Diatoms : fundamentals and applications 9781119370215, 1119370213, 9781119370734, 9781119370727

Table of contents :
Content: Foreword xviiPreface xxiii1 A Memorial to Frithjof Sterrenburg: The Importance of the Amateur Diatomist 1 Janice L. Pappas1.1 Introduction 11.2 Background and Interests 31.3 The Personality of an Amateur Diatomist 71.4 The Amateur Diatomist and the Importance of Collections 111.5 The Amateur Diatomist as Expert in the Tools of the Trade 121.6 The Amateur Diatomist as Peer-Reviewed Scientific Contributor 151.7 Concluding Remarks 20Acknowledgments 21References 212 Alex Altenbach - In Memoriam of a Friend 29 Wladyslaw AltermannReferences 313 The Beauty of Diatoms 33 Mary Ann Tiffany and Stephen S. Nagy3.1 Early History of Observations of Diatoms 333.2 Live Diatoms 353.3 Shapes and Structures 353.4 Diatom Beauty at Various Scales 363.5 Valves During Morphogenesis 373.6 Jamin-Lebedeff Interference Contrast Microscopy 393.7 Conclusion 40Acknowledgments 40References 414 Current Diatom Research in China 43 Yu Xin Zhang4.1 Diatoms for Energy Conversion and Storage 434.1.1 Introduction 434.1.2 Diatom Silica: Structure, Properties and Their Optimization 464.1.3 Diatoms for Lithium Ion Battery Materials 484.1.4 Diatoms for Energy Storage: Supercapacitors 514.1.5 Diatoms for Solar Cells 564.1.6 Diatoms for Hydrogen Storage 584.1.7 Diatoms for Thermal Energy Storage 594.2 Diatoms for Water Treatment 614.2.1 Support for Preparation of Diatomite-Based Adsorption Composites 614.2.2 Catalyst and Template for Preparation of Porous Carbon Materials 634.2.3 Modification of Surface and Porous Structure 664.2.4 Support for Preparation of Diatomite-Based Metal Oxide Composites 754.3 Study of Tribological Performances of Compound Dimples Based on Diatoms Shell Structures 86References 885 Cellular Mechanisms of Diatom Valve Morphogenesis 99 Yekaterina D. Bedoshvili and Yelena V. Likhoshway5.1 Introduction 995.2 Valve Symmetry 1005.3 Valve Silification Order 1025.4 Silica Within SDV 1035.5 Macromorphogenesis Control 1045.6 Cytoskeletal Control of Morphogenesis 1065.7 The Role of Vesicles in Morphogenesis 1075.8 Valve Exocytosis and the SDV Origin 1085.9 Conclusion 110References 1106 Application of Focused Ion Beam Technique in Taxonomy-Oriented Research on Ultrastructure of Diatoms 115 Andrzej Witkowski, Tomasz P?oci?ski, Justyna Grzonka, Izabela Zg?obicka, Ma?gorzata B?k, Przemys?aw D?bek, Ana I. Gomes and Krzysztof J. Kurzyd?owski6.1 Introduction 1166.2 Material and Methods 1176.3 Results 1176.3.1 Complex Stria Ultrastructure 1176.3.1.1 Biremis lucens (Hustedt) Sabbe, Witkowski & Vyverman 1995 1176.3.1.2 Olifantiella mascarenica Riaux-Gobin & Compere 2009 1206.4 Discussion 1236.4.1 Cultured Versus Wild Specimens 1246.5 Conclusions 124Acknowledgements 126References 1267 On Light and Diatoms: A Photonics and Photobiology Review 129Mohamed M. Ghobara, Nirmal Mazumder, Vandana Vinayak, Louisa Reissig, Ille C. Gebeshuber, Mary Ann Tiffany and Richard Gordon7.1 Introduction 1307.2 The Unique Multiscale Structure of the Diatom Frustules 1307.3 Optical Properties of Diatom Frustules 1397.3.1 The Frustule as a Box with Photonic Crystal Walls 1437.3.2 Light Focusing Phenomenon 1467.3.3 Photoluminescence Properties 1517.3.4 Probable Roles of the Frustule in Diatom Photobiology 1527.4 Diatom Photobiology 1537.4.1 Underwater Light Field 1537.4.2 Cell Cycle Light Regulation 1547.4.3 The Phototactic Phenomenon in Pennates 1547.4.4 Chloroplast Migration (Karyostrophy) 1567.4.5 Blue Light and Its Effects on Microtubules of Cells 1577.4.6 Strategies for Photoregulation Under High Light Intensity 1597.4.7 Strategies for Photoregulation Under Ultraviolet Radiation (UV) Exposure 1597.4.8 Diatoms and Low Light 1607.4.9 Diatoms and No Light 1617.4.10 Light Piping and Cellular Vision 1617.5 Diatom and Light Applications 1627.5.1 In Photocatalysis 1627.5.2 Bio-Based UV Filters 1647.5.3 In Solar Cells 1657.5.4 Applications Based on Luminescence Properties 1677.5.5 Cloaking Diatoms 1677.6 Conclusion 169Acknowledgement 169Glossary 169References 1718 Photosynthesis in Diatoms 191 Matteo Scarsini, Justine Marchand, Kalina M. Manoylov and Benoit Schoefs8.1 Introduction 1918.2 The Chloroplast Structure Reflects the Two Steps Endosymbiosis 1948.3 Photosynthetic Pigments 1968.3.1 Chlorophylls 1968.3.2 Carotenoids 1978.4 The Organization of the Photosynthetic Apparatus 1978.5 Non-Photochemical Quenching (NPQ) 2008.6 Carbon Uptake and Fixation 2028.7 Conclusions and Perspectives 204Acknowledgment 205References 2059 Iron in Diatoms 213 John A. Raven9.1 Introduction 2139.2 Fe Acquisition by Diatoms 2149.3 Fe-Containing Proteins in Diatoms and Economy of Fe Use 2149.4 Iron Storage 2199.5 Conclusions and Prospects 220Acknowledgements 220References 22010 Diatom Symbioses with Other Photoauthotroph 225 Rosalina Stancheva and Rex Lowe10.1 Introduction 22510.2 Diatoms with a N2-Fixing Coccoid Cyanobacterial Endosymbiont 22610.3 Diatoms with N2-Fixing Filamentous Heterocytous Cyanobacterial Endosymbionts 23310.4 Epiphytic, Endogloeic and Endophytic Diatoms 23510.5 Diatom Endosymbionts in Dinoflagellates 238Acknowledgements 239References 23911 Diatom Sexual Reproduction and Life Cycles 245 Aloisie Pouli?kova and David G. Mann11.1 Introduction 24511.2 Centric Diatoms 24711.2.1 Life Cycle and Reproduction 24711.2.2 Gametogenesis and Gamete Structure 25011.2.3 Spawning 25111.3 Pennate Diatom Life Cycles and Reproduction 25211.4 Auxospore Development and Structure 25711.4.1 Incunabula 25911.4.2 Perizonium 26011.5 Induction of Sexual Reproduction 261Acknowledgments 262References 26312 Ecophysiology, Cell Biology and Ultrastructure of a Benthic Diatom Isolated in the Arctic 273 Ulf Karsten, Rhena Schumann and Andreas Holzinger12.1 Introduction 27412.2 Environmental Settings in the Arctic 27412.3 Growth as Function of Temperature 27512.4 Growth After Long-Term Dark Incubation 27712.5 Cell Biological Traits After Long-Term Dark Incubation 27912.6 Ultrastructural Traits 28212.7 Conclusions 283Acknowledgements 284References 28413 Ecology of Freshwater Diatoms - Current Trends and Applications 289 Aloisie Pouli?kova and Kalina Manoylov13.1 Introduction 28913.2 Diatom Distribution 29213.3 Diatom Dispersal Ability 29213.4 Functional Classification in Diatom Ecology 29413.5 Spatial Ecology and Metacommunities 29613.6 Aquatic Ecosystems Biomonitoring 29913.7 Conclusions 301References 30114 Diatoms from Hot Springs of the Kamchatka Peninsula (Russia) 311 Tatiana V. Nikulina, E. G. Kalitina, N. A. Kharitonova, G. A. Chelnokov, Elena A. Vakh and O. V. Grishchenko14.1 Introduction 31114.2 Materials and Methods 31314.3 Description of Sampling Sites 31314.3.1 Malkinsky Geothermal Field 31414.3.2 Nachikinsky Geothermal Field 31714.3.3 Verkhnaya-Paratunka Geothermal Field 31714.3.3.1 Goryachaya Sopka Hot Spring 31814.3.3.2 Karimshinsky Hot Spring 31814.3.4 Mutnovsky Geothermal Field 31814.3.4.1 Dachny Hot Springs 31914.3.4.2 Verkhne-Vilyuchinsky Hot Spring 31914.4 Results 32014.4.1 Malkinsky Geothermal Field 32014.4.2 Nachikinsky Geothermal Field 32014.4.3 Verkhnaya-Paratunka Geothermal Field 32614.4.3.1 Goryachaya Sopka Hot Spring 32614.4.3.2 Karimshinsky Hot Spring 32614.4.4 Mutnovsky Geothermal Field 32614.4.4.1 Dachny Hot Springs 32614.4.4.2 Verkhne-Vilyuchinsky Hot Spring 32714.5 Summary 330References 33115 Biodiversity of High Mountain Lakes in Europe with Special Regards to Rila Mountains (Bulgaria) and Tatra Mountains (Poland) 335 Nadja Ognjanova-Rumenova, Agata Z. Wojtal, Elwira Sienkiewicz, Ivan Botev and Teodora Trichkova15.1 Introduction 33515.1.1 Factors Which Control the Diatom Distribution 33615.1.2 Biodiversity Assessment 33715.2 Recent Datom Biodiversity in High Mountain Lakes in bulgaria and Poland 33815.2.1 The Rila Lakes, Bulgaria 33815.2.2 The Tatra Lakes, Poland 33915.3 Diatom Community Changes in High-Mountain Lakes in Bulgaria and Poland from Pre-Industrial Times to Present Day 34015.3.1 The Rila Mts. 34015.3.2 Tatra Mts. 34215.4 Monitoring Data '2015' and Correlations Between the Data Sets of the Rila Mts. and the Tatra Mts. 34415.4.1 The Rila Lakes 34415.4.2 The Tatra Lakes 34615.5 Red-List Data: Cirque "Sedemte Ezera", Rila Mts. and Tatra Mts. 34915.5.1 Cirque "Sedemte Ezera", Rila Mts. 34915.5.2 Tatra Mts. 34915.6 Summary 349Acknowledgements 351References 35116 Diatoms of the Southern Part of the Russian Far East 355 Tatiana V. Nikulina and Lubov A. Medvedeva16.1 History of the Study of Freshwater Algae of the Southern Part of the Russian Far East 35516.1.1 The Primorye Territory 35716.1.1.1 Lakes and Reservoirs 35716.1.1.2 Rivers and Streams 35816.1.2 The Amur Region 36016.1.2.1 The Upper Amur 36016.1.2.2 The Middle Amur 36016.1.3 The Jewish Autonomous Region 36116.1.4 The Khabarovsk Territory 36116.1.4.1 The Middle Amur 36116.1.4.2 The Lower Amur 36116.1.5 The Sakhalin Region 36216.1.5.1 Sakhalin Island 36216.1.5.2 Moneron Island 36316.1.5.3 The Kuril Islands 36316.2 Diatom Flora of the Southern Part of the Russian Far East 363References 37717 Toxic and Harmful Marine Diatoms 389 Stephen S. Bates, Nina Lundholm, Katherine A. Hubbard, Marina Montresor and Chui Pin Leaw17.1 Introduction 39017.2 Harmful Diatoms 39117.2.1 How Diatoms May Cause Harm 39117.2.2 Diatom Oxylipins 39117.2.2.1 Polyunsaturated Aldehydes (PUAs) 39117.2.2.2 Oxylipin Production by Pseudo-nitzschia 39617.3 Toxic Diatoms 39717.3.1 Diatoms That Produce -N-Methylamino-L-Alanine (BMAA) 39717.3.2 Nitzschia navis-varingica 40017.3.3 Nitzschia bizertensis 40017.3.4 Pseudo-nitzschia spp 40117.3.4.1 New Species 40117.3.4.2 Distribution 40117.3.4.3 Sexual Reproduction 40117.3.4.4 Genomic Insights Into Pseudo-nitzschia and Its Population Genetic Structure 41017.3.4.5 New Knowledge of Pseudo-nitzschia 41117.3.5 Identification of Toxic Diatoms 41417.3.5.1 Classical Methods 41417.3.5.2 Molecular Approaches 41517.4 Gaps in Knowledge and Thoughts for Future Directions 417References 41818 Diatoms in Forensics: A Molecular Approach to Diatom Testing in Forensic Science 435 Vandana Vinayak and S. Gautam18.1 Introduction 43518.2 Postmortem Forensic Counter Measures 43818.3 Differences in Drowned Victims vs Those that Die of Other Causes 43918.4 Techniques to Identify Diatoms in Biological Sample 44018.4.1 Morphological Analysis of Water Samples 44118.4.2 Role of Site Specific Diatoms 44218.5 Case Studies 44318.5.1 Case 1 44318.5.2 Case 2 44318.5.3 Case 3 44418.6 Identification of Diatom Using Molecular Tools in Tissue and Water Samples 44618.7 Differentiation of Diatom DNA in the Tissue of a Drowned Victim 44718.8 Polymerase Chain Reaction (PCR) 44818.9 Diatom DNA Extraction from Biological Samples of a Drowned Victim 44818.9.1 Biological Samples 44818.9.2 Plankton/Diatom Isolation from Tissues Using Colloidal Silica Gradient and Phenol Chloroform Method for DNA Extraction 45418.10 Best Barcode Markers for Diatoms to Diagnose Drowning 45418.10.1 Cytochrome C Oxidase Subunit 1 (COI) 45518.10.2 Nuclear rDNA ITS Region 45618.10.3 Nuclear Small Subunit rRNA Gene 45718.11 DNA Sequencing 45718.12 Advancement in Sequencing Leads to Advancement of Data Interpretation 45818.13 Conclusion and Future Perspectives 459Acknowledgements 459List of Abbreviations Used 460References 46019 Diatomite in Use: Nature, Modifications, Commercial Applications and Prospective Trends 471 Mohamed M. Ghobara and Asmaa Mohamed19.1 The Nature of Diatomite 47119.1.1 Diatomite Formation 47219.1.2 Diatom Frustule's Resistance Against Dissolution (The Reason for Their Preservation Over Millions of Years) 47319.2 The History of Discovery and Ancient Applications 47519.3 Diatomite Occurrence and Distribution 47619.4 Diatomite Mining and Processing 47719.5 Diatomite Characterization 47919.6 Diatom Frustules Modifications 48019.7 Diatomite in Use 48119.7.1 Diatomite-Based Filtration 48219.7.1.1 Water Filtration 48319.7.1.2 Beer Filtration 48419.7.1.3 Recent Trends in Diatomite-Based Separation Techniques 48519.7.1.4 Reuse of Spent DE Filter Media 48519.7.2 Diatomite for Thermal Insulation 48519.7.3 Diatomite-Based Building Materials 48719.7.4 Diatomaceous Earth as an Insecticide 48819.7.5 Diatomaceous Earth as a soil amendment 48819.7.6 Diatomaceous Earth as a Filler 48919.7.7 Diatomaceous Earth as Abrasive Material 49019.7.8 Diatomaceous Earth as Animals' and Human's Food Additives 49019.7.9 Diatomaceous Earth and Nanotechnology 49119.7.9.1 Diatomaceous Earth in Solar Energy Harvesting Systems 49119.7.9.2 Diatomaceous Earth-Based Superhydrophobic Surfaces 49119.7.9.3 Diatomaceous Earth Composites as Catalysts 49219.7.9.4 Diatomaceous Earth-Based Supercapacitors 49219.7.9.5 Diatomaceous Earth-Based Pharmaceutical and Biomedical Applications 49219.7.9.6 Diatomaceous Earth-Based Lab-on-a-Chip 49419.7.10 Non-Industrial Applications 49419.8 Diatomite Fabrication and Future Aspects 49519.9 Conclusion 495Acknowledgements 496References 49620 Diatom Silica for Biomedical Applications 511 Shaheer Maher, Moom Sin Aw and Dusan Losic20.1 Introduction 51120.2 Diatoms: Natural Silica Microcapsules for Therapeutics Delivery 51320.2.1 Structure 51320.2.2 Surface Modification of Diatoms 51420.2.3 Diatoms Applications as Drug Carriers 51620.2.4 Diatoms as a Source of Biodegradable Carriers for Drug Delivery Applications 52220.2.4.1 Diatoms as a Source of Biodegradable Silicon Micro and Nano Carriers for Drug Delivery 52520.2.5 Diatom Silica for Other Biomedical Applications 52720.2.5.1 Tissue Engineering 52720.2.5.2 Haemorrhage Control 52820.3 Conclusions 530Acknowledgements 531References 53121 Diafuel (TM)(Diatom Biofuel) vs Electric Vehicles, a Basic Comparison: A High Potential Renewable Energy Source to Make India Energy Independent 537 Vandana Vinayak, Khashti Ballabh Joshi and Priyangshu Manab Sarma21.1 Introduction 53821.2 Debate on Relation of Green House Gas Emissions (GHG) with CO2 and Temperature 53921.3 Outcomes of Paris Agreement 2015 54121.4 Energy Demands for India 54221.5 Critics Talking About Entry of EV in Market 54521.6 Comparison Between Electric Vehicles vs Vehicles with Diafuel (TM) at Large 54621.6.1 Electric Vehicles 54621.6.1.1 Status of EV in India 54821.6.1.2 Predicted Impact of EV on Global and Indian Network Versus Their Energy Sources 54921.6.2 Diafuel (TM) 55021.6.2.1 Diafuel (TM) Industrial Production 55221.6.2.2 Designing an Energy Self-Sufficient Indian House Producing Diafuel (TM) 55421.6.2.3 Working Prototype of Diatom Panels for the Indian House 55521.6.2.4 Advantages of Diafuel (TM) 55621.7 Source for Generation of Electricity to Drive EVs 55721.7.1 Resources with Zero Carbon Emission 55821.7.1.1 Nuclear Power 55921.7.1.2 Solar Energy for Faster Adoption and Manufacturing of Electric & Hybrid Vehicles in India 55921.7.1.3 Wind Power 56021.7.1.4 Barriers for Wind and Solar Energy 56121.8 CO2 Emissions by Electric Vehicle vs Gasoline Driven Vehicles 56221.9 Depletion of Earth Metals to Run EV's vs Abundant Resources for Diafuel (TM) 56421.9.1 Can Diafuel (TM) be the Answer 56621.9.2 Harvesting Diafuel (TM) from Diatoms 56621.10 Current Status 56721.10.1 Data Analysis and Comparison Between EV and Diafuel (TM) 56921.11 Conclusions 569Acknowledgement 574List of Abbreviations Used 574References 57422 Bubble Farming: Scalable Microcosms for Diatom Biofuel and the Next Green Revolution 583 Richard Gordon, Clifford R Merz, Shawn Gurke and Benoit Schoefs22.1 Introduction 58422.1.1 The Bubble Farming Concept 58822.1.2 Bubble Injection, Sampling, Harvesting and Sealing, Maybe by Drones 59222.1.3 Approach 59422.2 Mechanical Properties 59422.2.1 Optimal Bubble Size 59622.3 Optical Properties 59722.4 Surface Properties 59922.4.1 Gas Exchange Properties 59922.5 Toxicity Restrictions 60922.5.1 Algal Oil Droplet Properties 61122.6 Biofilms 61122.7 Bacterial Symbionts 61222.7.1 Soil as a Source of CO2 61322.8 Demand 61422.8.1 The Choice of Diatoms vs Other Algae 61422.9 Exponential Growth vs Stationary Phase 61722.10 Carbon Recycling 61922.11 Packaging 61922.11.1 Crop Choice by Farmers 62022.11.2 Bubble Farming vs Photobioreactors and Raceways 62022.12 Summary 620Acknowledgements 626References 626Index 655

Citation preview

Diatoms: Fundamentals and Applications

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106

Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Diatoms: Fundamentals and Applications

Edited by

Joseph Seckbach and Richard Gordon

This edition first published 2019 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2019 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www. wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data Names: Seckbach, J. (Joseph), editor. Title: Diatoms : fundamentals and applications / edited by Joseph Seckbach and Richard Gordon. Description: Hoboken, New Jersey : Wiley ; Salem, Massachusetts : Scrivener, [2019] | Includes bibliographical references and index. | Identifiers: LCCN 2019003170 (print) | LCCN 2019003908 (ebook) | ISBN 9781119370734 (ePDF) | ISBN 9781119370727 (ePub) | ISBN 9781119370215 (hardcover) Subjects: LCSH: Diatoms. Classification: LCC QK569.D54 (ebook) | LCC QK569.D54 D54 2019 (print) | DDC 579.8/5--dc23 LC record available at https://lccn.loc.gov/2019003170 Cover image: Exhibition diatom mount of mixed marine and freshwater diatoms by Klaus D. Kemp. Photomicrograph in polarized darkfield illumination by Stephen S. Nagy, M.D. Photo copyright reserved by the photographer. Cover design: Russell Richardson Set in size of 11pt and Minion Pro by Exeter Premedia Services Private Ltd., Chennai, India Printed in the USA 10 9 8 7 6 5 4 3 2 1

Dedication to Lawrence Bogorad Lawrence Bogorad, born on August 21, 1921 in Tashkent, Uzbekistan, came to the USA at the age of two and passed away on December 28, 2003. He served as a professor in the Botany Department at the University of Chicago for 14 years (from 1953 to 1967) and in the Biology Department at Harvard University for 34 years. At the University of Chicago he pioneered the molecular biology of chloroplast biogenesis, and investigated the photosynthetic pigments. He studied (after the discovery and publication of DNA in chloroplasts), the endosymbiotic ancestor from cyanobacteria as the contributors of the chloroplast DNA. At Harvard, he continued his investigation on the molecular biology of the photosynthetic apparatus. Laurie (his nickname among his colleagues, although I preferred to always approach him by his University title,) was involved in many scientific societies, among them the American Academy of Arts and Sciences (where he served a term as President) and the National Academy of Sciences (Merchant, 2009), and served on the editorial board of Proceedings of the National Academy of Sciences. Bogorad received many awards for his studies. In the personal sphere, he was warm and friendly, full of optimism. His lab was home to five decades of graduate students, postdoctoral fellows, and visiting scientists—all benefiting from his training of them. Here are some recollections of the four years I spent under the ‘wings’ of Professor Lawrence Bogorad at the University of Chicago. He was my mentor for my MSc and PhD  (Seckbach, Bogorad and McIlrath, 1966; Rodermel, Viret and Krebbers, 2005). He welcomed me to the Botany Department at the end of 1961 when I first appeared at the gates of the University. During that interview, we planned my first-year program as a graduate student. Later on, he organized a research assistantship for me so I could continue with my graduate work. As a supportive professor close to his students, he shared with me the latest news on scientific updates in botany and even astrobiology. Later on, when I was involved in a project on the possibilities of “Life on Venus” at UCLA in 1968 with Professor W. F. Libby (Seckbach and Libby, 1970), I approached him, among others, for advice on growing algae under high CO2 and elevated temperatures. Bogorad suggested that I try the red alga Cyanidium caldarium (his “favorite alga”) for my Venus project, and his advice worked very well. Therefore, I have to give him great credit for his guidance and for my finally changing my focus from plant physiology to the new field of astrobiology, and asked him to write the foreword for a book on Cyanidium (Bogorad, 1994). During the subsequent years, I visited him at his lab in Harvard and he hosted evenings in his home in Lexington, Massachusetts. Sometimes he even waited for my arrival at the train station and took me to his home. When he visited Israel and presented a seminar at The Hebrew University of Jerusalem, he introduced me to his mother, and I drove his wife Rosalyn on a tour of Jerusalem. I had warm feelings toward Bogorad and his family, considered him a colleague and a dear friend and dedicate this volume to his memory.

v

vi Dedication to Lawrence Bogorad Joseph Seckbach, PhD University of Chicago, 1965; currently retired from The Hebrew University of Jerusalem; home address: P.O.B. 1132, Efrat, 90435, Israel [seckbach@huji. ac.il].

References Bogorad, L. (1994) Foreword. In: Evolutionary Pathways and Enigmatic Algae: Cyanidium caldarium (Rhodophyta) and Related Cells. J. Seckbach, (ed.) Springer Science +Business Media, B.V., Dordrecht: pp. ix–x. Merchant, S.S. (2009) Lawrence Bogorad, 1921-2003, A Biographical Memoir. U.S. National Academy of Science, Washington, DC, USA. Rodermel, S., Viret, J.-F. and Krebbers, E. (2005) Lawrence Bogorad (1921–2003), a pioneer in photosynthesis research: a tribute. Photosynthesis Research 83(1), 17–24. Seckbach, J., Bogorad, L. and McIlrath, W.J. (1966) Xanthinin deacylase: A new enzyme from xanthium. In: Plant Physiology. American Society of Plant Physiologists, Rockville, MD, USA: p. R67. Seckbach, J. and Libby, W.F. (1970) Vegetative life on Venus? Or investigations with algae which grow under pure CO2 in hot acid media at elevated pressures. Space Life Sciences 2(2), 121–143.

Contents Foreword Preface 1

2

3

4

A Memorial to Frithjof Sterrenburg: The Importance of the Amateur Diatomist Janice L. Pappas 1.1 Introduction 1.2 Background and Interests 1.3 The Personality of an Amateur Diatomist 1.4 The Amateur Diatomist and the Importance of Collections 1.5 The Amateur Diatomist as Expert in the Tools of the Trade 1.6 The Amateur Diatomist as Peer-Reviewed Scientific Contributor 1.7 Concluding Remarks Acknowledgments References

xvii xxiii 1 1 3 7 11 12 15 20 21 21

Alex Altenbach – In Memoriam of a Friend Wladyslaw Altermann References

29

The Beauty of Diatoms Mary Ann Tiffany and Stephen S. Nagy 3.1 Early History of Observations of Diatoms 3.2 Live Diatoms 3.3 Shapes and Structures 3.4 Diatom Beauty at Various Scales 3.5 Valves During Morphogenesis 3.6 Jamin-Lebedeff Interference Contrast Microscopy 3.7 Conclusion Acknowledgments References

33

Current Diatom Research in China Yu Xin Zhang 4.1 Diatoms for Energy Conversion and Storage 4.1.1 Introduction

43

31

33 35 35 36 37 39 40 40 41

43 43 vii

viii Contents

5

6

4.1.2 Diatom Silica: Structure, Properties and Their Optimization 4.1.3 Diatoms for Lithium Ion Battery Materials 4.1.4 Diatoms for Energy Storage: Supercapacitors 4.1.5 Diatoms for Solar Cells 4.1.6 Diatoms for Hydrogen Storage 4.1.7 Diatoms for Thermal Energy Storage 4.2 Diatoms for Water Treatment 4.2.1 Support for Preparation of Diatomite-Based Adsorption Composites 4.2.2 Catalyst and Template for Preparation of Porous Carbon Materials 4.2.3 Modification of Surface and Porous Structure 4.2.4 Support for Preparation of Diatomite-Based Metal Oxide Composites 4.3 Study of Tribological Performances of Compound Dimples Based on Diatoms Shell Structures References

46 48 51 56 58 59 61

Cellular Mechanisms of Diatom Valve Morphogenesis Yekaterina D. Bedoshvili and Yelena V. Likhoshway 5.1 Introduction 5.2 Valve Symmetry 5.3 Valve Silification Order 5.4 Silica Within SDV 5.5 Macromorphogenesis Control 5.6 Cytoskeletal Control of Morphogenesis 5.7 The Role of Vesicles in Morphogenesis 5.8 Valve Exocytosis and the SDV Origin 5.9 Conclusion References

99

Application of Focused Ion Beam Technique in Taxonomy-Oriented Research on Ultrastructure of Diatoms Andrzej Witkowski, Tomasz Płociński, Justyna Grzonka, Izabela Zgłobicka, Małgorzata Bąk, Przemysław Dąbek, Ana I. Gomes and Krzysztof J. Kurzydłowski 6.1 Introduction 6.2 Material and Methods 6.3 Results 6.3.1 Complex Stria Ultrastructure 6.3.1.1 Biremis lucens (Hustedt) Sabbe, Witkowski & Vyverman 1995 6.3.1.2 Olifantiella mascarenica Riaux-Gobin & Compére 2009 6.4 Discussion 6.4.1 Cultured Versus Wild Specimens 6.5 Conclusions Acknowledgements References

61 63 66 75 86 88

99 100 102 103 104 106 107 108 110 110

115

116 117 117 117 117 120 123 124 124 126 126

Contents ix 7

On Light and Diatoms: A Photonics and Photobiology Review 129 Mohamed M. Ghobara, Nirmal Mazumder, Vandana Vinayak, Louisa Reissig, Ille C. Gebeshuber, Mary Ann Tiffany and Richard Gordon 7.1 Introduction 130 7.2 The Unique Multiscale Structure of the Diatom Frustules 130 7.3 Optical Properties of Diatom Frustules 139 7.3.1 The Frustule as a Box with Photonic Crystal Walls 143 7.3.2 Light Focusing Phenomenon 146 7.3.3 Photoluminescence Properties 151 7.3.4 Probable Roles of the Frustule in Diatom Photobiology 152 7.4 Diatom Photobiology 153 7.4.1 Underwater Light Field 153 7.4.2 Cell Cycle Light Regulation 154 7.4.3 The Phototactic Phenomenon in Pennates 154 7.4.4 Chloroplast Migration (Karyostrophy) 156 7.4.5 Blue Light and Its Effects on Microtubules of Cells 157 7.4.6 Strategies for Photoregulation Under High Light Intensity 159 7.4.7 Strategies for Photoregulation Under Ultraviolet Radiation (UV) Exposure 159 7.4.8 Diatoms and Low Light 160 7.4.9 Diatoms and No Light 161 7.4.10 Light Piping and Cellular Vision 161 7.5 Diatom and Light Applications 162 7.5.1 In Photocatalysis 162 7.5.2 Bio-Based UV Filters 164 7.5.3 In Solar Cells 165 7.5.4 Applications Based on Luminescence Properties 167 7.5.5 Cloaking Diatoms 167 7.6 Conclusion 169 Acknowledgement 169 Glossary 169 References 171

8

Photosynthesis in Diatoms Matteo Scarsini, Justine Marchand, Kalina M. Manoylov and Benoît Schoefs 8.1 Introduction 8.2 The Chloroplast Structure Reflects the Two Steps Endosymbiosis 8.3 Photosynthetic Pigments 8.3.1 Chlorophylls 8.3.2 Carotenoids 8.4 The Organization of the Photosynthetic Apparatus 8.5 Non-Photochemical Quenching (NPQ) 8.6 Carbon Uptake and Fixation 8.7 Conclusions and Perspectives Acknowledgment References

191

191 194 196 196 197 197 200 202 204 205 205

x Contents 9

Iron in Diatoms John A. Raven 9.1 Introduction 9.2 Fe Acquisition by Diatoms 9.3 Fe-Containing Proteins in Diatoms and Economy of Fe Use 9.4 Iron Storage 9.5 Conclusions and Prospects Acknowledgements References

213 213 214 214 219 220 220 220

10 Diatom Symbioses with Other Photoauthotroph Rosalina Stancheva and Rex Lowe 10.1 Introduction 10.2 Diatoms with a N2-Fixing Coccoid Cyanobacterial Endosymbiont 10.3 Diatoms with N2-Fixing Filamentous Heterocytous Cyanobacterial Endosymbionts 10.4 Epiphytic, Endogloeic and Endophytic Diatoms 10.5 Diatom Endosymbionts in Dinoflagellates Acknowledgements References

225

11 Diatom Sexual Reproduction and Life Cycles Aloisie Poulíčková and David G. Mann 11.1 Introduction 11.2 Centric Diatoms 11.2.1 Life Cycle and Reproduction 11.2.2 Gametogenesis and Gamete Structure 11.2.3 Spawning 11.3 Pennate Diatom Life Cycles and Reproduction 11.4 Auxospore Development and Structure 11.4.1 Incunabula 11.4.2 Perizonium 11.5 Induction of Sexual Reproduction Acknowledgments References

245

12 Ecophysiology, Cell Biology and Ultrastructure of a Benthic Diatom Isolated in the Arctic Ulf Karsten, Rhena Schumann and Andreas Holzinger 12.1 Introduction 12.2 Environmental Settings in the Arctic 12.3 Growth as Function of Temperature 12.4 Growth After Long-Term Dark Incubation 12.5 Cell Biological Traits After Long-Term Dark Incubation 12.6 Ultrastructural Traits 12.7 Conclusions Acknowledgements References

225 226 233 235 238 239 239

245 247 247 250 251 252 257 259 260 261 262 263 273 274 274 275 277 279 282 283 284 284

Contents xi 13 Ecology of Freshwater Diatoms – Current Trends and Applications Aloisie Poulíčková and Kalina Manoylov 13.1 Introduction 13.2 Diatom Distribution 13.3 Diatom Dispersal Ability 13.4 Functional Classification in Diatom Ecology 13.5 Spatial Ecology and Metacommunities 13.6 Aquatic Ecosystems Biomonitoring 13.7 Conclusions References

289

14 Diatoms from Hot Springs of the Kamchatka Peninsula (Russia) Tatiana V. Nikulina, E. G. Kalitina, N. A. Kharitonova, G. A. Chelnokov, Elena A. Vakh and O. V. Grishchenko 14.1 Introduction 14.2 Materials and Methods 14.3 Description of Sampling Sites 14.3.1 Malkinsky Geothermal Field 14.3.2 Nachikinsky Geothermal Field 14.3.3 Verkhnaya-Paratunka Geothermal Field 14.3.3.1 Goryachaya Sopka Hot Spring 14.3.3.2 Karimshinsky Hot Spring 14.3.4 Mutnovsky Geothermal Field 14.3.4.1 Dachny Hot Springs 14.3.4.2 Verkhne-Vilyuchinsky Hot Spring 14.4 Results 14.4.1 Malkinsky Geothermal Field 14.4.2 Nachikinsky Geothermal Field 14.4.3 Verkhnaya-Paratunka Geothermal Field 14.4.3.1 Goryachaya Sopka Hot Spring 14.4.3.2 Karimshinsky Hot Spring 14.4.4 Mutnovsky Geothermal Field 14.4.4.1 Dachny Hot Springs 14.4.4.2 Verkhne-Vilyuchinsky Hot Spring 14.5 Summary References

311

15 Biodiversity of High Mountain Lakes in Europe with Special Regards to Rila Mountains (Bulgaria) and Tatra Mountains (Poland) Nadja Ognjanova-Rumenova, Agata Z. Wojtal, Elwira Sienkiewicz, Ivan Botev and Teodora Trichkova 15.1 Introduction 15.1.1 Factors Which Control the Diatom Distribution 15.1.2 Biodiversity Assessment 15.2 Recent Datom Biodiversity in High Mountain Lakes in bulgaria and Poland 15.2.1 The Rila Lakes, Bulgaria 15.2.2 The Tatra Lakes, Poland

289 292 292 294 296 299 301 301

311 313 313 314 317 317 318 318 318 319 319 320 320 320 326 326 326 326 326 327 330 331 335

335 336 337 338 338 339

xii Contents 15.3

Diatom Community Changes in High-Mountain Lakes in Bulgaria and Poland from Pre-Industrial Times to Present Day 15.3.1 The Rila Mts. 15.3.2 Tatra Mts. 15.4 Monitoring Data ‘2015’ and Correlations Between the Data Sets of the Rila Mts. and the Tatra Mts. 15.4.1 The Rila Lakes 15.4.2 The Tatra Lakes 15.5 Red-List Data: Cirque “Sedemte Ezera”, Rila Mts. and Tatra Mts. 15.5.1 Cirque “Sedemte Ezera”, Rila Mts. 15.5.2 Tatra Mts. 15.6 Summary Acknowledgements References

340 340 342 344 344 346 349 349 349 349 351 351

16 Diatoms of the Southern Part of the Russian Far East Tatiana V. Nikulina and Lubov A. Medvedeva 16.1 History of the Study of Freshwater Algae of the Southern Part of the Russian Far East 16.1.1 The Primorye Territory 16.1.1.1 Lakes and Reservoirs 16.1.1.2 Rivers and Streams 16.1.2 The Amur Region 16.1.2.1 The Upper Amur 16.1.2.2 The Middle Amur 16.1.3 The Jewish Autonomous Region 16.1.4 The Khabarovsk Territory 16.1.4.1 The Middle Amur 16.1.4.2 The Lower Amur 16.1.5 The Sakhalin Region 16.1.5.1 Sakhalin Island 16.1.5.2 Moneron Island 16.1.5.3 The Kuril Islands 16.2 Diatom Flora of the Southern Part of the Russian Far East References

355

17 Toxic and Harmful Marine Diatoms Stephen S. Bates, Nina Lundholm, Katherine A. Hubbard, Marina Montresor and Chui Pin Leaw 17.1 Introduction 17.2 Harmful Diatoms 17.2.1 How Diatoms May Cause Harm 17.2.2 Diatom Oxylipins 17.2.2.1 Polyunsaturated Aldehydes (PUAs) 17.2.2.2 Oxylipin Production by Pseudo-nitzschia 17.3 Toxic Diatoms 17.3.1 Diatoms That Produce Β-N-Methylamino-L-Alanine (BMAA)

