of aerosols containing microcystin. Mar. Drugs 5 (4), 136–150. Cheung, M., Liang, S., Lee, J., 2013. Toxin-producing cyanobacteria in freshwater: a review of the problems, impact on drinking water safety, and efforts for protecting public health. J. Microbiol. 51 (1), 1–10. Codd, G.A., Edwards, C., Beattie, K.A., 1992. Fatal attraction to cyanobacteria? Nature 359 (6391), 110–111. Codd, G.A., Metcalf, J.S., Beattie, K.A., 1999. Retention of Microcystis aeruginosa and microcystin by salad lettuce (Lactuca sativa) after spray irrigation with water containing cyanobacteria. Toxicon 37 (8), 1181–1185. Codd, G.A., Metcalf, J.S., Morrison, L.F., Krienitz, L., Ballot, A., Pflugmacher, S., et al., 2003. Susceptibility of flamingos to cyanobacterial toxins via feeding. Vet. Rec. 152 (23), 722–723. Codd, G.A., Lindsay, J., Young, F.M., Morrison, L.F., Metcalf, J.S., 2005. Harmful cyanobacteria: from mass mortalities to management measures. In: Huisman, J., Matthijs, H.C.P., Visser, P.M. (Eds.), Harmful Cyanobacteria. Springer, Berlin, pp. 1–23. Cook, W.O., Beasley, V.R., Lovell, R.A., Dahlem, A.M., Hooser, S.B., Mahmood, N.A., 1989. Consistent inhibition of peripheral cholinesterases by neurotoxins from the freshwater cyanobacterium Anabaena flos-aquae: studies of ducks, swine, mice and a steer. Environ. Toxicol. Chem. 8 (10), 915–922. Cox, P.A., Banack, S.A., Murch, S.J., Rasmussen, U., Tien, G., Bidigare, R.R., et al., 2005. Diverse taxa of cyanobacteria produce β-N-methylamino-lalanine, a neurotoxic amino acid. Proc. Natl. Acad. Sci. USA 102 (14), 5074–5078. Cox, P.A., Richer, R., Metcalf, J.S., Banack, S.A., Codd, G.A., Bradley, W.G., 2009. Cyanobacteria and BMAA exposure from desert dust: a possible link to sporadic ALS among Gulf War veterans. Amyotroph. Lateral Scler. 10 (S2), 109–117. Cox, P.A., Davis, D.A., Mash, D.C., Metcalf, J.S., Banack, S.A., 2016. Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain. Proc. R. Soc. B Biol. Sci. 283 (1823), 2015–2397. Cronberg, G., Annadotter, H., Lawton, L.A., Hansson, H.-B., Gothe, U., Skulberg, O.M., 1995. A large outbreak of gastroenteritis associated with the toxic cyanobacteria Planktothrix (Oscillatoria) agardhii. In: 1st International Symposium on Toxic Cyanobacteria, 20-24 August 1995, Ronne, Bornholm, Denmark. Devic, E., Li, D., Dauta, A., Henriksen, P., Codd, G.A., Marty, J.L., et al., 2002. Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases. Appl. Environ. Microbiol. 68 (8), 4102–4106. Devlin, J.P., Edwards, O.E., Gorham, P.R., Hunter, N.R., Pike, R.K., Stavric, B., 1977. Anatoxin-a, a toxic alkaloid from Anabaena flos-aquae NRC-44h. Can. J. Chem. 55 (8), 1367–1371. Dietrich, D., Hoeger, S., 2005. Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): a reasonable or misguided approach? Toxicol. Appl. Pharmacol. 203 (3), 273–289. Drews, G., Weckesser, J., 1982. Function, structure and composition of cell wall and external layers. In: Barr, N.G., Whitton, B.A. (Eds.), The Biology of Cyanobacteria. Blackwell Scientific Publications, Oxford, pp. 333–357. Dunlop, R.A., Cox, P.A., Banack, S.A., Rodgers, K.J., 2013. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation. PLoS One 8 (9), e75376. Edwards, C., Beattie, K.A., Scrimgeour, C.M., Codd, G.A., 1992. Identification of anatoxin-A in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh. Scotland. Toxicon 30 (10), 1165–1175. Falconer, I.R., Humpage, A.R., 2001. Preliminary evidence for in  vivo tumour initiation by oral administration of extracts of the blue-green alga Cylindrospermopsis raciborskii containing the toxin cylindrospermopsin. Environ. Toxicol. 16 (2), 192–195. Falconer, I.R., Burch, M.D., Steffensen, D.A., Choice, M., Coverdale, O.R., 1994. Toxicity of the blue-green alga (cyanobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal model for human injury and risk assessment. Environ. Toxicol. 9 (2), 131–139. Fastner, J., Beulker, C., Geiser, B., Hoffmann, A., Kroger, R., Teske, K., et al., 2018. Fatal neurotoxicosis in dogs associated with tychoplanktic, anatoxin-a producing Tychonema sp. in mesotrophic lake Tegel, Berlin. Toxins 10 (2), e60.



Cyanobacterial toxins and their effects on human and animal health Chapter | 35  571