389

355 357 357 358 360 360 360 361 361 361 361 362 362 363 363 363 377

390 391 391 391 391 396 397 397

Contents xiii 17.3.2 Nitzschia navis-varingica 17.3.3 Nitzschia bizertensis 17.3.4 Pseudo-nitzschia spp 17.3.4.1 New Species 17.3.4.2 Distribution 17.3.4.3 Sexual Reproduction 17.3.4.4 Genomic Insights Into Pseudo-nitzschia and Its Population Genetic Structure 17.3.4.5 New Knowledge of Pseudo-nitzschia 17.3.5 Identification of Toxic Diatoms 17.3.5.1 Classical Methods 17.3.5.2 Molecular Approaches 17.4 Gaps in Knowledge and Thoughts for Future Directions References 18 Diatoms in Forensics: A Molecular Approach to Diatom Testing in Forensic Science Vandana Vinayak and S. Gautam 18.1 Introduction 18.2 Postmortem Forensic Counter Measures 18.3 Differences in Drowned Victims vs Those that Die of Other Causes 18.4 Techniques to Identify Diatoms in Biological Sample 18.4.1 Morphological Analysis of Water Samples 18.4.2 Role of Site Specific Diatoms 18.5 Case Studies 18.5.1 Case 1 18.5.2 Case 2 18.5.3 Case 3 18.6 Identification of Diatom Using Molecular Tools in Tissue and Water Samples 18.7 Differentiation of Diatom DNA in the Tissue of a Drowned Victim 18.8 Polymerase Chain Reaction (PCR) 18.9 Diatom DNA Extraction from Biological Samples of a Drowned Victim 18.9.1 Biological Samples 18.9.2 Plankton/Diatom Isolation from Tissues Using Colloidal Silica Gradient and Phenol Chloroform Method for DNA Extraction 18.10 Best Barcode Markers for Diatoms to Diagnose Drowning 18.10.1 Cytochrome C Oxidase Subunit 1 (COI) 18.10.2 Nuclear rDNA ITS Region 18.10.3 Nuclear Small Subunit rRNA Gene 18.11 DNA Sequencing 18.12 Advancement in Sequencing Leads to Advancement of Data Interpretation 18.13 Conclusion and Future Perspectives Acknowledgements List of Abbreviations Used References

400 400 401 401 401 401 410 411 414 414 415 417 418

435 435 438 439 440 441 442 443 443 443 444 446 447 448 448 448 454 454 455 456 457 457 458 459 459 460 460

xiv Contents 19 Diatomite in Use: Nature, Modifications, Commercial Applications and Prospective Trends Mohamed M. Ghobara and Asmaa Mohamed 19.1 The Nature of Diatomite 19.1.1 Diatomite Formation 19.1.2 Diatom Frustule’s Resistance Against Dissolution (The Reason for Their Preservation Over Millions of Years) 19.2 The History of Discovery and Ancient Applications 19.3 Diatomite Occurrence and Distribution 19.4 Diatomite Mining and Processing 19.5 Diatomite Characterization 19.6 Diatom Frustules Modifications 19.7 Diatomite in Use 19.7.1 Diatomite-Based Filtration 19.7.1.1 Water Filtration 19.7.1.2 Beer Filtration 19.7.1.3 Recent Trends in Diatomite-Based Separation Techniques 19.7.1.4 Reuse of Spent DE Filter Media 19.7.2 Diatomite for Thermal Insulation 19.7.3 Diatomite-Based Building Materials 19.7.4 Diatomaceous Earth as an Insecticide 19.7.5 Diatomaceous Earth as a soil amendment 19.7.6 Diatomaceous Earth as a Filler 19.7.7 Diatomaceous Earth as Abrasive Material 19.7.8 Diatomaceous Earth as Animals’ and Human’s Food Additives 19.7.9 Diatomaceous Earth and Nanotechnology 19.7.9.1 Diatomaceous Earth in Solar Energy Harvesting Systems 19.7.9.2 Diatomaceous Earth-Based Superhydrophobic Surfaces 19.7.9.3 Diatomaceous Earth Composites as Catalysts 19.7.9.4 Diatomaceous Earth-Based Supercapacitors 19.7.9.5 Diatomaceous Earth-Based Pharmaceutical and Biomedical Applications 19.7.9.6 Diatomaceous Earth-Based Lab-on-a-Chip 19.7.10 Non-Industrial Applications 19.8 Diatomite Fabrication and Future Aspects 19.9 Conclusion Acknowledgements References 20 Diatom Silica for Biomedical Applications Shaheer Maher, Moom Sin Aw and Dusan Losic 20.1 Introduction 20.2 Diatoms: Natural Silica Microcapsules for Therapeutics Delivery

471 471 472 473 475 476 477 479 480 481 482 483 484 485 485 485 487 488 488 489 490 490 491 491 491 492 492 492 494 494 495 495 496 496 511 511 513

Contents xv 20.2.1 Structure 20.2.2 Surface Modification of Diatoms 20.2.3 Diatoms Applications as Drug Carriers 20.2.4 Diatoms as a Source of Biodegradable Carriers for Drug Delivery Applications 20.2.4.1 Diatoms as a Source of Biodegradable Silicon Micro and Nano Carriers for Drug Delivery 20.2.5 Diatom Silica for Other Biomedical Applications 20.2.5.1 Tissue Engineering 20.2.5.2 Haemorrhage Control 20.3 Conclusions Acknowledgements References 21 Diafuel™(Diatom Biofuel) vs Electric Vehicles, a Basic Comparison: A High Potential Renewable Energy Source to Make India Energy Independent Vandana Vinayak, Khashti Ballabh Joshi and Priyangshu Manab Sarma 21.1 Introduction 21.2 Debate on Relation of Green House Gas Emissions (GHG) with CO2 and Temperature 21.3 Outcomes of Paris Agreement 2015 21.4 Energy Demands for India 21.5 Critics Talking About Entry of EV in Market 21.6 Comparison Between Electric Vehicles vs Vehicles with Diafuel™ at Large 21.6.1 Electric Vehicles 21.6.1.1 Status of EV in India 21.6.1.2 Predicted Impact of EV on Global and Indian Network Versus Their Energy Sources 21.6.2 Diafuel™ 21.6.2.1 Diafuel™ Industrial Production 21.6.2.2 Designing an Energy Self-Sufficient Indian House Producing Diafuel™ 21.6.2.3 Working Prototype of Diatom Panels for the Indian House 21.6.2.4 Advantages of Diafuel™ 21.7 Source for Generation of Electricity to Drive EVs 21.7.1 Resources with Zero Carbon Emission 21.7.1.1 Nuclear Power 21.7.1.2 Solar Energy for Faster Adoption and Manufacturing of Electric & Hybrid Vehicles in India 21.7.1.3 Wind Power 21.7.1.4 Barriers for Wind and Solar Energy 21.8 CO2 Emissions by Electric Vehicle vs Gasoline Driven Vehicles

513 514 516 522 525 527 527 528 530 531 531

537 538 539 541 542 545 546 546 548 549 550 552 554 555 556 557 558 559

559 560 561 562

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Depletion of Earth Metals to Run EV’s vs Abundant Resources for Diafuel™ 21.9.1 Can Diafuel™ be the Answer 21.9.2 Harvesting Diafuel™ from Diatoms 21.10 Current Status 21.10.1 Data Analysis and Comparison Between EV and Diafuel™ 21.11 Conclusions Acknowledgement List of Abbreviations Used References 22 Bubble Farming: Scalable Microcosms for Diatom Biofuel and the Next Green Revolution Richard Gordon, Clifford R Merz, Shawn Gurke and Benoît Schoefs 22.1 Introduction 22.1.1 The Bubble Farming Concept 22.1.2 Bubble Injection, Sampling, Harvesting and Sealing, Maybe by Drones 22.1.3 Approach 22.2 Mechanical Properties 22.2.1 Optimal Bubble Size 22.3 Optical Properties 22.4 Surface Properties 22.4.1 Gas Exchange Properties 22.5 Toxicity Restrictions 22.5.1 Algal Oil Droplet Properties 22.6 Biofilms 22.7 Bacterial Symbionts 22.7.1 Soil as a Source of CO2 22.8 Demand 22.8.1 The Choice of Diatoms vs Other Algae 22.9 Exponential Growth vs Stationary Phase 22.10 Carbon Recycling 22.11 Packaging 22.11.1 Crop Choice by Farmers 22.11.2 Bubble Farming vs Photobioreactors and Raceways 22.12 Summary Acknowledgements References Index

564 566 566 567 569 569 574 574 574

583 584 588 592 594 594 596 597 599 599 609 611 611 612 613 614 614 617 619 619 620 620 620 626 626 655

Foreword Prof. Dr. Andrzej Witkowski Palaeoceanology Unit University of Szczecin Mickiewicza 16a PL-70-383 Szczecin, Poland http://www.marinebenthicdiatoms.univ.szczecin.pl tel. +48 91 4442465 [email protected] Already seven years have passed since the publication of The Diatom World, a review of progress in the field of diatom research, edited by Joseph Seckbach and John P. Kociolek (Seckbach & Kociolek 2011). Needless to say, this period was marked further with significant progress in studies on diatoms. In all certainty, the wealth of data acquired over this justifies the publication of this new book, “Diatoms: Fundamentals and Applications”. In addition to fundamental issues of diatom biology including valve morphogenesis, sexual reproduction and cell cycle, ecology and biodiversity, it includes numerous contributions on applied aspects of diatom research. The section on applied aspects begins with a review of diatomite applications, including commercial use and future trends (Ghobara 2019). A related chapter discusses photonic properties of biogenic silica brought into the intricate patterns of diatom valves, an inspiration for numerous generations of professional and self-taught diatomists, but also chemists and physicists (Ghobara et al. 2019). Progress in biomedical applications research is presented in chapters on drug delivery with diatomaceous silica as a potentially biodegradable drug carrier, for tissue engineering and hemorrhage control on the one hand (Maher, Aw & Losic 2019), and on the use of molecular methods in forensic science on the other (Vinayak & Gautama 2019). The latter chapter is a leap forward as it may spare the diatomists performing forensic examinations the burden of processing drowning victims’ internal organs. The application of metabarcoding will not solve the problem of the physical presence of diatoms, e.g., in lungs, but will support the identification of diatoms and thus facilitate the identification of the habitat where the victim drowned. This may be another sign of metabarcoding outcompeting the classic light microscope (LM) examination and counting of processed diatom valves on slides. To remind the reader, the United Kingdom gave up the use of LM-based diatom valve counting in water quality assessment in 2017 (Mann et al. 2017). Metabarcoding became the preferred alternative, let us hope, successfully. This hope is expressed on behalf of all skeptical practitioners of classic diatom indices in river and lake monitoring. Although diatom lipids are considered a very good source of biodiesel, and some species are even named as oleaginous forms (see Fistulifera solaris for an example), other microalgae xvii

xviii Foreword are still preferred in large scale biomass growth and oil production. The present book introduces a new term, and a corresponding trade mark, of Diafuel with Fistulifera saprophila valve graphics playing a central part (Vinayak, Joshi & Sharma 2019). Unlike other microalgae, diatom organelles and lipid drops are encased in a siliceous box-like frustule. Unlike the “soft bodied” microalgae, diatoms go undamaged unless the pressure applied to squeeze oil exceeds a critical strength. Shall this allow the process of milking diatoms? Let us wait and see. So far, however, a splendid field of research on nanoindentation is seeking to obtain a milking pressure that will not kill diatoms, and allow them to restore their lipid droplets. Considering human energy use forecasts, this shall be required within the time span of several human generations. A unique opportunity presented by the “diafuel” project is that the secondary product or “waste” of the technological process is the biogenic silica that - owing to its photonic properties – can be “recycled” in further production of energy, for instance in solar panels or as a component of new valuable materials with unique photonic properties (Ghobara et al. 2019). One drawback of using diatom mass cultures for biofuel production purposes is the necessity of choosing between open race ponds which can be contaminated with airborne mineral and microbial waste, and closed photobioreactors which are more expensive to maintain. Culturing diatoms in bubble wrap, proposed in the chapter by Gordon et al. (2019), could be an alternative to these two solutions. If successfully implemented, upon certain conditions, with “bubble farming” biofuel production costs could be lower than those of mineral fuels. In line with this is the review of the enormous scale of applied ongoing research on diatoms in China (Zhang 2019). Discussed in this latter chapter are examples of applied research involving diatoms in materials and biomaterials science, energy production and storage, waste water treatment, composites, diatom-based ceramics, etc. Given that numerous laboratories in China also work on fundamental aspects in diatom research including ecology, biology, taxonomy and phylogeny, the overall impression is tremendous. Framed prints of beautiful diatoms can generate substantial sums of money. However, it seems that the beauty of diatoms unspoiled with any commercial issues is a value in itself. It is quite common that professional diatomists maintain friendly relations with diatom enthusiasts who are experts in light microscopy, often using very sophisticated systems. Perhaps surprisingly, with the changing technologies there is also an increase in the number of non-professional diatomists who own scanning electron microscopes. I know at least a few in Europe. The diatom beauty chapter definitely hosts images that should rank within fine arts (Tiffany & Nagy 2019). Morphogenesis of the diatom frustule is the subject of another chapter  (Bedoshvili & Likhoshway 2019). Despite the progress in our understanding of the cellular mechanism of valve formation, its genetic controls remain largely unknown. Fundamental aspects of diatom research are represented by such highlights as a review of sexual reproduction and life cycle, with the latest perspective on these issues (Poulíčková & Mann 2019). This research, which involves experiments for scientists of Benedictine patience, is crucial for understanding numerous aspects in diatom taxonomy and systematics. However, despite its splendid reputation and importance, few young scholars are willing to learn the techniques and spend days, months and years at an inverted microscope isolating clonal cultures of similar strains to discover their sexual compatibility and perform successful crossing experiments. Certainly the use of molecular tools makes the search for potentially compatible clones easier, but does not guarantee offspring. Research on diatom symbiosis is less common. Fortunately, a chapter on endosymbionts in diatom cells

Foreword

xix

(cyanobacteria), and on diatoms as endosymbionts (in dinoflagellates) is published in this book (Stancheva & Lowe 2019). Any attempt to define a diatom usually involves a phrase like: diatoms are unicellular, photosynthetic organisms present in all habitats providing enough ambient light and a minimum of moisture. Diatom life and valve morphogenesis are intertwined with photosynthesis. Despite the fact that diatoms play such an important role in aquatic and terrestrial ecosystems, their photosynthesis at the organellar level, as emphasized by the authors of the chapter on diatom photosynthesis diatoms, is rather poorly known (Scarsini et al., 2019). A series of chapters presents reviews on biodiversity and comparisons of diatoms living in freshwater habitats including rivers and freshwater mountainous habitats on one hand and potentially toxic marine diatoms on the other. Research on inland diatom assemblages of the Russian Far East (Nikulina & Medvedeva 2019) and on those from hot springs in Kamchatka (Nikulina et al. 2019) is presented in two chapters. Included are also chapters on freshwater diatoms of the South and Central European Mountain Ranges, including Tatra (Poland) and Rila (Bulgaria) (Ognjanova-Rumenova et al. 2019). The review of freshwater diatom ecology provides a link between fundamental and applied aspects of diatom research (Poulíčková & Manoylov, 2019). Covered in this review are the most recent aspects of freshwater diatom ecology, dispersal, biodiversity and biogeography, with an emphasis on practical aspects of using freshwater diatoms, i.e., in biomonitoring of freshwater habitats. As in the chapter on Kamchatka hot springs, another extreme habitat is presented in a review on ecophysiology of the Arctic fjord diatom strain Navicula directa  (Karsten & Holzinger, 2019). The adaptation of this species to harsh environmental conditions related to low temperatures and lack of light during the long polar night has been proven in a series of experiments. The role of iron (Fe) in diatom physiology is reviewed by  (Raven 2019). Despite the importance of iron, the existing knowledge is scarce and mostly limited to marine planktonic taxa. This book is crowned with the most up to date review on diatoms as potential producers of toxins dangerous to humans and other living organisms (not only domoic acid). Aside from fairly numerous representatives of Pseudo-nitzschia, only two marine Nitzschia species have been detected as toxin producers. However, we should expect the list of toxins and their producers to increase. It is not always the case that bloom forming toxin producers appear in strongly human impacted environments (e.g. shrimp aquaculture). Some inhabit Arctic and Antarctic marine waters or cold oceanic currents. The chapter by Bates et al. (2019) presents the complex biology of toxic diatoms, their distribution, and detection methods. The present book is unique as it provides also an emotional component: it includes several chapters that commemorate those of our diatomist colleagues who recently passed away. Joseph Seckbach commemorates his friend Lawrence Bogorad, a late professor at the University of Chicago and dedicates the whole volume to his memory (Seckbach 2019). This is a tribute to Joseph Seckbach’s MSc and PhD mentor at the University of Chicago. They remained in very friendly contact after Joseph’s graduation. Lawrence Bogorad’s research on photosynthetic pigments made a considerable impact on our understanding of chloroplast origin and photosynthesis. Further, Wladyslaw Altermann summarizes the life and scientific career of Alex Altenbach, a renowned palaeontologist and protistologist (Altermann 2019). The third, touching text by Janice Pappas is dedicated to Frithjof Sterrenburg, a diatom enthusiast with whom many of us had collaborated in the past (Pappas 2019). Personally, I met Frithjof in Frankfurt am Main during his visit to Horst Lange-Bertalot. We spent a memorable few days with Frithjof. I cherish the memory of countless phone calls I made

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that were answered by Frithjof, when an answer to an urgent taxonomic issue was past due. It is a pity that our two joint efforts to get the Kinker collection project funded failed. One special recollection that I have is Frithjof ’s pride when he published a joint paper with his father (Sterrenburg & Sterrenburg 1990). Probably few diatomists know that Nitzschia nienhuisii Sterrenburg F.A.S. & Sterrenburg F.J.G. 1990 from the coast of Mauritania was described by son and father Sterrenburgs. This distinctive and beautiful diatom, common around African coasts of Atlantic and Indian Oceans, seems to require a transfer to a new (as yet unnamed) genus. Years after witnessing Frithjof ’s pride, I found myself moved by similar feelings when publishing a joint paper with my son (Dabek et al. 2015). This book will definitely be a connection between the fundamental and applied research on diatoms, and a connection between two scientific communities. Personally, I consider my professional contacts with materials science community and use of their tools, i.e. Focused Ion Beam (FIB, Witkowski (2019)) as very inspiring and fruitful in my own, principally fundamental, diatom research.

References Altermann, W. (2019) Alex Altenbach – in memoriam of a friend. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) WileyScrivener, Beverly, MA, USA: pp. 27–30. Bates, S.S., Lundholm, N., Hubbard, K.A., Montresor, M. and Leaw, C.P. (2019) Toxic and harmful marine diatoms. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 388–434. Bedoshvili, Y.D. and Likhoshway, Y.V. (2019) Cellular mechanisms of diatom valve morphogenesis. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 97–112. Dabek, P., Witkowski, J., Witkowski, A. and Riaux-Gobin, C. (2015) Morphology of Biddulphia seychellensis (Grunow in Van Heurck) FW Mills and the generic limits of Biddulphia Gray. Nova Hedwigia 144(Supplement), 97-105. Ghobara, M.M. and Mohamed, A. (2019) Diatomite in use: Occurrence, characterization, modification, and prospective trends. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 471–510. Ghobara, M.M., Mazumder, N., Vinayak, V., Reissig, L., Gebeshuber, I.C., Tiffany, M.A. and Gordon, R. (2019) On light and diatoms: A photonics and photobiology review. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 127–188. Gordon, R., Merz, C.R., Gurke, S. and Schoefs, B. (2019) Bubble farming: Scalable microcosms for diatom biofuel and the next Green Revolution. In: Diatoms: Fundamentals

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& Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) WileyScrivener, Beverly, MA, USA: pp. 583. Karsten, U., Schumann, R., and Holzinger, A. (2019) Ecophysiology, cell biology and ultrastructural anatomy of a benthic diatom isolated in the Arctic. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) WileyScrivener, Beverly, MA, USA: pp. 271–286. Maher, S., Aw, M.S. and Losic, D. (2019) Diatom silica for biomedical applications. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 511–536. Mann, D.G., Kelly, M.G., Walsh, K., Glover, R., Juggins, S., Sato, S., Boonham, N. and Jones, T. (2017) Development and adoption of a next-generation-sequencing approach to diatom-based ecological assessments in the UK [Abstract]. Phycologia 56(4, Supplement), 125-126. Nikulina, T.V., Kalitina, E.G., Kharitonova, N.A., Chelnokov, G.A., Vakh, E.A. and Grishchenko, O.V. (2019) Diatoms from hot springs of the Kamchatka Peninsula (Russia). In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 309–332. Nikulina, T.V. and Medvedeva, L.A. (2019) Diatoms of the southern part of the Russian Far East. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 353–386. Ognjanova-Rumenova, N., Wojtal, A.Z., Sienkiewicz, E., Botev, I. and Trichkova, T. (2019) Biodiversity of high mountain lakes in Europe with special regards to Rila Mountains (Bulgaria) and Tatra Mountains (Poland) In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 333–352. Pappas, J.L. (2019) A memorial to Frithjof Sterrenburg: The importance of the amateur diatomist. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 1–26. Poulíčková, A. and Mann, D.G. (2019) Diatom sexual reproduction and life cycles. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 243–270. Poulíčková, A. and Manoylov, K.M. (2019) Ecology of freshwater diatoms – current trends and applications. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 287–308. Raven, J.A. (2019) Iron and ferritin in diatoms. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard

xxii Foreword Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 211–222. Scarsini, M., Marchand, J., Manoylov, K.M. and Schoefs, B. (2019) Photosynthesis in diatoms. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 189–210. Seckbach, J. and Kociolek, J.P. (eds.) (2011) The Diatom World. Springer London, Limited. Seckbach, J. (2019) Dedication to Lawrence Bogorad. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) WileyScrivener, Beverly, MA, USA: pp. v–vi. Stancheva, R. and Lowe, R. (2019) Diatom symbioses with other photoauthotrophs. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 223–242. Sterrenburg, F.A.S. and Sterrenburg, F.J.G. (1990) An outline of the marine littoral diatom biocoenosis of the Banc-d’Arguin, Mauritania, West Africa. Botanica Marina 33(5), 459-465. Tiffany, M.A. and Nagy, S.S. (2019) The beauty of diatom cells in light and scanning electron microscopy. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 31–40. Vinayak, V. and Gautama, S. (2019) Diatoms in forensics: A molecular approach to diatom testing in forensic science. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 435–470. Vinayak, V., Joshi, K.B. and Sarma, P.M. (2019) Diafuel© (diatom biofuel) vs electric vehicles, a basic comparison: A high potential renewable energy source to make India energy independent. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 537–582. Witkowski, A. (2019) Application of focused ion beam in studies of ultrastructure of diatoms. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) Wiley-Scrivener, Beverly, MA, USA: pp. 113–126. Zhang, Y.X. (2019) Current diatom research in China. In: Diatoms: Fundamentals & Applications [DIFA, Volume 1 in the series: Diatoms: Biology & Applications, series editors: Richard Gordon & Joseph Seckbach]. J. Seckbach and R. Gordon, (eds.) WileyScrivener, Beverly, MA, USA: pp. 41–96.

Introduction to Diatoms: Fundamentals and Applications Joseph Seckbach

Diatoms: Fundamentals and Applications is the first volume in the series Diatoms: Biology and Applications, published by Wiley-Scrivener and edited by Richard Gordon and Joseph Seckbach. Diatoms: Fundamentals and Applications complements adds further to the knowledge presented in our first volume (The Diatom World, Joseph Seckbach and J. Patrick Kociolek, eds., Springer, 2011). Moreover, the current volume includes some topics not covered in the previous volume. What are the diatoms? They are fascinating microscopic unicellular or colonial, micro scopic eukaryotic algae. They are ubiquitously distributed in aqueous habitats, considered a major part of phytoplankton. They are present in fresh water, saline environments, brackish water and marine areas, and they are a source of biofuel. They live in high and low temperatures, and at different pH values. Their cells are divided into 2 halves. Their cell wall is silicified. This volume presents many facets of diatoms that you have never encountered and had no inkling of their existence. Diatoms utilize 20% of the atmospheric CO2 and release (via their photosynthesis process) our atmospheric O2 vital to all life. (See chapter by Matteo et al.). Their chloroplasts are uniquely composed and differ from other green algae and from higher plants by not possessing chlorophyll b as is present in other green plants. Our contributors are from quite a few countries, including Canada, China, Egypt, France, Germany, India, Israel, Poland, Russia, South Africa, UK and USA.

The Topics Covered in This Volume are Varied Ecology; cell biology; biodiversity, distribution in nature; photosynthesis, light and diatoms; iron and ferritin; toxic diatoms, sexual reproduction, biofuel; ion beams; diatom uses; external uses; forensic use; medical silica; and diatom research in China. It is assumed that an endosymbiotic event (see Stancheva and Lowe), took place in the past during the evolution of diatoms. According to this theory, a host eukaryotic primitive cell absorbed a cyanobacteria type of cell and used this guest (or penetrator) as part of its eukaryotic plastid entity with some exchange of genetic material with the host nucleus. For full photosynthetic activity iron is required (chapter by Raven). Otherwise, with a lack of iron in its nutrition, the green algae and higher plants turn pale and chlorotic - green-less). Algal-ferritin presents as a storehouse of iron for the photosynthetic reaction and for other requirements for cellular iron. xxiii

xxiv Introduction to Diatoms: Fundamentals and Applications Cytological studies of the silicon diatomic cell wall, exhibiting the most beautiful diatoms appearance, are presented by Karsten and Holzinger, by Witkowski, and by others. Furthermore, the “ideal beauty” of the diatomic walls is admired specifically in the chapter by Tiffany and Nagy. From among the ecological environments, diatoms are ubiquitous, living in fresh water, cold Polar water, hot spring water, and mountain lakes (see Poulickova and Mann, Nikulina et al., Ognjanova-Rumenova et al.) This book is dedicated to the memory of three close colleagues; Lawrence Bogorad, -who was my mentor for my MSc and PhD at the University of Chicago. My colleague Alex Altenbach, whom I met when I was a DAAD in the Department of Geology at Ludwig Maximilian University in Munich. Frithjof Sterrenburg, a colleague who was an electron microscopist and an amateur diatomist from The Netherlands. All three of the above were involved in algae and diatom research.

Acknowledgment We, the editors thank our authors for their contributions, specifically our Russian authors, and the reviewers of the chapters.

1 A Memorial to Frithjof Sterrenburg: The Importance of the Amateur Diatomist Janice L. Pappas Department of Mathematics, University of Michigan, Ann Arbor, USA

Abstract Frithjof A.S. Sterrenburg was an amateur diatomist who became an expert in taxonomy, nomenclature, microscopy, and photomicrography. He is best known for his work on Gyrosigma Hassall and Pleurosigma W. Smith but contributed in many areas of diatom research. Because of who he was and the nature of his interactions with the diatom community, he has had and will continue to have a lasting impact. His contributions to diatom research have influenced our understanding and usage of diatom taxonomy and nomenclature and how taxonomy informs ecological, biostratigraphic and other biological studies. His affable yet incisive character enabled him to be a teacher and mentor to professionals and amateurs and to be respected and appreciated internationally. Keywords: Diatoms, Gyrosigma, Pleurosigma, microscopy, amateur diatomists, photomicrography, museum collections, taxonomy, valve morphogenesis, constructal morphology

1.1

Introduction

Throughout science, amateurs have contributed to the body of knowledge in many disciplines. Amateurs dedicate themselves to the pursuit of knowledge concerning their specific interests, and when it comes to diatoms, this is no exception. During the 1800s, diatom research was conducted by hobbyists who had occupations in very different fields (Bahls 2015; Gordon et al. 2009). For example, Friedrich Traugott Kützing was a pharmacist and school teacher who became a diatomist. He discovered that diatoms were composed of silica and had two parts to their shells, one was “primary” and the other “secondary,” and was aided in publishing his findings by C.G. Ehrenburg, the preeminent zoologist and diatomist of the time (Werner 1977). Some amateurs formed microscopical societies or clubs as enthusiasts pursuing their common interest in the microscopic world (e.g., The Quekett Microscopical Club). In the post-Victorian world, diatom research has become professionalized, and currently, individuals can find Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (1–28) © 2019 Scrivener Publishing LLC

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Figure 1.1 Frithjof A. S. Sterrenburg at his microscope. (courtesy of H. van Dam)

professional mentors for formalized training to become a diatom researcher as one’s occupation. Having said this, modern amateur or citizen scientists are still making contributions to diatom research. Because so few individuals are formally trained, credentialed, paid, and have a career specifically in diatom research, and because diatom research is at a stage where the enormity of necessary work involves potentially hundreds of thousands of species, amateurs have a vital role to play. Amateurs who engage in this role seriously soon realize that contributing requires associating oneself with professionals, especially concerning technical matters in taxonomy, nomenclature, microscopy, and photomicrography. The quintessential example of the modern amateur diatomist was Frithjof Sterrenburg (Figure  1.1). He died on March, 11, 2016 and left his mark on diatom research. He had passion and dedication to diatom research that earned him recognition and respect from professionals internationally. Frithjof’s work in diatom taxonomy and nomenclature as well as microscopy and photomicrography has benefited professional and amateur diatomists alike, and his contributions have continued to be influential in the way diatom researchers engage in and pursue their studies. No matter how one perceives what it means to be an amateur, Frithjof transcended the notion because of who he was and how he lived his life. Historically, amateur diatomists were unpaid and typically worked in various occupations (Bahls 2015; Werner 1977). A prime example was Astrid Cleve-Euler. She was the first female to be awarded a doctorate in science at Uppsala University, Sweden in the late 19th century. Yet, throughout her life, she was never employed as a scientist despite her contributions to chemistry, botany, geology, and diatom research (Swedish

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Natural History Museum). Even van Leeuwenhoek (17th to 18th century) who was a tradesman and politician did not get paid to study the microscopic world (Pedrotti accessed on 2016; Sterrenburg 1982). Other dedicated amateurs engaged in diatom research. Pleurosigma angulatum, originally named Navicula angulata by John Thomas Quekett (Sterrenburg 1990a), an amateur diatomist who was a microscopist and histologist, was typified by the Reverend William Smith, an amateur turned academic who was a prominent diatom researcher in the Victorian era (Werner 1977). As a common leisure activity, diatoms were mounted in various configurations, slides were either purchased or prepared by the individual, and mounts were viewed with microscopes situated in Victorian parlors (Lynk accessed on 2016). Diatom mounters such as Johann Diedrich Möller (originator of the art of diatom mounting) (Walker 2009) and dentist William Gatrell (Stevenson 2009) were much in demand to produce intricate arrangements for viewing. The advent of the microscope and its common usage paved the way for such activities and induced the proliferation of amateur diatomists interested in the natural history of these eye-catching, exquisite microorganisms. Frithjof was a throwback, in a sense, to this ilk of diatom researcher. Yet, he developed his own style and went beyond amateurs such as surgeon and physician John Redmayne (Stevenson 2013) and teacher John Albert Long (Walker 2012). Like Quekett, Redmayne and Long plied their trade as amateur diatomists in the late 19th and early 20th century. They bought and sold diatom samples as well as made mounts and relied on popular publications such as Hardwicke’s Science Gossip for the latest information on diatoms (e.g., Taylor 1885). In Hardwicke’s volume XXI from 1885, an article on Jacques-Joseph Brun’s publication, “The Diatoms of the Alps and the Jura,” was presented in which Brun, a pharmacologist and diatomist (JStor Global Plants accessed on 2016), talked about the deposition of diatoms over time and the formation of Kieselgühr (Taylor 1885). His taxonomic work like that of his contemporaries on fossil diatoms provided the impetus for the commencement of Adolph Schmidt’s Atlas in 1874 (Schmidt et al. 1874–1959) that was continued with contributions from others until 1959—including Friedrich Hustedt, a teacher turned professional diatomist who has had a great influence on diatom research (Alfred Wegener Institute 2015). Frithjof’s trajectory into diatom research was more along the lines of Smith, Brun, and Hustedt. Like these predecessors, Frithjof progressed from amateur to expert, garnering respect for his expertise. Frithjof elevated his contributions to the level of scientific peer-review rather than being only presentable in popular publications, and like many of his predecessors, he has had a lasting impact on diatom research.

1.2

Background and Interests

Frithjof A. S. Sterrenburg was born in 1934. Originally, he studied medicine at Amsterdam University. His autodidactic nature conflicted with a formalized education approach, and inevitably, he would venture out on his own. He was well-read and able to learn various subjects at many levels of difficulty and did so as a life-long endeavor. For Frithjof, life and learning was an adventure.

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Figure 1.2 Frithjof playing the trumpet with trombonist Bill Rank from the Paul Whiteman orchestra (1968). (permission from C.J.Sterrenburg)

Figure 1.3 Frithjof in his backyard, extolling a friend to see into the night sky (circa 1980). She and her biologist husband were hosts of the Sterrenburgs in Sulawesi, Indonesia. (permission from C.J. Sterrenburg)

Frithjof showed a propensity for immersing himself in a wide range of learning experiences, and some of his emerging talents were expressed in such divergent fields as music (e.g., Sterrenburg 1967) and astronomy. In his youth, Frithjof played trumpet (Figure 1.2), saxophone, clarinet, and piano in big-band style orchestras, and he also arranged compositions for such orchestras. He was a jazz musician for many years (de Wolf, personal communication). He owned many telescopes (Figure  1.3). Some of Frithjof’s first publications in the early 1980’s described how to see the stars more clearly (Sterrenburg 1983a, b). He liked to share conversations about the stars with many people, including long-time friends Michael Stringer and Wulf Herwig, as he relished being able to see beyond our immediate world. Frithjof was a tinkerer and had a knack for all things electronic, electrical and mechanical (e.g., Sterrenburg 1979). Frithjof shared an interest in electrical devices with

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Wulf and long-time friend Hein de Wolf. He loved airplanes (van Dam, personal communication) and loved to restore vintage cars and motorbikes as well as military radio receivers, which he collected to round out his fascination with the military. He authored manuals on receivers (de Wolf, personal communication; Sterrenburg 1970a) and was on the editorial board of professional electronics journals (Sterrenburg et al. 2007). He was an avid motorbike rider noted Michel Poulin while on a visit to the Sterrenburg home. Hein was always surprised when Frithjof would show up at his office. Would Frithjof be attired in his motorcycle outfit or an extravagant suit (de Wolf, personal communication)? His knowledge and interest in history (Hargraves 2016) as well as documenting and saving historical collections (Hamilton, personal communication), included military history (e.g., Sterrenburg 1970b; Weaver & Sterrenburg 1968) specifically about World War II (WWII). The decoding of the German Peenemünde documents contained in the Oslo Report was amassed in a perceptive compendium written by Frithjof (Sterrenburg year unknown; Stringer, personal communication). Much of Frithjof’s outlook on life was influenced by his experiences as a youth. Frithjof came of age during a time when the world was in the grip of economic depression and followed by WWII, having family members that suffered during the times of Nazi Germany (Herwig, personal communication). He grew up during the 1930s and 1940s, and his personal recollections were compiled in 2009 in a web forum on WWII aircraft (Hargraves, personal communication; Sterrenburg 2009a). He knew much about that period in human history, and although he was sensitive about the effect that era had on himself (Poulin, personal communication), he was interested in how other individuals were affected as well (Sterrenburg & Toonder 1972). Over the years, Frithjof and his dear wife, José (Figure 1.4), built a good life together. They enjoyed their largest restoration project of an 1890 Victorian house in the small farming village of Sijbekarspel, North Holland. It was a labor of love where they lived for 35 very happy years. As Michel Poulin recalls, their home was “very well maintained and had a nice garden.” For over 40 years, Frithjof was self-employed in communications, advertising and public relations as a consultant to research centers and hi-tech industries in the fields of communications, medical instrumentation (e.g., Sterrenburg 1983c), aviation, defense, pharmaceuticals, energy production, and environmental sciences. He enjoyed interacting with clients, and with his effusive manner, he completed each project with the close attention to detail it deserved. Frithjof and José loved to travel throughout Europe, especially in the Balkans, Greece and Portugal, the Pacific Islands (Rarotonga and Western Samoa) Indonesia, Australia, New Zealand, and the Western United States which included California (especially San Francisco), Nevada, Utah, Colorado, Arizona, and Florida. Holidays were spent in Germany and England, once on a vintage motorbike, and once along Australian's East coast on a modern motorbike. For work purposes, Frithjof travelled to the United Arab Emirates. Frithjof and José got to see many parts of the world and experience many cultures and ways of living. As a colorful individual who had a gift for storytelling (Figure  1.5), Frithjof was forthright, strong-willed, and not afraid to engage in conversations or debates on controversial subjects (e.g., science vs. religion, formally unveiled posthumously in Sterrenburg 2018). Frithjof was eloquent and forceful in his opinions, explications, and disquisitions. There was no mistake about where Frithjof stood on any given subject (e.g., Sterrenburg 1976, 1977), and he acted on his strong convictions. Although

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Figure 1.4 Frithjof and José relaxing amongst the trees and flowers (circa 1978). (permission from C.J. Sterrenburg)

Figure 1.5 Frithjof telling a tall tale (1985). (permission from C.J. Sterrenburg)

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Frithjof and José were in the process of moving to Heiloo at the time of the 2003 North American Diatom Symposium (Gordon, personal communication), Frithjof was also reticent about traveling to the U.S. because of his opposition to the 1990 Gulf War; instead, he sent a video presentation with a “cheerful toast at the end” for viewing at the meeting (Hamilton, personal communication). Frithjof was a cosmopolitan individual, bearing witnessing to various modes of life and immersing himself in the plethora of ideas he encountered. He was of nimble mind, turning those ideas into self-taught knowledge and assembling that knowledge into a finely-honed result. This attitude served him well in his scientific pursuits involving diatoms.

1.3

The Personality of an Amateur Diatomist

Whether engaging a client, friend, or diatom enthusiast, Frithjof always had time to attend to your inquiry, and he did it with his usual affability, a bit of flare, intensity of focus, and a dry, sharp sense of humor (e.g., Stringer, personal communication; Hargraves, personal communication). His command of and facility for languages (Hargraves 2016) was always on display with a deftness in elocution that embellished your sensibilities about the subject being discussed. When engaging in a conversation with Frithjof, sometimes you were taken on a “ride” that you had no idea was about to happen. When corresponding with Frithjof during the compilation of the special issue on diatom nanotechnology and the difficulties encountered in trying to get the issue published in a timely fashion, I found myself drawn into a discussion of metaphorical representations from ancient Greek history and mythology to deal with the situation. We exchanged musings on Helen of Troy, Medea and Aegeus. One of the regular editorial board members let me know that I was the impetus for restarting the process of publication, so I ended up being the protagonist, with a transformation from Helen of Troy to Medea, as determined by Frithjof. The antagonist? Those involved with the special issue may remember the exchange of e-mails that occurred during this “tragicomedy” and may surmise who might be the antagonist. Frithjof and I determined that he was not Aegeus, and therefore was no match for Medea! When Paul Hargraves e-mailed Frithjof and told him he could not recall meeting him at a conference, Frithjof sent a picture of “himself” that Paul quickly determined was Austrian Admiral Hermann Freiherr von Spaun (Austrian Admiral Hermann Freiherr von Spaun accessed on 2016). Disappointed that the ruse was quickly dispelled, Frithjof received an unidentified picture of royal dignitaries from which he was asked to identify Paul. Frithjof knew the picture was of the nine sovereigns at Windsor for the funeral of King Edward VII, and he chose Albert the 1st of Belgium to be Paul (Albert the 1st of Belgium accessed on 2016). However, as Paul stated, he is unaware of any relation to Albert (Hargraves, personal communication). Frithjof’s sense of humor was always on display as a source of amusement for anyone who communicated with him. Frithjof possessed admirable knowledge of the Old Testament as he exchanged quotes in English or German with Joseph Sechback whom he called Methuselah.

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During a visit to Edam, Joseph and his wife were treated to a ride in Frithjof’s sports car on their way to a tour of a cheese factory (Sechback, personal communication). Richard Gordon found out from Frithjof that being an itinerant biologist who has a small number of degrees of separation from Erdös (a sort of “close encounter of the Erdös kind”), may induce metamorphosis into an Erdös-like being (Gordon 2011)! In an eloquent speech at the Dutch-Flemish Association of Diatomists in the Spring of 2016, Herman van Dam memorialized Frithjof. During the advent of the Nederlands – Vlaamse Kring van Diatomisten (NVKD), Herman met Frithjof, and they had a long and prosperous friendship. He talked about Frithjof’s love of diatoms and reiterated the nature of the eclectic mix of interests, endeavors and knowledge that characterized Frithjof. He spoke of Frithjof’s contributions, along with Hein de Wolf, to the Diatomededelingen, for which Frithjof penned six articles. That Frithjof contributed many publications on Gyrosigma and Pleurosigma, among other taxa, was highlighted by Herman, as well as stating that Frithjof was recognized as an international expert in diatom research. Herman noted that Frithjof was a person of strong opinions and principles (van Dam, personal communication). Herman also noted that Frithof possessed the quality of magnanimity, for example, in his willingness to help and counsel individuals, including himself, on diatom taxonomic and nomenclatural matters. Paul Hargraves said, “he gave most generously of his time and expertise (on identifying a Gyrosigma), and it was included in one of his many publications on the genus, graciously including me as a co-author, as he did with our subsequent collaborations on Haslea.” Wulf Herwig found Frithjof to be extremely intelligent, wise and humorous and always helpful. Jackie White-Reimer remembers Frithjof as a warm, gracious person. When she and her family were house guests, Frithjof and José “went out of their way to make us comfortable and show us around. … They even rented a car big enough to accommodate all of us because theirs was too small.” The Reimers and Sterrenburgs spent time at each other’s homes, where Frithjof was a “considerate house guest.” Frithjof even made an indelible impression on the Reimer’s daughters, Laura and Emilie, ages 12 and 10 at the time. They remember him having a British accent and having an easy, friendly, nice, and happy personality. Emilie remembered that Frithjof gave them a tin of gingerbread cookies on the sly, which delighted them to no end (White-Reimer, personal communication). Some individuals were fortunate to receive holiday well-wishes with wonderful displays of Frithjof’s photographic talents (Figures 1.6–9). There were dazzling displays of diatoms and otherworldly entities. His generous nature extended to identifying diatoms and instructive comments on microscopy to professionals and amateurs directly or via the web. Often, his name appeared in the acknowledgments for his contribution toward correct identification and/or correct nomenclature of the diatom taxa considered. He was especially beneficent with his time as people sought his expertise concerning Gyrosigma and Pleurosigma species. Michael Stringer was especially effusive in his sentiments about Frithjof with whom he shared many interests. As Michael appreciatively said, “he was always there to help me,” and “he transformed my study of diatoms that exists today.” Michael also said that Frithjof’s favorite sweets were English licorice, Christmas puddings, and Christmas cakes laced with whisky and brandy (Stringer, personal communication). Frithjof definitely had a taste for the fruits of life!

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Happy Christmas from Frithjof and a fine 2007!

Figure 1.6 Digital Christmas card sent to friends in 2006.

Merry christmas and a happy 2009! Josephine and Frithjof Sterrenburg

Figure 1.7 Digital Christmas card sent to friends in 2008.

Frithjof embodied a many-faceted personality (de Wolf, personal communication). He had many friends that remember those qualities that made him a unique person (Herwig, personal communication; Stringer, personal communication). Klaus Kemp and Phil Basson gave tribute to Frithjof on the diatom-l website as one “who has contributed so much to the study of Diatoms and was so supportive of the amateur fraternity with encouragement and donations of material from Sulawesi and other locations,” and that Frithjof “was a true gentleman.” (Kemp & Basson 2016)

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Figure 1.8 Digital Christmas card sent to friends in 2010.

Figure 1.9 Digital Christmas card sent to friends in 2011.

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The Amateur Diatomist and the Importance of Collections

Frithjof was a Research Associate at the National Natural History Museum “Naturalis” in Leiden, The Netherlands. He visited and used collections at various museums and understood their value. An advocate for museums, Frithjof was vociferous on this score (Sterrenburg 1990b), both in print and on the web, extolling the virtues of collections, access to collections, documenting collections, and having someone in charge of the stewardship of and communications about collections. In 1990, he explicated the meaning collections have as a basis for taxonomy, morphology and ecology research, especially in diatom research (Sterrenburg 1990b, 2002a). In 1991, Hein de Wolf and Frithjof undertook the task of updating Fryxell’s 1975 paper by conducting an international survey of diatom collections to enhance access (de Wolf & Sterrenburg 2003). As Frithjof said, Taxonomy is not stamp collecting, but (re)defining the biological individuality of the taxa (“names”) described. This can only be done reliably if you examine the original materials to define an unequivocal standard of reference (the “type”) and this shows the great value of Museum collections and the stalwarts who look after them! (Sterrenburg 2011a)

and For such studies to make any sense at all, good taxonomy is an indispensable requirement and, despite financial constraints, diatom taxonomy has flourished in the past 25 years, just as it did in the second half of the 19th century. It is now universally accepted that taxonomy must be based on the investigation of the original materials from which species were originally described, by the process called typification, as in the course of time misidentifications have accumulated, leading to an erroneous shift in the species paradigm. Hence the crucial importance of collections of original materials. (Sterrenburg & de Wolf 2004)

Herman van Dam stated that Frithjof was dedicated to the preservation of collections (van Dam, personal communication). With Hein de Wolf, Frithjof published papers on the Kinker collection (Sterrenburg & de Wolf 1993, 2004; de Wolf & Sterrenburg 1993). Johannes Kinker was a stockbroker by profession and a Victorian-era amateur diatomist of high repute who amassed an extensive diatom collection (Sterrenburg & de Wolf 1993). He did not publish, but Henri-Ferdinand van Heurck, the industrialist and chemist as well as amateur diatomist, microscopist and botanist (Robbrecht 2007), had stated that Kinker’s collection contained “masterpieces” (Stevenson 2009). The discovery of the collection was a testament to the dedication of Frithjof and Hein to enable recognition of Kinker’s contribution to diatom research as an amateur and the importance of museum collections. They gave homage to the amateur diatomist more generally by stating that, It has been fashionable to picture these Victorian investigators as quaint Daddy Longlegs irrelevantly chasing after obscure creatures, but that image is false. Laying the foundations of science is never irrelevant, and the current classification of diatoms and

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practical scientific applications of diatom studies were given a sound basis by the work of these earlier investigators. Kinker need not have been modest because he was “only an amateur.” So were most of his contemporaries, and the position of “amateur-savant” was a respectable and even distinguished one in his time. In fact, if modern trends in financing of Science continue, we may well see a return to that situation in our lifetime. (Sterrenburg & de Wolf 1993)

1.5

The Amateur Diatomist as Expert in the Tools of the Trade

From a very young age, Frithjof was interested in the natural world, especially wild flowers, insects, and diatoms. His inquisitive nature and fascination with electronics and physics was but a precursor for his developing interests. As chronicled in a brief webpage (Sterrenburg 2014a), when Frithjof was 12 years old, he had a Victorian drum microscope. At the time, his family lived in a house where the cellar had a foot of standing sewage water. Being a lad with a curiosity about bacteria, Frithjof sampled some of the odoriferous concoction, placed it on a coverslip, and mounted it in Canada balsam. He brought the slide to his family doctor, who had a Zeiss microscope, and upon viewing, discovered that Spirillium was present. His father had introduced him to the microscope and gave Frithjof his window into a whole new world. Frithjof was articulate, witty, erudite and incisive, self-confident, and with his eclectic knowledge immersed himself in any topic that suited his interest. With his fascination of the microscopic world, Frithjof developed his love of diatoms. His abilities with light microscopy, sample and slide preparation, and photomicrography were the result of much trial and error, and he was rewarded repeatedly for his determination and dedication while studying diatoms. Frithjof saw that there were many facets to diatom research. Field work and collecting water samples were just the beginning (Figure 1.10). Preparing those samples for viewing in the microscope was a sizable consideration, and Frithjof learned quickly about the link between handling samples and pretreatment of the materials to minimize damage or the loss of diatoms. Whether using water samples, mica fragments, or dry materials, Frithjof was cognizant of the various protocols for the most appropriate treatment (Sterrenburg (Year unknown) accessed on 2016). Frithjof developed detailed protocols concerning the mounting process to produce the best results. His tutelage on making and viewing morphologically-meaningful diatom slide mounts was clear. As Frithjof would remind us, “NEVER, repeat NEVER, make a preparation on the slide, always on the cover-slip. Preparation on the slide results in serious deterioration of image quality” (Sterrenburg 2002b, 2006a). Frithjof’s expertise in microscopy was second to none (Sterrenburg 1975, 2002b, 2013a), and he was cogently instructive on the concepts for basic, sound usage of the light microscope (Sterrenburg 2011b). In e-mails with Paul Hamilton, Frithjof was even concerned about the position of the arm of the microscope (i.e., facing toward or away from the user) because this had an impact on how to read slide location scales

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Figure 1.10 Frithjof and a student taking samples in Florida (circa 1982). (permission from C.J. Sterrenburg)

(Hamilton, personal communication). He did image quality comparisons among objectives (Sterrenburg 2012a, b), and he was always interested in optics (Sterrenburg 2009b, 2012a, b), including weighing the thickness of a diatom’s silicification and what effect the refractive index of mounting media would have on image resolution. In response to queries on the diatom-l listserv, Frithjof summarized some “salient points” on microscope optics, including phase contrast (Sterrenburg 2009b) and differential interference contrast or Nomarski (Sterrenburg 1978): 1. check whether your microscope has “infinity corrected” objectives, or objectives corrected for a tube length of 160 mm by looking at the engraving on your objectives. It either mentions “160” or gives the infinity symbol. You cannot combine objectives that differ in this respect. 2. the usual phase contrast objectives give better contrast, but lower resolution 3. plan objectives are not relevant for diatoms, they give flat images to the edge, but diatoms usually cover only the centre of the image. Also, diatoms themselves are not flat! 4. a condenser of more than NA 1.0 MUST always be oiled to the slide with immersion oil. This is messy and inconvenient so 90% of people use their condenser “dry”. In that case, its effective NA will never be greater than 1.0 even if the specification says it has NA 1.4. 5. also, contrast will decrease with higher effective condenser NA. The best option for low-contrast diatoms is DIC but that requires special optics (objectives and condenser) and is expensive. 6. You need not aim at the highest possible resolution if you can also do SEM. That solves all problems. Many genera are not even identifiable without SEM.

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Diatoms: Fundamentals and Applications 7. to summarize: unless your work aims at critical taxonomic separation of difficult and very finely structured taxa, I'd recommend a good modern NA 1.3 objective. If you go for the ultimate, DIC supported by SEM is the best option. (Sterrenburg 2012c)

Frithjof was very methodical in his approach to understanding microscope optics and microscopy. Illumination was an important factor in stellar microscopic technique, and Frithjof, being a keen observer, recognized annular oblique illumination as one way to obtain a phase contrast effect (Sterrenburg 1975, 1978, 2010a). Frithjof, being at the forefront of knowledge about lighting options, he tested the utility of LEDs with various microscope optics arrangements, and in another diatom-l post, referring to the case since 2004: …since then, more suitable LEDs have appeared. I have tested some white LEDs of 1W (circa3.5V, 0.3A) and smaller, which have a luminescent area of about 3 mm. For Köhler illumination, this may be too small if the microscope condenser has a large bottomlens, with the smaller diameter lenses of Leitz condensers, including the Heine, it’s sufficient. However, one need not use Köhler, critical illumination is quite satisfactory. In that case, very low powers can be used without any problem simply by unscrewing the top lens. Objectives of the highest NA can also be used without any problem, in contrast to what the article referred to states. … Even the small LEDs, combined with a good collector, are far too bright to use at full power visually in simple brightfield! As for uneven illumination of the field of view: this is simply remedied nowadays by using image-processing software that permits background subtraction. (Sterrenburg 2009c)

The right kind of optics and proper illumination enabled light microscopists to develop skill to see at the limits of resolution using diatom strew mounts. Frithjof was masterful in this craft, and as he said, “… microscope users will be familiar with such mellifluous names as Pleurosigma angulatum or Frustulia rhomboides because these have been standard test objects to check the quality of their lenses - and, especially, the quality of their microscope expertise… (Sterrenburg 2002a), and “Diatoms are commonly used to test the quality of the objectives - and even more so, the expertise of the microscopist.” (Sterrenburg 2012b) As a natural outcome of his interest and ability in microscopy, Frithjof, along with Peter Höbel (2011), elucidated the technique of using ultraviolet or blue light photomicrography to obtain a crisp, 3D effect when material is not available for scanning electron microscopy (SEM) determination (Höbel & Sterrenburg 2011). Another technique was developed by Frithjof along with Hamilton and Williams (2012) to locate specimens on microscope slides regardless of the brand of microscope, and this technique circumvents the need to scribe or ink rings around specimens or purchase an England Finder (Hamilton, Williams & Sterrenburg 2013; Sterrenburg, Hamilton & Williams 2012). He used his knowledge and creative thinking in concocting whatever piece of equipment, microscope lens (Sterrenburg 2006b, Sterrenburg 2011b) or illuminator (Sterrenburg 2002c) that was necessary to view diatoms with clarity. He embraced technological improvements and applied the fine details of his amassed

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knowledge to microscopic and slide preparation techniques in his quest to do exemplary diatom taxonomic studies. Frithjof saw taxonomic puzzles as his opportunity to meet the challenge of doing scientific sleuthing with all the twists and turns one would encounter in finding solutions (e.g., Sterrenburg 2011a).

1.6

The Amateur Diatomist as Peer-Reviewed Scientific Contributor

Frithjof was resourceful in solving problems, utilizing his many talents. He was mindful of the role of past amateurs in diatom research and knowledgeable about the history that intertwined the advent of microscopy and popular interest in diatoms. Diatoms became his special interest after looking at the Reverend William Smith’s description of Gyrosigma tenuissimum in 1853 and found it to be wonting. The puzzle to be solved—the original description stated that the valves had deeply curved sides; other descriptions stated it had straight sides. Smith’s material was not accessible for decades, but when it was, with his keen eye, nimble mind and technical ability, Frithjof solved the puzzle by finding straight sided valves, contrary to Smith’s original description. The road to discovery was fraught with twists and turns, but along with Myriam de Haan and Wulf Herwig, a “lucky break” (Sterrenburg Sterrenburg, de Haan, & Herwig 2014) emerged so that the puzzle could be solved. With this, Frithjof was hooked and on his way to making a lasting presence with his love of studying diatoms. Frithjof first published on diatoms in the 1970s. He was instrumental in advancing diatom taxonomy and nomenclatural studies of many taxa (e.g., Sterrenburg 1988). In one tabulation derived from AlgaeBase (Guiry & Guiry accessed on 2017), Frithjof named, either alone or with colleagues 39 species (and/or varieties): 16 Gyrosigma, 8 Pleurosigma, 8 Haslea, two Frustulia (with Horst Lange-Bertalot) (Lange-Bertalot & Sterrenburg 2004), two Anorthoneis, one Nitzschia, one Surirella, one Petrodictyon, and the holotype Talaroneis furcigerum (Grunow) Sterrenburg (Kooistra, Forlani, Sterrenburg, & Stefano 2004). Many other taxa were subjected to Frithjof’s careful assessment. As a testament to Frithjof, Gyrosigma sterrenburgii Stidolph, Pleurosigma sterrenburgii Stidolph, Neidium sterrenburgii Metzeltin and Lange-Bertalot, and Rhopalodia sterrenburgii Krammer were named after him (Guiry and Guiry accessed on 2017). Frithjof’s most notable contributions in diatom taxonomy and nomenclature are his work on Gyrosigma and Pleurosigma, with publications spanning the decades from 1989 to 2013 as sole author (e.g., Sterrenburg 1989, 1990c, 1991a, b, 1992, 1993a, b, 1994a, b, 1995a, b, 1997, 2000, 2001a, 2002d, 2003a, 2003b, 2007) or with collaborators (Jahn & Sterrenburg 2003; Jahn et al. 2005; Sterrenburg & Tiffany 2004; Sterrenburg & Underwood 1997; Sterrenburg et al. 2000, 2003a, b; Sunesen et al. 2013; Sar et al. 2012, 2013; Liu et al. 2015). As Paul Hamilton observed, “Frithjof was thorough and precise in his work. I think I can say that he was ‘part of the old school’ where attention to detail was critical and worth the extra time in getting the research right before publishing.” Frithjof was diligent and meticulous in naming and correcting taxon misidentifications and in devising new

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morphological characters to aid in identifications in Gyrosigma and Pleurosigma (e.g., Sterrenburg 1991b, 1993b). Frithjof’s taxonomic insightfulness and “eagle-eye” was evident in his comments in a diatom-l post concerning a 2012 publication on Pleurosigma intermedium from Guam. Frithjof stated:

… (on) the recent publication on Guam diatoms: (Lobban, C.S., Schefter, M., Jordan, R.W., Arai, Y., Sasaki, A., Theriot, E.C., Ashworth, M., Ruck, E.C. & Pennesi, C. (2012). Coral-reef diatoms (Bacillariophyta) from Guam: new records and preliminary checklist, with emphasis on epiphytic species from farmerfish territories. Micronesica 43(2): 237-479.) showed itself to be helpful when I checked some provisional identifications in my own samples from the Pacific. As an update, here are some data on Pleurosigma intermedium W. Smith, because the specimens the authors present are not that species… In LM, P. intermedium has a broader apex and (arrow) a “calcar”, absent in the Guam specimens. This calcar corresponds to an accessory fissure in SEM. In SEM, P. intermedium has a row of internal marginal duplex areolae that continues around the apex. In the Guam specimen, the apical margin is unperforated. In P. intermedium the internal central raphe nodule is surrounded by a rather wide nonareolated field, in the Guam specimen the areolae continue right up to the central nodule. Note also the difference in shape for the central internal raphe fissure endings. These differences are morphogenetically completely independent and the differentiation is thus based on polythetic characters. The Guam specimens do not appear to match the type of P. acus Mann 1925 as this shows a different stria density ratio. (Sterrenburg 2012d)

Frithjof displayed his consternation about correct nomenclature as distinct from taxonomic issues with comments about Haslea brittanica in two diatom-l posts (2014 b, c): Thanks for the several replies to my question re Haslea brittannica. All respondents refer to Iconographia Diatomologica, vol. 7 (2000). I have this, and that's just the problem, as follows:

there is one LM figure, which shows perpendicularly arranged striae this is NOT from the type material

Perpendicularly arranged striae are seen in Hasleas, but also in non-Hasleas, e.g. Hustedt's section of Naviculae orthostichae. Would not SEM be required to verify the presence of the generic characters (e.g. as given in Round et al.), which are invisible in LM? Does generic transfer not require verification in the type material? There might be a look-alike.

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Although I agree that Haslea may be possible, I wonder if transfer is formally valid without reference to type material specimens, and SEM in this case. (Sterrenburg 2014b)

and To summarize the many replies I got (thanks, folks!): The transfer is formally valid according to the Code (which only requires citation of the basionym and the reference). But whether it's taxonomically correct is an entirely different matter (investigation of type, SEM). (Sterrenburg 2014c)

A comparative study of Haslea with Gyrosigma and Pleurosigma included definitive description and argument for the identification of morphological characters and separation of these diatom genera (Sterrenburg Tiffany Hinz Herwig & Hargraves 2015). Sometimes, nomenclature revisions do not make their way into the common vernacular of professionals and amateurs who want to identify their diatom specimens. Frithjof was always on hand to see that taxa were named correctly and make such revisions available in publications and on the web (Sterrenburg 2012b). The transfer of Surirella patrimonii Sterrenburg to Petrodictyon is a case in point that illustrated Frithjof’s ability to meld phase-contrast microscopic investigation with examination of type material for the purposes of naming the appropriate genus, and subsequent identity of the taxon in the marine littoral habitat off the coast of Sulawesi, Indonesia (Sterrenburg 2001b). Frithjof was very clear on the problems associated with unresolved issues in the appropriate naming of diatom taxa (Sterrenburg 2010b) or the status of a taxon’s name. The trials and tribulations of naming taxa are dependent on one’s microscope and optics capability, slide preparation technique, and a discerning eye in being able to see fine structural details upon examination of diatoms. Frithjof would painstakingly go through the steps to produce valid naming of a diatom taxon (Sterrenburg 2002d). His work on a project with colleagues Stuart Stidolph, Kathryn Smith, and Alexandra Kraberg culminated in the U. S. Geological Survey publication of Stidolph’s Atlas of 1000 photomicrographs of over 400 marine littoral marine diatom species (Sterrenburg 2012e; Stidolph, Sterrenburg, Smith, Kraberg, & Stuart 2012). As always, Frithjof made sure the taxonomy and nomenclature were impeccable and up to date. Frithjof saw the value in making the atlas as well as other resources available to the diatom community because he understood that there were few large compilations of information on diatoms (e.g., Sterrenburg 2013b), and the literature tends to be scattered and sometimes difficult to access. Frithjof said that the result of good taxonomic practice is “the elimination of established spurious taxa,” but the “chronic state of renovation” regarding the introduction of new genera has induced a multitude of nomenclatural changes. Documentation of taxonomic and nomenclatural changes are scattered throughout the literature, and Frithjof was an advocate of a web-based approach to amassing such information and making it readily accessible to all (Sterrenburg 2002e). At one time, Frithjof was a researcher at the Netherlands Institute of Ecology, Centre for Estuaring and Coastal Ecology. He recognized that ecological studies were

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only as good as the soundness of the names of the taxa. He understood the multipurposed value of taxonomic studies and was involved in studies on the epiphytic diatom flora of seagrasses from various localities (e.g., Sterrenburg et al. 1995). He published papers with his father, F. J. G. Sterrenburg, on marine littoral taxa, including Navicula orthoneoides (Sterrenburg & Sterrenburg 1991) and the naming of a new species, Nitzschia nienhuisii (Sterrenburg & Sterrenburg 1990). The diatom flora is cosmopolitan. However, determining specific epithets for the diatom flora, not previously known from intertidal seagrass habitats off the coast of Mauritania, enabled Frithjof and his father to characterize the ecology as uninfluenced by aeolian deposits (Sterrenburg & Sterrenburg 1990). Frithjof, along with Michael Stringer, was interested in studying the diatom population dynamics of “The Car Park Lagoon,” one lagoon among a series of marine tidal lagoons on Two Tree Island, Essex, United Kingdom. Diatom populations had collapsed; however, by the winter of 2010, diatom blooms had returned. Frithjof and Michael documented the changes using slide mounts from samples, with Frithjof identifying the taxa present. Frithjof said, that finding diatoms in such cold conditions was not unheard of because “numerous diatom species rank among the principal coldloving ‘extremophilic’ organisms ...” (Stringer & Sterrenburg 2011) Diatom environmental tolerances would be revisited to form a thought-provoking paper by Frithjof and colleagues. Larger issues were always on Frithjof’s mind concerning species designations and biodiversity studies. Environmental conditions can affect diatom morphology. Teratogenetic malformations in valves including not previously detected asymmetry, abnormal bends, distorted margins, and irregular striae patterns were observed such that Frithjof wondered what effect this has on the naming of species (Sterrenburg 1973). The effect of environment on diatom diversity, and biodiversity more generally, is a pervasive topic in scientific circles and the political sphere, and Frithjof was quite aware of the importance that impeccable diatom taxonomy would have as a lasting impact on identifying what is present and determining how threatened those taxa might become (Sterrenburg 2005a). In many ways, Frithjof’s attention to details and complexities of taxonomic determinations was his stock-in-trade as a diatomist; he stated that, …there is the widespread misconception that taxonomy consists of the description of new species or varieties. The realization that taxonomy may instead have to start with the elimination of established but invalid ‘pseudospecies’ may come as a surprise to workers not specializing in the subject.

Along the same line, Only when the identity of an organism has been unambiguously fixed does it become possible to determine its biological characteristics: its physiology, nutritional requirements, habitat and biogeography. Only when these are known, the organism can become a source of information on the Earth’s history. This is especially the case for diatoms, whose siliceous exoskeleton is both taxonomically informative and durable, permitting conclusions over many millions of years. (Sterrenburg 2005a)

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Diatom cytology (Sterrenburg 1994b) and morphogenesis were just as interesting to Frithjof as taxonomy, nomenclature and ecology. He conducted studies on raphe fissure deflection and handedness as morphogenetic characters in Gyrosigma (Sterrenburg 1993b). Along with Mary Ann Tiffany and María Esther Meave del Castillo, Frithjof was involved in valve morphogenesis studies of Pleurosigma (Sterrenburg, Tiffany & del Castillo 2005, Sterrenburg, Meave del Castillo & Tiffany 2003b), with an eye toward determining that the valves have nanostructure oval pillars in internal and external layers that are sandwiched together and not loculate in structure. Frithjof saw the importance of silica deposition in terms of the construction of the frustules and its connection to morphology and how this should be reflected in the taxonomy. Valve morphogenesis and its relation to constructal morphology was explored further by Frithjof. With his interest in the “engineering” of diatom silica opaline frustules and their morphology, Frithjof was led to idea of “crystal palaces” and their role in nanotechnology (Sterrenburg 2005b). Because of the link between diatom research and the desire to use technological advances, especially regarding SEM, diatoms could be studied in terms of their structure, function and morphogenetic steps in silica nanostructure formation in detail. Frithjof and colleagues Tiffany and Meave del Castillo continued studies of Pleurosigma valve morphogenesis to document the steps in valve formation (Sterrenburg et al. 2005). Gyrosigma valve formation was also found to have the non-loculate sandwich configuration, albeit slightly differently from Pleurosigma, and that these structures were found to be weaker than the loculate nanoconstructions of other diatoms (Sterrenburg et al. 2005). Frithjof and colleagues found that Pleurosigma and potentially Gyrosigma, have “speed bumps” constructed between adjacent pillars to compensate for the drawback of the non-loculate design (Sterrenburg et al. 2005). Studying diatom morphogenesis and pattern formation illustrated how Frithjof had the ability to go beyond what he already knew and how he continued to challenge himself. Along with Richard Gordon and Kenneth Sandhage, Frithjof was also an editor of the special volume on diatom nanotechnology in the Journal of Nanoscience and Nanotechnology published in 2005 where his nanostructure publications also appear (Gordon et al. 2005a,Sterrenburg 2005b; Gordon, Sterrenburg & Sandhage 2005b). To take the “constructal paradigm” further, Frithjof, Richard Gordon, Mary Ann Tiffany, and Stephen Nagy wrote a chapter for a volume on astrobiology (edited by Joseph Seckbach), examining diatom constructal morphology and its bearing on whether diatoms are extremophiles (Sterrenburg et al. 2007). Frithjof and colleagues described the various habitats, including hot springs, peat bogs, highly saline or polluted waters, and on mudflats in marine littoral zones and wondered whether such species also found in less extreme habitats would qualify as extremophilic. At the nanostructure level from their geometric design, diatoms can withstand the stresses of various physical and chemical environmental conditions impinging on their frustules. They exhibit rigid morphogenetic control and a constancy within a species. Frithjof knew that this “constructal paradigm” was still not sufficiently known to explain diatom morphological diversity of pattern formation, yet that diversity is a testament to diatom survival in all manner of habitats. Despite this, Frithjof and colleagues at this juncture were reluctant to call diatoms extremophiles (Sterrenburg et al. 2007).

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From the various subjects covered and their interrelationship, Frithjof exhibited his development and growth as an amateur scientist. He contributed to microscopy, sample and slide preparation methods, photomicrography, and by expounding the intricacies and interrelationships of a variety of biological fields and how they impinge on diatom research. Undoubtedly, he has had the most direct and long-lasting impact via his work on his favorite diatom genera, Gyrosigma and Pleurosigma. In his approach to diatom studies, he had the ability to communicate the essence of a problem, the rationale for engaging in such studies, and how to go about finding the solution. Frithjof is the epitome of why amateurs are important in diatom research. His eloquent manner, engaging personality, and affable character made Frithjof Sterrenburg not only a unique contributor, but also one who transcended the title “amateur” to become a professional as teacher, mentor, author, and expert in diatom research. As Frithjof said, “diatom taxonomy is a specialism (Sterrenburg 2012b),” especially for those who become serious diatom researchers, as he did.

1.7

Concluding Remarks

Frithjof was the consummate amateur who became a professional, in the very best sense of the word, whether he knew you as a colleague, instructor, friend, or acquaintance concerning the study of diatoms. His legacy is many-fold in that he understood the intricacies of excellent microscopy practice along with slide preparation and photomicrography. More expansively, Frithjof understood and espoused the conceptual basis of the importance of taxonomy and resolving nomenclatural issues and how these studies provided legitimacy for diatom ecological and biostratigraphic studies as well as resulting from using cytological evidence. He was always working on diatom research, having manuscripts and involvement in projects that have yet to be published (Sterrenburg unpublished manuscripts 1996, 2002f, 2004, 2013c, d). Specifically, Frithjof is known as the authoritative source on the diatom genera Gyrosigma and Pleurosigma. For the benefit of all of those who study diatoms, his collections have been donated to and are housed in Bremerhaven, Germany at the Alfred Wegener Institute at the Friedrich Hustedt Center for Diatom Research. For 60 years, he was a very dear, loving, always cheerful and optimistic husband, as José said. In accordance with Frithjof’s wishes, as José told me, “his ashes now rest among his tiny friends, the diatoms, on the ocean floor.” Frithjof always said that his epitaph should read “Never a dull moment,” and as José absolutely agreed, his wishes were granted. As Hein de Wolf said in a diatom-l post (de Wolf 2016) with a message from José: Never a dull moment. This is how Frithjof Sterrenburg would like to be remembered. A malignant tumour attacked his brilliant brain. It put an end to his happy and valuable life on the eleventh day of March. But most importantly: he died with a satisfied mind (his own words). José Sterrenburg

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We can say unequivocally, that we are all richer persons for having known Frithjof A. S. Sterrenburg. We miss you and are thankful that your work lives on.

Acknowledgments All of Frithjof Sterrenburg’s publications are in the References section of this chapter. I would like to thank Dick Gordon and Joe Seckbach for the opportunity to write this memorial chapter and for all their help and advice along the way. I would also like to express my sincere gratitude to Herman van Dam, Wulf Herwig, Paul Hargraves, Paul Hamilton, Jackie White-Reimer (including Emilie and Laura), Hein de Wolf, and last, but not least, Michael Stringer for their heartfelt musings and wonderful stories that helped me to imbue the right kind of flavor into the memorial chapter that it deserved. While communicating with Michael Stringer in November, 2017, he let me know how grateful he was for this tribute to his dear friends, Frithjof and José, as he “‘topped up’ my Christmas pudding with brandy, they are 10 years old! One will be served with brandy custard and clotted cream.” He said his discovery of Haslea pallidum in 2006, identified for him by Frithjof, will go on the U.K. diatom register in 2018. He was rightly proud of this accomplishment and was grateful for his camaraderie with Frithjof. He died on February 23, 2018. I would like to express my extra special gratitude to José Sterrenburg for her exceptional help and contribution of most of the personal information and personal snapshots. We communicated often during the autumn of 2016, and without her generosity, this work would not have come to full fruition. Unfortunately, José Sterrenburg died on December 1, 2016. Although she did not get to see the completed work, I think she would have been very happy with this memorialization of Frithjof, and so, I dedicate this memorial chapter to José as well.

References Albert the 1st of Belgium, https://en.wikipedia.org/wiki/Funeral_of_Edward_VII#/media/File: The_Nine_Sovereigns_at_Windsor_for_the_funeral_of_King_Edward_VII.jpg, accessed on 19 December 2016. Alfred Wegener Institute. (2015). FriedrichHustedt, http://www.awi.de/en/science/biosciences/ polar- biological- oceanography/ main- research- focus/ hustedt- diatom- study- centre/ friedrich-hustedt.html. Austrian Admiral Hermann Freiherr von Spaun, https://en.wikipedia.org/wiki/Hermann_von_ Spaun#/ media/ File: Admiral_ Hermann_ Freiherr_ von_ Spaun_( Sport_ und_ Salon_ 1905). png, accessed on 19 December 2016. Bahls, L.L. (2015). The role of amateurs in modern diatom research, Diatom Research, 30(2), 209–210. de Wolf, H. (2016). Frithjof Sterrenburg, https://list.indiana.edu/sympa/arc/diatom-l. de Wolf, H., Sterrenburg, F.A.S. (1993). The legacy of the Dutch diatomist J. Kinker (1823–1900), Quekett Journal of Microscopy 37. de Wolf, H., Sterrenburg, F.A.S. (2003). International Survey of Diatom Collections, http:// home.planet.nl/~wolf0334/.

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Gordon, R., Kling, H.J., Sterrenburg, F.A.S. (2005a). A guide to the diatom literature for diatom nanotechnologists, J. Nanosci. Nanotechnol., 5(1). Gordon, R. (2011). Cosmic Embryo #1: My Erdös Number Is 2i, http://www.science20.com/ cosmic_embryo/cosmic_embryo_1_my_erd%C3March6s_number_2i. Gordon, R., Losic, D., Tiffany, M.A., Nagy, S.S., Sterrenburg, F.A. (2009). The Glass Menagerie: diatoms for novel applications in nanotechnology, Trends Biotechnol., 27(2). Gordon, R., Sterrenburg, F.A.S., Sandhage, K.H. (2005b). A Special Issue on Diatom Nanotechnology, J. Nanosci. Nanotech., 5(1). Guiry, M.D., Guiry, G.M. (2017) AlgaeBase. World-wide electronic publication, National University of Ireland, Galway, http://www.algaebase.org. Hamilton, P.B., Williams, D.M., Sterrenburg, F.A.S. (2013). Some notes on locating specimens in the microscope, Diatom Research, 28(4). Hargraves, P.E. (2016). Frithjof Sterrenburg, https://list.indiana.edu/sympa/arc/diatom-l. Höbel, P., Sterrenburg, F.A.S. (2011). UV photomicrography of diatoms, Diatom Research, 26(1–2). Jahn, R., Sterrenburg, F.A.S. (2003). Gyrosigma sinense (Ehrenberg) desikachary: typification and emended species description, Diatom Research, 18(1). Jahn, R., Sterrenburg, F.A.S., Kusber, W.-H. (2005). Typification and taxonomy of Gyrosigma fasciola (Ehrenberg) J W. Griffith et Henfrey, Diatom Research, 20(2). JStorGlobal Plants, Jacques-Joseph Brun. (1826–1908). http://plants.jstor.org/stable/10.5555/al. ap.person.bm000392757, accessed on 2 December 2016. Kemp, K., Basson, P. (2016). Never a dull moment, https://list.indiana.edu/sympa/arc/diatom-l. Kooistra, W.H.C.F., Forlani, G., Sterrenburg, F.A.S., Stefano, M.D. (2004). Molecular phylogeny and morphology of the marine diatom Talaroneis posidoniae gen. et sp. nov. (Bacillariophyta) advocate the return of the Plagiogrammaceae to the pennate diatoms, Phycologia, 43(1). Lange-Bertalot, H., Sterrenburg, F.A.S. (2004). New Frustulia species (Bacillariophyceae) from fossil freshwater deposits in Florida, U.S.A, Nova Hedw., 78(3–4). Liu, B., Sterrenburg, F.A.S., Huang, B. (2015). Gyrosigma xiamenense sp. nov. (Bacillariophyta) from the middle intertidal zone, Xiamen Bay, southern China, Phytotaxa, 222(4). Lynk, H. (2016). A Cabinet of Curiosities: A Selection of Antique Microscope Slides from the Victorian Era c. 1830s ~ 1900, http://www.victorianmicroscopeslides.com/history.htm, accessed on 28 November 2016. Pedrotti, P.W. (2016). Thonis Philipszoon, “Antonj van Leeuwenhoek” 1632-1723 A.D, http:// www.vanleeuwenhoek.com/, accessed on 2 December 2016. Robbrecht, E. (2007). Botanic Garden Meise History, Henri Van Heurck (Antwerp 1839-1909), http://www.plantentuinmeise.be/PUBLIC/GENERAL/HISTORY/vanheurck.php. Sar, E.A., Hinz, F., Sterrenburg, F.A.S., Lavigne, A.S., Lofeudo, S., Sunesen, I. (2012). Species of Pleurosigma (Pleurosigmataceae) with lanceolate or slightly sigmoid valve outlines: analysis of type material, Diatom Research, 27(4). Sar, E.A., Sterrenburg, F.A.S., Lavigne, A.S., Sunesen, I. (2013). Diatoms from marine coastal environments of Argentina. Species of the genus Pleurosigma (Pleurosigmataceae, Boletin De La Sociedad Argentina De Botanica 48(1). Schmidt, A., Schmidt, M., Fricke, F., Heiden, H., Müller, O., Hustedt, F. (1874–1959). Atlas der Diatomaceenkunde. Aschersleben, Leipzig. Sterrenburg, F.A.S. (Year unknown). The Oslo Report 1939—Nazi Secret Weapons Forfeited, http://www.v2rocket.com/start/chapters/peene/oslo_report.html. Sterrenburg, F.A.S. (1967). Fifty Years of Jazz Records (in Dutch). Stichting IVIO, Amsterdam.