Figgatt, M., Hyde, J., Dziewulski, D., Wiegert, E., Kishbaugh, S., Zelin, G., et al., 2017. Harmful algal bloom-associated illnesses in humans and dogs identified through a pilot surveillance system—New York, 2015. MMWR Morb. Mortal. Wkly Rep. 66 (43), 1182–1184. Fogg, G.E., Stewart, W.D.P., Fay, P., Walsby, A.E., 1973. The Blue-Green Algae. Academic Press, London. Francis, G., 1878. Poisonous Australian lake. Nature 18, 11–12. Froscio, S.M., Humpage, A.R., Burcham, P.C., Falconer, I.R., 2001. Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin. Environ. Toxicol. 16 (5), 408–412. Fujiki, H., Mori, M., Nakayasu, M., Terada, M., Sugimura, T., Moore, R.E., 1981. Indole alkaloids: dihydroteleocidin B, teleocidin, and lyngbyatoxin A as members of a new class of tumor promoters. Proc. Natl. Acad. Sci. USA 78 (6), 3872–3876. Fujiki, H., Sugimura, T., Moore, R.E., 1983. New classes of environmental tumor promoters: indole alkaloids and polyacetates. Environ. Health Perspect. 50, 85–90. Gaget, V., Humpage, A.R., Huang, Q., Monis, P., Brookes, J.D., 2017. Benthic cyanobacteria: a source of cylindrospermopsin and microcystin in Australian drinking water reservoirs. Water Res. 124, 454–464. Giannuzzi, L., Sedan, D., Echenique, R., Andrinolo, D., 2011. An acute case of intoxication with cyanobacteria and cyanotoxins in recreational water in Salto Grande Dam, Argentina. Mar. Drugs 9 (11), 2164–2175. Glover, W.B., Baker, T.C., Murch, S.J., Brown, P.N., 2015. Determination of β-N-methylamino-L-alanine, N-(2-aminoethyl)glycine, and 2,4-­diaminobutyric acid in food products containing cyanobacteria by ultra-performance liquid chromatography and tandem mass spectrometry: single-laboratory validation. J. AOAC Int. 98 (6), 1559–1565. Gugger, M., Lenoir, S., Berger, C., Ledreux, A., Druart, J.C., Humbert, J.F., et al., 2005. First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin—a associated with dog neurotoxicosis. Toxicon 45 (7), 919–928. Handeland, K., Ostensvik, O., 2010. Microcystin poisoning in roe deer (Capreolus capreolus). Toxicon 56 (6), 1076–1078. Hastie, C.J., Borthwick, E.B., Morrison, L.F., Codd, G.A., Cohen, P.T.W., 2005. Inhibition of several protein phosphatases by a non-covalently interacting microcystin and a novel cyanobacterial peptide, nostocyclin. Biochim. Biophys. Acta 1726 (2), 187–193. Hawkins, P.R., Runnegar, M.T., Jackson, A.R., Falconer, I.R., 1985. Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir. Appl. Environ. Microbiol. 50 (5), 1292–1295. He, X., Liu, Y.L., Conklin, A., Westrick, J., Weavers, L.K., Dionysiou, D.D., et al., 2016. Toxic cyanobacteria and drinking water: impacts, detection and treatment. Harmful Algae 54, 174–193. Henegan, P., Andrew, A., Kuczmarski, T., Michaelson, N., Storm, J., Atkinson, A., et al., 2017. Aerosol exposure to cyanobacteria as a potential risk factor for neurological disease (P5.086). Neurology 88 (16S), P5.086. Henriksen, P., Carmichael, W.W., An, J.S., Moestrup, O., 1997. Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds. Toxicon 35 (6), 901–913. Hilborn, E.D., Beasley, V.R., 2015. One health and cyanobacteria in freshwater systems: animal illnesses and deaths are sentinel events for human health risks. Toxins 7 (4), 1374–1395. Hindman, H.S., Carson, L.A., Favero, M.S., Petersen, N.J., Schonberger, L.B., Solano, J.T., 1975. Pyrogenic reactions during haemodialysis caused by extramural endotoxin. Lancet 306 (7938), 732–734. Horner, R.D., Kamins, K.G., Feussner, J.R., Grambow, S.C., Hoff-Lundquist, J., Harati, Y., et al., 2003. Occurrence of amyotrophic lateral sclerosis among Gulf War veterans. Neurology 61, 742–749. Humbert, J.F., Fastner, J., 2017. Ecology of cyanobacteria. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 9–18. Hunter, P.D., Matthews, M.W., Kutser, T., Tyler, A.N., 2017. Remote sensing of cyanobacterial bloom in inland, coastal, and ocean waters. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 89–99. Ibelings, B.W., Backer, L.C., Kardinaal, W.E.A., Chorus, I., 2014. Current approaches to cyanotoxin risk assessment andrisk management around the globe. Harmful Alg. 40, 63–74. Islam, M.S., Drasar, B.S., Sack, R.B., 1994. Probable role of blue-green algae in maintaining endemicity and seasonality of cholera in Bangladesh: a hypothesis. J. Diarrhoeal Dis. Res. 12 (4), 245–256. James, K.J., Furey, A., Sherlock, I.R., Stack, M.A., Twohig, M., Caudwell, F.B., et al., 1998. Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection. J. Chromatogr. 798 (1–2), 147–157. Jochimsen, E.M., Carmichael, W.W., An, J.S., Cardo, D.M., Cookson, S.T., Holmes, C.E., et al., 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N. Engl. J. Med. 338 (13), 873–878. Kittler, K., Schreiner, M., Krumbein, A., Manzei, S., Koch, M., Rohn, S., et al., 2012. Uptake of the cyanobacterial toxin cylindrospermopsin in Brassica vegetables. Food Chem. 133 (3), 875–879. Knoll, A.H., 2003. Life on a Young Planet: The First Three Billion Years of Evolution on Earth. Princeton University Press, Princeton, NJ. Kokocinski, M., Gagala, I., Jasser, I., Karosiene, J., Kasperoviciene, J., Kobos, J., et al., 2017. Distribution of invasive Cylindrospermopsis raciborskii in the East-Central Europe is driven by climatic and local environmental variables. FEMS Microbiol. Ecol. 93 (4). fix035. Konur, O. (Ed.), 2020a. Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020b. The scientometric analysis of the research on the algal science, technology, and medicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020c. 100 citation classics in the algal science, technology, and medicine: a scientometric analysis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam.