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Sterrenburg, F.A.S. (1970a) Receivers. Instructions for the Advanced Amateur on the Art of Receivers, Amplifiers, Antennas, Measuring and the Like, 1st edition (in Dutch), De Muiderkring, Bussum, (2nd through 5th editions (in Dutch) published after 1970 until 1980). Sterrenburg, F.A.S. (1970b). Electronica en de Battle of Britain, Radio Bulletin, Aug, 1970, 321. Sterrenburg, F.A.S. (1973). Extreme malformation and the notion of species, Microscopy, 32. Sterrenburg, F.A.S. (1975). Guidance on Microscopy (in Dutch). Kluwer, Deventer. Sterrenburg, F.A.S. (1976). Silence around a Nobel investigation (in Dutch), Organorama, 13(1). Sterrenburg, F.A.S. (1977). Harvest the Past (in German), Organorama, 14(3), 11. Sterrenburg, F.A.S. (1978). Enhancing the visibility of diatoms, Microscopy, 33(6), 384. Sterrenburg, F.A.S. (1979). Report on medical electronics (in Dutch). Kluwer, Deventer. Sterrenburg, F.A.S. (1982). Anton van Leeuwenhoek: Pioneer or Loner (in Dutch, Organorama, 19(2). Sterrenburg, F.A.S. (1983a) IRAS - Mission Invisible, Astronomy, 11, 66. Sterrenburg, F.A.S. (1983b). A Phased Approach to Astronomy, Astronomy, 11(6), 24. Sterrenburg, F.A.S. (1983c). Computers in medicine, Organorama, 20. Sterrenburg, F.A.S. (1988). Observations on the genus Anorthoneis Grunow, Nova Hedwigia, 47(3–4), 363. Sterrenburg, F.A.S. (1989). Studies on tube-dwelling Gyrosigma populations, Diatom Research, 4(1). Sterrenburg, F.A.S. (1990a). The quest for Quekett – in search of Navicula angulata Quekett 1848, Microscopy, 468. Sterrenburg, F.A.S. (1990b). Diatom collections—legacy or legend? Diatom Research, 5(2). Sterrenburg, F.A.S. (1990c). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). A new phenomenon: co-existence of dissimilar raphe structures in populations of several species. In: Ouvrage dedié la Mémoire du Professeur Henry Germain, M. Ricard (ed.). Koeltz Scientific Books, Königstein, Germany. Sterrenburg, F.A.S. (1991a). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). The typus generis of Pleurosigma, some presumed varieties and imitative species, Botanica Marina, 34(6), 561. Sterrenburg, F.A.S. (1991b). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Light-microscopical criteria for taxonomy, Diatom Research 6(2), 367. Sterrenburg, F.A.S. (1992). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). The type of the genus Gyrosigma and other Attenuati sensu Peragallo, Diatom Research, 7(1), 137. Sterrenburg, F.A.S., de Wolf, H. (1993). The Kinker collection: preliminary investigation, Quekett Journal of Microscopy, 37, 35. Sterrenburg, F.A.S. (1993a). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). The identity of Pleurosigma obscurum, W Smith. Diatom Research, 8(2). Sterrenburg, F.A.S. (1993b). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Rules controlling raphe fissure morphogenesis in Gyrosigma, Diatom Research, 8(2). Sterrenburg, F.A.S. (1994a). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). The species of Sullivant & Wormley 1859, synonymy and differentiation from other Gyrosigma taxa, Proceedings of the Academy of Natural Sciences of Philadelphia, 145, 217. Sterrenburg, F.A.S. (1994b). Terpsinoe musica Ehrenberg (Bacillariophyceae, Centrales), with emphasis on protoplast and cell division, Netherlands Journal of Aquatic Ecology, 28(1). Sterrenburg, F.A.S. (1995a). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Gyrosigma acuminatum (Kützing) Rabenhorst, G. spenceri (Quekett) Griffith et Henfrey and G. rautenbachiae Cholnoky, Proceedings of the Academy of Natural Sciences of Philadelphia, 146, 467.

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Sterrenburg, F.A.S. (1995b). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Gyrosigma balticum (Ehrenberg) Rabenhorst, G. pensacolae sp. n. and simulacrum species, Botanica Marina, 38(1–6), 401. Sterrenburg, F.A.S. (1996). Cytoplasmic inheritance in diatoms, unpublished manuscript. Sterrenburg, F.A.S. (1997). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Gyrosigma kutzingii (Grunow) Cleve and G. peisonis (Grunow) Hustedt, Proceedings of the Academy of Natural Sciences of Philadelphia, 148, 157. Sterrenburg, F.A.S. (2000). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Gyrosigma reversum (Gregory) Hendey and G. naja (Meister) Sterrenburg, nov. comb, Proceedings of the Academy of Natural Sciences of Philadelphia, 150, 301. Sterrenburg, F.A.S. (2001a). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). The types of Shadbolt and related taxa, Proceedings Academy of Natural Sciences of Philadelphia, 151, 121. Sterrenburg, F.A.S. (2001b). Transfer of Surirella patrimonii Sterrenburg to the genus Petrodictyon, Diatom Research, 16(1). Sterrenburg, F.A.S. (2002a). A second look at some well-known test diatoms, http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artjul02/ fsdiatom.html. Sterrenburg, F.A.S. (2002b). Microscopy primer, http://www.microscopy-uk.org.uk/index.html? http://www.microscopy-uk.org.uk/primer/. Sterrenburg, F.A.S. (2002c). A heavy caliber microscope lamp, http://www.microscopy-uk.org. uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artoct02/fslamp.html. Sterrenburg, F.A.S. (2002d). Nulla vestigia retrorsum. The case of Pleurosigma aequatoriale Cleve, Constancea, 83(1), 14. Sterrenburg, F.A.S. (2002e). Taxonomy and the web? Diatom Research, 17(2). Sterrenburg, F.A.S. (2002f). Taxonomy – ecology – microscopy. A true triad, unpublished manuscript.. Sterrenburg, F.A.S. (2003a). Studies on the diatom genera Gyrosigma and Pleurosigma (Bacillariophyceae). Pleurosigma strigosum W. Smith and some presumptive relatives. Celebrating Norman I. Hendey’s Centennial, Micropaleontology, 49(2). Sterrenburg, F.A.S. (2003b). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Pleurosigma obscurum W. Smith revisited, Diatom Research, 18(2), 323. Sterrenburg, F.A.S. (2004). Habitat specificity of diatoms, unpublished manuscript. Sterrenburg, F.A.S. (2005a). Taxonomy and ecology – an inseparable pair, Proceedings of the California Academy of Sciences, 56, 156. Sterrenburg, F.A.S. (2005b). Crystal palaces - diatoms for engineers, J. Nanosci. Nanotechnol., 5(1). Sterrenburg, F.A.S. (2006a). Cleaning diatom samples, http://www.microscopy-uk.org.uk/mag/ indexmag.html?http://www.microscopy-uk.org.uk/mag/artaug06/fs-diatoms.html. Sterrenburg, F.A.S. (2006b). A compilation of LOMO microscope resources on Micscape. Supplemental page to Micscape article (4: Optics, correction collar maintenance), http:// www.microscopy-uk.org.uk/mag/artoct06/iw-JenaObjsup.html. Sterrenburg, F.A.S. (2007). Basionym of Gyrosigma scalprum, Diatom Research, 22(2), 495. Sterrenburg, F.A.S. (2009a). Personal recollections of WW2, https://ww2aircraft.net/forum/ search/133066/. Sterrenburg, F.A.S. (2009b). Zernike’s colour phase-contrast, http://www.microscopy-uk.org. uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artjul09/fs-phase.html. Sterrenburg, F.A.S. (2009c). LED lighting, https://list.indiana.edu/sympa/arc/diatom-l.

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Sterrenburg, F.A.S. (2010a). Extreme annular illumination, http://www.microscopy-uk.org. uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artapr10/fs-pseudo-phase. html. Sterrenburg, F.A.S. (2010b). Nitzschia singalensis vs N. firthii. What’s in a diatom name? http:// www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/ artapr10/fs-nitzschia.html. Sterrenburg, F.A.S. (2011a). Pandora’s box. The diatoms of Sullivant & Wormley 1859, http:// www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/ artsep11/fs-pandora.html. Sterrenburg, F.A.S. (2011b). Advanced Techniques for visualization of diatom structures? Micros. Today 19. Sterrenburg, F.A.S. (2012a). What price optics? Micscape Magazine, 198, http://www.microscopy-uk.org.uk/mag/artapr12/fs-optics.html Sterrenburg, F.A.S. (2012b). Diatoms and microscope optics – some thoughts, http://www.microscopy-uk.org.uk/mag/artjan13/fs-diatom-micro.html. Sterrenburg, F.A.S. (2012c). Objectives, https://list.indiana.edu/sympa/arc/diatom-l. Sterrenburg, F.A.S. (2012d). Guam diatoms, https://list.indiana.edu/sympa/arc/diatom-l. Sterrenburg, F.A.S. (2012e). Stidolph Diatom Atlas, https://list.indiana.edu/sympa/arc/diatom-l. Sterrenburg, F.A.S. (2013a). An imaging conundrum in diatoms, http://www.microscopy-uk. org.uk/mag/artfeb13/fs-diatom-conundrum.html. Sterrenburg, F.A.S. (2013b). Book review: An introduction to the microscopical study of diatoms. Robert B. McLaughlin. Edited by John Gustav Delly & Steve Gill, http://www.microscopy-uk.org.uk/mag/artfeb13/fs-review.html. Sterrenburg, F.A.S. (2013c). The search of fine detail: the history of diatom imaging. In: Diatoms are Forever: Growing your Nanotechnology, R Gordon, F.A.S Sterrenburg, M.A Tiffany, S.S.Nagy & I.C Gebeshuber (eds.), unpublished manuscript. Sterrenburg, F.A.S. (2013d). The naming of the beasts: making an inventory of the realm of diatoms. In Diatoms are Forever: Growing your Nanotechnology R. Gordon, F.A.S. Sterrenburg, M.A. Tiffany, S.S. Nagy and I.C. Gebeshuber (eds.), unpublished manuscript. Sterrenburg, F.A.S. (2014a). My favourite slide, http://www.microscopy-uk.org.uk/mag/artfeb14/fs-favourite.html. Sterrenburg, F.A.S. (2014b). Haslea brittanica, https://list.indiana.edu/sympa/arc/diatom-l. Sterrenburg, F.A.S. (2014c). Haslea brittanica (follow-up message), https://list.indiana.edu/sympa/arc/diatom-l. Sterrenburg, F.A.S. (Year unknown). How to prepare diatom samples, https://www.scribd.com/ document/155323338/Clean-Diatoms, accessed on 16 December 2016. Sterrenburg, F.A.S. (2018). Science vs religion - the fallacious conflict. In: Theology and Science: Discussions about Faith and Facts, pp. 181–195. J. Seckbach & R. Gordon. World Scientific Publishing. Sterrenburg, F.A.S., de Haan, M., Herwig, W.E. (2014). A lucky break. How past vandalism favoured modern diatom research, www.microscopy-uk.org.uk/mag/artjul14/fs-lucky-break.docx. Sterrenburg, F.A.S., De Souza-Mosimann, R.M., Fernandes, L.F. (2002). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Rediscovery of a "lost" species: Gyrosigma spectabile (Grunow ex Peragallo) Cleve, Cryptogam. Algol., 23(2), 179. Sterrenburg, F.A.S., de Wolf, H. (2004). The Kinker diatom collection: discovery – exploration – exploitation. In: VII International Symposium ‘Cultural Heritage in Geosciences, Mining and Metallurgy: Libraries – Archives – Museums’: “Museums and their collections”, C.F. Winkler Prins & S.K. Donovan (eds.), pp. 253–260. 19–23 May 2003, Scripta Geologica Special Issue, Leiden, The Netherlands.

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Sterrenburg, F.A.S., Erftemeijer, P.L.A., Nienhuis, P.H. (1995). Diatoms as epiphytes on seagrasses in South Sulawesi (Indonesia). Comparison with growth on inert substrata, Botanica Marina, 38(1). Sterrenburg, F.A.S., Gordon, R., Tiffany, M.A., Nagy, S.S. (2007). Diatoms: living in a constructal environment. In Algae and Cyanobacteria in Extreme Environments. Series: Cellular Origin, Life in Extreme Habitats and Astrobiology. Vol. 11, J. Seckbach (ed). Dordrecht, The Netherlands: Springer. Sterrenburg, F.A.S., Hamilton, P., Williams, D. (2012). Universal coordinate method for locating light-microscope specimens, Diatom Research, 27(2). Sterrenburg, F.A.S., Meave del Castillo, M.E., Tiffany, M.A. (2003a). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Pleurosigma species in the plankton from the Pacific coast of Mexico, with the description of P. gracilitatis sp nov, Cryptogam. Algol., 24(4), 291. Sterrenburg, F.A.S., Meave del Castillo, M.E., Tiffany, M.A. (2003b). Valve morphogenesis in the diatom genus Pleurosigma W. Smith (Bacillariophyceae): an engineering paradigm. In NADS2003. E Gaiser (ed). North American Diatom Society (abstract). Sterrenburg, F.A.S., Sterrenburg, F.J.G. (1990). An outline of the marine littoral diatom biocoenosis of the Banc-d'Arguin, Mauritania, West Africa, Botanica Marina, 33(5), 459. Sterrenburg, F.A.S., Sterrenburg, F.J.G. (1991). Studies on a widely distributed marine littoral diatom - Navicula orthoneoides Hust (Bacillariophyceae, Nova Hedwigia, 52(3–4), 411. Sterrenburg, F.A.S., Tiffany, M.A. (2004). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Two new species from the Red Sea: Gyrosigma schmidianum nov sp and Gyrosigma tubicolum nov sp, Diatom Research, 19(2). Sterrenburg, F.A.S., Tiffany, M.A., Lange, C.B. (2000). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). Species from the Salton Sea, California, USA, including Pleurosigma ambrosianum, nov sp, Proceedings of the Academy of Natural Sciences of Philadelphia, 150, 305. Sterrenburg, F.A., Tiffany, M.A., del Castillo, M.E. (2005). Valve morphogenesis in the diatom genus Pleurosigma W. Smith (Bacillariophyceae): Nature’s alternative sandwich, J. Nanosci. Nanotechnol., 5(1). Sterrenburg, F.A.S., Tiffany, M.A., Hinz, F., Herwig, W.E., Hargraves, P.E. (2015). Seven new species expand the morphological spectrum of Haslea. A comparison with Gyrosigma and Pleurosigma (Bacillariophyta), Phytotaxa, 207(2), 143. Sterrenburg, F.A.S., Toonder, M. (1972). Letters. F.A.S. Sterrenburg to Marten Toonder (1912–2005). Sterrenburg, F.A.S., Underwood, G.J.C. (1997). Studies on the genera Gyrosigma and Pleurosigma (Bacillariophyceae). The marine "Gyrosigma spenceri" records: Gyrosigma limosum Sterrenburg et Underwood nov. sp, Proceedings Academy of Natural Sciences of Philadelphia, 148, 165. Stevenson, B. (2009). William Gatrell (1864–1902), Victorian Era Microscope Specimen Mounter, http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopyuk.org.uk/mag/artjan09/bs-gatrell.html. Stevenson, B. (2013). John thomas redmayne, 1846–1880, http://microscopist.net/RedmayneJT. html. Stidolph, S.R., Sterrenburg, F.A.S., Smith, K.E.L., Kraberg, A., Stuart, R. (2012). Stidolph diatom atlas, U.S. Geological Survey Open-File Report 2012–1163, http://pubs.usgs.gov/of/2012/ 1163/.

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Stringer, M.J., Sterrenburg, F.A.S. (2011). Phoenix rising: eclipse and resurrection of a diatom sanctuary, http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopyuk.org.uk/mag/artjan11/ms-fs-phoenix.html. Sunesen, I., Sterrenburg, F.A.S., Sar, E.A. (2013). Pleurosigma guarreranum, sp. nov. (Pleurosigmataceae, Bacillariophyta), from the Gulf of San Matías, Argentina, and comparison with the allied species Pleurosigma exemptum and P. obesum, European Journal of Phycology, 48(4). Swedish natural history museum, astrid cleve von euler, http://www.nrm.se/forskningochsaml ingar/vaxter/kryptogambotanik/virtuellautstallningar/astridclevevoneuler.1601.html [30 November 2016]. Taylor, J.E. (ed.) (1885). Hardwicke’s science-gossip; an illustrated medium of interchange and gossip for students and lovers of nature, Vol. XXI. Chatto and Windus, Piccadilly, London. The Quekett Microscopical Club (2016), http://www.quekett.org/, accessed on 30 November 2016. Walker, D. (2009). Enjoying a Möller 80 form diatom type-slide with a microphotograph setting, http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/ mag/artjan09/dw-moller.html. Walker, D. (2012). The diatomist John Albert Long (1863–1945*): Notes on aspects of his life and work with examples of his prepared slides, http://www.microscopy-uk.org.uk/mag/artdec11/dw-long.html. Weaver, W., Sterrenburg, F.A.S. (1968). Science and imagination: a selection from the work of Warren Weaver (in Dutch). Wetenschappelijke Uitgeverij, Amsterdam. Werner, D. (ed.) (1977). The Biology of Diatoms, Botanical Monographs, Vol. 13. University of California Press, Berkeley and New York.

2 Alex Altenbach – In Memoriam of a Friend Wladyslaw Altermann Department of Geology, University of Pretoria, Pretoria, South Africa

Alexander Volker Altenbach passed away in 2015, after a long illness. Almost two years after Alex’s premature death, I was disclined and reluctant to write this obituary on the invitation of another friend, Joseph Seckbach. I felt that after two years and publications in memoriam of Alex (Schiebel, Schönfeld, & Struck 2016), this would not be necessary and might even be misplaced. But Joseph insisted and after all, it was me that brought Joseph and Alex together, during the visit of Joseph on a (DAAD) Deutscher Akademischer Austauschdienst scholarship to my Department at the Ludwig Maximilians University of Munich (LMU), in 2006. An interesting and praised book resulted from this relationship; edited by Alex, Joan M Bernhard and Joseph (Anoxia; Evidence for Eukaryote Survival & Paleontological Strategies, Altenbach et al. 2012a). Alex was born in 1953, close to Frankfurt, was only few months older than me, but far more advanced in his career and in science. When we first met at the LMU, in 1994, he had just become a contracted professor in palaeontology and was already internationally renowned for his work on forams. I was busy with my habilitation (DSc) on Archean stromatolites, while Alex’s habilitation procedure was just about to be completed. We easily found a common language and soon, together with another new young professor at the LMU, Wolfgang Heckl, a specialist in nanotechnology and today the Director General of the famous and world’s largest museum of science and technology, the “Deutsches Museum” in Munich, we discovered a joint interest in Precambrian life and in finding new methods of investigation of the Archean biosphere. This was a very productive time and Alex, although later less interested in Archean palaeobiology, was always a stimulating part and the deliverer of great ideas in our working group. Alex was an infinite source of ideas. With his profound experience in computing, programming, statistical methods and, of course, marine biology and sedimentology, he was a universal academic and scholar, interested in all disciplines and deeply engaged in the work of his colleagues and his students. He studied geology and palaeontology at Goethe University, in Frankfurt and thereafter gained experience on an oil rig in Denmark and in hydrocarbon exploration in the North Sea, but he was also engaged in environmental geology and was even involved in a private consulting and a geo-software company, based in Hamburg. His later experience encompassed mainly work in Spain where he remained active until his late years. He joined the University Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (29–32) © 2019 Scrivener Publishing LLC

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Figure 2.1 Alexander Volker Altenbach (1953–2015).

of Kiel in the 1980s, where he became interested in the sea and seafloor interactions, a project at the newly formed Marine Geosciences and the Geomar Research Center at Kiel University. His PhD thesis, in 1985, was on the biomass of benthic foraminifera (Altenbach 1985) and his work on the measurement of organic carbon and biomass in foraminifera, was an international breakthrough for Alex (Altenbach 1987). He spent months investigating the relationships between foraminifera and sedimentation along many coasts of tropical and polar seas and on research cruises and campaigns between Patagonia, Namibia and Australia, and published extensively on foraminifera of various stratigraphic periods to recent, on carbon flux, stable isotope fractionation and biomass calculations. Some focussed on the the Namibian diatomaceous mud belt (Altenbach et al. 2012b; Leiter & Altenbach 2010; Schönfeld & Altenbach 2005; Struck et al. 2002). Alex was offered a full professorship at the LMU in 1995, where for a while, since 1994, he substituted for the former, also prematurely departed micropalaeontologist, Prof Konrad Weidich. As a new staff member he immediately started to spin connections, search for new partners and for new contacts. This was how we met. Unconventionally, Alex did not wait for invitations but made his rounds through the different institutes in the Faculty of Geosciences and knocked on doors. This is how he found me late one night, working on the habilitation thesis. He sat down, introduced himself and started to ask and answer questions. Later, he introduced me to his research and his young colleagues and students who he valued and loved so much, Ulli Struck, Christoph Mayr, Carola Leiter, and many more. He was always interested in

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the progress of others, in learning and teaching and in support of the Institutes and the Faculty of Geosciences at LMU. Alex’s research was driven by curiosity and by the desire to discover things outside of conventional thinking. His publications were sometimes controversial and, as he wrote himself, reviews that rejected his new findings because they contradicted accepted models, drove him to new activities and stimulated more work on the subject. Thus, a rejection of his manuscript on foraminifera thriving under sulphidic conditions, by an international journal (“…anoxic foraminifera don’t seem reasonable…”), led him to the book on anoxic strategies in eukaryotes (Altenbach et al. 2012a). In this book we have published together a speculative essay on the relevance of anoxic and agglutinated foraminifera to Archean eukaryote evolution. This was more a philosophical concept than scientific work and data, but it was a lot of fun to write and certainly educational (Altermann et al. 2012). In the same book, Alex published on Proterozoic foraminifera and redox conditions with his wife Maren (Altenbach & Gaulke 2012), and on carbon and nitrogen fractionation in foraminifera (Altenbach et  al. 2012b). The book included excellent science by leaders in their disciplines, like Tom Fenchel, Aharon Oren, Jürgen Schieber and others, just to mention a few, and a large number of young researchers, including students of Alex and his colleagues. He established a stable isotope laboratory and later became the Dean of the Faculty for one term, a work he found necessary and interesting, but he hated the bureaucracy involved. Alex was not necessarily a politician and became very disappointed when the Faculty became divided and new departments were formed, all ending in unhealthy competition replacing multidisciplinary cooperation and leading to egoistic fights between colleagues. He withdrew from departmental politics and concentrated on science and the support of the Bavarian State Collection and the Institute of Paleontology and Historical Geology at Ludwig Maximilians University, where he was an enthusiastic professor. He worked with his many students and with his wife Maren. With Maren he published a travel guide to the exotic nature of Australia and on the ecology and behaviour of lizards and other reptiles of Indonesia and Philippines (Gaulke & Altenbach 1994, 2007). Soon he became ill and started his long fight against cancer. I was already at the University of Pretoria, South Africa, but we continued to meet and to cooperate. Each time we met in Munich, I could see the devastating changes in his health and his body. But Alex was hard on himself and a strong fighter, still working and in the office with his research and students as often as possible. He still dreamed of visiting me with his wife Maren and investigating the large lizards of the Karoo and the Orange River that I have photographed for him in the field. Unfortunately this dream was never realised. Alex lost the fight for his life on 24th of August 2015. He is deeply missed. Wlady Altermann, Pretoria, July 2017

References Altenbach, A.V. (1985). Die Biomasse der benthischen Foraminiferen: Ph.D. Thesis Kiel University, 167.

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Altenbach, A.V. (1987). The measurement of organic carbon in foraminifera, The Journal of Foraminiferal Research, 17(2), 106–109. Altenbach, A.V., Gaulke, M. (2012). Did redox conditions trigger test templates in Proterozoic Foraminifera? In: Anoxia: Evidence for Eukaryote Survival and Paleontological Strategies. Cellular Origin, Life in Extreme Habitats and Astrobiology (COLE), Vol. 21, A.V. Altenbach, J.M. Bernhard & J. Seckbach (eds.), pp. 592–614. Springer, Dordrecht. Altenbach, A.V., Bernhard, J.M., Seckbach, J. (eds.). (2012a). Anoxia: Evidence for Eukaryote Survival and Paleontological Strategies. Cellular Origin, Life in Extreme Habitats and Astrobiology (COLE). vol. 21, Dordrecht : Springer. Altenbach, A.V., Leiter, C., Mayr, C., Struck, U., Hiss, M., Radic, A. (2012b). Carbon and nitrogen isotopic fractionation in foraminifera: Possible signatures from anoxia. In: Anoxia: Evidence for Eukaryote Survival and Paleontological Strategies. Cellular Origin, Life in Extreme Habitats and Astrobiology (COLE), Vol. 21, A.V. Altenbach, J.M. Bernhard & J Seckbach (eds.), pp. 518–535. Springer, Dordrecht. Altermann, W., Altenbach, A.V., Leiter, C. (2012). The relevance of anoxic and agglutinated benthic foraminifera to the possible Archean evolution of eukaryotes. In: Anoxia: Evidence for Eukaryote Survival and Paleontological Strategies. Cellular Origin, Life in Extreme Habitats and Astrobiology (COLE), Vol. 21, A.V Altenbach, J.M. Bernhard & J. Seckbach (eds.), pp. 615–630. Springer, Dordrecht. Gaulke, M., Altenbach, A.V. (1994). Contribution to the knowledge of the snake fauna of Masbate (Philippines) (Squamata:Serpentes), Herpetozoa, 7, 63–66. Gaulke, M., Altenbach, A. V. (2007) Australia: Natur und TierVerlag Gmbh. p. 391. Münster, Germany. Leiter, C., Altenbach, A.V. (2010). Benthic foraminifera from the diatomaceous mud belt off namibia: Characteristic species for severe anoxia, Palaeontol. Electronica, 13(2). Schiebel, R., Schönfeld, J., Struck, U. (2016). Memorial to Alexander Volker Altenbach (1953–2015), Journal of Foraminiferal Research, 46(1), 4–6. Schönfeld, J., Altenbach, A.V. (2005). Late Glacial to Recent distribution pattern of deep-water Uvigerina species in the north-eastern Atlantic, Mar. Micropaleontol., 57(1–2), 1–24. Struck, U., Altenbach, A.V., Emeis, K.-C., Alheit, J., Eichner, C., Schneider, R. (2002). Changes of the upwelling rates of nitrate preserved in the δ15N-signature of sediments and fish scales from the diatomaceous mud belt of Namibia, Geobios, 35(1), 3–11.

3 The Beauty of Diatoms Mary Ann Tiffany1,* and Stephen S. Nagy2 1

Department of Biology, San Diego State University, San Diego, CA, USA 2 Montana Diatoms Helena, Montana, USA

Abstract From the early eighteenth century to the present anyone with a microscope could admire very small creatures. Because of their attractiveness, one of the favorite choices of objects to observe has been diatoms. They are ubiquitous in fresh, brackish and marine systems and so are easy to sample. A drop of water or material scraped from rock from a pond, lake or sea can contain a multitude of species of diatoms. They can be admired using a number of techniques including brightfield, darkfield, phase contrast, differential interference microscopy, electron microscopy and Jamin-Lebedeff interference contrast microscopy. Keywords: Diatoms, history, beauty, SEM, brightfield microscopy, darkfield microscopy, Jamin-Lebedeff images, living cells, valve morphogenesis, arranged diatom slides

3.1

Early History of Observations of Diatoms

The beauty of microscopic diatoms has been noted since the microscope was first utilized. In 1703 Mr. C., a fellow of the Royal Society of London observed, in describing what was likely Tabellaria, they were “many pretty branches, compos’d of rectangular oblonga and exact squares” (Round et al. 1990). Since then, especially in Victorian times, it was a popular fashion among some gentlemen in European countries to study and draw diatoms from around the world. These early amateur diatomists made permanent slides, now understood to be valuable antiques, from many gatherings. For examples see: https://www.antiquemicroscop esandslides.com/diatoms. Strew slides of cleaned diatom frustules illuminated with darkfield and viewed under low power can show unexpected colors (F.B. Taylor 1929): “On examining certain diatoms under low and medium powers with dark ground illumination, it will be noticed that they show most lovely shades of color, exquisite blues or a glowing iridescence; e.g., many forms of Actinocyclus, Actinoptychus, Auliscus, Climacosphenia, Lepidodiscus, Pleurosigma and Podosira. These colors are not inherent in the shell, but are the result of diffraction, or interference with the light passing *Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (33–42) © 2019 Scrivener Publishing LLC

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Diatoms: Fundamentals and Applications through the minute markings of the diatom, as if through a diffraction grating; they disappear as the markings are resolved into dots and spherules under higher powers.”

At one time microscope manufacturers provided darkfield condensers for lowerpower objectives with narrower cones of illumination than those available now. The modern darkfield condensers typically have cones of illumination of NA of 1.2–1.4, which are designed for use with medium- and high-power achromatic objectives. Although they are difficult to locate, darkfield condensers with NAs of 0.7–0.85, 0.8– 0.95 can be specifically useful when searching strews for rare forms, which may then be located in the strew field simply by looking for their refraction color. The best darkfield/ oblique illumination tool may be a Leitz Heine Condenser, with an infinitely-variable cone of illumination which can provide darkfield to circular oblique illumination, and be adjusted for each individual objective. Although these older darkfield condensers may have been manufactured for a frame or stand different from the one in use, in most cases devising an adapter to mount on a stand different from the manufacturer’s is not difficult. Carpenter observed in 1856: “In another group, that of Diatomacece (also still regarded as Animalcules, not only by Ehrenberg, but by many other Naturalists), not only are the forms of the plants often very remarkable but their surfaces exhibit markings of extraordinary beauty and symmetry, which are among the best ‘test-objects’ that can be employed for the higher powers of the instrument [the microscope].”

Additionally, intricate arrangements were made with diatom valves in fantastic and geometric designs. These take extreme patience and skill to create. Since the slides were sold commercially, diatom mounters kept their methods a secret, and with the exception of several papers which detail methods and techniques by Meakin and Swatman published originally in the 1930s. Klaus Kemp, who started working at Flatters and Garnett at age 16, after emigrating to England at age 10, had the privilege of being hired by Wilfred Garnett and placed in charge of the preservation department, he also took him under his tutelage and learned biological taxonomy. He examined slides created by and owned by the company with Gordon McKechne , and found himself “absolutely amazed at how beautiful the individual slides were.” When he was shown a 400-form Exhibition slide by Moeller, he decided “I’m going to do that,” as he saw the tiny dot illuminated by sunlight as he held the slide in his hand. He worked for years in the Preservation Department, and stayed with the firm until it closed. When the firm was dissolved, Wilfred Garnett authorized him to retain the diatom collection which had been returned by the Reverend Calloway who made Diatom slides for the Company but failing eyesight caused him to give this up, the collection was in fact originally from J.A. Long. Klaus charged with conserving the collection for posterity and personal use. He has spent a lifetime re-inventing the craft of collecting, identifying, and mounting diatoms. He went on to reinvent the materials and methods of making exhibition slides, and made copies of Moeller’s ornate arrangements, making a substitution of one form in the arrangement to illustrate that this was a copy, and not made by Moeller. He has

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Figure 3.1 Arranged slides, also known as Exhibition Mounts. (a) Brightfield Light Microscopy (LM), scale bar = 200 μm. (MAT) (b) Polarized Darkfield image. (SSN) Slides prepared by Klaus Kemp. Copyright reserved by the photographer.

since made a circular arrangement of some 1500 forms (Personal communication to SSN.) (Figures 3.1a and 3.1b). He also has in corporation also found and described new species to Science. He clearly appreciates the beauty of diatoms as evidenced by the following video: https://youtu.be/qxkbSk--EUY In modern times some authors emphasize the utility of diatom study in ecological and evolutionary investigations (e.g., Cox 2012; Stoermer & Smol 1999) but if these authors are queried they will invariably state that the captivating aesthetics of diatoms also intrigue them. The human eye appreciates symmetry and purity of geometric design, qualities that diatoms clearly possess.

3.2

Live Diatoms

Living diatoms have an intrinsic beauty and the complete frustule can be imaged in an ordinary brightfield microscope. They are often a lovely golden color, the plastids being pigmented with fucoxanthin and carotenoids as well as the usual xanthophyll (Kuczynska, Jemiola-Rzeminska, & Strzalka 2015). Diatom cells are often relatively thick. Using a focus stacking technique and Photoshop on 2–6 images at different levels of focus, the inherent limited depth of field of the light microscope can be overcome and valves and chloroplasts can be shown simultaneously in the same image (Figures  3.2a–3.2d). This technique is often used in macrophotography of insects and flowers. Differential interference microscopy (DIC, Figures 3.2e and 3.2f) shows the cell organelles clearly and is especially useful in imaging lightly silicified species (Hasle & Syvertson 1996). Comparing the same species as in Figures 3.2c and 3.2d to Figure 3.2e, Figure 3.2f, the silica frustule, composed of girdle bands and valves, shows more contrast and detail than in brightfield.

3.3

Shapes and Structures

Diatoms come in an incredible variety of shapes and sizes, from the tiny Minidiscus spp. (about 3 μm in diameter) to some that are almost visible to the naked eye. The scanning electron microscope allows a three-dimensional view of diatom frustules, valves and girdle bands. Many diatom valves are simple, but decorated sticks (Figure 3.3a). Others are

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Figure 3.2 Live diatoms. (a) Coscinodiscus wailesii, scale bar = 10 μm (LM). (b) Actinoptychus senarius, scale bar = 10 μm. (c) Ditylum brightwellii, scale bar = 10 μm (LM). (d) Odontella longicruris, scale bar = 10 μm (LM). (e) Ditylum brightwellii, Differential Interference Contrast (DIC, courtesy of Charles Krebs). (f) Odontella longicruris, (DIC, courtesy of Charles Krebs). Copyright reserved by the photographer.

undulating in various planes (Figures 3.3b and 3.3c). Several genera, e.g., Pleurosigma and Gyrosigma (favorites of Frithjof Sterrenburg), are gently sigmoid (Figure 3.3d). An especially unusual fossil diatom, Kittonia elaborata from Oamaru New Zealand, has tubular spines resembling so-called Martian antennae (Figure 3.3e). Another diatom with spines is Ditylum brightwellii which also sports a glass fringe-like skirt (Figure 3.3f). The shape of Surirella fastuosa resembles a boat under construction with ribs and keel (Figure 3.3g). Didymosphenia geminata has the unusual shape of a sarcophagus (or a classic bottle of Coca Cola) (Figure 3.3h). The heavy valve of Cerataulus turgidus has a twisted appearance with large ocelli and spines (Figure 3.3i). The geometry of diatom valves is often circular or football shaped but some species are polygonal with three or more sides (Figures 3.3j–l). Adolph Schmidt considered a Melosira from Mors to be the most beautiful diatom, but Klaus Kemp prefers Mastogloia for its complex internal structure, unique to this genera.

3.4

Diatom Beauty at Various Scales

Diatom valves have two sides, the internal and external faces. Some even have the “typical” petri dish structure where the sides of the dish are called the mantle. We will use Arachnoidiscus ehrenbergi as an example of how diatoms are beautiful on both sides at different scales. The internal view of the whole valve (Figure 3.4a) shows a structure much like a stained glass “rose” window with radiating ribs. One of the two heterovalvar shells has central radial slits (Figures 3.4b and 3.4d). The other is more plain but still attractive (not

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Figure 3.3 Various attractive shapes of diatoms, scanning electron microscopy (SEM). (a) Thalassionema nitzschioides. (b) Terpsinoë musica. (c) Glyphodiscus stellatus. (d) Pleurosigma ambrosianum. (e) Kittonia elaborata. (f) Ditylum brightwellii. (g) Surirella fastuosa. (h) Didymosphenia geminata. (i) Cerataulus turgidus. (j) Trigonium arcticum. (k) Triceratium pentacrinus. (l) Trigonium formosum fo. quadrangularis. Scale bars = 5 μm (a) Scale bars = 10 (g) Scale bars = 20 μm (b, c, d, f, j, k ) Scale bar = 50 μm (e, h, i, l). All photos by MAT. Copyright reserved by the photographer.

shown). The costae interconnect the ribs leading to a spider web-like appearance (hence the generic name). In SEM the exterior valve does not show the ribs but can have the central slits (Figures 3.4c and 3.4d). The mantle is steep with smaller volae (term used by Round et al. 1990 for the pore occlusions) than on the surface of the valve (Figure 3.4e). At high magnification the individual vola is exquisite with curly slits and tiny spheres decorating the edges. Additionally, the surface of the valve exhibits a fine net-like decoration (Figure 3.4f).