572  PART |IX  Algal toxicology

Konur, O., 2020d. The scientometric analysis of the research on the algal structures. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020e. The scientometric analysis of the research on the algal genomics. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020f. The scientometric analysis of the research on the algal photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020g. The scientometric analysis of the research on the algal ecology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020h. The scientometric analysis of the research on the algal bioenergy and biofuels. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020i. The scientometric analysis of the research on the algal biomedicine. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020j. The scientometric analysis of the research on the algal foods. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020k. The scientometric analysis of the research on the algal toxicology. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020l. The scientometric analysis of the research on the algal bioremediation. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020m. The pioneering research on the cyanobacterial photosystems and photosynthesis. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020n. The pioneering research on the bioethanol production from green macroalgae. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Konur, O., 2020o. The pioneering research on the wound care by alginates. In: Konur, O. (Ed.), Handbook of Algal Science, Technology and Medicine. Elsevier, Amsterdam. Krienitz, L., Ballot, A., Kotut, K., Wiegand, C., Putz, S., Metcalf, J.S., et al., 2003. Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya. FEMS Microbiol. Ecol. 43 (2), 141–148. Krienitz, L., Ballot, A., Casper, P., Codd, G.A., Kotut, K., Metcalf, J.S., et al., 2005. Contribution of toxic cyanobacteria to massive deaths of Lesser Flamingos at saline-alkaline lakes of Kenya. Verh. Int. Ver. Theor. Angew. Limnol. 29 (2), 783–786. Lantigua, J., 2017. Tainted Waters: Threats to Public Health and the People’s Right to Know. American Civil Liberties Union, Florida. Lawton, L.A., Edwards, C., Beattie, K.A., Pleasance, S., Dear, G.J., Codd, G.A., 1995. Isolation and characterization of microcystins from laboratory cultures and environmental samples of Microcystis aeruginosa and from an associated animal toxicosis. Nat. Toxins 3 (1), 50–57. Magalhaes, V.F., Soares, R.M., Azevedo, S.M.O., 2001. Microcystin contamination in fish from the Jacarepagua Lagoon (Rio de Janeiro, Brazil): ecological implication and human health risk. Toxicon 39 (7), 1077–1085. Mahmood, N.A., Carmichael, W.W., 1986. The pharmacology of anatoxin-a(s), a neurotoxin produced by the freshwater cyanobacterium Anabaena flosaquae NRC 525-17. Toxicon 24 (5), 425–434. Mahmood, N.A., Charmichael, W.W., Pfahler, D., 1988. Anticholinesterase poisonings in dogs from a cyanobacterial (blue-green algae) bloom dominated by Anabaena flos-aquae. Am. J. Vet. Res. 49 (4), 500–5003. Metcalf, J.S., Codd, G.A., 2012. Cyanotoxins. In: Whitton, B.A. (Ed.), Ecology of Cyanobacteria II: Their Diversity in Space and Time. Springer, Dordrecht, pp. 651–675. Metcalf, J.S., Beattie, K.A., Pflugmacher, S., Codd, G.A., 2000. Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR. FEMS Microbiol. Lett. 189 (2), 155–158. Metcalf, J.S., Bell, S.G., Codd, G.A., 2001. Colorimetric immuno-protein phosphatase inhibition assay for specific detection of microcystins and nodularins of cyanobacteria. Appl. Environ. Microbiol. 67 (2), 904–909. Metcalf, J.S., Barakate, A., Codd, G.A., 2004. Inhibition of plant protein synthesis by the cyanobacterial hepatotoxin, cylindrospermopsin. FEMS Microbiol. Lett. 235 (1), 125–129. Metcalf, J.S., Morrison, L.F., Krienitz, L., Ballot, A., Krause, E., Kotut, K., Putz, S., Wiegand, C., Pflugmacher, S., Codd, G.A., 2006. Analysis of the cyanotoxins anatoxin-a and microcystins in Lesser Flamingo feathers. Toxicol. Environ. Chem. 88, 159–167. Metcalf, J.S., Banack, S.A., Lindsay, J., Morrison, L.F., Codd, G.A., 2008. Co-occurrence of β-N-methylamino-L-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies, 1990-2004. Environ. Microbiol. 10 (3), 702–708. Metcalf, J.S., Richer, R., Cox, P.A., Codd, G.A., 2012. Cyanotoxins in desert environments may present a risk to human health. Sci. Total Environ. 421–422, 118–123. Metcalf, J.S., Banack, S.A., Kotut, K., Krienitz, L., Codd, G.A., 2013. Amino acid neurotoxins in feathers of the Lesser Flamingo, Phoeniconaias minor. Chemosphere 90 (2), 835–839. Metcalf, J.S., Lobner, D., Banack, S.A., Cox, G.A., Nunn, P.B., Wyatt, P.B., et al., 2017. Analysis of BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and toxicity of D-BMAA. Amino Acids 49 (8), 1427–1439. Metcalf, J.S., Banack, S.A., Powell, J.T., Tymm, F.J.M., Murch, S.J., Brand, L.E., et al., 2018. Public health responses to toxic cyanobacterial blooms: perspectives from the 2016 Florida event. Water Policy 20 (5), 919–932. Mez, K., Beattie, K., Codd, G., Hanselmann, K., Hauser, B., Naegeli, H., et al., 1997. Identification of a microcystin in benthic cyanobacteria linked to cattle deaths on alpine pastures in Switzerland. Eur. J. Phycol. 32 (2), 111–117.



Cyanobacterial toxins and their effects on human and animal health Chapter | 35  573

Mhlanga, L., Day, J., Cronberg, G., Chimbari, M., Siziba, N., Annadotter, H., 2006. Cyanobacteria and cyanotoxins in the source water from Lake Chivero, Harare, Zimbabwe, and the presence of cyanotoxins in drinking water. Afr. J. Aquat. Sci. 31 (2), 165–173. Miller, A.P., Tisdale, E.S., 1931. Public health engineering: epidemic of intestinal disorders in Charlestion, W. Va., occurring simultaneously with unprecedented water supply conditions. Am. J. Public Health Nations Health 21 (2), 198–200. Molica, R.J.R., Oliveira, E.J.A., Carvalho, P.V.C., Costa, A.N.S.F., Cunha, C.C., Melo, G., et al., 2005. Occurrence of saxitoxins and an anatoxin-a(s)-like anticholinesterase in a Brazilian drinking water supply. Harmful Algae 4 (4), 743–753. Monteiro, S., Santos, R., Blaha, L., Codd, G.A., 2017. Lipopolysaccharide endotoxins. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 109–126. Murch, S.J., Cox, P.A., Banack, S.A., 2004. A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam. Proc. Natl. Acad. Sci. USA 101 (133), 12228–12231. Mynderse, J.S., Moore, R.E., Kashiwagi, M., Norton, T.R., 1977. Antileukemia activity in the osillatoriaceae: isolation of debromoaplysiatoxin from Lyngbya. Science 196 (4289), 538–540. Nishiwaki-Matsushima, R., Ohta, T., Nishiwaki, S., Suganuma, M., Kohyama, K., Ishikawa, T., et al., 1992. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res. Clin. Oncol. 118 (6), 420–424. Norris, R.L., Eaglesham, G.K., Pierens, G., Shaw, G.R., Smith, M.J., Chiswell, R.K., et al., 1999. Deoxycylindrospermopsin, an analog of cylindrospermopsin from Cylindrospermopsis raciborskii. Environ. Toxicol. 14 (1), 163–165. Norris, R.L., Seawright, A.A., Shaw, G.R., Senogles, P., Eaglesham, G.K., Smith, M.J., et al., 2002. Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse. Toxicon 40 (4), 471–476. Ohta, T., Sueoka, E., Lida, N., Komori, A., Suganuma, M., Nishiwaki, R., et al., 1994. Nodularin, a potent inhibitor of protein phosphatases 1 and 2A, is a new environmental carcinogen in male F344 rat liver. Cancer Res. 54 (24), 6402–6406. Ohtani, I., Moore, R.E., Runnegar, M.T.C., 1992. Cylindrospermopsin: a potent hepatotoxin from the blue-green alga Cylindrospermopsis raciborskii. J. Am. Chem. Soc. 114, 7942–7944. Osborne, N.J., Shaw, G.R., 2008. Dermatitis associated with exposure to a marine cyanobacterium during recreational water exposure. BMC Dermatol. 8, 5. Paerl, H.W., Huisman, J., 2008. Blooms like it hot. Science 320 (5872), 57–58. Paerl, H.W., Huisman, J., 2009. Climate change: a catalyst for global expansion of harmful cyanobacterial blooms. Environ. Microbiol. Rep. 1 (1), 27–37. Paerl, H.W., Paul, V.J., 2012. Climate change: links to global expansion of harmful cyanobacteria. Water Res. 46 (5), 1349–1363. Papadimitriou, T., Katsiapi, M., Vlachopoulos, K., Christopoulos, A., Laspidou, C., Moustaka-Gouni, M., et al., 2018. Cyanotoxins as the “common suspects” for the Dalmatian pelican (Pelecanus crispus) deaths in a Mediterranean reconstructed reservoir. Environ. Pollut. 234, 779–787. Pelley, J., 2016. Taming toxic algae blooms. ACS Cent. Sci. 2 (5), 270–273. Pilotto, L., Douglas, R., Burch, M., Cameron, S., Beers, M., Rouch, G., et al., 1997. Health effects of exposure to cyanobacteria (blue-green algae) during recreational water-related activities. Aust. N. Z. J. Public Health 21 (6), 562–566. Pouria, S., de Andrade, A., Barbosa, J., Cavalcanti, R.L., Barreto, V.T.S., Ward, C.J., et al., 1998. Fatal microcystin intoxication in haemodialysis unit in Caruaru, Brazil. Lancet 352 (9121), 21–26. Puschner, B., Hoff, B., Tor, E.R., 2008. Diagnosis of anatoxin-a poisoning in dogs from North America. J. Vet. Diagn. Investig. 20 (1), 89–92. Puschner, B., Bautista, A., Wong, C., 2017. Debromoaplysiatoxin as the causative agent of dermatitis in a dog after exposure to freshwater in California. Front. Vet. Sci. 4, 50. Pybus, M.J., Hobron, D.P., Onderka, D.K., 1986. Mass mortality of bats due to probable blue-green algal toxicity. J. Wildl. Dis. 22 (3), 449–450. Recknagel, F., Orr, P., Swanepoel, A., Joehnk, K., Anstee, J., 2018. Operational forecasting in ecology by inferential models and remote sensing. In: Recknagel, F., Michener, W.K. (Eds.), Ecological Informatics: Data Management and Knowledge Discovery. Springer, Cham, pp. 319–339. Richer, R., Anchassi, D., El-Assaad, I., El-Matbouly, M., Ali, F., Makki, I., et al., 2012. Variation in the coverage of biological soil crusts in the State of Qatar. J. Arid Environ. 78, 187–190. Rinehart, K.L., Harada, K.I., Namikoshi, M., Chen, C., Harvis, C.A., Munro, M.H.G., et al., 1988. Nodularin, microcystin, and the configuration of Adda. J. Am. Chem. Soc. 110 (25), 8557–8558. Rodger, H.D., Turnbull, T., Edwards, C., Codd, G.A., 1994. Cyanobacterial (blue-green algal) bloom associated pathology in brown trout, Salmo trutta L., in Loch Leven, Scotland. J. Fish Dis. 17 (J2), 177–181. Roy-Lachapelle, A., Solliec, M., Bouchard, M.F., Sauve, S., 2017. Detection of cyanotoxins in algae dietary supplements. Toxins 9 (3), e76. Saker, M.L., Thomas, A.D., Norton, J.H., 1999. Cattle mortality attributed to the toxic cyanobacterium Cylindrospermopsis raciborskii in an outback region of North Queensland. Environ. Toxicol. 14 (1), 179–182. Schantz, E.J., Mold, J.D., Stanger, D.W., Shavel, J., Riel, F.J., Bowden, J.P., et al., 1957. Paralytic shellfish poison. VI. A procedure for the isolation and purification of the poison from toxic clam and mussel tissues. J. Am. Chem. Soc. 79 (19), 5230–5235. Schopf, J.W., 2000. The fossil record: tracing the roots of the cyanobacterial lineage. In: Whitton, B.A., Potts, M. (Eds.), The Ecology of Cyanobacteria: Their Diversity in Time and Space. Springer, Dordrecht, pp. 13–35. Scott, L.L., Downing, T.G., 2018. β-N-methylamino-L-alanine (BMAA) toxicity is gender and exposure-age dependent in rats. Toxins 10 (1), e16. Seawright, A.A., Nolan, C.C., Shaw, G.R., Chiswell, R.K., Norris, R.L., Moore, M.R., et al., 1999. The oral toxicity for mice of the tropical cyanobacterium Cylindrospermopsis raciborskii (Woloszynska). Environ. Toxicol. 14 (1), 135–142. Simmons, J., 1998. Algal control and destratification at Hanningfield reservoir. Water Sci. Technol. 37 (2), 309–316. Sinha, R., Pearson, L.A., Davis, T.W., Burford, M.A., Orr, P.T., Neilan, B.A., 2012. Increased incidence of Cylindrospermopsis raciborskii in temperate zones—is climate change responsible? Water Res. 46 (5), 1408–1419.