3.5

Valves During Morphogenesis

Diatoms have two valves and a number of girdle bands comprising the frustule. During asexual division each new daughter cell retains an original valve and fashions a new

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Figure 3.4 Example of eye-appeal of a diatom, Arachnoidiscus ehrenbergi, at different scales (SEM). (a) Interior view of entire valve. (b) Central part of internal valve, note 3-dimensionality of the radial ribs. (c) External view of entire valve. (d) Central part of external valve with radial slits. (e) View of edge of external valve. (f) Highly magnified view of external cribrum with tiny silica spheres. Scale bars are 1 μm (f), 10 μm (d), 20 μm (b), 50 μm (a, c). All photos by MAT. Copyright reserved by the photographer.

one. As part of this process silica is added to a basal layer in a distinct pattern (e.g., Schmid & Volcani, 1984, Tiffany 2008, 2015). Forming valves do not yet have their full complement of silica and thus appear different from the mature counterpart. These unfinished structures can have a beauty of their own. Asterolampra is a genus with rays and an exquisite star-like pattern (Tiffany & Hernandez-Becerril 2005). Usually seven rays occur in modern Asterolampra marylandica (Figure 3.5a and 3.5b), occasionally six rays are seen (e.g., Round et al. 1990, fig b, page 210). When the outer surface of immature valves at different stages is examined in SEM at high magnification, intricate designs emerge (Figures 3.5c–f). The earliest stage arising from the basal layer is a hexagonal wall pattern -each hexagon a little less than one micron across (see Plate 3.6, Figure 3.1 in Tiffany & Hernandez-Becerril 2005). The hexagons can be discerned below curious triangular structures in Figures 3.5c and 3.5d. As the silicification progresses and the triangles merge, the designs become floral and these might make fascinating wallpaper patterns (Figures 3.5e and 3.5f). The surface of the mature valve is much less interesting as the designs are almost complete covered over with silica (see Figure 3.6, Figure 3.8 in Tiffany and Hernandez-Becerril 2005).

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Figure 3.5 Beauty of the external surface of immature valves of Asterolampra marylandica at increasing stages and high magnification (SEM). (a) Internal view of whole mature valve. (b) External view of immature valve. (c)-(f) Surface of valve as it matures. Scale bars = 20 μm (a and b), scale bars = 1 μm (c-f). All photos by MAT. Copyright reserved by the photographer.

3.6

Jamin-Lebedeff Interference Contrast Microscopy

Using specialized microscopic illumination techniques, spectacularly beautiful photographs of diatoms may be made. Combining darkfield with polarized light techniques can reveal beautiful but subtle interference colors at medium power magnification. Phase contrast images of diatoms may be manipulated using digital editing programs to bring out latent colors which are the product of refraction and interference, but no technique really compares to images which can be created using JaminLebedeff interference contrast equipment with white light. A number of English and German microscope manufacturers made this equipment early in the 20th century, but the equipment is rare, often mishandled and thus unusable, and at best it is difficult to use. The polarized illuminating beam of light from the condenser is split into two parallel light bundles by a calcite plate in the condenser, one passing adjacent to the diatom and the other passing through the frustule. The two beams are recombined in the objective, which is fitted with an identical but opposite calcite plate, and the resulting image can be manipulated to generate interference colors which vary according to the optical path length of the part of the diatom being illuminated. The second author of this article has made a specialty of imaging diatom using this

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Figure 3.6 Comparison of Jamin-Lebedeff images and SEM of the same species. Actinoptychus heliopelta, scale bar = 20 μm (a and b). Trigonium graeffeanum, scale bar = 50 μm (c and d). Aulacodiscus janischii, scale bar = 100 μm (e and f). SEM by MAT; JL by SSN. Copyright reserved by the photographer.

technique. Figures 3.6a–f (Figure 3.6) compares colorful Jamin-Lebedeff images of three species with those obtained using SEM.(Figure  3.7) Figures 3.7a–f compare three additional species. The same structures may be observed in both; the SEM images show more 3D effects but the Jamin-Lebedeff are arguably prettier.

3.7

Conclusions

The beauty of diatoms will surely continue to attract scientists and laypersons alike. Even though many modern investigators are using genomic techniques to study these organisms, the splendor of their siliceous shells will continue to intrigue, and likely will be both the first, and the most long-lasting, of their attractions.

Acknowledgments The authors thank Charles Krebs for use of the Differential Interference Contrast Images. We are also grateful to Steve Barlow for the generous use of the Electron Microscope Facility at San Diego State University. We thank Klaus Kemp for his inspiring story of

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Figure 3.7 Comparison of Jamin-Lebedeff images and SEM of the same species. Didymosphenia geminata, scale bar = 50 μm (a and b). Tetracyclus emarginatus, scale bar = 10 μm (c and d). Coscinodiscus sp., scale bar = 50 μm (e and f). SEM by MAT; JL by SSN. Copyright reserved by the photographer.

choosing and achieving a seemingly-impossible goal, for his generosity in sharing his hard-earned knowledge, and his patience in teaching, over many years.

References Carpenter, W.B. (1856) The Microscope and its Revelations, First Edition. Cox, E.J. (2012). Ontogeny, homology, and terminology—wall morphogenesis as an aid to character recognition and character state definition for pennate diatom systematics, J. Phycol., 48(1), 1–31. Klaus Kemp. http://www.diatoms.co.uk/. Kuczynska, P., Jemiola-Rzeminska, M., Strzalka, K. (2015). Photosynthetic pigments in diatoms, Mar. Drugs, 13(9), 5847–5881. Meakin, S.H. (1939). The study of diatoms. The Microscope, Vol III, 152; 187; 209; 239. Vol. IV, 8; 50, London, U.K. Round, F.E., Crawford, R.M., Mann, D.G. (1990). The diatoms, biology & morphology of the genera. Cambridge University Press, New York. Stoermer, E.F., Smol, J.P. (1999) The diatoms: applications for the environmental and earth sciences. Cambridge University Press, Cambridge.

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Swatman, C.C. (1937). Cleaning diatoms for microscopical use, The Microscope, I(11), 43. Revised publication. (1949). The Microscope Vol VII, 132, p. 149. Taylor, F.B. (1929) Notes on Diatoms: An Introduction to the Study of the Diatomaceae. Bournemouth,UK. Tiffany, M.A. (2008). Valve development in Aulacodiscus, Diatom Research, 23(1), 185–212. Tiffany, M.A. (2015). Valve and girdle band morphogenesis in the pseudocellate diatom species Biddulphia biddulphiana J.E. Smith (Boyer) and Isthmia nervosa Kütz, Nova Hedwigia, Beiheft, 144, 61–95. Tiffany, M.A., Hernandez-Becerril, D.U. (2005). Valve morphogenesis in the diatom family Asterolampraceae H. L. Smith, Micropaleontology, 15, 217–258.

4 Current Diatom Research in China Yu Xin Zhang State Key Laboratory of Mechanical Transmissions, College of Materials Science and Engineering, Chongqing University, Chongqing, P.R. China

Abstract Diatom silica, a three-dimensional (3D) natural biomaterial generated from single cell algae with unique patterns of nano-sized features such as pores, ridges, spikes and spines, is shown to have several exceptional structural, mechanical, optical, photonics, transport, and chemical properties optimized through 200 million of years of evolution. In the past, the applications of diatom silica were quite limited in China. However, in recent years, the unique shapes and ordered porous structures of diatoms at the micro- and nanoscale, high specific surface area, thermal stability, and cost-effectiveness provide enormous advantages and opportunities to use diatom-based material for many applications. Considerable research from China is devoted to modify or convert diatom silica into other materials but with preserved diatom structure. The application of diatom-based composite materials in energy storage has attracted extensive attention and has great potential. Three-dimensional porous materials have a wide range of applications in the environmental field. By modifying the surface of diatoms, excellent performance of the three-dimensional porous material can be achieved. A great deal of research has been done on diatom-based composite materials in the environmental field in China by depositing functional materials to achieve functional composite materials. Keywords: Diatom, porous materials, energy storage, environmental field

4.1 4.1.1

Diatoms for Energy Conversion and Storage Introduction

New synthetic materials with nanoscale dimensions and unique properties have been extensively explored in last two decades for development new solutions for energy production and storage. (Schlapbach & Zuttel 2001; Arico, Bruce, Scrosati, Tarascon, & Van Schalkwijk 2005) Among them porous nanostructures with different morphologies made from materials including carbon, silicon, inorganic oxides and polymers were increasingly explored providing some unique properties such as high surface area available and structural architecture for ion transport, and favourable mechanical, electrical, electrochemical, magnetic and optical properties or their Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (43–98) © 2019 Scrivener Publishing LLC

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combination required for energy conversion or storage processes. (Schlapbach et al. 2005; Nakajima & Volcani 1969; Sumper, Lorenz, & Brunner 2003; Hirscher 2004; Klaine et al. 2008) The main disadvantage of these synthetically produced materials is their high production cost, long production time with batch process, lack of scalability, the use of toxic chemicals and producing of hazardous waste with significant environmental footprint which are not acceptable for sustainable production and established climate protection standards. (Biswas & Wu 2005; Cerneaux et al. 2007) No only environmental issues are critical for their production, their use also for instance, superfine transition metals particles (e.g., Fe and vanadium) coated with redox-cycling organic chemicals (e.g., quinones), or carbon nanotubes with metal impurities could cause negative health impact. (Nel, Xia, Madler, & Li 2006) To address these problems there is strong attention to replace these hazardous materials using natural or biomaterials with low processing cost and minor impact on environment as an alternative solution for energy conversion and storage. Nature developed a unique biomolecular machinery of a biosystemability to make many biomaterials with unprecedented complexity and multiple functionality that outperforms man-made manufacturing technologies. (Tibbitt, Dahlman, & Langer 2016; Wang, Santos, Evdokiou, & Losic 2015) The natural molecular self-assembly process developed from millions years of evolution is able to create these unique biological structures with high precision and reproducibility under mild environmental conditions and lower energy consumption. These biological materials attracted enormous interests for the biomimetic engineering of new nanostructured materials with complex architecture and unique properties, but also as source of low cost natural materials ready to be used with minimal processing. Most of living organisms such as bacteria, algae, fish, insects, plants, animals and humans (bone) are able to synthesize this type of inorganic structures or their organic composites into intricate architectures with ordered micro-to nanoscale features that are not possible be replicated through existing engineering or chemical synthetic processes. (Wang et al. 2015; Maher et al. 2016; Aw, Simovic, Addai-Mensah, & Losic 2011) Among them, the amorphous silica exoskeletons (frustules) of the single-celled algae called diatoms are one of the most spectacular examples of biologically derived nanostructured materials. (Maher et  al. 2015) Each of the estimated 100.000 diatom species has a specific 3-d silica shell called frustule with distinctive shape decorated with a unique pattern of nano-sized features such as pores, ridges, spikes and spines. (Maher et al. 2015; Mann 1999; De Stefano & De Stefano 2005; Losic Mitchell & Voelcker 2009) Each frustule structure has several hierarchical layers of porous membranes or structures differing in shapes, size and patterns. A remarkable diversity of diatom shapes and pore architectures can be gained from Figure 4.1 which includes several most typical diatom shapes. These variety of shapes and ordered porous structures irrefutably demonstrates precision and brilliance of natural design at the micro- and nanoscale providing enormous opportunity to use this material for broad applications. New term called diatom nanotechnology was recently created to describe new emerging field on exploration these outstanding materials and their applications across different disciplines including molecular biology, materials science, biotechnology, nanotechnology, and photonics. (Losic et al. 2009) Variety of potential applications for diatom silica, including optics, photonics, catalysis, biosensosors,

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Figure 4.1 Extraordinary diversity of shapes and 3-d structures in diatoms build by silica.

drug delivery, microfluidics, molecular separation, filtration, adsorption, bioencapsulations and immunoisolations and template synthesis of nanomaterials have been proposed and explored. (Yu, Addai-Mensah, & Losic 2011; Yu, Addai-Mensah, & Losic 2010; Bao et al. 2011) It is worth noting that diatoms silica can be obtained by cultivation of diatoms at considerable quantity but their enormous quantities area available from low cost fossilized diatoms called diatomaceous earth, white mineral powder composed of pure diatom frustules available in thousands tonnes from mining industry. Therefore it is not surprising that these promising natural materials based on their unique structures has been considered for energy conversion and storage. This review presents the recent progress on the applications of diatom-based nanostructures (native diatom, diatomite (diatom frustules), diatom replica, and their composites) in energy-related fields such as lithium ion battery materials, supercapacitors, solar cells, hydrogen storage properties and thermal energy storage properties. Even the field is still in early stage, it is progressing fast with strong expectation to spur strong contribution of diatom nanotechnology to this field.

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4.1.2 Diatom Silica: Structure, Properties and Their Optimization Diatom silica or frustule structure composed of pure silica to protect cells is composing of two valves called that are banded together by girdle bands which wrap around and holds them together. The valve consists of piled hexagonal chambers divided by silica plates. The overall structure of a valve can be summarized as follows: lines of silica called costae diverge and occasionally branch from a nucleation site, the linear midrib in pennate diatoms or the circular mid ring in centre. (Gordon & Drum 1994; Lewin 1990; Losic Short Mitchell Lal & Voelcker 2007b; Nassif & Livage 2011; Sumper 2002) These complex hierarchical silica structures of diatoms were developed and optimized by evolution to provide many functions including cell protecting with strong mechanical structure and strength against predators, easy movement in water environment, pore structures for molecular sieving for nutrients uptake, specific nano to micro scale pore patterns with optical and photonic properties for light and energy harvesting and possible sensing and communications. Diatom silica walls (frustules), are appeared in various textures and shapes, which are mostly symmetrical. The micro-scale size of the diatom frustules and the nano-scale size of the pore structures are similar to the wavelengths of light that make them enhance the optical scattering properties and be used as photoelectric devices. (Li, Wang, & Yu 2011; Zhang, Chou, Russo, Jenekhe, & Cao 2008; Jeffryes, Gutu, Jiao, Rorrer 2008; Noll, Sumper, & Hampp 2002; Anderson, Holmes, Hanif, & Cundy 2000) More importantly, photosynthetic receptors of diatoms are located in chloroplasts close to the silica wall, thus the light channelling and focusing properties of their silica structures can help the transmission and collection of more light into the photoreceptors, developing the silica frustules which help to increase the rate of photosynthesis. (Jeffryes et  al. 2008; Tachibana, Akiyama, & Kuwabata 2007; Kooistra, Gersonde, Medlin, & Mann 2007; Medarevic, Losic, & Ibric 2016; Gordon, Losic, Tiffany, Nagy, & Sterrenburg 2009a; Weatherspoon, Allan, Hunt, Cai, & Sandhage 2005) Regarding that properties of diatom silica have many limitations such as high resistivity which are not favourable for energy conversion and storage and other applications considerable research is devoted to modify or convert silica into other materials but with preserved diatom structure. These modifications involved many materials including metals, semiconductors, carbon and polymers. (Losic et al. 2009b) Several approaches based on metal (Au, Ag, Pt) and nanoparticle coatings, using hydrothermal conversion, sol-gel chemical vapor deposition and atomic layer deposition have been presented to convert silica surface into composite materials with new and higher efficient optical, electrical and magnetic properties. (Losic et al. 2009b) Hydrothermal treatment in combination with thermal annealing were employed for conformal coating of diatoms by ZnFe2O4/SiO2. These coatings showed green photoluminescence resulting from the 4G–6S transition in Mn2+ ions. (Weatherspoon et al. 2005; Zhao, Gaddis, Cai, & Sandhage 2005; Ernst, Church, Gaddis, Snyder, & Sandhage 2007) Besides, sol-gel surface-coating processes, combining with structure-directing agents, have afforded conformal coating of diatoms by various oxides. (Ernst et al. 2007; Liu et al. 2007) Moreover, reducing the size of diatom-membrane pores while preserving the shape of the pores was exhibited to produce photocatalytically active diatom silica though atomic-layer deposition of titania. (Losic et al. 2006) The hierarchically porous

Current Diatom Research in China

47

Table 4.1 Studies on the energy-related application of diatom biosilicastructures-based devices. Deviceapplications

Modificationprocess

Improvement

Lithiumbattery

magnesiothermicreduction, impregnation and carbonization

excellentcycle performance and high capacity retention

Supercapacitors

basedon metal-oxide

highspecific capacitance and high cyclic stability

Dyesensitized solar cells

TiO2embedding or deposition

increasingphotovoltaic conversion efficiency

Hydrogenstorage

acid-thermaltreatment

hydrogenadsorption capacity

Thermalenergy storage

materialmodification

hightensile ductility and heat storage capacity

diatoms with transition metal oxides modified structures are proposed to exhibit excellent specific capacitance and capacitance retention during cycling, and commonly are with elaborately detailed and patterned nanoscale features. (Losic et al. 2009b; Losic et al. 2010; Toster et al. 2013; Yu, Addai-Mensah, & Losic 2010; Jantschke, Herrmann, Lesnyak, Eychmuller, & Brunner 2012; Rosi, Thaxton, & Mirkin 2004) Another strategy is to completely convert diatom silica into another material where several approaches where demonstrated with conversion into non-natural metals (Au, Ag), polymers and Si without changing the bioassembled 3D morphologies. (Losic et al. 2009b) This strategy called as BaSIC (bioclastic and shape preserving inorganic conversion) is pioneered by Snandhage and his team and includes gas/silica displacement reactions, conformal coatings, or a combination of those. Gas-silica displacement with an oxidation/reduction reaction using elemental gas reactants or a metathesis reaction using halide-gas reactants were used to convert silica-based frustules into MgO and TiO2 replicas, respectively. (Cai & Sandhage 2005; Sandhage et al. 2002; Unocic, Zalar, Sarosi, Cai, & Sandhage 2004; Cai et al. 2007) A combination of the displacement-reaction and solution-coating methods has been shown to generate a series of composite replicas with a variety of functional chemistries, including MgO/BaTiO3, MgO/BaTiO3 (Eu3+-doped), BaTiO3, and SrTiO3. Boron nitride was conformally coated on diatom frustules. Afterwards, the underlying frustules yielded free-standing boron nitride structures, which can hold the promise for large-scale fabrication of nanostructured nonoxide ceramics for many applications including energy not yet explored. One of the most famous diatom modification processes was achieved by magnesiothermic reduction using gaseous magnesium as a reducing agent where the diatom silica was converted into diatom silicon with precise preservation their 3-d porous structure. In the first step diatom silica is converted into a continuous nanocrystalline mixture of silicon and magnesia by thermal treatment at 650 °C, followed by selective

48

Diatoms: Fundamentals and Applications

magnesia dissolution, which yielded an interconnected network of silicon nanocrystals with form of initial diatom structure. (Bao et al. 2007) This process is used for the fabrication of porous silicon providing many advantages in low cost, time, advanced properties and scalability compared with porous silicon prepared by electrochemical process. Subsequently, combination of diatom silicon with carbon coating to gain better electrochemical performances attracted considerable attention to design new electrode materials for many energy applications which few are already demonstrated. (Shen, Wang, & Chen 2014) From these presented approaches we learned how many new materials and properties can be designed and fabricated from diatom silica materials. However, their only limited number was explored for energy production and conversion applications. Table  4.1 summarize currently developed concepts and the energy-related application of diatom biosilica structures-based devices. Considering enormous potential and properties of these materials we expect in future enormous progress on their applications including translation into real devices and energy production system.

4.1.3 Diatoms for Lithium Ion Battery Materials The performance of low cost, high energy density and long lasting rechargeable lithium-ion batteries greatly depends on lithium storage materials. In the past, graphite has been considered as potential candidate for the anode material in lithium-ion batteries (LIBs) due to its excellent cycling performance. However, the theoretical capacity of graphite is only 372 mAh g−1. It is crucial to develop electrode materials exhibiting higher power and energy densities. Silicon is one of the attractive anode materials for lithium-ion batteries due to its high capacity of 4200 mAh g−1. (Dahn, Zheng, Liu, & Xue 1995; Etacheri, Marom, Elazari, Salitra, & Aurbach 2011; Poizot, Laruelle, Grugeon, Dupont, & Tarascon 2001; Wen & Huggins 1981) In past years many different forms of silicon were explored for Li battery applications including silicon films, nanoparticles, electrochemically produced porous silicon etc. (Etacheri et al. 2011; Poizot et al. 2001) Among them, a porous silicon with an unique hierarchical structure and high surface area was found to be very attractive electrode material having ability to facilitate the fast transport of lithium ions due to its highly accessible surface area for liquid electrolytes providing excellent rate properties and maintaining good electronic conductivity during charging/discharging cycles. To improve the performances of porous silicon electrodes it is critically important to develop electrodes with new porous nano architectures able to greatly enhance performance by decreasing crystal strain and increasing the surface area available for ion transport. Through the lithium-ion insertion and extraction processes, silica as anode material is significantly hindered by severe volume expansion and fast capacity fading, leading to the pulverization of electrode structure and the poor cycling performance. (Losic et al. 2007) Thus, to enhance electrical conductivity, charge storage ability, electronic transport ability as well as suppress of the particle pulverization, it is necessary to improve the performances of the silicon anode by including new nanoscale morphologies with increased surface and porosity such as nanowires, nanotubes (Gordon & Drum 1994), nanosheets and nanospheres (Lewin 1990). The following discharge capacity performance of prepared Li ion batteries were obtained using different silicon

Current Diatom Research in China

49

structures, such as 3D porous Si particles (around 2600 mAh g−1), Si nanotube arrays (about 1800 mAh g−1), Si nanotubes (around 1000 mAh g−1). (Jeffryes, Campbell, Li, Jiao, & Rorrer 2011) Another approach to improve performance of Si based anodes was to perform coating using Si-containing polymers, (Yu et al. 2011) silicohydride. (Nassif & Livage 2011) However, the reaction conditions of them are very laborious, time consuming and need complicated processing such as high pressure or temperature and expensive raw materials. Carbon coating was regarded as the most the most optimal solution because carbon-coated hierarchically porous silicon provide sufficient room to accommodate the volume change of silicon, improve cycling stability, and improve electronic conductivity compared to the bare porous silicon, leading to facilitate the formation of a stable solid electrolyte interphase (SEI) layer. (Baranauskas et al. 2000) The hierarchically porous Si/C composite was shown to exhibit the highest reversible capacity of about 1628 mAh g−1 at the first cycle with excellent capacity retention in the following cycles. (Bao et al. 2011) One of the biggest limitations of silicon and porous silicon anodes for Li ion battery applications is high production costs of these materials which makes them less attractive for making batteries for emerging electric car industry. To improve these problems of synthetically produced Si for Li ion batteries diatom silica is recognised as a promising raw material for the fabrication of porous silicon anodes. Subsequently, combine silicon with carbon coating which can alleviate the volume change of silicon and maintain the electric contact between the porous Si particles. (Shen et al. 2014) The porous silicon particles were obtained by magnesiothermically reducing commercial diatomite. With the void space between the silicon particles and the pores, each silicon particles are designed to supply sufficient room to accommodate the volume change of silicon in the charge and discharge processes, thus indicating much improved cycling stability. Campbell et al. (Campbell et al. 2016) showed in recent work one of the first demonstration of carbon coated and converted diatom silicon as a High Rate Capable Li-ion Battery Anode. Diatom silicon was prepared by reduction process from diatom silica using magnesiothermic process followed by carbonization with polyacrylic acid (PAA) which is summarized in Figure  4.2. The resulting diatom converted nano Si exhibited a high BET specific surface area of

Diatoms frustule (SiO2)

Mg-reduction

C-Coated NanoSi

NanoSi CVD C-Coat

Frustule-like NanoSi

Figure 4.2 Schematic illustration of the process of obtaining C-coated, DE-derived, frustule-like nanoSi structures for use as Li-ion anode active material. (Campbell et al. 2016)

Diatoms: Fundamentals and Applications

50

60 1000 40 Charge Discharge CE

500 0

(a)

0

10

20 2 mgSi cm−2 20

30

40

50

Specific capacity (mAh g–1)

1500

C/30

800

60 C/10

C/5

C/2

600

C/30 1C

40

2C

4C

400

20

200 0

Cycle 50

1.2

Cycle 25

Cycle 1

0

10

20

30

40

50

60

70

0

Cycle #

1 0.8

Delithiation

0.6 0.4

0

500

1000

1500

Specific capacity (mAh g–1)

2000

C/5 4C 2C 1C C/2 C/10 C/30

1 0.8 0.6 0.4 0.2

Lithiation

0.2

1.6–3.2 mgSi cm−2

1.2 Voltage (V vs. Li+/Li)

Voltage (V vs. Li+/Li)

1000

80

1.4

1.4

(c)

1200

(b)

1.6

0

Charge Discharge CE

1400

0

Cycle #

100

1600

Coulombic efficiency (%)

80

Coulombic efficiency (%)

Specific capacity (mAh g–1)

1800 100

2000

2500

0

(d)

0

200

400

600

800

1000

1200

1400

Specific capacity (mAh g–1)

Figure 4.3 Electrochemical characterization of DE-derived nanoSi-based electrodes, including chargedischarge cycling performance for 50 cycles at C/5 based on Si (a), C-rate testing for 75 cycles at C-rates from C/30–4C (b), voltage profiling of the charge-discharge data at C/5 for cycles 1, 25 and 50 (c), voltage profiling of various C-rates (d), CV for cycles 1–10. (Campbell et al. 2016)

162.6 cm2 g−1, compared to a value of 7.3 cm2 g−1 for the original DE. DE contains SiO2 architectures that make ideal bio-derived templates for nanoscaled silicon. The DE-based nanoSi anodes exhibit good cyclability, with a specific discharge capacity of 1102.1 mAh g−1 after 50 cycles at a C-rate of C/5 (0.7 A g−1Si) and high areal loading (2 mg cm-2) (Figure 4.3). This work also demonstrates the first rate capability testing for a DE-based Si anode; C-rates of C/30–4C were tested. The anode maintained a specific capacity of 654.3 mAh g−1 - nearly 2x higher than graphite’s theoretical value (372 mAh g−1). In another work by Wang et al. the specific discharge and charge capacities are about 1700 mAh g−1 at the current density of 0.1 A g−1 was observed on porous Si/C composites anodes obtained from Si converted diatoms. The anode also exhibits much better cyclability than that of the bare porous silicon (205 mAh g−1). The result indicated that the restriction of the particle pulverization becomes the key to the improvement of the performances of the silicon-anode based lithium ion batteries. (Shen et al. 2014; Wang, Fan, Huang, Li, & Qu 2012; Kang, Ryou, Choi, & Lee 2012; Liu, Kopold, van Aken, Maier, & Yu 2015; Lisowska-Oleksiak, Nowak, & Wicikowska 2014; Wu, Shi, Wang, & Jin 2015; Chen, Lu, Sun, & Xu 2015; Liang, Li, Zhu, Guo, & Qian 2015; Wang, Li, Ostrikov, Yang, & Zhang 2015) These examples indeed, demonstrated that silicon converted diatoms are promising anode materials for lithium ion batteries and we can expect more work on further advancing these electrodes to improve capacity and cyclability.

Current Diatom Research in China

4.1.4

51

Diatoms for Energy Storage: Supercapacitors

Supercapacitors have become some of the most promising candidates for next-generation power devices owing to their high power density, fast charging/discharging rate, sustainable cycling life (millions of cycles), and excellent cycle stability. In particular, pseudocapacitors based on transition metal oxides exhibit much higher specific capacitance than those based on carbonaceous materials and conducting polymers on account that they can provide a variety of oxidation states for efficient redox charge transfer. (Conway 1999; Bao, Zang, & Li 2011; Brezesinski, Wang, Tolbert, & Dunn 2010; Hou, Cheng, Hobson, & Liu 2010; Chen et al. 2011; Rakhi, Chen, Cha, & Alshareef 2012; Simon & Gogotsi 2008; Wang, Zhang, & Zhang 2012; Wang, Yang, Huang, & Kang 2013; Wang et al. 2013) In particular, pseudocapacitors based on transition metal oxides exhibit much higher specific capacitance than those based on carbonaceous materials and conducting polymers on account that they can provide a variety of oxidation states for efficient redox charge transfer. The transition metal oxides as electrode materials for supercapacitors including CuO (Zhang et al. 2013), MnO2 (Santhanagopalan, Balram, & Meng 2013; Huang, Li, Dong, Zhang, & Zhang 2015), NiO (Aravindan et al. 2013), Fe2O3 (Wang, Ma, Wang, Liu, & Hao 2013), MoO3 (Liang, Cao, & Qian 2011), V2O5 (Qu, Zhu, Gao, & Wu 2012), Co3O4 (Wang et al. 2012), have been shown to enhance energy and power densities of supercapacitors. However, the majority of metal oxides also have a large portion of bulk and suffer from low electronic conductivity, low ionic diffusion constant and structural susceptibility which limit their applications. (Xiong, Hembram, Reifenberger, & Fisher 2013; Wu, Liu, & Hu 2013) It is crucial to maximize utilization of the pseudocapacity of metal oxides though providing reliable templates with high porosity becomes one of the essential criteria in designing high-performance electrodes for metal-oxide-based electrochemical supercapacitors. Electrochemical supercapacitors based on manganese dioxides (MnO2) have attracted intensive interest due to low fabrication cost, high specific capacitance (theoretical capacity is 1370 F g−1), abundant availability, environmental compatibility, as well as high cyclic stability in alkaline/neutral media. (Wei, Cui, Chen, & Ivey 2011; Peng et al. 2013) The manganese oxides supercapacitors with various structures and morphologies such as nanowires (Yang et  al. 2014), nanosheets (Kai et  al. 2012), nanotubes(Li et al. 2012), nanoflowers (Su et al. 2013), and hollow nanospheres (Liu et al. 2014) have been fabricated via electrochemical and chemical routes, and their electrochemical properties have been investigated. (Wei, Cui, Chen, & Ivey 2011) The concept to use diatomite 3D structure combined with manganese and Ni oxides and make composites electrode for electrochemical capacitors application was pioneered by Zhang team indicating valuable potential of these developments. (Zhang et al. 2014; Guo et al. 2016; Li et al. 2015; Wen et al. 2016; Zhang et al. 2014) Figure 4.4 show typical morphology of MnO2-modified diatom structure used for making supercapacitor electrodes showing confirmed coating of the MnO2 layer with nanofiber structures on diatom silica structure preserving their pores and entire shapes. (Zhang et al. 2014) Purified diatoms exhibit the capability of 8 F g−1, while the hierarchical and porous MnO2-modified diatoms obtained via a one-pot hydrothermal method show a higher power capability of 202.6 F g−1. Because of unique structure of diatom surface, the

52

Diatoms: Fundamentals and Applications

200 nm

3 m

(a)

300 nm

(c)

(b)

300 nm

(d)

Figure 4.4 SEM images of MnO2-modified diatomite composite used as electrode for supercapacitors. (Zhang et al. 2014)

MnO2-modified nanosheets are observed to grow vertically on the purified diatomite increasing the specific surface area of electrode, thus building hierarchical architecture. Besides, the MnO2 nanostructures after etching the diatomite with a higher power capability (297.8 F g−1) and good cycle stability (95.92% retention after 5000 cycles). Subsequently, they presented that the Melosira-type MnO2 pattern exhibited the specific capacitance of 371.2 F g−1 at a scan rate of 0.5 A g−1 and good cycle stability (93.1% capacitance retention after 2000 cycles at a scan rate of 5 A g−1 (Figure 4.5). (Li et al. 2015) Based on the these results, the hierarchical and porous MnO2-modified diatomite composite is shown clearly to be a promising active material for low-cost, environmentally friendly, and electrochemically stable supercapacitors. Furthermore, combining MnO2-modified diatoms with other materials was shown that is possible to improve the performance of supercapacitors. For instance, hollow diatom silica structures, TiO2 nanospheres and MnO2 mesoporous nanosheets were synthesized and explored for application in high-performance supercapacitors. The concept is presented in Figure 4.6. (Guo et al. 2016) The mixture demonstrated a high power capacitance of 425 F g−1 at a scan rate of 0.2 A g−1 and a long cyclic stability (retain 94.1% after 2000 cycling). On account that abundant interfaces and openpore channels provided with TiO2 nanospheres and layer on diatom structures the electronic transport of MnO2 nanosheets is increasing (Figure  4.7). Additionally, a unique combination of MnO2-nanostructures, grapheme oxide nanosheets (GO) and porous diatomaceous earth (DE) microparticles showed a larger specific capacitance of 152.5 F g−1 at 160 °C and a relatively better cycle stability (83.3% capacitance retention after 2000 cycles at a scan rate of 2 A g−1). (Wen et al. 2016) These studies indicated that unique diatom structures mixture have great prospects to be an active material for supercapacitors which are summarised in Table 4.2. Nickel oxide (NiO) is well studied for supercapacitors because of high specific capacitance, high chemical/thermal stability, ready availability, environmentally

Current Diatom Research in China 20

Coscinodiscus Melosira Navicula

15

0.8

5 0 –5 –10 –15 –20 0.0

0.2 0.4 0.6 Potential (V) vs. SCE

0.4 0.2 0.0 –0.2

0.8

–1 Specific capacitance (F g )

15

–Z” (Ohm)

90 –Z” (Ohm)

0

200

400 600 Time (S)

800

1000

280

120

60

Coscinodiscus Melosira Navicula

10

5

30 0

0

Coscinodiscus Melosira Navicula

0.6

Potential (V) vs. SCE

Current (A g–1)

10

–0.2

53

0

5

10

15

30

60

90

200 160

–120

Z” (Ohm) 0

Coscinodiscus Melosira Navicula

240

120

0

1

2

3

4

5

Current density (A g–1)

Z’ (Ohm)

120 0.8

0.8

0.6

0.6

0.6

0.4 0.2 0.0

–0.2

0

0

100

200

300

400

500

600

Potential (V) vs. SCE

40

0.8 Potential (V) vs. SCE

80

Potential (V) vs. SCE

Specific capacitance (F g–1)

160

0.4 0.2 0.0

–0.2

400

0.2 0.0

–0.2 0

100

Time (S)

0

0.4

200

300 400 Time (S)

500

800

600

0

100

1200

200

1600

300 Time (S)

400

500

2000

Cycle numbers

Figure 4.5 The electrochemical performance of the diatom/MnO2 core-shell structure electrodes measured in 1 M Na2SO4 solution. (Li et al. 2015)

Air SiO2 TiO2 MnO2 TiF4

Diatom

KMnO4

Diatom@TiO2

Diatom@TiO2@MnO2

Figure 4.6 Schematic diagram showing the process of synthesis of diatom@TiO2, diatom@TiO2@MnO2 3-D composite supercapacitors with diatom’s morphology. Cross section of pore structure is presented to show coating internal and external diatom surface with TiO2 and MnO2 nanocomposites. (Guo et al. 2016)

54

Diatoms: Fundamentals and Applications

6 m

600 nm

(a)

(b)

3 m

2 m

(c)

(d)

1 m

200 nm

(e)

(f)

Figure 4.7 (a and b) SEM images of purified diatomite, (c and d) NiO-modified diatomite composites and (e and f) the pores in the centre of diatomite shell. (Zhang et al. 2014)

Table 4.2 Comparison of the electrochemical properties of MnO2 diatom based supercapacitors.

Materials

%/cycles numbers

Purified diatom

Csp (F g−1)

Conditions

8

Reference Zhang et al. 2014

93/1000

371.2

0.5 A g−1

Li et al. 2015

NiO/diatom

90.61/1000

218.7

0.25 A g−1

Zhang et al. 2014

MnO2/diatom

95.92/5000

202.6

0.25 A g−1

Zhang et al. 2014

MnO2(etch diatom)

90.48/5000

297.8

0.25 A g−1

Zhang et al. 2014

diatom@TiO2@MnO2

94.1/2000

425

0.2 A g−1

Guo et al. 2016

DE@MnO2

83.3/2000

152.5

2 A g−1

Wen et al. 2016

MnO2/diatom

Current Diatom Research in China MnO2 nanosheets MnO2 nanorods

Mg-thermic reduction

Diatom

Si-diatom

Outside

Outside

Inside

Inside

Hydrothermal reaction

55

Calcination

Si-diatom@MnO2 Si-diatom@MnO2 nanosheets nanorods

Figure 4.8 Schematic illustration of the synthesis of Si-diatom@MnO2 material for supercapacitors application. (Zhang’s group)

benign nature and lower cost. (Hu, Qu, Chen, & Li 2013; Mai, Tu, Xia, Gu, & Wang 2011) However, a large portion of bulk NiO suffers from low ionic diffusion constant and structural susceptibility which limit their applications. (Wu, Hu, & Liu 2013; Marcinauskas, Kavaliauskas, Valincius 2012) To improve the specific capacitance of supercapacitors with NiO nanowires, the hierarchically porous NiO moss-decorated diatomite was prepared. The high magnification images show that hundreds of large pores are regularly arrayed on one side of the frustules and there are scarcely any discrete impurities in the pores. It was found that unique NiO-modified diatomite structures exhibit a specific capacitance of 218.7 F g−1 and excellent cycling stability (90.61% retention after 1000 cycles). (Zhang et al. 2014) Based on these electrochemical results it was concluded that hierarchical structure has influence for electrolyte diffusion on improvement of electrochemical performances making NiO-modified diatomite as an attractive electrode for high-performance supercapacitors. Previous studies using MnO2 and NiO modified diatoms for electrode applications indicates an importance of controlling morphology of these oxides on diatom surface that have significant impact on performance. Results showed that metal oxides/ hydroxides decorated on diatom surface with precise control of morphology and component have a high power density, faster charging/discharging rate, sustainable cycling life, excellent cycle stability. In principle, such unique nanostructures may solve the aggregation and volume expansion problem of electrode materials during long-term cycles, which is beneficial for the structural stability of the nanostructure. Further improvement in development of Diatom based supercapacitors was demonstrated by YuXin and Losic team based on diatom silicon morphology made by the conversion of diatom silica into silicon using the magnesiothermic reduction followed growth of MnO2 nanostructured layer using hydrothermal process. The concept of fabrication of these a new 3D Si-diatom@MnO2 electrode for high performance supercapacitor applications is presented in Figure 4.8. (Zhang’s group) The performances of Si-diatom@MnO2 nanosheets showed the best electrochemical performance, which delivered a high specific capacitance of 341.5 F g−1 at a current density of 0.5 A g−1, good rate capability (47.7% retention with current increases around 20 times) together with a steady cycle property (84.8% remained after 2000 cycles). An asymmetric supercapacitor based on the Si-diatom@MnO2 nanosheets as the positive electrode and AGO as the negative electrode delivered a maximum power density of

56

Diatoms: Fundamentals and Applications

2.22 kW kg−1 and energy density of 23.2 Wh kg−1. These outstanding electrochemical properties can be attributed to a good electrical conductivity of silicon diatom and its unique nanostructure, which enlarged the surface area and increased the active sites exposed in the electrolyte. Considering that silicon diatoms can be obtained from inexpensive and available natural resources using scalable conversion process, these results suggest that Si-diatom@MnO2 electrodes showed great potential to be used as lowcost and high-performance electrode material for supercapacitors. The combination with other composites is expected to further improve these performances and possible translate these electrodes into real practical applications.