574  PART |IX  Algal toxicology

Sipia, V., Kankaanpaa, H., Peltonen, H., Vinni, M., Meriluoto, J., 2007. Transfer of nodularin to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus L.), and salmon (Salmo salar L.) in the northern Baltic Sea. Ecotoxicol. Environ. Saf. 66 (3), 421–425. Sipia, V., Neffling, M.L., Metcalf, J.S., Nybom, S.M.K., Meriluoto, J., Codd, G.A., 2008. Nodularin in feathers and liver of eiders (Somateria mollissima) caught from the western Gulf of Finland in June–September 2005. Harmful Algae 7 (1), 99–105. Sivonen, K., Jones, G., 1999. Cyanobacterial toxins. In: Chorus, I., Bertram, J. (Eds.), Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management. E & FN Spon, London, pp. 41–111. Smith, V.H., 1983. Low nitrogen to phosphorus ratios favor dominance by blue-green algae in lake phytoplankton. Science 221 (4611), 669–671. Soll, M., Williams, M.C., 1985. Mortality of a white rhinoceros (Ceratotherium simum) suspected to be associated with the blue-green alga Microcystis aeruginosa. J. S. Afr. Vet. Assoc. 56 (1), 49–51. Spoof, L., Catherine, A., 2017. Appendix 3, tables of microcystins and nodularins. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 526–537. Stewart, W.D.P., 1972. Heterocysts of blue-green algae. In: Desikachary, T.V. (Ed.), Taxonomy and Biology of Blue-Green Algae. University of Madras, Madras, pp. 227–235. Stewart, W.D.P., 1980. Some aspects of structure and function in N2-fixing cyanobacteria. Annu. Rev. Microbiol. 34, 497–536. Svircev, Z., Drobac, D., Tokodi, N., Vidovic, M., Simeunovic, J., Miladinov-Mikov, M., et al., 2013. Epidemiology of primary liver cancer in Serbia and possible connection with cyanobacterial blooms. J. Environ. Sci. Heal. C 31 (3), 181–200. Teixeira, M.G., Costa, M.C.N., Carvalho, V.L.P., Peireira, M.S., Hagner, E., 1993. Gastroenteritis epidemic in the area of the Itaparica Dam, Bahia, Brazil. Bull. Pan Am. Health Organ. 27 (3), 244–253. Terao, K., Ohmori, S., Igarashi, K., Ohtani, I., Watanabe, M.F., Harada, K.I., et al., 1994. Electron microscopic studies on experimental poisoning in mice induced by cylindrospermopsin isolated from blue-green alga Umezakia natans. Toxicon 32 (7), 833–843. Testai, E., Buratti, F.M., Funari, E., Manganelli, M., Vichi, S., Arnich, N., et al., 2016. Review and analysis of occurrence, exposure and toxicity of cyanobacteria toxins in food. Eur. Food Saf. Auth. Support. Publ. 13 (2), 998E. Thomas, A.D., Saker, M.L., Norton, J.H., Olsen, R.D., 1998. Cyanobacterium Cylindrospermopsis raciborskii as a probable cause of death in cattle in northern Queensland. Aust. Vet. J. 76 (9), 592–594. Townsend, S.A., Boland, K.T., Wrigley, T.J., 1992. Factors contributing to a fish kill in the Australian wet/dry tropics. Water Res. 26 (8), 1039–1044. Turner, P.C., Gammie, A.J., Hollinrake, K., Codd, G.A., 1990. Pneumonia associated with contact with cyanobacteria. Br. Med. J. 300 (6737), 1440–1441. Ueno, Y., Nagata, S., Tsutsumi, T., Hasegawa, A., Watanabe, M.F., Park, H.D., et al., 1996. Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis 17 (6), 1317–1321. Van Halderen, A., Harding, W.R., Wessels, J.C., Schneider, D.J., Heine, E.W.P., Van der Merwe, J., et al., 1996. Cyanobacterial (blue-green algae) poisoning of livestock in the Western Cape Province of South Africa. J. S. Afr. Vet. Assoc. 66 (4), 260–264. Vega, A., Bell, E.A., 1967. α-Amino-β-methylaminoproprionic acid, a new amino acid from seeds of Cycas circinalis. Phytochemistry 6 (5), 759–762. Veldee, M.V., 1931. An epidemiological study of suspected water-borne gastroenteritis. Am. J. Public Health Nations Health 21 (11), 1227–1235. Vidal, F., Sedan, D., D’Agostino, D., Cavalieri, M.L., Mullen, E., Varela, M.M.P., et al., 2017. Recreational exposure during algal bloom in Carrasco Beach, Uruguay: a liver failure case report. Toxins 9 (9), e267. Ward, C.J., Beattie, K.A., Lee, E.Y.C., Codd, G.A., 1997. Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria: comparisons with high-performance liquid chromatographic analysis for microcystins. FEMS Microbiol. Lett. 153 (2), 465–473. Weiss, J.H., Koh, J.Y., Choi, D.W., 1989. Neurotoxicity of β-N-methylamino-L-alanine (BMAA) and β-N-oxalylamino-L-alanine (BOAA) on cultured cortical neurons. Brain Res. 497 (1), 64–71. Wimmer, K.M., Strangman, W.K., Wright, J.L.C., 2014. 7-Deoxy-desulfo-cylindrospermopsin and 7-deoxy-desulfo-12-acetylcylindrospermopsin: two new cylindrospermopsin analogs isolated from a Thai strain of Cylindrospermopsis raciborskii. Harmful Algae 37, 203–206. Wood, S.A., Selwood, A.I., Rueckert, A., Holland, P.T., Milne, J.R., Smith, K.F., et al., 2007. First report of homoanatoxin-a and associated dog neurotoxicosis in New Zealand. Toxicon 50 (2), 292–301. Wynne, T.T., Stumpf, R.P., 2015. Spatial and temporal patterns in the seasonal distribution of toxic cyanobacteria in Western Lake Erie from 2002-2014. Toxins 7 (5), 1649–1663. Yamamoto, Y., 1976. Effect of some physical and chemical factors on the germination of akinetes of Anabaena cylindrica. J. Gen. Appl. Microbiol. 22 (6), 311–323. Yu, S.Z., 1995. Primary prevention of hepatocellular carcinoma. J. Gastroenterol. Hepatol. 10 (6), 674–682.

Further reading Du Preez, H.H., Swanepoel, A., Cloete, N., 2017. The occurrence and removal of algae (including cyanobacteria) and their related organic compounds from source water in Vaalkop Dam with conventional and advanced drinking water treatment processes. Water SA 43 (1), 67–80. Metcalf, J.S., Codd, G.A., 2017. Immunoassays and other antibody applications. In: Meriluoto, J., Spoof, L., Codd, G.A. (Eds.), Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Chichester, pp. 263–266.

Chapter 36

Different intoxication mechanism between paralytic shellfish toxin (PST)- and/or tetrodotoxin-contaminated xanthid crabs and PST-contaminated edible shore crabs in Japan and their food poisonings Tamao Noguchia, Osamu Arakawab, Kinue Daigo, Hiroshi Oikawac, Manabu Asakawad, Keisuke Miyazawa a

Tokyo Health University, Tokyo, Japan, bNagasaki University, Nagasaki, Japan, cNational Research Institute of Fisheries Research Agency, Yokohama, Japan, dHiroshima University, Hiroshima, Japan

36.1 Introduction Outbreaks of human intoxication by toxic crabs have been reported in various parts of the world (Halstead, 1965; Guinot, 1967; Holthuis, 1968; Sommer, 1932). Only the toxicity of the sand crab, Emerita analoga, was experimentally confirmed to be due to the occurrence of a toxic dinoflagellate plankton red tide in 1932 by Sommer, although more than 20 species of crabs had been suspected to be toxic. In the Ryukyu Islands, toxic crabs were believed to be the primary source of toxin causing fish to become ciguateric. Sporadic outbreaks of human intoxication due to toxic crabs have been reported and crabs were, in fact, not usually, eaten in these areas (Hashimoto, 1979). Three species in the family Xanthidae, ‘umore-ougi-gani’ Zosimus aeneus, ‘subesubemanjyu-gani’ Atergatis floridus, and ‘tsubuhiraashi-ougi-gani’ Platypodia granulosa were found to be toxic by a screening test for toxic crabs using 1000 specimens in 72 species of 8 families collected from the Ryukyu Islands (Hashimoto et al., 1969; Konosu et al., 1969). Other suspected species were non-toxic. The toxin was identical to the known paralytic shellfish toxin (PST), ‘saxitoxin’ (STX) (Noguchi et al., 1969). There are great individual and regional variations in the toxicity of this crab (Konosu et  al., 1969; Noguchi et  al., 1983a,b, 1984; Asakawa et al., 2018), suggesting that the toxin is of exogenous rather than endogenous origin. However, there have been no signs of a red tide composed of PST-producing dinoflagellate plankton, and no toxic organisms and substances found from contents of digestive glands of toxic xanthid crabs (Daigo, 1989). It is strange that the toxin is found in only three species of the many members of the family Xanthidae and that the composition of the toxin depends on the collection place (Noguchi et al., 1986, 2011). Edible crabs, carnivorous ‘togekuri-gani’ Telmessus acutidens and ‘ishi-gani’ Charybdis japonica were found to contain PST in each hepatopancreas (digestive organ) in Japan (Oikawa et al., 2002). Their PST intoxication mechanism was shown to result from feeding on PST-contaminated bivalves (green mussel and/or short-necked clam). Several food poisoning cases due to ingestion of the detritus-feeder horseshoe crab Carcinoscorpius rotundicauda belonging to the family Xihosuridae occurred in Thailand (Noguchi, 1996). The causative agent was shown to be PST and/or tetrodotoxin (TTX) (Kungsuwan et al., 1987; Kanchanapongkul and Krittayapoositpot, 1995).

36.2  Food poisoning cases with xanthid crabs Since 1960s, 20 cases of crab poisoning have occurred in the Ryukyu Islands. Z. aeneus might have been the causative organism in many food poisoning cases, since it is large in size, greater in toxicity and possesses notoriety on various islands Handbook of Algal Science, Technology and Medicine. https://doi.org/10.1016/B978-0-12-818305-2.00036-X © 2020 Elsevier Inc. All rights reserved.