4.1.5 Diatoms for Solar Cells Solar cells are classified by silicon solar cells, dye-sensitized nanocrystal solar cells, plastic solar cells, organic solar cells and so on. (Baxter et al. 2009; Bruchez & Hotz 2007; Hernandez-Alonso, Fresno, Suarez, & Coronado 2009; Calzaferri 2010; Chen & Mao 2007; Inoue 2009; Goncalves, Bermudez, Ribeiro, & Mendes 2008) Among them, the silicon solar cells with the unique tunable optical and electronic properties become the most dominant in the solar energy application. Silicon semiconductors are not good conductors for electricity leading to the large resistance and simultaneously with a mass loss. It is not employed in large scale since high cost and immature technology. In 1991, Grätzel published a remarkable report that present a novel type of solar cell known as a dye-sensitized solar cell (DSSC) and has a 7% efficiency based on nanocrystalline TiO2, subsequently improving the efficiency to 10%. (Nazeeruddin et al. 1993; Oregan & Gratzel 1991) Since then, DSSCs were attracted considerable interests on researchers on account that they provided the possibility of enhancing efficient solar energy conversion and low cost. Metal oxide materials like TiO2 (Chen & Mao 2007), ZnO (Zhang, Dandeneau, Zhou, & Cao 2009), SnO2 (Duong, Choi, He, Le, & Yoon 2013), and Nb2O5 (Barea et al. 2011) are applied in DSSCs. Among them, TiO2 is crucial due to its attractive optical, electrical and biological properties, which can yield optimal performance and be activated by UV light. However, a thin film of TiO2 lacks the high surface area that nanoparticles provide. (Losic et al. 2006) However, some of synthetic processing involves complex deposition systems or chemically toxic elements. It is essential to develop the biological materials with environmental friendliness and low toxicity. Furthermore, the hybrid functional nanostructured materials have contributed to enhancing efficiency and reducing production cost. (Cerneaux et al. 2007; Oregan & Gratzel 1991; Grätzel 2003) The specific surface area of diatoms can promote solar cell efficiency due to the large number of dye molecules adsorbed onto the nanostructured surfaces. (Pan et al. 2004; Schlapbach & Zuttel 2001; Toster et al. 2013) Diatom has a refractive index of 1.43 (Fuhrmann, Landwehr, El Rharbi-Kucki, & Sumper, 2004) while porous TiO2 thin films have refractive indexes ranging from 1.7 to 2.5 (Tachibana et al. 2007), which is giving TiO2-diatom layers high dielectric layer contrast and light scattering in the pore array. That is, TiO2-diatom compound DSSCs have improved the efficiency of the DSSC. (Huang et al., 2015) The application of diatoms in solar cell did not have a long history. Chandrasekaran et al. (Chandrasekaran et  al. 2014) showed for the first time in 2014 that the semiconducting and high surface area 3D silicon replicas prepared from diatom sustain

Current Diatom Research in China

57

Si

SiO2 Mg-thermic reduction

SH

Diatom frustule

SH

SH SH SH SH SH SH SH SH

Photocurrent density/A cm2

Potentiostat

RE

Time/sec

CE Electrochemical set-up

Au Au-Si diatoms electrode

Figure 4.9 Schematic representation of magnesiothermic conversion of diatom frustules to nanostructured silicon and their application for photoelectrochemical energy conversion. In the second step, thiol-modified silicon diatom frustules are attached to a gold electrode surface and used in a threeelectrode cell set up to perform photocurrent measurements. (Chandrasekaran et al. 2004)

photocurrents and enable solar energy conversion. After that, many scientists started their research in this area, including researchers in China. Huang et al. prepared a TiO2-diatom paste mixture though spin-coating and hightemperature sintering techniques. They demonstrated that when the DSSCs were coated with three layers, that was one layer of diatom paste and two layers of TiO2 paste, yielded an optimal power conversion efficiency of 5.26%. That had a 38% increase in efficiency of DSSCs coated with three layers of TiO2 paste. Furthermore, a high-speed centrifugal processing technology and sedimentation-rate separation technique were creatively applied to obtain the diatom frustules. In consequence, the results demonstrated that mixing diatom frustules with the TiO2 paste can enhance light-trapping effect and optical scattering properties and increase the dye loading and surface area of working electrodes due to micro-scale size and nano-sized pore structures of the diatom (Figure 4.10). Chen et al. also made a theoretical and experimental study to understand the nanophotonic light-trapping surcutre of diatom frustule for enhanced solar energy. He found that the enhanced absorption efficiency occurs within the 400–500 nm and 650–700 nm region and diatom can effectively manipulate the incoming light by coupling into the localized waveguiding modes, which would enhance the solar energy conversion efficiency in diatiom. In previous studies, diatoms were not only applied in DSSCs, but also in other types of solar cells. Chen et al. presented the effects of a chlorophyll extract from diatom algae with better thermal stability as a spin-coating anti-reflection layer on surface textured silicon solar cells. They found that depositing a thin diatom extract layer slightly can reduce the reflection by up to 13% over spectral regions of 350–1100 nm. However, application of diatoms in solar cells is a promising orientation due to its low cost, natural abundance and

58

Diatoms: Fundamentals and Applications Light

FTO substrate TiO2+diatom

(a)

2 m

(b)

2 m

(c)

Figure 4.10 (a) The schematic illustration of TiO2-diatom working electrode. (b) Diatoms and (c) diatom frustules observed under an optical microscope. (Huang et al. 2015)

environmentally benign nature, we still need to enhance conversion efficiency of diatom solar cells.

4.1.6

Diatoms for Hydrogen Storage

Hydrogen energy has been proposed as an alternative fuel for the clean and highly efficient energy production. However, hydrogen storage is particularly challenging as establishing a safe and efficient system which is difficult at room temperature and atmospheric pressure. Natural minerals as hydrogen storage materials such as metal–organic framework-5 (MOF-5) of composition Zn4O(BDC)3 (Rosi et  al. 2003), microporous metal coordination materials (MMOMs), single-walled carbon nanotubes (SWNT) (Pan et al. 2004), silicate nanotubes, magnesium hydride (MgH2) have been extensively explored. (Schlapbach & Zuttel 2001; Mu, Mu, & Yuan 2005) The high dissociation temperature, slow hydriding-dehydriding kinetics and reactivity to oxidation limit the use some of these materials for hydrogen-related applications. Surprisingly, the high porosity with large surface area, small particle size, strong adsorbability and excellent thermal stability actuate diatomite a perfect candidate for hydrogen storage. (Karatepe, Erdogan, Ersoy-Mericboyu, & Kucukbayrak 2004) In another study, Jin et al. (Jin, Zheng, & Yang 2014) found that hydrogen adsorption capacity of pristine diatomite is 0.463 wt% at 2.63 MPa and 298K, which is the highest of the known sorbents. The hydrogen adsorption capacity of activated diatomite with acid-thermal could reach up to 0.833 wt% because of the appropriate pore properties by activation. Results confirmed important funding that the hydrogen adsorption capacity strongly depends on the natural pore characteristics of diatomite. To do the further research, they create an effective metal-modified method which has been developed to disperse Pd and Pt nanoparticles on diatomite. By incorporation

Current Diatom Research in China

59

with 0.5 wt% of Pt and Pd, hydrogen adsorption capacities are enhanced to 0.696 wt% and 0.980 wt%, respectively. Hence, diatomite mineral with large surface area, appropriate pore volume and small pore size is the promising physisorption-based material for hydrogen storage at room temperature. The influence on the hydrogen desorption behavior under the diatomite-specific porous microstructure opens window to further improve performance of these devices.

4.1.7

Diatoms for Thermal Energy Storage

Thermal energy storage (TES) as an intermediate step to versatile, clean and efficient use of energy, has received more and more worldwide attention and increasing research interest. (Dincer 2002; Khudhair & Farid 2004; Tyagi, Kaushik, Tyagi, & Akiyama, 2011; Liu 2010) Thus, in order to obtain a comfortable living environment, it is needed to control the indoor environment temperature change using the air conditioning system, which also leads to a lot of energy consumption. Among various energy storage methods, thermal energy storage is recognized as one of the key technologies is an effective method for adjusting the time difference between power supply and demand in the future. There are three types of TES methods: sensible heat storage, latent heat storage, and reversible chemical reaction heat storage. (Zhou, Zhao, & Tian, 2012; Regin, Solanki, & Saini, 2008) Among the TES methods, latent heat energy storage realized using a phase change material (PCM) is the most effective technique due to its obvious advantages of high energy storage density and narrow temperature variation during the thermal energy charge/discharge process. (Memon 2014; Regin et al. 2008; Zhou et al. 2012) There are mainly two kinds of PCMs, inorganic PCMs and organic PCMs. The inorganic PCMs refer to inorganic salt hydrates based on the latent heat storage during dehydration and hydration, which have a high energy storage density and a high thermal conductivity. (Dincer 2002; Khudhair et al. 2004; Liu 2010; Memon 2014; Regin et al. 2008; Sarier & Onder 2012; Tyagi et al. 2011; Zhou et al. 2012) However, they also have several drawbacks which limit their application, such as leakage problem of PCM during solid-liquid change process. (Sarier & Onder 2012; Zhang, Shi, Wang, Fang, & Liu 2013) To overcome this problem, shape stabilization supports are introduced to fabricate form-stable composite PCM. (Kenisarin & Kenisarina 2012) Stabilization supports of PCM usually include microencapsulation containers, polymer microencapsulation shells, and porous materials (Nomura, Okinaka, & Akiyama, 2009), such as expanded graphite (Zhang et al. 2013; Lafdi, Mesalhy, & Elyafy 2008), fatty acid esters (Sari & Bicer 2012), paraffin bentonite (Li, Wu, Kao, & Tan 2011), granular (Zhang, Zhou, Wu, & Li 2005), perlite (Jiao, Ji, & Fang 2012), gypsum (Li, Wu, & Chen 2011), diatomite (Li, Kao, Wu, & Tan 2011; Karaman, Karaipekli, Sari, & Bicer 2011), vermiculite (Karaipekli & Sari 2009; Karaipekli & Sari 2010), attapulgite (Li, Wu, & Kao, 2011) and clay minerals (Dong, Li, Zhou, & Wu 2004). Remarkably, porous materials can absorb or release water vapor when the relative humidity in the living environment changes. So the porous materials can regulate the relative humidity of the indoor environment, which makes people feel comfortable and reduces energy consumption. (Chen, Su, Qin, & Fang 2015) Considering

60

Diatoms: Fundamentals and Applications

Table 4.3 Melting point and latent heat of PCMs and PCM/diatomite composites. Melting point (°C)

Latent heat (J/g)

References

Paraffin/calcined diatomite

33.04

89.54

156

paraffin

27.47

201.50

PCM-DP600-CNTs

27.12

89.40

Capric acid

31.5

155.5

Lauric acid

44

175.8

Palmitic acid

63

212.1

Stearic acid

69.6

222.2

Paraffin/diatomite

41.11

70.51

GHM

45.98

172.80

GHL

40.21

157.61

GHM/diatomite

45.86

96.21

GHL/diatomite

39.03

63.08

PEG (50 wt%)/diatomite

27.7

87.09

hexadecane/diatomite

23.68

120.1

graphite nanoplatelets(xGnP)

22.09

120.8

Octadecane/diatomite

31.29

116.8

Octadecane/diatomite/xGnP

30.20

126.1

PCM samples

158

160

161

143

149

hexadecane/diatomite/exfoliated 162

this, diatomite is a feasible candidate for an economical and light-weight material for incorporating PCM for thermal energy storage. (Jeong, Jeon, Lee, & Kim 2013) Over the past two decades, the incorporation of PCMs with diatoms has been investigated as potential technology for minimizing energy consumptions. SEM morphologies of paraffin/diatomite composite PCMs (2000×). (Sun, Zhang, Zheng, Park, & Frost 2013) Xu et al. (Xu & Li 2014) reported a paraffin/diatomite composite phase change material featuring high tensile ductility and heat storage capacity, which was used as fine aggregate in the production of novel thermal energy storage engineered cementitious composites (Table 4.3). Their teams also fabricated new paraffin/diatomite/multi-wall carbon nanotubes composite PCM with a melting temperature of 27.12 °C and latent

Current Diatom Research in China

61

heat of 89.40 J/g. Moreover, this composite PCM shows good chemical compatibility and thermal stability. (Xu & Li 2014) Moreover, Li et al. (Li, Sanjayan, & Wilson 2014) showed semblable phase change material composites compounded diatomite with paraffin. Karaman et al. (Karaman et al. 2011) prepared a kind of polyethylene glycol (PEG)/diatomite composite as a novel form-stable phase change material for thermal energy storage. The results showed that the melting temperature and latent heat of the composite PCM were 27.70 °C and 87.09 J/g. The thermal conductivity of compound material was improved by adding expanded graphite. Li et al. (Li, Wu, & Kao 2011) prepared several kinds of binary fatty acid/diatomite shape-stabilized phase change materials using the fusion adsorption method. The results indicated that the latent heat of capric-lauric acid/diatomite decreased to 57% of that of capric-lauric acid PCMs, and the phase-transition temperature rises lightly from 16.36 °C to 16.74 °C. In summary, modified diatomite composite phase change materials have the following remarkable features: large apparent specific heat for the phase-transition temperature region, suitable thermal conductivity, keeping shape-stabilized in the phase change process and no need for containers.

4.2

Diatoms for Water Treatment

Diatomite (diatomaceous earth), a natural biogenetic mineral, is mainly composed of amorphous silica (belonging to opal-A in mineralogy). Diatomite processes the developed porosity, especially macropores, and rich surface silanols (Si-OHs), showing promising in utilization of adsorption and support. Understanding surface properties of diatomite is essential to improve its application as the functional materials. Researches on surface hydroxyl species of diatomite and on their transformation under thermal treatment conditions are investigated by combining 1H MAS NMR and Fourier transform infrared spectrometer (FTIR) methods. The results showed that surface hydroxyl groups of diatomite include isolated hydroxyl groups and hydrogen-bonded hydroxyl groups, and the hydroxyl groups originated from water adsorbed on diatomite surface. The adsorbed water are desorbed after thermal treatment and the signals of protons of isolated hydroxyl groups and hydrogenbonded hydroxyl groups in NMR and FTIR curves increase and reach their maxima at 1000 °C. After 1100 °C calcination, the hydroxyl groups that classified to strongly hydrogen-bonded ones and the isolated hydroxyl groups condense basically. But some weakly hydrogen-bonded hydroxyl groups may still persist in the micropores. Moreover, on the basis of the confirmation of surface and structural properties of diatomite, the direct applications and the modification to improve the adsorption and loading capacity of guest molecules/ions are focused on.

4.2.1 Support for Preparation of Diatomite-Based Adsorption Composites Diatomite-supported/unsupported magnetite nanoparticles were prepared by co-precipitation (sample denote MagDt-P) and hydrosol methods (MagDt-H). The average sizes of the unsupported and supported magnetite nanoparticles are around 25 and

62

Diatoms: Fundamentals and Applications

Si

H O H

Si

H O H

Si

H

O

O H

Si

O Si

O

O (c)

(b)

Si

V

H

–H2O

Si

Si O

H

H O

Si

Si Si

–H2O O

Si

–H2O III

H

II

H

(a) H

Si H

IV

O

O H

O

Si H

–H2O

O

Si

I

H

H

Si O

Si

O

Si

Figure 4.11 Schematic representation of the hydroxyl structure and the dehydration process of diatomaceous silica.

Magnetite particles

Magnification

100 nm

(a)

(b)

70 nm

1000 nm

(c)

(d)

1000 nm

100 nm

(e)

(f)

(g)

(h)

800 nm

600 nm

600 nm

20 nm

Figure 4.12 TEM images of (a) Magnetite nanoparticles; (b) Diatomite; (c and d) two diatom shells in MagDt-P; (e and f) two diatom shells in MagDt-H; (g and h) sample MagDt-H with high resolution.

15 nm, respectively. The supported magnetite nanoparticles exist on the surface or inside the pores of diatom shells, with better dispersing and less coaggregation than the unsupported ones. The uptake of hexavalent chromium Cr(VI) on the synthesized magnetite nanoparticles was mainly governed by a physico-chemical process, which included an electrostatic attraction followed by a redox process in which Cr(VI) was reduced into trivalent chromium Cr(III). The adsorption of Cr(VI) was highly pH dependent and the kinetics of the adsorption followed a pseudo-second-order model. The adsorption data of diatomite-supported/unsupported magnetite fit well with the Langmuir isotherm equation.

MicroMag

40 20 0

(a) Removal efficiency (%)

Cr uptake (mg/g Fe3O4)

80 60

1

2

3

4

5 6 pH value

(b)

7

8

9

Mag MicroMag MagDt-H

50 20

MagDt-H Mag MicroMag 0

20

40

60 80 t (min)

40

60 80 t (min)

100

120

100

120

Mag MicroMag

20 10

MagDt-H 0

(d) 0

10 5 0

(c)

Dt

100

0

63

Mag

100

Qe (mg/g)

Removal efficiency (%)

Current Diatom Research in China

0

20

40

60

80 100 120 140 Ce (mg/L)

Figure 4.13 (a) Effect of initial solution pH values on the removal of Cr(VI) on Mag, MicroMag and Dt; (b) effect of agitation time on the Cr(VI) removal efficiency of Mag, MicroMag and MagDt-H; (c) effect of agitation time on the normalized Cr uptake to actual Fe3O4 content; and (d) the Cr(VI) adsorption isotherms of Mag, MicroMag and MagDt-H.

The diatomite-supported magnetite synthesized via hydrosol method exhibited a higher adsorption capacity per unit mass of magnetite (qe–s, 11.4 mg/g), calculated from the pseudosecond-order kinetics equation, than the unsupported nanoscaled magnetite (10.6 mg/g) and the micron-scaled magnetite (5.3 mg/g). The monolayer adsorption capacities per unit mass (g) of magnetite content (Qm–s) of these materials, obtained from the simulation with the Langmuir equation, followed a similar order of diatomite-supported nanoscaled magnetite (69.2 mg/g)>unsupported nano-scaled magnetite (21.7 mg/g)>micron-scaled magnetite (14.6 mg/g). These results indicate that diatomite support is useful on improving the Cr(VI) adsorption capacity. Furthermore, supporting of diatomite is of help for the storage and pelletization of the magnetite nanoparticles. These fundamental results demonstrate that the diatomite-supported/unsupported magnetite nanoparticles are readily prepared, enabling promising applications for the removal of Cr(VI) from aqueous solution.

4.2.2 Catalyst and Template for Preparation of Porous Carbon Materials Diatomite was used as a template to synthetize macroporous carbons. However, in the preparation of diatomite-based porous carbon, sulfuric acid was always used as the catalyst to catalyze the carbon precursor. The addition of sulfuric acid is potentially harmful to the environment and also increases the cost of the preparation. In our previous study, we have found that diatomite itself contains solid acid sites: the Brønsted acid sites are sourced from some H-bonded silanols, and the Lewis acid sites result from very small clay mineral particles strongly adhered to the diatom shells. Therefore, these inherent acid sites of diatomite (sourced from genus Coscinodiscus Ehrenberg) instead of the additive acid were utilized to catalyze the carbon precursor, and the key effects of the solid acidity on the final structure of porous carbon were investigated.

64

Diatoms: Fundamentals and Applications

10 m

(a)

100 nm

(e)

1 m

(b)

200 nm

(f)

1 m

(c)

50 nm

(g)

100 nm

(d)

5 nm

(h)

Figure 4.14 Scanning electron microscopy (SEM) images of (a) diatom shell; (b) edge macropores of the diatom shell; (c) cross-sectional view of carbon pillars; (d) ordered macropores of C/DtCE. Transmission electron microscopy (TEM) images of (e) ordered macropores of C/DtCE; (f) carbon pillar (inset: selected area electron diffraction (SAED) pattern); (g) mesopores in the carbon pillar; (h) micropores of the carbon wall.

The results shows that the solid acidity makes raw/activated diatomite a catalyst for the generation of porous carbon (the obtained carbon denoted as C/DtCE), and the porous parameters of the carbon products are strongly dependent on the solid acidity of diatomite templates. The morphology of diatomite also dramatically affects the textural structure of porous carbon. Two types of macroporous structures in the carbon product, the partially solid pillars and the ordered hollow tubes, derive from the replication of the central and the edge pores of diatom shell, respectively (Figure 4.14). The mesopores and size-uniform wormhole-like micropores resulted from the stacking of the graphite microcrystals and the structure-reconfiguration of the carbon film during the removal of templates, respectively. The acidity of diatomite dramatically affected the porosity of the carbons. More acid sites of diatomite template resulted in higher surface area and pore volume of the carbon products, showing a positive correlation between the acidity of the diatomite template and the pore parameters of diatomitetemplated carbons. The hierarchically porous carbon shows high specific surface area (up to 426 m2/g) and pore volume (up to 0.195 cm3/g) and good capability for the adsorption of solvent naphtha and H2, enabling potential applications in adsorption and gas storage. Diatomites sourced from different diatom genus show different morphology and porosity, resulting in various structures and adsorption capacity for methylene blue (MB) of the obtained diatomite-templated carbons. The end-closed carbon tubes were derived from the replication of diatom shell of genus Synedra Ehrenberg (the obtained carbon denoted as C/DtSE, (Figure 4.15). The Langmuir monolayer adsorption capacityfor three adsorbents follow an order of C/DtSE (333 mg/g)>C/DtCE = commercial activated carbon (CAC, 250 mg/ g). The diatomite-templated carbons possess higher

Current Diatom Research in China Furfurly alcohol Polymerization

65

Carboniziation

Central pores

Diatom+silica

Edge pores

Polymer+silica

Carbon+silica Partially solid pore

Furfurly alcohol Polymerization

Carboniziation Hollow pore

Figure 4.15 Schematic representation of the possible formation mechanism of the hierarchically porous carbon (C/DtCE). 250

CAC C/DtCE C/DtSE

qe (mg/g)

200

Carbon tubes

150 100

Carbon tubes Carbon pillers

C/DtSE

50 100 nm

0 0

250

500

1 m

750

1000

C/DtCE 1250

1500

Ce (mg/L)

Figure 4.16 MB adsorption isotherms of diatomite-templated carbons.

MB adsorption capacity than commercial activated carbon, showing promising applications in the fields of adsorption and purification. However, it is must be pointed that etching of the diatomite template is of the potential threats to environment by using HF as the etchant. Therefore, KOH was selected in our study to etch the diatomite template and we found that KOH also acted as the activation agent to enhance the porosity of the diatomite-templated carbon. The separated carbon products were loaded by KOH on its surface and the porosity of the carbons improved after once more carbonization in the tubular oven under N2 atmosphere. KOH-activated diatomite-templated carbons possessed the higher specific surface area (988 m2/g), total pore volume (0.675 cm3/g) and the MB adsorption capacity (645.2 mg/g) than those of the original carbons and CO2-activated carbons. Moreover, zeolite K-H (Z/Dt) byproduct was prepared by the silicon- and potassium-containing solutions derived from the KOH-etched diatomite template served as the silicon and potassium sources. The zeolite products possessed a stick-like morphology and nanosized

66

Diatoms: Fundamentals and Applications

Z/Dt

10 (a)

20

30

40

50

60

70

80

2 (°)

10 nm

100 nm (b)

50 nm (c)

Figure 4.17 (a) XRD pattern, (b) SEM image and (c) TEM image (the inset: porous morphology) of zeolite K-H.

particles with a mesopore-predominant porous structure. Therefore, diatomite is here used not only the catalyst and template but also the silicon source to prepare the carbon materials and zeolite, providing a facile economically feasible method for recycling and further utilization of the waste solution.

4.2.3

Modification of Surface and Porous Structure

Naturally occurring diatomite with a mesoporous/macroporous structure has been used for the adsorption of metal ions or organic molecules in aqueous solution. However, diatomite exhibited an ordinary adsorption capacity for these molecules or ions that was close to or even lower than those of natural zeolite or clay minerals, such as kaolinite and montmorillonite. This is due to the fact that the surface silanols (i.e., Si–OH) of diatom frustules do not possess a strong adsorption affinity for the aforementioned adsorbates, and these adsorbates are too small to be filtered by the micron- or nanometer-sized channel of diatomite; moreover, the ionic exchange capacity of diatomite is much smaller than that of zeolite or montmorillonite.

Current Diatom Research in China Si

R

Hydrogen bond

X CH3CH2 or H R (CH2)3NH2

67

Si Silicon of diatomite Si Silicon of APTES

R Si O O Si Si O Si O OX Oligomerized APTES R Si O Si O O Directly grafted APTES Si H O O O R R R OX R O R XO Si O Si Si O Si O Si O Si Si O OX O O R R R R R R Si H O R O OX O Si R R Si H O Si H Si O Si H H O O O O O O O O O O O Si Si Si Si Si Si Si Si Si Si H

O

O

Diatomite surface (a)

Diatomite surface (b)

Figure 4.18 Schematic presentation for the related mechanisms of the APTES modification: (a) oligomerization-dominant silylation; (b) grafting-dominant silylation.

Surface Silylation To increase the adsorption capacity of ions, silylation is used to modify the surface properties of diatomite.

1) Surface Silylation for Adsorption of Cu(II) Ion Organosilane with metal-affinitive groups has been applied to several types of synthetic silicas to improve their heavy-metal adsorption, which is proven to be an efficient way to modify diatomite surfaces. The covalent bonding between the organic components and the diatom frustules enables a durable immobilization of the organic moieties on the silylated products, and prevents their leaching into the surrounding solutions. By introducing special functional groups in the organosilane agents used, the adsorption selectivity of the resultant materials to contaminants could be significantly improved. γ-aminopropyltriethoxysilane (APTES) is selected for surface silylation of diatomite and the derivatives show high adsorption capacity for Cu(II). The hydrolyzed APTES is oligomerized and then attached to the diatomite surface by hydrogen bonding or Van der Waals interaction, resulting in a cross-linked structure containing a high content of APTES species. Heating at a high temperature (800 °C) leads to the removal of physically adsorbed water and the hydrogen-bonded capping water, exposing the surface isolated silanols of diatomite (Figure 4.18). These silanols act as the substrates for the grafting of the APTES molecules. In grafting dominant structures, a relatively thin APTES layer is formed, and the grafted APTES exhibits a very high thermal stability. The decomposition temperature of grafted APTES is approximately 540 °C. The Cu(II) adsorption on APTES-modified diatomite is 13 times greater than that on unmodified diatomite because of the high affinity of the amino groups for Cu ions. The grafting-dominant modified diatomite has a dramatically higher efficiency for Cu(II) adsorption than the oligomerization-dominant diatomite. The main reason for

Diatoms: Fundamentals and Applications Dt-800/M

100

Qe/(mg/g)

18

Dt-800/M

500 0 0

Dt 0

400

800

0 0

240

(a)

(c)

Dt

t/qt

Dt-M

50

Dt-M

36

1000

t/(min)

480

720

240

480 t/(min)

50

Dt

100

10 0

0 0

0 0

50

150 100 Ce/(mg/L)

200

(d)

0

50

100 150 Ce/(mg/L)

Dt-M

4 2

50

Dt

720

6

200

Dt-800/M Dt-M

20

0

(b)

Ce/Qe

Removal efficiency (%)

68

200

Dt-800/M 30

60

90

Ce/(mg/L)

120

Figure 4.19 (a) Effect of agitation time on the Cu(II) removal efficiency of Dt (diatomite), Dt/M (APTES-grafted diatomite) and Dt-800/M (APTES-grafted derivatives with diatomite heated at 800 °C before silylation); (b) linear fitting plots based on pseudo-second-order kinetic model for the adsorption of Cu(II); (c) the Cu(II) adsorption isotherms of Dt, Dt/M and Dt-800/M; (d) linear fitting plots based on Langmuir isotherm model for the adsorption of Cu(II).

this greater efficiency is that the nitrogen atoms in the cross-linked network formed by oligomerized APTES are strongly affected by hydrogen bonding, which weakens their coordination with the copper species.

2) Surface Silylation for Adsorption of Some Organic Molecules To improve the adsorption of organic molecules, phenyltriethoxysilane (PTES, C6H5Si(OCH2CH3)3) with phenyl functional groups was used to modify diatomite surface. After silylation, a functional group (-C6H5, phenyl) was successfully introduced onto the surface of Diatomite (Dt, and the silylated product denoted as PTES-Dt). PTES-Dt exhibited hydrophobic properties with a water contact angle (WCA) as high as 120°±1°, whereas Dt was superhydrophilic with a WCA of 0. The benzene adsorption data on both Dt and PTES-Dt fit well with the Langmuir isotherm equation. The Langmuir adsorption capacity of benzene on PTES-Dt is 28.1 mg/g, more than 4-fold greater than that on Dt. Moreover, the adsorption kinetics results show that equilibrium was achieved faster for PTES-Dt than for Dt, over the relative pressure range of 0.118–0.157. The excellent benzene adsorption performance of PTES-Dt is attributed to strong p-system interactions between the phenyl groups and the benzene molecules as well as to the macroporosity of the PTES-Dt.

Modification of Porous Structure for Benzene Adsorption To increase the porous structure, micropores/macropores are introduced onto diatomite, for the preparation of composites with hierarchically porous structures for the adsorption capacity of organic molecules.

Current Diatom Research in China

69

Macroporous/mesoporous diatom frustule

Qm (mg/g)

30

O R O R Phenyl groups R Si O Si O Si OX O R R R R R Si OX R Si O Si Si O O Si O Si OX O O O O O O Si O Si Si Si Si Si Si Si Si

5 m

iatomite

20

Silylated d

10

Unmodified diatom

Silylated diatomite H H O O Si Si

ite

0 0

20 40 60 80 100 120 /P (mar) Benzene adsorption isotherms

H O Si

H O Si

H H OO Si

H H H H O O O O Si Si Si Si

Unmodified diatomite

Figure 4.20 Schematic conformation of PTES on the surface of diatomite and the adsorption capacity of benzene of the derivatives.

500 nm 200 nm

(a1)

1 m

(b1)

0 nm 100

1 m

360 10 m

(a)

(a2)

0.5 m

10 m

(b2)

nm

1 m

(b)

Figure 4.21 (a) SEM images of Dt; (b) SEM image of Dt-E.

1) Surface Etching and Microporous Modification A facile NaOH etching method was employed to treat the frustule support, followed by hydrothermal growth of MFI-type zeolite at the surface of frustules previously seeded with nanocrystalline silicalite-1, for the preparation of hierarchically porous diatomite/ MFI-type (Dt-Z) composites. NaOH etching yielded dual effects on optimising the porosity parameters of the Dt/Z composites: (i) more of the support macroporosity was preserved; (ii)substantial microporosity was introduced via a well-distributed coating of zeolite without blocking the macropores in the frustules due to the enlargement of the pores on frustules by etching. As indicated by the SEM images (Figure 4.21b), the disk-shaped morphology of the diatom frustule was well preserved after NaOH etching. And the diameters of the central macropores increased to 400–1000 nm. The morphological changes occurring during the preparation steps of the composites with or without NaOH etching (Dt/Z or Dt-E/Z) were revealed by SEM images in Figure 4.22 respectively. After the seeding process, nano-sized silicalite−1 particles were coated homogeneously on the surface of diatom frustules as well as on the inner wall of central macropores (Figure 4.22a , d). The sizes of the central macropores of both diatomite seeded (Dtseeded) and NaOH etching seeded (Dt-Eseeded) decreased by

Diatoms: Fundamentals and Applications

70

280 nm

1 m

350 nm 2 m

100 nm

(a)

(b)

3 m

(c)

550 nm

250 nm 700 nm (d)

2 m

1 m

(e)

3 m

(f)

002 2 1/ m

2 1/ m

1 m

(a)

20 0

Figure 4.22 (a),(b),(c) SEM images of Dtseeded, Dt/Z2, Dt/Z4 respectively and (d), (e), (f) SEM images of Dt-Eseeded, Dt-E/Z2, Dt-E/Z4 respectively.

1 m

(b)

Figure 4.23 TEM images of (a) Dt-E/Z2 (inset: SAED pattern of the area in the dashed circles); (b) Dt-E/ Z4 (inset: SAED pattern of the area in the dashed circles).

approximately 160 nm due to the nanocrystals coating the surface of the inner pore wall (Figure 4.22a, d). Hydrothermal treatment for 2 days clearly enlarged the MFItype particles and their sizes in the composites (Dt/Z2 and Dt-E/Z2 respectively) grew to approximately 150 nm. Hydrothermal treatment for 4 days further enlarged the zeolite particles. A continuous zeolite film was formed on the surface of the diatom frustules by the inter-growth of zeolite crystals. The macroporosity of the diatoms was completely lost for Dt/Z4 (Figure 4.22c), while both the disk morphology and the macroporosity were preserved for Dt-E/Z4 (Figure 4.22f). The TEM image of Dt-E/Z2 (Figure 4.23a) reveals that the diatom frustule surface was completely and homogeneously covered with nanoparticles and that the macroporous structure of diatomite remained clearly visible. The circular streaking in the SAED pattern (inset of Figure  4.23a) indicates that randomly orientated, crystalline

Current Diatom Research in China

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1.0

0.8

C / C0

0.6 (a) (b) (c) (d) (e) (f) (g)

0.4

0.2

0.0 0

50

100

150

200

t / min

250

Dt Sil-1 Dt-E/Z1 Dt-E/Z4 Dt-Z1 Dt-Z4 Model 300

350

Figure 4.24 Breakthrough curves and mathematic models for (a) Dt; (b) Sil-1; (c) Dt-E/Z1; (d) Dt-E/ Z2; (e) Dt/Z2; and (f) Dt-E/Z4(normalized to 0.2 g solid) at 25 C. Dry nitrogen with benzene vapor (C0 = 1.51 mmol/L, P/P0 = 0.27) was passed through the column at 1.00 mL/min during sorption.

Macro-D Macroporous diffusion Micro-A Microporous adsorption Meso-A Mesoporous adsorption

Micropore

O

m Sur od fa PDDA(+) ific ce at ion

Macropore In situ coating Crystallization

Si

Silicalite-1

Micro-A

Benezene Adsorption

Macro-D Meso-A

Diatom shell (Dt)

PDDA-modified Dt

M-Dt/Sil-1nano composite

Channel effect

Figure 4.25 Schematic presentation for the synthesization and benzene adsorption evaluation process of the diatomite/silicalite−1 composite.

zeolite nanocrystals coated the surface of the diatom frustules. A TEM image of Dt-E/ Z4 shows a dense film (dark regions) of intergrown zeolite particles (Figure 4.23b). The SAED patterns of a crystal (inset of Figure 4.23b) matched the SAED patterns of a previously reported MFI zeolite crystal, further confirming that the MFI-type zeolite was coated on the surface of the diatom frustules. The breakthrough measurement is a direct method designed to clarify the dynamic adsorption performance of VOCs at low concentration. The Dt/Z composites exhibited higher benzene adsorption capacity per unit mass of zeolite and less mass transfer resistance than pure silicalite-1 nanoparticles, evaluated from the breakthrough curves in Figure 4.24.

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350 nm

250 nm 5 m (a1)

Macropores

1 m

(a2)

200 nm

200 nm (c)

Macropores

Macropores Sil-1 (80 nm)

Sil-1

200 nm 500 nm

200 nm

(b1)

(b2)

(c)

Figure 4.26 SEM images of (a1), (a2) Dt; (b1), (b2) M-Dt/Sil-1nano; and (c) Dt/Sil-1nano; and TEM image of (b3) M-Dt/Sil-1nano.

2) Surface Charging and Microporous Modification A novel, hierarchically porous diatomite/silicalite-1 (M-Dt/Sil-1nano) composite with a high benzene removal efficiency was prepared via a facile coating process, as shown in Figure 4.25. Surface charge of the diatomite was modified initially, followed by the in situ synthesization of silicalite-1 nanoparticles (Sil-1nano) on the surface of the pre-modified diatomite support under the mild, low-temperature reflux reaction conditions. As shown by the SEM images in Figure  4.26b1and b2, surface of the obtained M-Dt/Sil-1nano composite after the in-situ coating process was coated with silicalite-1 nanoparticles (80 nm). Macropores of the M-Dt/Sil-1nano composite were clearly visible having pore sizes of 100–200 nm. The transmission electron microscope result in Figure 4.26b3 shows that the macropores of the diatomite support were distinctly in the M-Dt/Sil-1nano composite with a layer of silicalite-1 nanoparticles coated on the inner edge of the pores. The inserted SEAD patterns of a crystal in Figure 4.26b3, matching the crystalline structure previously reported for MFI zeolite, further confirmed the coating of Sil-1nano onto diatomite. The SEM image of the Dt/Sil-1nano composite without the premodification of the diatomite (Figure 4.26c) revealed that only a small amount of Sil-1nano were inhomogeneously coated onto the diatomite, indicating the superiority of the surface charging premodification method. The diatomite/silicalite-1 composite exhibited considerably higher static and dynamic benzene adsorption capacities (94.9 mg/g (Sil-1nano) and 246.0 mg/g (Sil-1nano) respectively) per unit mass of zeolite than did the synthesized Sil-1nano and commercial ZSM-5 (Figure 4.27). The breakthrough curve results in Figure 4.28 indicate that the

Amount adsorbed (mg/g)

Current Diatom Research in China 100 90 80 70 60 50 40 30 20 10 0

73

Sil-1nano

ZSM-5

M-Dt/Sil-1nano

Dt 0

20

40 60 80 100 Absolute pressure (mbar)

120

Figure 4.27 Static benzene adsorption–desorption isotherms of various samples.