575

576  PART | IX  Algal toxicology

of the Pacific during the field survey in the Ryukyu Islands. However, this species is considered non-toxic in Espritu Santo and consumed by inhabitants without any harmful effects (Hashimoto, 1979). On Serua Island near the New Hebrides, two young sisters died from eating this crab in 1968 (Schwierner, 1968). Typical food poisoning cases due to xanthid crabs are as follows. Case 1 (Hashimoto, 1979): A woman and her son in Yanagi-cho, Naze, Amami-oshima Island, were poisoned by a kind of coral crab, ‘segani’ on April 22, 1965. The crabs were bought from a peddler. Five crabs were served for breakfast immediately after having been boiled in miso soup. About 15 min after the meal, the woman who drank only the broth, complained of numbness of the tongue, paralysis in the mouth, and vomited vigorously. Subsequently, she developed paralysis of the limbs and fell down. She received medical treatment for about 5 days before she recovered completely. Her son, who ate a small amount of meat and perhaps the viscera in addition to the broth, developed numbness of the limbs, severe vomiting, and was hospitalized. He was more severely affected than his mother. The causative crab was suspected of being Z. aeneus. Case 2 (Minoda, 1989): A woman and her husband in Sakieda, Ishigaki Island, ingested a cup of miso-soup, and soup including the whole body of a small sized Z. aeneus crab respectively and soon after ingestion in December 1984, they suffered from numbness of the tongues, followed by the lips, fingers, and whole bodies, as well as weakness, dizziness and repeated vomiting and were hospitalized. Three days after, they returned home. The causative agent was identified as PST (STX). Typical signs and symptoms of crab poisoning were shown as follows. Within 15 min to a few hours after ingestion of crabs, numbness of the lips and limbs and wobbling gait developed. These symptoms are followed by severe vomiting, stupor, aphasia, and respiratory difficulty. Finally, the victim fell unconscious and respiration failed. Death took place within 4 or 6 h after ingestion. The fatality rate was extremely high. These symptoms coincided with those induced by STX.

36.3  Toxicity of xanthid crabs As described in the introduction section, screening tests on a total of about 1000 specimens, covering 72 species and 8 families that were collected at random, mainly from shallow waters of coral reefs in the Ryukyu Islands were carried out (Hashimoto et al., 1969). From the epidemiology study of crab poisoning, the toxin was found to be thermostable, watersoluble, and cause paralysis. All toxic specimens were identified by Professor S. Miyake of Kyushu Sangyo University to be Z. aeneus, A. floridus, and P. granulosa.

36.3.1  Z. aeneus Z. aeneus is the largest species of the three toxic crabs, with a maximum carapace length of 55 mm and carapace breadth 88 mm (Hashimoto, 1979). The color of the live animal is greenish blue with irregular white, brown, and yellow patches. The fingers of the chelipeds are black. This species is commonly found on live coral reefs in the Indo-Pacific, from the Red Sea to the Ryukyu and Hawaii. A total of 309 specimens were collected from 11 places, Amami Islands, Ishigaki Island, Marcus Island, Hachijojima Island, Bonin Islands, Espiritu Santo (New Hebrides), American Samoa, Rangiroa Atoll (Tuamotu Islands), Cebu Island, Tokara Island, and Wakayama (Konosu et al., 1968, 1969; Sagara et al., 2009; Asakawa et al., 2018). They were examined for toxicity after being extracted with distilled water or 0.1% acetic acid solution (Table 36.1). About 70% of the specimens were toxic. Almost all specimens in Ishigaki Island, Hachijojima Island, Bonin Islands, Rangiroa Atoll and Wakayama were toxic. The value of toxicity was the highest in Ishigaki Island (1700 MU/g in which one mouse unit (MU) is equivalent to 0.18 μg STX) followed by Amami-oshima Island (350 MU/g), Rangiroa Atoll (130 MU/g), Bonin Islands (50 MU/g), Wakayama (15.4 MU/g), and Marcus Island (5 MU/g). However, none of the samples collected from Espiritu Santo and American Samoa were toxic. All samples collected from Tokara Islands, Japan were also found to be non-toxic or weakly toxic. The inhabitants in Espiritu Santo and Tokara Islands eat this crab, but no intoxication has occurred. It was on Serua Island, near the New Hebrides, that the two young sisters died from eating this crab in 1968. The toxicity of Z. aeneus shows great individual and regional variations. Therefore, the toxin may be of exogenous rather than endogenous origin. However, there has been no sign of a red tide of PST-producing dinoflagellate plankton such as certain species of the genera Alexandrium, Gymnodinium, and Pyrodinium, and Z. aeneus does not live in brackish or fresh water where PST-producing cyanobacteria inhabit. This crab inhabits living coral reef in subtropical and tropical zone and its predatory habit is omnivorous. It grazes mostly on microorganisms such as turf algae and small red algae like Jania sp. growing on the surface of large coral and rocks. Although Jania sp. was reported to show weak PST toxicity (Kotaki et al., 1983), it was also found from the stomach contents of many non-toxic crabs. Accordingly, PST contamination of Z. aeneus by Jania sp. remains uncertain (Daigo, 1989).

PST/TTX intoxication mechanism of toxic crabs Chapter | 36  577



TABLE 36.1  Toxicity of Z. aeneus crabs collected from various regions of the Pacific Ocean. Collection place

No. of samples

No. of toxic specimens

Average value of toxicity (MU/g)a

126

125b

1700

Japan Ishigaki Island Amami-oshima

c

71

48

Tokara Islands

16

1

d

350

Marcus Island

28

5c

4

Hachijojima Island

1

1

30

Bonin Island

5

5

50

Wakayama

2

2

15 c

Espiritu Santo, New Hebrides

38

0

American Samoa

2

0c

Rangiroa, Tuamotu Islands

9

9

Cebu Island, Philippines

11

11

130

a

Toxicity level of all specimens was calculated as STX.

b

> 2 MU/g.

c

> 20 MU/g.

d

>10 MU/g.