1.0

Ct/Co

0.8 0.6 Dt M-Dt/Silnano ZSM-5 Silnano Model of Dt Model of M-Dt/Silnano Model of ZSM-5 Model of Silnano

0.4 0.2 0.0 0

25

50

75 t (min)

100

125

150

Figure 4.28 Breakthrough curves of Dt, M-Dt/Sil-1nano, ZSM-5 and Sil-1nano for benzene adsorption.

composite displayed a lower mass transfer resistance and considerably faster penetration rate than Sil-1nano and commercial ZSM-5, arising from the macroporosity in the composite reducing the resistance to gas penetration.

3) Diatomite-Based Ceramic/silicalite-1 Composite The above-mentioned methods provide effective ways for the improvement benzene adsorption performance of the diatomite. However, in previous studies, the as-synthesized composites were in powder form. And their gas adsorption performance is readily disturbed by fluid resistance, leading to the low mass transfer efficiency. Therefore, a novel composite (Sil-PCS) incorporating advantages from both hierarchically macroporous ceramic supports with unique 3D reticulated structures and a microporous silicalite-1 nanoparticles (Sil-1) coating was synthesized using a facile method. Firstly, the porous ceramic supports with three-dimensional reticulated structures were first prepared using the polymeric sponge method in which diatomite was used as the ceramic framework and polyurethane foam was used as the sacrificial template. This

74

Diatoms: Fundamentals and Applications First order macropore Second order macropore

Micropore O Si

Sil-1 200 m

80 nm

Porous nanocomposite

500 nm 15 m

In situ loading of Sil-1

Diatomite framework

Figure 4.29 Hierarchically porous structures of the diatomite-based ceramic/silicalite−1composite.

100 Amount adsorbed (mg/g)

90

(d)

80 70 60

(c)

50 40 (e)

30 20 10

(b)

0 0

20

80 100 40 60 Absolute pressure (mbar)

(a) 120

Figure 4.30 Benzene adsorption (filled symbols) and desorption (open symbols) isotherms of (a) Dt, (b) PCS, (c) ZSM-5, (d) SilSYN, and (e) Sil-PCS.

process was followed by facile in situ homogeneous coating of Sil-1 on the surface of the ceramic under mild conditions. The hierarchical porosity of the nanocomposites was due to the inherent micropores of Sil-1, the mesopores resulting from the stacking of Sil-1, and the hierarchical macropores of ceramic supports (Figure 4.29). As the in situ silicalite−1 coating process facilitates the stability and dispersity of Sil1 on the modified surface of the ceramic supports, and the hierarchically porous monolithic structure of the composites which is beneficial to the mass transfer efficiency for benzene adsorption, the composites (Sil-PCS) exhibited a much higher benzene adsorption capacity (133.3 mg/g(Sil-1)) compared with that of a commercial micron-sized ZSM-5 product (66.5 mg/g) and a synthesized Sil-1 (SilSYN, 94.7 mg/g) (Figure 4.30). In addition, the dynamic process for benzene adsorption of the composite fit well with the linear driving force (LDF) model, and adsorption–desorption rate

Current Diatom Research in China

75

Rate constant (s−1)

0.03 ZSM-5 SilSYN Sil-PCS 0.02

0.01

0.00 0.0

0.2

0.4 0.6 0.8 Relative pressure (P/P0)

1.0

Figure 4.31 Rate constant variations for benzene adsorption (filled symbols) and desorption (open symbols) with relative pressure on ZSM-5 (red symbols), SilSYN (green symbols) and Sil-PCS (blue symbols).

constants of the composites were 3 and 5 times higher than those of the ZSM-5 and SilSYN, respectively, as evaluated via the IGA instrument (Figure 4.31).

4.2.4

Support for Preparation of Diatomite-Based Metal Oxide Composites

Diatomite-Fe2O3 hybrid material, honeycomb structure with circular pores synthesized as a heterogeneous catalyst for photo-Fenton degradation of organic contaminants by a novel method. The catalytic activities of the diatomite-Fe2O3 catalyst were evaluated by the degradation of organic dye under visible light irradiation (>420 nm) in the presence of hydrogen peroxide. The results showed that the catalyst exhibited excellent catalytic property for 99.14% decoloration and 73.41% TOC removal of Rhodamine B, which could be attributed to the synergetic effects of the adsorptive power of diatomite and the hydroxyl radicals produced by heterogeneous photo-Fenton reactions. In addition, the decoloration efficiency was still higher than 90% after the catalyst being used for 5 cycles. Hence, the simplicity and cost-effectiveness of the hybrid material could be very promising for synthesizing for high-efficiency visible-light photocatalyst in the degradation of organic pollutants. The Pd nanoparticles immobilized on natural diatomite were achieved by a simple procedure. The catalysts are highly active for Heck and Suzuki reactions and can be recovered and reused many times. The catalytic process was also investigated. The synthesis of the diatomite-supported Pd nanoparticles was carried out using a slight modification of the procedure previously reported for the synthesis of silver nanoparticles on silica spheres (Scheme 1). The Pd nanoparticles were formed with a size in the range of 20–100 nm. 3.66 wt % of Pd was found in the diatomite supported Pd nanoparticles catalyst. The diatomite-supported Pd prepared above was first used in Heck coupling reactions, which is a versatile method for carbon-carbon bond formation in organic synthesis.

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Diatoms: Fundamentals and Applications

(a)

(d)

50 nm Fe2O3

(c)

(f)

400 nm

400 nm

(e)

(b)

Figure 4.32 SEM images of diatomite(a,b,c) and diatomite-Fe2O3(d,e,f).

Diatomite

Coated

Diatomite-Fe2O3

vis

Impregnation

·OH Adsorption

Degradation

Rhodamine B

Fe2O3

H2O

CO2

Figure 4.33 The mechanism of RhB photo-Fenton degradation in the diatomite-Fe2O3/H2O2/vis system.

Current Diatom Research in China Diatomite

SnCl2.2H2O

H2PdCI4

CF3COOH

PVP

77

Diatomite-supported Pd

Figure 4.34 Preparation of Diatomite-Supported Palladium Nanoparticles.

(111)

(200) (220)

10

20

500 nm

30

40

50

60

70

80

2 (degree)

Figure 4.35 (left) TEM image of the diatomite-supported Pd nanoparticles (mean particle size: 55 nm). (right) XRD pattern of the diatomite-supported Pd.

47.8 nm 53.4 nm

39.4 nm

43.2 nm

Figure 4.36 SEM image of nanosilver diatomite.

Nanosilver/diatomite nanocomposites were developed with a facile, effective in-situ reduction method. The as-prepared nanosilver/diatomite nanocomposites demonstrated amazing antibacterial properties to gram-positive and gram-negative bacteria. The in-situ generated silver nanoparticles, are found to disperse evenly in the diatomite. It could kill > 99.999% of the bacterial only in 5 min (0.5 g nanosilver diatomite to treat 100 ml of water) with minimum silver ions leaching into the treated water, leading a promising application in water purification industry.

78

Diatoms: Fundamentals and Applications Ag+

Si

O H

Si

O H

Si

+

Ag aq. Ion exchange

O

Diatomite surface

Diatomite surface

H

Si

O Ag+

Si

O Ag+

Si

O

Figure 4.37 Schematic diagram describing the ion exchange process occurred on silanols.

(a)

(b)

(c)

(d)

2 m

Figure 4.38 SEM images of natural diatomite (a) and pillared diatomite prepared by pillaring solution with different concentrations: (b-d) Pillaring solution: 0.1 mol/L; 0.2 mol/L; 0.5 mol/L. Ion exchange

Aging

Na+-diatomite

Intercalated diatomite

Na+

Pillar presursor

Pillared diatomite Pillar

Figure 4.39 Principle of pillaring and main steps in preparation of pillared diatomite.

In order to greatly improve adsorption capacity, the diatomite was pillared by polyhydroxyl-aluminum. The adsorption capacities of Pb2+ and Cd2+ on pillared diatomite increase by 23.79% and 27.36% compared with natural diatomite, respectively. The surface property of pillared diatomite is more favorable for ion adsorption than natural diatomite. The result suggests that diatomite can be modified by pillaring with polyhydroxyl-aluminum to improve its adsorption properties greatly. Flower-, wire-, and sheet-like MnO2-deposited diatomites have been prepared using a hydrothermal method with Mn(Ac)2, KMnO4 and/or MnSO4 as Mn source

Current Diatom Research in China

4 m

(a)

100 nm

(b)

(a)

4 m

(c)

5 m

500 nm

(b)

79

200 nm

(c)

Figure 4.40 SEM images of (a, b) flower-like MnO2/diatomite, (c, d) wire-like MnO2/diatomite, and (e, f) sheet-like MnO2/diatomite.

and diatomite as support. The different shapes of MnO2 were generated due to the use of different preparation conditions (manganese sources, reactants, reaction temperature and reaction time). The diatomite sample possessed a disk-like morphology with a uniform ordered porous structure, with the diameter of each disk about 25 μm. There were a large number of flower-, wire-, and sheet-like MnO2 nanoparticles randomly deposited on the surface of the diatomite. It is shown that the MnO2-deposited diatomite samples with different morphologies possessed high surface areas and abundant surface hydroxyl groups (especially the wire-like MnO2/diatomite sample). The wirelike MnO2/diatomite sample showed the best performance in the removal of Cr(VI), giving the maximum Cr(VI) adsorption capacity of 101 mg/g. A new iron oxide-diatomite system was synthesized in one pot by mixing natural diatomite with a ferrous sulfate solution at room temperature. Iron (29% w/w) was crystallized under goethite phase (α-FeOOH). Analysis conducted by SEM further highlighted the preferential presence of goethite on the surface of diatomite, which involves hydrogen bonding between diatomite surface silanol groups and ion species. The mechanism of formation of goethite is suggested to occur through the precipitation of ferrous ions by hydroxyls ions followed by the oxidation of the iron species to yield first to lepidocrocite (γ-FeOOH) then goethite that is thermodynamically more stable. The remediation properties of Diatom-Fe towards arsenite species cations were investigated in batch jar tests for 24 hr at auto-equilibrium pH. The materials showed a noticeable higher As (III) sorption capacity (16 mg/g) than the native diatomite (0.5 mg/g). These results can be explained by the strong affinity of arsenites to goethite

80

Diatoms: Fundamentals and Applications

Maqn 1523x

1 m

Maqn 5980x

(a)

Maqn 23920x

2 m

(b)

500 nm

Maqn 47841x

1 m

(c)

(d)

Figure 4.41 SEM images of Diatom-Fe at different magnifications.

Contaminants

Adsorption −1 0

GNTD

N-TiO2 CB

1

3

Products O2

2.5 eV N 2p

2 Contaminants

Ti 2p

− 2

e−

VB

Adsorption

0.7 eV O 2p

h+

H 2O

Products

H

Figure 4.42 The mechanism diagram of photocatalytic activity of GNTD.

which is further emphasized by the dispersion of the iron oxide crystals on diatomite surface. Granulated N-doped TiO2/diatomite (GNTD) composite combine nitrogen-doping with particle immobilization for both water treatment and bacteria inactivation. Pretreated diatomite was found to be a good TiO2 supporter, due to its high adsorption capability to enrich target molecules around TiO2 nanoparticles, which improves its photo-oxidation efficiency, and granulation increases its recoverability. N dopant showed its effect of extending the light response region of TiO2 to visible light region. This structure could effectively narrow the band gap of TiO2 and broaden its adsorption edge, thus make it visible-light responsive. The contaminants (including RhB and bacteria) were first adsorbed onto the surface of diatomite, and then degradated by the active species generated by N-TiO2, as illustrated by Figure 4.42. The whole process showed good efficiency under visible light irradiation, thanks to the adsorption ability of pretreated diatomite and the photoactivity of N-TiO2.

Current Diatom Research in China

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70 60

80

50

MnFe2O4/DE-NaOH

MnFe2O4/DE-NaOH

DE-NaOH

Qt/(mg/g)

Qt/(mg/g)

DE-NaOH DE MnFe2O4/DE

30

MnFe2O4

60

MnFe2O4

40

DE MnFe2O4/DE

40

20 20 10 0

0 0

(a)

60

120

180 t/(min)

240

0

300

(b)

60

120

180 t/(min)

240

300

Figure 4.43 Effect of agitation time on the adsorption of MB (a) and BF (b) by the MnFe2O4/DE-NaOH, MnFe2O4, DE-NaOH, DE and MnFe2O4/DE composite with an initial concentration of 50 mg/L.

The novel magnetic MnFe2O4/diatomite composites were synthesized via a precipitation / hydrothermal method combined with alkali treatment. The magnetic MnFe2O4 nanoparticles were successfully immobilized on purified diatomite by the hydrothermal method under mild conditions. The sequential alkali treatment was to further improve its adsorption performance. The adsorption isotherms well followed the Langmuir isotherm model, and the maximal adsorption capacity for methylene blue and basic fuchsine can reach 104.06 and 284.09 mg/g at 318 K, respectively. The adsorption behavior of the MnFe2O4/diatomite composite is an endothermic and spontaneous process. The magnetic MnFe2O4/diatomite composite can be a promising candidate as an adsorbent for the removal of cationic dyes from wastewater. The MnO2 nanowires-deposited diatomite samples were prepared using the hydrothermal method with KMnO4 and (NH4)2S2O8. These MnO2 nanowires/diatomite samples showed high surface areas and superb Cr(VI) and As(V) adsorption behaviors, with the maximum Cr(VI) and As(V) adsorption capacities being 197.6 and 108.2 mg/g, respectively. The Cr(VI) or As(V) adsorption mechanism was confirmed as ion exchange between hydroxyl groups on the surface of MnO2 nanowires/diatomite and Cr(VI) or As(V) species. TiO2-diatomite photocatalysts were prepared by sol–gel process with various premodified diatomite. The photocatalytic activities of different samples were evaluated by their degradation rate of methyl orange (MO) dye under UV and visible-light irradiation. The samples prepared by phosphoric acid pretreatment method exhibit the highest photocatalytic activity. After 90 min of UV irradiation, about 90% of MO is decomposed by the best effective photocatalyst. And after 8 hr visible-light irradiation, nearly 60% of MO is decomposed by the same sample. Further mechanism investigation reveals that the H3PO4 pretreatment process can obviously change the surface features of diatomite carrier, cause the formation of Si–O–Ti bond, increase the binding strength between TiO2 and diatomite, restrain crystal growth of loaded TiO2, and thus form thermal-stable mesoporous structure at the granular spaces. It helps to build micro-, meso- and macro-porous hierarchical porous structure in TiO2-diatomite, and improves the charge and mass transfer efficiency during catalyzing process, resulting

Diatoms: Fundamentals and Applications

82

1 m

400 nm

(a)

(b)

1 m

300 nm

(e)

(f)

1 m

400 nm

(c)

(d)

1 m

400 nm

(g)

(h)

1.0

1.0

0.8

0.9 0.8

0.6

C/C0

C/C0

Figure 4.44 SEM images of the MnO2/diatomite samples obtained after hydrothermal treatment at 90 °C for (a, b) 6 hr, (c, d) 8 hr, (e, f) 10 hr, and (g, h) 12 hr.

0.4

MO TiO2 TiO2-Dt-500 TiO2-500-Dt-500 TiO2-5P-Dt-500 TiO2-15P-Dt-500

0.2 0.0 (a)

0.7 0.6

MO Dt TiO2-Dt-500 TiO2-500-Dt-500 TiO2-5P-Dt-500 TiO2-15P-Dt-500

0.5 0.4

0

20

40 60 Time (min)

80

0 (b)

2

4 Time (h)

6

8

Figure 4.45 The time profiles of C/C0 for MO in the presence of Dt, TiO2 and TiO2-Dt under (a) visible light and (b) UV light.

in the significantly increased photocatalytic activity of TiO2-diatomite pretreated by phosphoric acid. The TiO2 nanoparticles were immobilized on diatomite via a typical hydrolysis precipitation process using TiCl4 as precursor. TiO2 nanoparticles with the average grain size of around 7–14 nm were well deposited on the surface of diatomite. The photocatalytic activity toward the reduction of aqueous Cr (VI) was demonstrated under UV light. Compared with the commercial TiO2 (P25, Degussa), the TiO2/diatomite composites had better reactive activity because of their relatively higher adsorption capacity. Furthermore, the prepared photocatalyst exhibited relatively good photocatalytic stability depending on the reusability tests. V-doped TiO2/diatomite composite photocatalysts with different vanadium concentrations were synthesized by a modified sol-gel method. The diatomite was responsible

Current Diatom Research in China 004

100

83

105

200

5 1/nm

0.193 nm 200

1 m

100 nm

(a)

10 nm

(b)

Figure 4.46 TEM images of the samples (a) diatomite, (b) TiO2-diatomite.

TiO2

TiO2

TiO2

TiO2

(a)

100 nm

100 nm

(b)

Figure 4.47 TEM micrographs of pure TiO2 (a) and TiO2/diatomite (b).

for the well dispersion of TiO2 nanoparticles on the matrix and consequently inhibited the agglomeration. V-TiO2/diatomite hybrids showed red shift in TiO2 absorption edge with enhanced absorption intensity. Most importantly, the dopant energy levels were formed in the TiO2 bandgap due to V4+ ions substituted to Ti4+ sites. The 0.5% V-TiO2/ diatomite photocatalyst displayed narrower bandgap (2.95 eV) compared to undoped sample (3.13 eV) and other doped samples (3.05 eV) with higher doping concentration. The photocatalytic activities of V doped TiO2/diatomite samples for the degradation of Rhodamine B under stimulated solar light illumination were significantly improved compared with the undoped sample. In our case, V4+ ions incorporated in TiO2 lattice were responsible for increased visible-light absorption and electron transfer to oxygen – , while molecules adsorbed on the surface of TiO2 2 5+ V species presented on the surface of TiO2 particles in the form of V2O5 contributed to e––h+ separation. In addition, due to the combination of diatomite as support, this hybrid photocatalyst could be separated from solution quickly by natural settlement and exhibited good reusability. Novel visible-light-responsive diatomite/g-C3N4 composite was successfully synthesized via a facile impregnation-calcination method. Figure 4.50 shows the size and morphology of g- C3N4, raw diatomite, alkali treatment diatomite and diatomite/gC3N4 composites. It can be seen that diatomite sample has disk-like and highly developed macroporous structure. After NaOH washing, the morphology of raw diatomite was preserved, while the size of the central macropores increased (Figure 4.50(a)-(b)). After diatomite loading onto the surface of layered g- C3N4, the diatomite/g- C3N4 composite can be observed with abundant fluffy sheets (Figure 4.50(c)). Figure 4.50(d) is the TEM image of 2.32 wt% diatomite/g- C3N4. It can be clearly observed that some

Diatoms: Fundamentals and Applications (101)

84

(105) (211)

(004)

Intensity (a.u.) 10

(200)

Quartz

(f)

(f)

(e)

(e)

(d)

(d)

20

30

40 2 Theta (°)

50

(c)

(c)

(b)

(b) (a)

(a) 70 24.0

60

24.5

25.0 25.5 2 Theta (°)

26.0

Figure 4.48 XRD patterns for the diatomite, TiO2/diatomite, and V-TiO2/diatomite composites: (a) DE, (b) TD, (c) 0.25% V/TD, (d) 0.5% V/TD, (e) 1.0% V/TD, and (f) 1.5% V/TD. 6

1.4 TD 0.25%-V/TD 0.5%-V/TD 1.0%-V/TD 1.5%-V/TD

1.0

5 [F(R)hv]1/2

Absorbance (a.u.)

1.2

0.8 0.6

0.0 200

3 TD 0.25%-V/TD 0.5%-V/TD 1.0%-V/TD 1.5%-V/TD

2

0.4

1

0.2

(a)

4

300

400 500 600 Wavelength (nm)

700

0 2.0

800 (b)

2.5

3.0

3.5 hv (eV)

4.0

4.5

5.0

Figure 4.49 (a) UV-vis diffuse reflectance spectra (DRS) of the undoped and V-doped TiO2/diatomite composites and (b) the plot of transformed Kubelka-Munk function versus the energy of light.

diatomite particles was loaded on the surface of layered structure g-C3N4. In HR-TEM image(Figure  4.50(f)), the border of diatomite and g- C3N4 can be observed clearly, suggesting diatomite layers combining well with g- C3N4. The diatomite/g-C3N4 composite exhibits high efficiency for the degradation of RhB. The photoreaction kinetics constant value is about 1.9 times as high as that of g-C3N4 under visible light irradiation. The enhanced photoactivity is mainly attributed to the electrostatic interaction between g-C3N4 and negatively charged diatomite, synergistic effect lead to the efficient migration of the photogenerated electrons and holes of g-C3N4. TiO2 colloids with the most probably particle size of 10 nm were deposited on the surface of macroporous diatomite by a layer-by-layer (LBL) assembly method with using phytic acid as molecular binder. The basis of LBL assembly procedure is primarily the electrostatic attraction between oppositely charged species deposited from solution onto colloidal spheres, lends itself well to the task of producing colloidally stable, homogeneously coated particles. This flexible and facile procedure permits the coating of colloids of various shapes and sizes with uniform layers of diverse composition. In order to continue the deposition process and to form a three-dimensional coating of TiO2 nanoparticles, a “binder” molecule can be employed. Here, phytic acid has been selected as the ideal

Current Diatom Research in China

(a)

(b)

85

(c)

Diatom g-C3N4

Diatomite

200 nm

(d)

51 nm

5 nm

(e)

(f)

Figure 4.50 FE-SEM and TEM images of samples (a) FE-SEM image of diatomite; (b) FE-SEM image of alkali washed diatomite; (c) FE-SEM image of diatomite/g- C3N4 composite; (d) TEM of diatomite/gC3N4 composite; (e) SEAD pattern of g- C3N4; (f) Conjunction edge between flake-like g- C3N4 and diatomite particles.

10 m (a)

500 nm (b)

Figure 4.51 SEM images of TiO2 coated diatomite.

“binder” molecule for TiO2. Phytic acid is a well-known naturally occurring acid with six phosphate functional groups attached symmetrically to a cyclohexanehexol ring. A composite system for the sustained release of potassium ferrate(VI) (sustainedrelease K2FeO4) was prepared and applied for water treatment. The objective of this research was to maximize the effectiveness of K2FeO4 for water treatment by enhancing its stability using diatomite. It is found that the decomposition rate of K2FeO4 was obviously decreased, which greatly improved the contact rate between released K2FeO4 and pollutants. Via degradation of methyl orange, which was used as a model pollutant, the influential factor of K2FeO4 content within the complete sustained-release K2FeO4 system was studied. The optimal K2FeO4 content within the sustained-release K2FeO4 system was approximately 70%. In natural water samples, sustained-release K2FeO4 at a dosage of 0.06 g/L and with a reaction time of 20 min removed 36.84% of soluble microbial products

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0.1 m (a)

50 nm (b)

Figure 4.52 TEM images of (a) initial diatomite, (b) TiO2 coated diatomite.

Figure 4.53 SEM images of (a) K2FeO4; (b) diatomite; (c, d) diatomite/ K2FeO4).

and 17.03% of simple aromatic proteins, and these removal rates were better than those observed after traditional chlorine disinfection.

4.3

Study of Tribological Performances of Compound Dimples Based on Diatoms Shell Structures

With the increasing demand for reducing the friction and energy consumption, the surface texturing has attracted intensive attentions due to its hydrodynamic action as a lubricant. With development of the bionic technologies, it is possible to obtain an advanced surface

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Diatom shell

(a)

(b)

Figure 4.54 SEM images of Coscinodiscus sp., (a) shell of Coscinodiscus sp. (b) two-layer porous structure of Coscinodiscus sp.: the view from inside showing the second layer and outer layer of pores (see online version for colours).

texturing mechanism by simulating superior structures of biological systems. Researches available indicate that more than 25% productivities in nature are provided by diatoms (Werner 1977), whose frustules (i.e., shells) are made up of multi-level pore structures (Bhatta, Kong, & Rosengarten 2009; Gordon et al. 2009a). Even at the bottom of the same pore, one or more pores sometimes appear. This can be demonstrated with the two-level cylindrical pores of representative Coscinodiscus sp. Shown in Figure  4.54, where the diameter of the outermost cylindrical pro is about 1.212 μm, while at its bottom, 2.23 μm is approximately the diameter of the second level cylindrical pore. The diatom pore shapes are mainly cuboidal, cylindrical or hexagonal (Noyes Sumper & Vukusic 2008; Roselli, Stanca, Paparella, Mastrolia, & Basset 2013). It is the refined multi-level pore structures that make the diatom with good mechanical properties such as higer resilience and tensile properties so as to survive in evolution. For example, the elastic moduli of every layer of pores for the representative Coscinodiscus sp. Are separately up to 3.4, 1.7, and 15.61 GPa (Losic et al., 2009b; Losic et al., 2007b). A test showed that the hardness of Coscinodiscus sp. reaches up to 0.12 GPa (Subhash, Yao, Bellinger, & Gretz 2005). Further, it was verified that the diatom frustules can endure higher stresses ranging from 150 to 680 N·mm–1 (Hamm et al. 2003). So far, a very few researches have been conducted about tribological properties of diatoms through experiments. With an atomic force microscopy, it was found that the friction and wear between the diatom components can be overcome by its self-lubrication, and the bands of Aulacoseira granulate and Ellerbeckia arenaria (i.e., two kinds of diatoms) might act as a ball bearing or solid lubricant (Gebeshuber et  al. 2003; Gebeshuber, Stachelberger, & Drack, 2005). In spite of the researches above about simple dimple effects and diatom shells, little work has involved in simulating the tribological performances for the multi-level pore structure of a diatom, especially its bionic applications I engineering surfaces. As an extended exploration of the simple dimple used in the above researches about the surface texturing, the two-level pores of a diatom structure and thereof extended shapes are used for texturing parallel sliding surfaces, which are termed as the compound dimple in the present study. Every compound dimple consists of two-level pores, whose influences on the tribological performances of the surfaces are investigated with fluid-solid interaction (FSI) method. The application of this method is out of consideration for diatom frustule deformation due to the ambient high water

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pressure when it moves about in the water. In using this method, the diatom frustule deformation is incorporated into the water film thickness in solving water film pressure, and the obtained film pressure is considered in the further analysis of the frustule deformation which is a two-way coupling process. Fanming Meng et al. studied the physical models with different compound and simple dimple shapes are established first. Based on these models, the hydrodynamic lubrication action of the compound dimple is solved with the Navier-Strokes equation since it can overcome limitations of Reynolds equation in predicting lubrication performances for tribological pairs at conditions with obvious lubrication film inertia forces or small film thickness ratios (i.e., the ratio of lubrication film thickness to the clearance between the matching surfaces) where the turbulence flow of the lubricant tends to occur (Arghir, Roucou, Helene, & Frene 2003; Brajdic-Mitidieri, Gosman, Ioannides, & Spikes 2005; Sahlin, Glavatskih, Almqvist, & Larsson 2005). Meanwhile, constitutive equations of solid are used to solve mechanical performances of the texturing surfaces such as the deformation and tress. Next, comparisons towards the tribological performances for the parallel sliding surfaces are made between representative compound and simple dimple shapes and among the compound dimple shapes to find optimal compound dimples.

References Anderson, M.W., Holmes, S.M., Hanif, N., Cundy, C.S. (2000). Hierarchical Pore Structures through Diatom Zeolitization, Angew. Chem. Int. Ed., 39(15), 2707–2710. Aravindan, V., Suresh Kumar, P., Sundaramurthy, J., Ling, W.C., Ramakrishna, S., Madhavi, S. (2013). Electrospun NiO nanofibers as high performance anode material for Li-ion batteries, J. Power Sources, 227, 284–290. Arghir, M., Roucou, N., Helene, M., Frene, J. (2003). Theoretical Analysis of the Incompressible Laminar Flow in a Macro-Roughness Cell, J. Tribol., 125(No. 2), 309–318. Aricò, A.S., Bruce, P., Scrosati, B., Tarascon, J.-M., van Schalkwijk, W. (2005). Nanostructured materials for advanced energy conversion and storage devices, Nat. Mater., 4(5), 366–377. Aw, M.S., Simovic, S., Addai-Mensah, J., Losic, D. (2011). Silica microcapsules from diatoms as new carrier for delivery of therapeutics, Nanomedicine, 6(7), 1159–1173. Bao, Z., Song, M.-K., Davis, S.C., Cai, Y., Liu, M., Sandhage, K.H. (2011). High surface area, micro/mesoporous carbon particles with selectable 3-D biogenic morphologies for tailored catalysis, filtration, or adsorption, Energy Environ. Sci., 4(10), 3980–3984. Bao, Z., Weatherspoon, M.R., Shian, S., Cai, Y., Graham, P.D., Allan, S.M. et al. (2007). Chemical reduction of three-dimensional silica micro-assemblies into microporous silicon replicas, Nature, 446(7132), 172–175. Bao, L., Zang, J., Li, X. (2011). Flexible Zn2 SnO4 /MnO2 core/shell nanocable−carbon microfiber hybrid composites for high-performance supercapacitor electrodes, Nano Lett., 11(3), 1215–1220. Baranauskas, V., Chang, D.C., Li, B.B., Peterlevitz, A.C., Trava-Airoldi, V.J., Corat, E.J. et  al. (2000), Journal of Porous Materials, 7(1/3), 401–405. Barea, E., Xu, X., González-Pedro, V., Ripollés-Sanchis, T., Fabregat-Santiago, F., Bisquert, J. (2011). Origin of efficiency enhancement in Nb2O5 coated titanium dioxide nanorod based dye sensitized solar cells, Energy Environ. Sci., 4(9), 3414–3419.

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Baxter, J., Bian, Z., Chen, G., Danielson, D., Dresselhaus, M.S., Fedorov, A.G. et  al. (2009). Nanoscale design to enable the revolution in renewable energy, Energy Environ. Sci., 2(6), 559–588. Bhatta, H., Kong, T.K., Rosengarten, G. (2009). Diffusion through Diatom Nanopores, JNanoR, 7(7), 69–74. Biswas, P., Wu, C.-Y. (2005). Nanoparticles and the Environment, J. Air Waste Manage. Assoc., 55(6), 708–746. Brajdic-Mitidieri, P., Gosman, A.D., Ioannides, E., Spikes, H.A. (2005). CFD analysis of a low friction pocketed pad bearing, J. Tribol., 127(4), 803–812. Brezesinski, T., Wang, J., Tolbert, S.H., Dunn, B., Ordered mesoporous alpha-MoO3 with isooriented nanocrystalline walls for thin-film pseudocapacitors. Nat. Mater., 9(2), 146–151, 2010. Bruchez, M., Hotz, C. (2007). Humana. Cai, Y., Dickerson, M.B., Haluska, M.S., Kang, Z., Summers, C.J., Sandhage, K.H. (2007). Manganese-Doped Zinc Orthosilicate-Bearing Phosphor Microparticles with Controlled Three-Dimensional Shapes Derived from Diatom Frustules, J. American Ceramic Society, 90(4), 1304–1308. Cai, Y., Sandhage, K.H. (2005). Zn2SiO4-coated microparticles with biologically-controlled 3D shapes, phys. stat. sol., 202(10), R105–R107. Calzaferri, G. (2010). Artificial photosynthesis, Top. Catal., 53(3-4), 130–140. Campbell, B., Ionescu, R., Tolchin, M., Ahmed, K., Favors, Z., Bozhilov, K.N. et al. (2016), Sci Rep-Uk, 6. Cerneaux, S., Zakeeruddin, S. M., Pringle, J. M., Cheng, Y.-B., Grätzel, M., Spiccia, L. (2007). Novel nano-structured silica-based electrolytes containing quaternary ammonium iodide moieties, Adv. Funct. Mater., 17(16), 3200–3206. Chandrasekaran, S., Sweetman, M.J., Kant, K., Skinner, W., Losic, D., Nann, T. et  al. (2014). Silicon diatom frustules as nanostructured photoelectrodes, Chem. Commun., 50(72), 10441–10444. Chen, Y., Liu, K. (2016). Preparation of granulated N-doped Tio2 /diatomite composite and its applications of visible light degradation and disinfection, Powder Technology, 303, 176–191. Chen, C.-T., Hsu, F.-C., Huang, J.-Y., Chang, C.-Y., Chang, T.-Y., Lin, H.-M. et al. (2015). Effects of a thermally stable chlorophyll extract from diatom algae on surface textured Si solar cells, RSC Adv., 5(44), 35302–35306. Chen, J., Lu, X., Sun, J., Xu, F. (2015). Si@C nanosponges application for lithium ions batteries synthesized by templated magnesiothermic route, Mater. Lett., 152, 256–259. Chen, X., Mao, S.S. (2007). Titanium dioxide nanomaterials: synthesis, properties, modifications, and applications, Chem. Rev., 107(7), 2891–2959. Chen, W., Rakhi, R.B., Hu, L., Xie, X., Cui, Y., Alshareef, H.N. (2011). High-performance nanostructured supercapacitors on a sponge, Nano Lett., 11(12), 5165–5172. Chen, Z., Su, D., Qin, M., Fang, G. (2015). Preparation and characteristics of composite phase change material (CPCM) with Sio2 and diatomite as endothermal-hydroscopic material, Energy and Buildings, 86, 1–6. Conway, B.E. (1999). Kluwer-Plenum Publishing Corp. Dahn, J.R., Zheng, T., Liu, Y., Xue, J.S. (1995). Mechanisms for lithium insertion in carbonaceous materials, Science, 270(5236), 590–593. Danil de Namor, A.F., El Gamouz, A., Frangie, S., Martinez, V., Valiente, L., Webb, O.A. (2012). Turning the volume down on heavy metals using tuned diatomite. A review of diatomite and modified diatomite for the extraction of heavy metals from water, J. Hazard. Mater., 241–242, 14–31.

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De Stefano, M., De Stefano, L. (2005). Nanostructures in diatom frustules: functional morphology of valvocopulae in cocconeidacean monoraphid taxa, J. Nanosci. Nanotechnol., 5(1), 15–24. Dehestaniathar, S., Khajelakzay, M., Ramezani-Farani, M., Ijadpanah-Saravi, H. (2016). Modified diatomite-supported CuO–Tio2 composite: Preparation, characterization and catalytic CO oxidation, Journal of the Taiwan Institute of Chemical Engineers, 58, 252–258. Dincer, I. (2002). On thermal energy storage systems and applications in buildings, Energy and Buildings, 34(4), 377–388. Dong, Z., Li, Z.J., Zhou, H.M., Wu, K. (2004), Cement Concrete Res, 34, 927–934. Du, Y., Fan, H., Wang, L., Wang, J., Wu, J., Dai, H. (2013). α-Fe2O3 nanowires deposited diatomite: highly efficient absorbents for the removal of arsenic, J. Mater. Chem. A, 1(26), 7729. Du, Y., Wang, L., Wang, J., Zheng, G., Wu, J., Dai, H. (2015). Flower-, wire-, and sheet-like Mno2-deposited diatomites: Highly efficient absorbents for the removal of Cr, Journal of Environmental Sciences, 29, 71–81. Duong, T.-T., Choi, H.-J., He, Q.-J., Le, A.-T., Yoon, S.-G. (2013). Enhancing the efficiency of dye sensitized solar cells with an Sno2 blocking layer grown by nanocluster deposition, J. Alloys Compd., 561, 206–210. Ernst, E.M., Church, B.C., Gaddis, C.S., Snyder, R.L., Sandhage, K.H. (2007). Enhanced hydrothermal conversion of surfactant-modified diatom microshells into barium titanate replicas, J. Mater. Res., 22(05), 1121–1127. Etacheri, V., Marom, R., Elazari, R., Salitra, G., Aurbach, D. (2011). Challenges in the development of advanced Li-ion batteries: a review, Energy Environ. Sci., 4(9), 3243–3262. Feng, F.U., Jie, Z., Xiao-Mei, H.E., G.U.O., L., Hui-Dong, S., Dan-Jun, W. (2016), Journal of Inorganic Materials, 31, 881. Fuhrmann, T., Landwehr, S., El Rharbi-Kucki, M., Sumper, M. (2004). Diatoms as living photonic crystals, Appl. Phys. B, 78(3–4), 257–260. Gebeshuber, I.C., Kindt, J.H., Thompson, J.B., Del Amo, Y., Stachelberger, H., Brzezinski, M.A. et  al. (2003). Atomic force microscopy study of living diatoms in ambient conditions, J. Microsc., 212(3), 292–299. Gebeshuber, I.C., Stachelberger, H., Drack, M. (2005). Diatom bionanotribology--biological surfaces in relative motion: their design, friction, adhesion, lubrication and wear, J. Nanosci. Nanotechnol., 5(1), 79–87. Gonçalves, L.M., de Zea Bermudez, V., Ribeiro, H.A., Mendes, A.M. (2008). Dye-sensitized solar cells: A safe bet for the future., Energy Environ. Sci., 1(6), 655–667. Gordon, R., Drum, R.W. (1994), Int. Rev. Cytol., 150, 243–372. Gordon, R., Losic, D., Tiffany, M.A., Nagy, S.S., Sterrenburg, F.A. (2009a). The Glass Menagerie: diatoms for novel applications in nanotechnology, Trends Biotechnol., 27(2), 116–127. Gordon, R., Losic, D., Tiffany, M.A., Nagy, S.S., Sterrenburg, F.A.S. (2009b). The Glass Menagerie: diatoms for novel applications in nanotechnology, Trends Biotechnol., 27(2), 116–127. Grätzel, M. (2003). Dye-sensitized solar cells, Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 4(2), 145–153. Guo, X.L., Kuang, M., Li, F., Liu, X.Y., Zhang, Y.X., Dong, F. et al. (2016). Engineering of three dimensional (3-D) diatom@TiO2 @MnO2 composites with enhanced supercapacitor performance, Electrochim. Acta, 190, 159–167. Hamm, C.E., Merkel, R., Springer, O., Jurkojc, P., Maier, C., Prechtel, K. et al. (2003). Architecture and material properties of diatom shells provide effective mechanical protection, Nature, 421(4925), 841–843. Hernández-Alonso, M.D., Fresno, F., Suárez, S., Coronado, J.M. (2009). Development of alternative photocatalysts to TiO2: Challenges and opportunities, Energy Environ. Sci., 2(12), 1231–1257.