36.3.2  A. floridus A. floridus has a smooth carapace, its maximum carapace length is 35 mm and its carapace breadth is 62 mm (Hashimoto, 1979). The upper surface of the carapace breadth is 52 mm. The upper surface of the carapace appears dark brownish purple or greenish purple with light color blotches. The claws of chelipeds are dark brown. It is commonly found on rocky shores and on live coral reefs. In Japan the crab is caught by traps set for spiny lobsters on the ocean floor at a depth of 50–150 m. A. floridus is distributed from the Red Sea to Hawaii and along the south coast of Japan. Three hundred and twenty specimens of A. floridus collected from Ishigaki Island, Miura Peninsula, Shikinejima Island, Hachijojima Island, Taiwan, Guam and Wakayama were submitted to the screening test for the crab toxin (Inoue et al., 1968; Konosu et al., 1969; Noguchi et al., 1983a,b, 1984, 1986; Asakawa et al., 2018). The result is shown in Table 36.2. Almost all specimens were found to be toxic. Toxin of A. floridus crab on live coral reefs in the Ryukyu Islands is generally PST and its toxicity score is very high, while the toxin is TTX or something similar to TTX in crabs inhabiting the rocks of Japan Proper inclusive of Wakayama, and the toxicity score is comparative low. This species inhabits not only the same territory as Z. aenues on the coral reefs in the Ryukyu Islands, but also found between rocks in temperate areas, where Z. aeneus is generally not. Its predatory habit is omnivorous, the same as Z. aenues.

36.3.3  P. granulosa P. granulosa attains a carapace length of 30 mm and a carapace breadth of 42 mm (Hashimoto, 1979). It is commonly dark yellowish green, rarely yellow or purplish brown. The claws of the chelipeds are black. It is commonly on rocky shores and on dead coral reefs in the Indian and Pacific Oceans (Indo-Pacific Ocean). Thirty-three specimens of P. granulsoa collected from Ishiagki Island, Hachijojima Island and Taiwan were submitted to the screening test for the crab toxin (Noguchi, 1996; Daigo, 1989). The result is shown in Table 36.3. Almost all specimens were found to be toxic. Nineteen of them showed values of toxicity over 1000 MU/g and were collected from Ishigaki Island. On the other hand, two samples collected from Hachijojima Island and Taiwan showed low values of toxicity between 2 and 49 MU/g as shown in Table 36.3. The toxicity of P. granulosa also shows the great individual and regional variation. P. granulosa has not been found on living coral reefs, but dead ones in Ishigaki Island.

578  PART | IX  Algal toxicology

TABLE 36.2  Toxicity of A. floridus crabs collected from various regions of the Pacific Ocean. Collection place

No. of samples in the indicated toxicity ranges (MU/g)a

Total no. of samples

2–49

50–99

100–499

500–999

1000 +

Ishigaki Island

91

11

3

24

17

36

Miura

215

139

39

36

1

0

Shikinejima Island

1

0

0

0

0

0

Hachijojima Island

6

6

0

0

0

0

Wakayama

2

0

2

0

0

0

Taiwan

3

3

0

0

0

0

Guam

2

0

0

0

2

0

Japan

a

Toxicity of specimens from Miura, Shikinejima Is., Hachijojima Is., and Wakayama was calculated as TTX and from Ishigaki Is., Taiwan, and Guam as STX.

TABLE 36.3  Toxicity of P. granulosa crabs collected from various regions of the Pacific Ocean. No. of samples in the indicated toxicity ranges (MU/g)a Collection place

Total no. of samples

2–49

50–99

100–499

500–999

1000 +

Ishigaki Island

31

2

2

6

2

19

Hachijojima Island

1

1

0

0

0

0

Taiwan

1

1

0

0

0

0

Japan

a

Toxicity level of all specimens was calculated as STX.

36.3.4  Anatomical distribution of toxicity Distribution of the toxin in various parts of the body is shown in Table 36.4 (Hashimoto, 1979). Characteristically, the appendages (chelae and walking legs) were usually more toxic than the cephalothorax (trunk), and appreciable amounts of the toxin are detected in the exoskeleton (carapace) of both appendages and cephalothorax, while the muscles of the cephalothorax, the gills and the endophragm were weakly toxic or non-toxic. The lethal dose of STX when consumed orally by human (60 kg body weight) has been estimated to be 0.5 mg (equivalent to about 3000 MU). The amount corresponds to a 0.5 g portion of the muscle of the chelae of the Z. aeneus specimen that showed the maximal toxicity score in the Ishigaki specimens. The ovaries were weakly toxic and no marked variability of toxicity by sex was observed.

36.4  Origin of crab toxin In earlier work on crab toxin, Noguchi et al. (1969) isolated and identified STX as the sole toxic principle of Z. aeneus as follows. The crab toxin was extracted from strongly toxic Z. aeneus specimens collected from Ishigaki Island and Amamioshima Island using distilled water. The aqueous extract was purified by column chromatography using Amberlite IRC-50 followed by chromatography twice on Amberlite IRP-64 and once on activated alumina resulting in 7.6 mg pure toxin as shown in Table 36.5. It showed toxicity of 5100 MU/mg and was identified as STX by the IR-spectrum and optical density

PST/TTX intoxication mechanism of toxic crabs Chapter | 36  579



TABLE 36.4  Distribution of toxin in the body of toxic crabs. Body part

Toxicity (MU/g)

Body part

Z. aeneus

A. floridus

Appendages

Appendages

Toxicity (MU/g)

Exoskeleton of chelae

6000

Exoskeleton

150

Muscle of chelae

2000

Muscle

400

Exoskeleton of walking legs

2000

Cephalothorax

Muscle of walking legs

3500

Exoskeleton

65

Muscle

45

Cephalothorax Exoskeleton

2000

Viscera

25

Muscle

40

Gills