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Hirscher, M. (2004). Nanoscale materials for energy storage, Materials Science and Engineering: B, 108(1-2), 1–1. Hoshikawa, T., Ikebe, T., Yamada, M., Kikuchi, R., Eguchi, K. (2006). Preparation of silicamodified TiO2 and application to dye-sensitized solar cells, Journal of Photochemistry and Photobiology A: Chemistry, 184(78-85), 78, SV7–85. Hou, Y., Cheng, Y., Hobson, T., Liu, J. (2010). Design and synthesis of hierarchical MnO2 nanospheres/carbon nanotubes/conducting polymer ternary composite for high performance electrochemical electrodes, Nano Lett., 10(7), 2727–2733. Hu, L., Qu, B., Chen, L., Li, Q. (2013). Low-temperature preparation of ultrathin nanoflakes assembled tremella-like NiO hierarchical nanostructures for high-performance lithium-ion batteries, Mater. Lett., 108, 92–95. Huang, D.-R., Jiang, Y.-J., Liou, R.-L., Chen, C.-H., Chen, Y.-A., Tsai, C.-H. (2015). Enhancing the efficiency of dye-sensitized solar cells by adding diatom frustules into TiO2 working electrodes, Appl. Surf. Sci., 347, 64–72. Huang, M., Li, F., Dong, F., Zhang, Y.X., Zhang, L.L. (2015). MnO2 -based nanostructures for high-performance supercapacitors, J. Mater. Chem. A, 3(43), 21380–21423. Inoue, Y. (2009). Photocatalytic water splitting by RuO2-loaded metal oxides and nitrides with d0- and d10 -related electronic configurations, Energy Environ. Sci., 2(4), 364–386. Jantschke, A., Herrmann, A.-K., Lesnyak, V., Eychmüller, A., Brunner, E. (2012). Decoration of diatom Biosilica with noble metal and semiconductor nanoparticles (NO2->NH4+,urea (Kustka Sañudo-Wilhelmy Carpenter Capone & Raven 2003; Quigg et al. 2011; Twining & Baines 2013). The cyanobacterial symbionts are Richelia inside the frustule but outside the protoplasts of the marine diatoms Hemiaulus and Rhizosolenia, Rivularia on the frustule surface of the marine diatom Chaetoceros, and the non-photosynthetic cyanobacterial endosymbionts of freshwater diatoms in the Rhopalodiaceae (Epithemia and Rhopalodia) (Hilton et al. 2013; Kneip, Voss, Lockhart, & Maier 2008; Nakayama et al. 2014; Wouters et al. 2009). H+-pumping rhodopsin occurs in diatom genotypes adapted to low Fe environments. This allows light energy transduction with a very low Fe cost (Marchetti et al. 2012, 2015; Raven 2009). However, proteorhodopsin-based energy transduction has a quotient of H+ pumped per photon absorbed (1.0), the same as a water-water cycle involving only PSII and PTOX (1.0) but lower than that of the water-water cycle of linear electron flow involving both PSI and PSII (1.5) and of cyclic electron flow lacking (as in diatoms) the H+-pumping NADdh, i.e. a H+:photon absorbed quotient of 2.0 (Larkum et al. 2017; Raven 2009). The additional energy storage of linear electron flow coupled to NADP+ reduction is not relevant to the comparison with proteorhodopsin with chlorophyll-based H+ pumping, since the energy transduction is an add-on to that stoichiometric with NADP+ reduction. Furthermore, the probable plasmalemma location means H+ gradient generated by proteorhodopsins cannot phosphorylate ATP, except in the cyanobacterium Gloeobacter that lacks thylakoids and so that photosynthetic-, respiratory- and proteorhodopsin-based proton fluxes as well as the CFOCF1 ATP synthase (Larkum et al. 2018).

Iron in Diatoms 219

9.4

Iron Storage

Ferritin is a widespread, but not universal. means of intracellular Fe storage in Archaea, Bacteria and Eukarya (Arosio et  al. 2015; Botebol et  al. 2015; Marchetti et al. 2009; Marchetti et al. 2012; Masuda et al. 2015; Seckbach 1969). 2012, Marchetti et  al. (2009) showed from molecular genetic evidence that ferritin occurred in the raphid pennate diatoms Fragilariopsis cylindrica, Phaeodactylum tricornutum, Pseudo-nitzschia australis, Pseudo-nitzschia multiseries and other open ocean Pseudonitzschia spp, (Bacillariophyceae sensu stricto) but not in the centric diatom Thalassiosira pseudonana (Coscinodistophyceae). However, Groussman et al. (2015), using transcriptomic evidence, found ferritin in some species of all four classes of diatom. 17 of 18 species examined in the Bacillariophyceae sensu stricto, 4 of 9 in the Fragilariophyceae, all 6 of the Mediophyceae, and 6 of 22 of the Coscinodiscophyceae (Groussman et al. 2015). The obvious function of ferritin is in storing Fe in times of growth-saturating Fe supply, with use of stored Fe in growth when external Fe is growth limiting. Marchetti et al. (2009, Figure 4) showed that the ferritin-containing Pseudo-nitzschia underwent 4 more rounds of cell division (binary fission) after external Fe was withdrawn than in Thalassiosira oceanica, which lacks ferritin. Marchetti et al. (2009, Table S3) used the Fe content of Fe-replete and Fe-depleted cells to predict the number of cell divisions that can be supported for 4 strains of Pseudonitzschia (3.7–5.1 cell divisions) and two of Thalassiosira oceanica (1.6 and 3.2 cell divisions). Raven (1984) suggests that the capacity for storage (number of doublings supported by the stored element) is inversely proportional to the critical content (the content just sufficient to give the minimum doubling time); the data on Fe in diatoms give limited support to the data quoted on pp. 431–432 of Raven (1984) on N, P, K, Mg and Ca in other aquatic photolithotrophs. Iron storage in the vacuole of the coastal diatoms Thalassiosira pseudonana and Thalassiosira weissflogii, lacking ferritin, appears to use polyphosphate-associated Fe (Nuester et  al. 2012). In oceanic diatoms in high nutrient (N,P) – low chlorophyll (HNLC), Fe-deficient oceans there is an excess of P that could allows P to be used in Fe storage from brief periods of Fe availability. In another stramenopile, the brown alga Ectocarpus siliculosus, a major Fe storage compound resembles the amorphous Fe-P mineral core of bacterial and plant ferritins (Bőttger et al. 2012). A further role of ferritin in the prasinophycean marine picoplankter Ostreocccus is in photoperiod/scotoperiod iron homeostasis (Botebol et  al. 2015). While modulation by Fe availability of the diel variations in transcriptome and metabolome of the ferritincontaining pennate diatom Phaeodactylum (Smith et al. 2016), there is no mention of ferritin. While the evidence discussed above clearly shows that ferritin clearly acts as an Fe store, Pfaffen et  al. (2015) point out that the ferritin of Pseudo-nitzschia multiseries functions in Fe2+ oxidation and in buffering of Fe as well as Fe storage. While Pfaffen et al. (2015) suggest that this ferritin is optimised for Fe2+ oxidation rather than Fe storage should be considered in the context of falsification of claims of optimisation. A review that mentions diatom ferritin in relation to the functions of non-plant ferritins is Arosio et al. (2015).

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9.5

Conclusions and Prospects

Diatoms have several mechanisms of Fe uptake. Diatoms have the typical range of Fe catalysts in electron transport in photosynthesis and respiration, although Fe-containing cytochrome c6 is genotypically replaced by Fe-free plastocyanin, and Fe-free flavodoxin phenotypically replaces Fe-containing ferredoxin, in diatoms from low-Fe habitats. Diatoms lack the H+-pumping, high Fe pathway from ferredoxin to PQ component of cyclic electron flow, so diatom cyclic electron flow has a lower H+:electron ratio than in cyanobacteria, some green algae and most embryophytes. Some diatoms from low Fe habitats have Fe-free H+-pumping rhodopsins as a additional means of light energy conversion. Based on a limited number of investigations, pennate diatoms store Fe as ferritin, while centric diatoms use an FeS complex. None of the data on Fe interactions with diatoms have involved freshwater diatoms.

Acknowledgements Discussions with John Beardall, Philip Boyd, Paul Falkowski, Richard Geider, Zoe Finkel, Mario Giordano, Stephen Maberly, Adrian Marchetti, Francois Morel, Neil Price, Robert Strzepek and Bill Sunda have been very useful, The University of Dundee is a registered Scottish charity, No. 015096.

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10 Diatom Symbioses with Other Photoautotroph Rosalina Stancheva1,* and Rex Lowe2,3 1

Department of Biological Sciences, California State University, San Marcos, California, USA Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, USA 3 Biological Station, University of Michigan, Pellston, Michigan, USA

2

Abstract Symbioses between unicellular organisms are now accepted as the evolutionary source of organelles in eukaryotic cells and also the tremendous diversity of organisms previously classified as protists. In this review we explore the endosymbioses of cyanobacteria within diatoms and the endosymbioses of diatoms within other eukaryotes, specifically dinoflagellates. We also review the ecological importance of the diatom symbiotic relationships with prokaryotic and eukaryotic photoauthotrophs in nutrient limited aquatic habitats where nitrogen-fixing diatom symbioses are prevalent along with other diatom associations, resulting in a subsequent enhancement of aquatic food webs. Keywords: Endosymbionts, epithemia , rhopalodia, rhopalodiaceae, richelia endosymbionts

10.1

Introduction

The term symbiosis, i.e. “the living together of differently named organisms” introduced by de Bary (1878) as a neutral word for whole range of associations between organisms, today is more narrowly used to describe those relationships in which both partners benefit. Typically, the uni-or multicellular host provides the symbiont with access to nutrients and a stable environment and in return benefits from metabolic products of the symbiont (Schönfeld 2012). Symbioses range from temporary facultative associations in which the symbiont re-colonizes the host within every host generation, to a permanent obligate relationship, where at least one of the partners is not autonomous and the symbiont is vertically transmitted to the next host generation, thus both organisms evolve together to accommodate each other (Moran 2006). The evolutionary importance of symbiosis is manifested by the endosymbiont theory for the origin of plastids in eukaryotic cells (Merezhkowsky 1905). Mereschkowski’s theory was largely ignored at the time but was later resurrected by Margulis (1970). Diatoms are one of the most diverse groups of unicellular eukaryotic algae, whose evolution involved the capture of a red algal plastid implying more recent origin date in *Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (225–244) © 2019 Scrivener Publishing LLC

225

226 Diatoms: Fundamentals and Applications the early Mesozoic (Medlin 2016). Diatoms are distributed in virtually all freshwater and marine habitats, and are responsible for nearly one-quarter of the global primary production and 40% of marine primary production (Falkowski et al. 1998). They are highly competitive under variable environmental conditions partially due to their flexibility in forming associations with other organisms. Some genera of diatoms contain intracellular nitrogen-fixing (N2-fixing) cyanobacteria, which is beneficial for their nutrition. This endosymbiosis is important from an evolutionary and ecological prospective. The intracellular symbioses of N2-fixing cyanobacteria with primarily freshwater diatoms belonging to the family Rhopalodiaceae and with marine members of Rhizosoleniaceae or Hemiaulaceae represent new model systems to study molecular adaptations of genome evolution that accompany a switch from free-living to intracellular existence (Hilton et al. 2013; Nakayama & Inagaki 2017). Over the years, many reviews of the biology, evolution and to a limited degree the ecology of these diatom-cyanobacteria symbioses have appeared (e. g., Adler et al. 2014; Foster et al. 2011; Janson 2002; Kociolek & Hamsher 2017; Villareal 1992). Here we will summarize diatom-cyanobacteria symbioses in the light of their ecological impact, because they play an important role in nitrogen supply within oligotrophic ecosystems. Further, we will highlight some ecological aspects of diatoms in epiphytic, endogloeic and endophytic “loose symbiotic” relationship with freshwater and marine macroalgae in low nutrient ecosystems. Lastly, the diatom tertiary endosymbiosis with dinoflagellates will be briefly discussed. Other interesting diatom symbioses, such as with larger foraminifera (Lee 1994) and diatom epizoic associations with invertebrates and vertebrates (Denys 1997; Wetzel et al. 2012) are not subject of this summary, because comprehensive recent reviews on the topics are available (see Lee 2011; Robinson et al. 2016; Totti et al. 2011).

10.2

Diatoms with a N2-Fixing Coccoid Cyanobacterial Endosymbiont

The association of endosymbiotic cyanobacteria and the diatom Rhopalodia was first reported by Drum and Pankratz (1965) who described the cellular fine structure of ovoid inclusions, 4 to 6 microns wide, 5 to 7 microns long and separated from the diatom cytoplasm by a five-layered wall. Authors suggested that the inclusions were neither pyrenoids nor oil bodies and that they may be unique cell organelles whose function is unknown or possibly modified coccoid cyanobacteria. Geitler (1977) referred to endosymbionts in Epithemia, Rhopalodia and Denticula vanheurckii Brun as “sphäroidkörper” suspecting that they were cyanobacteria. Lowe et al. (1984) demonstrated that the fine structure of the cyanobacterial endosymbiont in Epithemia adnata (Kütz.) Bréb. is morphologically similar to the endosymbiont in Rhopalodia. This symbiosis seems to be universal and obligate across all diatom species belonging to the genera Rhopalodia and Epithemia in the family Rhopalodiaceae, which are primarily distributed in freshwater, but with some taxa abundant in brackish and marine environments (Kociolek et  al. 2015). However, illustrations of the cyanobacterial endosymbionts in literature are available only for freshwater taxa (listed in Table  10.1), which raises uncertainty about their existence in marine species

4−6 × 5–7 4−5 × 5–6 N/A N/A 4.4–6.3 × 5.5–13 N/A N/A 4.9–5.5 × 6–80.7

2 1–2 (4) 4 2 1–4 1–2 2 1–2

Rhopalodia gibba (Ehrenb.) O. Müll.

2.6–3.2 3–7.1 × 3.9–7.9

2.7–3.8 × 3.6–4.8

1–2 (4) 1–2

Epithemia sorex Kütz.

3.2−4 × 4–50.2

1–2

4–8 (16)

Epithemia adnata var. porcellus (Kütz.) R. Ross

N/A 3.2−4 × 4–50.2

Rhopalodia brebissonii Krammer

4–8 (16)

Epithemia adnata var. saxonica (Kütz.) R. M. Patrick

3.2–7.6 × 5.5–7.7 5−6 × 5.3–6.6

5 × 5–6 4.1–8.1 × 5.6–8.7

2–6 2–6

Epithemia adnata (Kütz.) Bréb.

CE size range (μm)

Epithemia turgida (Ehrenb.) 4–8 (16) Kütz. 2–12

CE per host cell

Diatom host

1, 4–7 (TEM) 3E (D) 1, 2 (LM, TEM) 3–2 (LM) 1A (LM) 1 (CLM), 2 (TEM) 1E (LM) 10 (LM) Drum and Pankratz (1965) Geitler (1977) Prehtl et al. (2004) Schönfeld 2012 Stancheva et al. (2013) Adler et al. (2014) Kociolek et al. (2015) This paper

(Continued)

1–4 (LM)

4 (D) 1F (LM)

3A-D (D) 1E (LM)

2 (D)

116 (D) 1 (D)

N/A 9 (LM)

Figure

This paper

Geitler (1977) Stancheva et al. (2013)

Geitler (1977) Stancheva et al. (2013)

Geitler (1977)

Geitler (1932) Geitler (1977)

Stancheva et al. (2013) This paper

Reference

Table 10.1 Cyanobacterial Endosymbionts in Rhopalodiacea Documented in the Literature. Abbreviation: CE – Cyanobacterial Endosymbiont; D – Drawing; LM – Light Microscopy Photomicrograph; CLM – Confocal Light Microscopy Photomicrograph; TEM – Transmission Electron Microscopy Photomicrograph, N/A – Not Available.

Diatom Symbioses with Other Photoautotroph 227

N/A

1–2 1–2

Rhopalodia vermicularis O. Müll.

Tetralunata vanheurckii (Brun) Hamsher, Graeff, Stepanek and Kociolek N/A 2.8–3.4 × 3–40.8

N/A

N/A

N/A

Rhopalodia hirudiniformis O. Müll.

4−6 × 5–7 3.2–4.8 × 4–60.4

2

Rhopalodia gibberula (Ehrenb.) O. Müll.

CE size range (μm)

Rhopalodia musculus (Kütz.) 1–2 (4) O. Müll.

CE per host cell

Cont.

Diatom host

Table 10.1

Geitler (1932) Geitler (1977)

Furey et al. (2017)

Furey Richardson-Coy Munubi Vadeboncoeur and Lowe (2017)

This paper

Drum and Pankratz (1965)

Reference

118 (D) N/A

Plate 3: 6 (LM)

Plate 2: 2, 5, 7 (LM)

5–8 (LM)

2, 3 (TEM)

Figure

228 Diatoms: Fundamentals and Applications

Diatom Symbioses with Other Photoautotroph 229

1

5

2 6

3 7

4

8

Figure 10.1–10.8 Light microscope images of Rhopalodia brebissonii (1-4) and Rhopalodia musculus cells (5-8) containing coccoid cyanobacterial endosymbionts (arrows); Figs 1, 2, 5, 6 valve views; 3, 4, 7, 8 girdle views. 1, 2 acid-cleaned valves. 2–6 gluthardehyde preserved cells. Material collected from brackish habitat in southern California (salinity 1.25 ppt, conductivity 2019 μS cm-1). Scale bar 10 μm.

(see Kociolek & Hamsher 2017). Therefore, we illustrate here endosymbionts in Rhopalodia brebissonii Krammer and R. musculus (Kütz.) O. Müll. (Figure  10.110.8), which inhabit brackish and marine coastal waters (Kociolek & Hamsher 2017; Witkowski et al. 2000). The presence of cyanobacteria in species within the family Rhopalodiaceae must have been a relatively early evolutionary event since it seems to only appear in two closely related genera. Nakayama et al. (2011) compared DNA sequences of cyanobacterial endosymbionts from two species of Epithemia and one species of Rhopalodia and concluded that the “endosymbionts in diatoms within Rhopalodiaceae were acquired by a common ancestor of rhopalodiacean diatoms and have been retained during host speciation”. Further, Hamsher et al. 2014 investigated the valve morphology of species of the genus Denticula from Sumatra including D. vanheurckii that had been described from Java. Authors concluded that these taxa differ significantly from the type species of Denticula and transferred them to a new genus Tetralunata in the Rhopalodiaceae. This corroborates Nakayama’s conclusion that the N2-fixing cyanobacterial endosymbiont is unique to genera in the family Rhopalodiaceae. Indeed, the only evidence for the presence of “sphäroidkörper” in Denticula (now Tetralunata) vanheurckii cells is a drawing by Geitler (1932, Fig. 118). A vertical transmission of cyanobacterial

230 Diatoms: Fundamentals and Applications

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Figure 10.9 and 10.10 Light microscope images of Epithemia adnata (9) and Rhopalodia gibba (10) sexually reproducing cells showing the vertical transmission of coccoid cyanobacterial endosymbionts (arrows). Material collected from streams in northern California. Scale bar 10 μm.

endosymbionts to the next diatom host generation during sexual reproduction has been observed in several species of Epithemia and Rhopalodia (Figure 10.9 and 10.10) and eventually in D. vanheurckii (Geitler 1932). The endosymbionts have subsequently been a target of several physiological and molecular investigations (Bothe et al. 2010; Floener & Bothe 1980; Kneip et al. 2008; Prechtl et al. 2004). These studies have shown that the endosymbionts are closely related to the free-living diazotrophic cyanobacterium Cyanothece ATCC 51142, which fix elemental nitrogen (N2) and are photosynthetically inactive, despite the presence of thylakoids. In most other instances of endosymbioses the algae furnish reduced carbon (C) products to the host (Taylor 1984), but in the Rhopalodiaceae the cyanobacteria are obligate symbionts getting their energy needs from the diatom while supplying fixed N to the diatom host. The number and biovolume of N2-fixing endosymbionts within the diatom cell is variable and appears to be a function of the availability of external sources of inorganic nitrogen. DeYoe et al. 1992 observed in a growth chamber experiment that decreases in nitrate and the N/P ratio leads to an increase in endosymbiont cell numbers and biovolume in Epithemia turgida (Ehrenb.) Kütz. and R. gibba. Accordingly, Stancheva et al. (2013) recorded decreasing endosymbiont biovolume in E. adnata, E. sorex Kütz., E. turgida and R. gibba with increasing ambient nitrate concentrations in streams in southern California, USA. In addition, Stancheva et al. (2013) provided molecular evidence for nitrogenase gene expression in Epithemia endosymbionts from environmental stream samples. The genome of the R. gibba endosymbiont shows loss of individual genes during the process of genome reduction (Kneip et al. 2008), which results in permanent and obligate relationship for the endosymbiont depending on the metabolites of the diatom cell. Adler et al. (2014) analyzed the current genomic and morphological data available for the R. gibba endosymbiont and concluded that this symbiosis is in a relatively early stage in its molecular and morphological characteristics compared to other model systems for investigation of endosymbiont to organelle conversion. Similarly, the complete endosymbiont genome sequence of E. (Nakayama et al. 2014) and R. gibberula (Nakayama & Inagaki 2017) reveal apparent reductive nature of their genomes lacking most of the genes involved in photosynthesis, but possessing conserved genes related to N2-fixation. However, the comparative genomic analyses indicate that the endosymbiont in R. gibberula exhibits slower progression in gene loss related to photosynthetic

Diatom Symbioses with Other Photoautotroph 231 metabolic pathways compare to the endosymbiont in E. turgida, suggesting that a certain degree of diversity exists among genomes of obligate endosymbionts in rhopalodiacean diatoms (Nakayama & Inagaki 2017). According to Cavalier-Smith and Lee (1985) symbiogenesis involves not only the symbiont gene loss, but the transfer of symbiont genes into the nucleus of the host with subsequent relocation of the gene product into the organelles, which requires evolution of a protein import system. The host-controlled organelles import most of the required proteins encoded in the nucleus from the host cytoplasm (Cavalier-Smith & Lee 1985). Despite the reductive development of the cyanobacterial endosymbiont in rhopalodiacean diatoms, their evolution is in the early stage of organellogenesis (Nakayama & Inagaki 2017). Epithemia and Rhopalodia often dominate the benthic communities in low N freshwater and brackish environments, where together with free-living N2-fixing cyanobacteria, they significantly contribute the N supply of the ecosystem. According to Howarth et  al. 1988, benthic N2-fixation in oligotrophic systems is mediated by cyanobacteria, in contrast to heterotrophic bacteria dominance in mesotrophic and eutrophic lakes and estuaries. The benthic N2-fixation is a major source of N for many oligotrophic lakes and lagoons, despite the moderate fixation rates, because other N inputs tend to be low (Howarth et al. 1988). Fairchild and Lowe (1985) studied aspects of the nutrient-niche of diatoms by deploying nutrient diffusing substrates (NDS) in the littoral zone of Douglas Lake, Michigan, USA. The NDS released potassium salts of nitrate, phosphate, and nitrate plus phosphate. They found that phosphate-releasing NDS stimulated an algal assemblage dominated by Anabaena sp., R. gibba and E. adnata and concluded that when phosphate levels are high relative to nitrate, N2fixing species (including members of Rhopalodiaceae) dominate benthic algal communities. The relationship between nitrate availability and proliferation of Epithemia has also been investigated experimentally in two western US streams. In late summer incubations in the South Fork Eel River in California, epiphyte assemblages on filaments of the green alga Cladophora incubated in ambient N-poor stream water (7 μg L-1) were dominated by Epithemia species (Figure 10.11), while epiphytes in chambers experimentally enriched with N (300 μg L-1) were dominated by the non-N2-fixing diatoms Achnanthidium minutissimum (Kütz.) Czarn. and Nitzschia palea (Kütz.) W. Sm. (Marks & Power 2001). Similarly, early successional communities on unenriched

11

Figure 10.11 Scanning electron microscope image of Cladophora glomerata colonized by epiphytic Epithemia sorex and E. turgida cells. Material collected from the Eel River, California, USA. Scale bar 10 μm.

232 Diatoms: Fundamentals and Applications experimental substrates in desert stream in Arizona, USA were dominated by E. sorex (Peterson & Grimm 1992). Grimm and Fisher (1986), Bahls and Weber (1988) published corroborating evidence in an investigation of diatom assemblages in streams in Montana, USA. They recorded in stream assemblages dominated by E. sorex the mean N/P ratio was 3.2, a value well below Redfield’s ratio (N/P=16) (Redfield 1958) which indicates that N becomes limiting to many algal species. Similarly, Stancheva et  al. (2013) showed that the relative proportion of Epithemia and Rhopalodia taxa decreased with increasing ambient inorganic N concentrations within the low end of the N gradient and N/P ratio below 15 in streams in southern California. The consequences of this symbiosis to the community structure of periphyton and its importance to aquatic food webs has been documented by several investigators (Furey et al. 2012; Power et al. 2009, 2013). The Eel River in northern California, epitomizes western rivers in the USA in watersheds influenced by a Mediterranean climate (Power et al. 2013). The Eel River, like many aquatic ecosystems in the Pacific Northwest, drains a watershed made up of recently uplifted marine sedimentary deposits (Lock et al. 2006). These rocks, along with those of volcanic origin in other basins of the Pacific Northwest, produce soils and runoff that are phosphorus-rich but relatively low in available N, leading to N limitation of photoautotrophs. N2-fixing diatoms in the Rhopalodiaceae are important members of the food web in these aquatic ecosystems. Power et al. (2009) studied the role of Epithemia living epiphytically on the green alga Cladophora enhancing food webs in the Eel River. Following winter storms on the Eel River, Cladophora (which dominates summer green algal growth) proliferates in streams several meters in length. The coarse cell wall of Cladophora provides a stable habitat for Epithemia sorex and E. turgida, each with endosymbiotic cyanobacteria. They found that insects (Chironomidae) grazing on Cladophora infested with Epithemia had a three to 25 times greater emergence rate than insects grazing on Cladophora without Epithemia dominance or on other filamentous algae that lacked these epiphytes. Additionally, the biomass of insects emerging from these mats had an eight to ten times greater biomass than insects emerging from areas in the stream lacking dense Epithemia populations. The aquatic insects in turn enter both aquatic and terrestrial food webs (Power & Dietrich 2002). Furey et al. (2012) further showed the close trophic relationship between dominant grazers (Pseudochironomus richardsonii Malloch) and Epithemia, documenting the importance of this resource to the success of the grazer. Indeed, more research on ecological aspects of diatom-cyanobacteria symbiosis is needed, because the importance of cyanobacterial N2-fixation in freshwater benthic food webs is not well understood at ecosystem level, particularly its potential to alleviate system-wide N limitation (Scott & Marcarelli 2012). In marine environments similar intracellular coccoid cyanobacteria have been observed in planktonic chain-forming non-pennate diatom Climacodium frauenfeldianum Grunow (Carpenter & Janson 2000). The diatom host cells contain 20 to 30 cyanobacterial endosymbiotic cells with diameter 2.5–3.5 microns. Phylogenetic analysis revealed that the Climacodium endosymbiont is closely related to the free-living diazotrophic cyanobacterium Cyanothece ATCC 51142 (Carpenter & Janson 2000), but is unlikely to be identical with the Rhopalodia gibba endosymbionts due to ultrastructural differences, e.g. presence of large starch-granules (Janson 2002). Recent reevaluation of the partial 16S rRNA sequence of the Climacodium cyanoendosymbiont

Diatom Symbioses with Other Photoautotroph 233 showed complete sequence identity to the free-living diazotrophic cyanobacterium Crocosphaera watsonii 8501 (a synonym of Cyanobium waterburyi Komárek) (Foster et  al. 2011). Further, authors demonstrated N2-fixation by the Climacodium cyanoendosymbiont and consequent N transfer to the diatom cell (Foster et  al. 2011). Occasionally, endosymbiotic coccoid cyanobacteria have been reported in other marine chain-forming planktonic diatoms, i.e. Streptotheca and Neostrepthotheca, and were assumed to be similar to the endosymbiont in Rhopalodia gibba (Villareal 1992 and references therein), but had not been studied further.

10.3

Diatoms with N2-Fixing Filamentous Heterocytous Cyanobacterial Endosymbionts

The diatom symbioses with intracellular filamentous cyanobacteria, some of which contain heterocytes, evolved in marine planktonic environments. Marine diatoms that have been reported to harbor filamentous cyanobacterial endosymbionts belong to lineages from the more primitive branches of the non-pennate diatom tree and their taxonomic diversity has been recently summarized (see for species list Kociolek & Hamsher 2017). However, many of these symbioses are only reported occasionally and are poorly known. The best characterized and ecologically significant symbiosis is between the filamentous heterocytous cyanobacterium Richelia intracellularis J. Schmidt and its diatom planktonic hosts belonging to the genera Rhizosolenia, Hemiaulus, Chaetoceros, Bacteriastrum and Guinardia (for host species list see Villareal 1992). The diatom symbioses with Richelia are often abundant in oligotrophic tropical and subtopical oceans, where they play a role in N cycle, and are rarely noted in coastal water (Villareal 1992). The cyanobacterial endosymbiont R. intracellularis has a short trichome with terminal heterocytes, and was first observed inside the cells of Rhizosolenia (Ostenfeld & Schmidt 1902). Typically one to several Richelia trichomes are located at the apex of the large host Rhizosolenia cell (Figure 10.12) in the periplasmic space between the cell membrane and the silica cell wall (Villareal 1990). In the smaller cells of Hemiaulus, the host cytoplasmic mass can obscure the Richelia trichomes, which are hardly visible

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Figure 10.12 and 10.13 Light microscope images of endosymbiotic cyanobacterium Richelia intracellularis in diatom cells of Rhizosolenia (12) and Hemiaulus (13). Bright-field microscopy shows the diatom host cells with endosymbionts (arrow); phycoerythrin fluorescence shows magnified cyanobacterial trichome with terminal heterocytes. Scale bars 30 μm. Reprinted with permission from Zeev et al. (2008), ISME Journal (2008) 2: 911–923, Figure 10.3.

234 Diatoms: Fundamentals and Applications with bright field microscopy. Epifluorescence microscopy revealed one or two Richelia endosymbionts per Hemiaulus cell (Figure  10.13), but their cellular location is not exactly known (Hilton et al. 2013). The primary diatom-Richelia interaction has been assumed to be the supply of fixed N to the diatom cell, due to the presence of specialized heterocytes where the N2-fixation is localized. Several lines of evidence demonstrate the N2-fixation by endosymbiotic Richelia, i.e. high rates of acetylene reduction in field and culture conditions (Mague et al. 1974; Villareal 1990); molecular genetic studies (Foster & Zehr 2006); and direct experimental evidence of N2-fixation and the transfer of fixed N products to diatom host cell measured by a high-resolution nanometer scale secondary ion mass spectrometry (Foster et al. 2011). In the latter study, authors estimated that Richelia fixes 81–744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners (Foster et al. 2011). Richelia endosymbionts are transferred vertically from host diatom to daughter cells during host cell division (Villareal 1990; Taylor 1982; Villareal 1989). However, epiphytic (Carpenter 2002; Gómez Furuya & Takeda 2005; Villareal 1992) and free-living trichomes of Richelia have been observed (Foster et al. 2007, Foster et al. 2011; Gómez et al. 2005; White, Prahl, Letelier, & Popp 2007), which suggests additional horizontal transmission of endosymbionts from the environment. Hilton et al. (2013) attribute some of the reports of free-living Richelia filaments (i.e., Lyimo et al. 2011; Zhang et al. 2011) to broken diatom valves. Richelia trichome division within host cell is uncoupled with the diatom cell division, which results in asymbiotic Rhizosolenia cells in culture even under N-limiting conditions (Villareal 1989). Since there are reports of Rhizosolenia-Richelia blooms with only 94–98% of the Rhizosolenia cells with cyanoendosymbionts (Taylor 1982), it was assumed that the reduced N limitation during blooms may permit asymbiotic Rhizosolenia cells to persist for some time (Villareal 1990). Similarly, approximately 80% of the Hemiaulus cells collected off Hawaii, and over 98% of Hemiaulus cells from Southwest North Atlantic Ocean contained Richelia endosymbionts when observed with epifluorescence microscopy (Heinbokel 1986; Villareal 1994). Hence, this symbiosis may not be obligatory for both partners. Another filamentous heterocytous cyanobacterium, Calothrix rhizosoleniae Lemmermann, is recorded as an epiphyte on the diatom hosts (typically on Chaetoceros), containing Richelia endosymbionts. Both cyanobacteria are morphologically similar resulting in taxonomic confusion. Comprehensive morphological and molecular analysis of Richelia and Calothrix (Foster & Zehr 2006; Janson et  al. 1999) showed that they differ in their phylogeny, trichome morphology, location and number per diatom partner in each diatom association studied, implying they are distinct taxa. A recent study demonstrated that that genome size and content of symbiotic intracellular Richelia and extracellular Calothrix rhizosoleniae strains differed substantially, suggesting that the cellular location may have determined varying evolutionary paths and different mechanisms of maintaining the symbiosis (Hilton et al. 2013). The Richelia intracellularis genome showed reduction, marked by its size, percent coding and GC content, indicating that the cyanobacterium is dependent on its host, in contrast to the genome of closely related extracellular Calothrix, which is more similar to those of free-living heterocytous cyanobacteria (Hilton et al. 2013). Authors proposed that Richelia-diatom symbiosis could have implications for the evolution of N2-fixing symbiosis and potentially for manipulating plant–cyanobacterial interactions.

Diatom Symbioses with Other Photoautotroph 235 Planktonic diatom– heterocytous cyanobacterial symbioses play an important and still underestimated role in the biogeochemistry of the ocean’s surface as source of N to support primary production in nutrient-poor tropical waters. In general, the Richelia association with Rhizosolenia is more commonly reported from the North Pacific central gyre, while the symbiosis with Hemiaulus have higher abundances in the Atlantic Ocean, Caribbean Sea, and Bahaman Islands (Foster et al. 2011 and references therein). Diatom-cyanobacterial symbioses have a patchy distribution throughout the world’s open oceans probably because diatom hosts require silicon to build their frustules (Sohm et  al. 2011). To date, the largest densities of these associations are identified in the Amazon River plume, which delivers high concentrations of silicate and other nutrients to the tropical North Atlantic Ocean (Subramaniam et  al. 2008). In spite of their patchy distribution and abundance, evidence suggests that diatom symbioses with Rhichelia and Calothrix are highly efficient at exporting C and N (Foster et  al. 2011; Subramaniam et al. 2008), and therefore could be very important part of biological pump where they occur (Sohm et al. 2011). During blooms of the diatom hosts Rhizosolenia and Hemiaulus, this symbiosis could supply up to 15% of the total phytoplankton N demand in the central North Pacific gyre (Villareal 1994 and reference therein). N2-fixation during summer bloom of Rhizosolenia associated with Richelia is estimated to provide as much as 35% to 48% of the phytoplankton-based N demand in the central basins of the Gulf of California (White et al. 2007). In the subtropical North Atlantic, Carpenter et al. (1999) encountered an extensive bloom of Hemiaulus-Richelia association, and estimated that the N supply by N2-fixation in the symbioses exceeded that of nitrate flux from deep water. Diatom-Richelia symbiosis is reported from oligotrophic waters of the Mediterranean Sea, but the lack of large-scale blooms and low N2-fixation rates are attributed to the P-starved conditions (Zeev et al. 2008). Richelia and Calothrix inhabiting diatoms are recorded also in the Indian Ocean (Norris 1961), the Red Sea (Kimor et al. 1992), and the western China Seas (Gómez et al. 2005), which makes them the most widespread marine diatom-cyanobacteria association described.

10.4

Epiphytic, Endogloeic and Endophytic Diatoms

Epiphytism by diatoms on macroalgae and other plant substrata has many benefits for the colonizers, although the chemical interactions between both organisms are rather complicated, including allelopathy. In freshwater ecosystems (in contrast to marine environments) there are currently no recognized algal colonizers with specific preferences to only one particular plant host (Burkholder 1996), which would classify algal epiphytisms as nonobligate associations (“loose symbiosis”) between both organisms. However, there is an evidence that the plant host serves as a major nutrient source for epiphytic algae, especially in oligotrophic and mesotrophic habitats where markedly different epiphyte communities were observed on natural as opposed to artificial plants (Burkholder & Wetzel 1989). Macrophytes have been found to leach dissolved organic C and a small amount of their tissue phosphorus (P) to epiphytes (Moeller et al. 1988). In contrast, in eutrophic conditions, loosely attached algal epiphytes do not exhibit substratum preferences, so the water column probably plays a major role in supplying their nutrients (Burkholder 1996 and reference therein).

236 Diatoms: Fundamentals and Applications

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Figure 10.14–10.17 Light and scanning electron microscope images of diatom Nitzschia palea (circled cells) inhabiting mucilage of green algae Chaetophora elegans (14) and Draparnaldia glomerata (15). 16 N. palea external valve view. 17 N. palea internal valve view. Material collected from non-perennial stream in southern California. Scale bar 50 μm (14, 15) and 2 μm (16, 17).

The diatom, Nitzschia palea (Kütz.) W. Sm., is a good example of discussed ecological distinction. This eutrophic motile species with fast maximal growth rates (Marks & Lowe 1993) was observed to inhabit the top layer of the epiphytic biofilm on Cladophora glomerata (L.) Kütz. under elevated ambient N and P concentrations, presumably obtaining nutrients from the water (Marks & Power 2001). Interestingly, N. palea and other Nitzschia species had been reported also as endogloeic, inhabiting the mucilage of the green macroalgae Chaetophora incrassata Hazen (Round 1984). Similarly, we recorded N. palea in large numbers within the mucilage of Chaetophora elegans (Roth) A. Agardh and Draparnaldia glomerata (Vaucher) C. Agardh in low nutrient non-perennial streams in southern California (Figure 10.14–10.17). The green algae Chaetophora and Draparnaldia possess alkaline phosphatase located on the surface of long apical hair cells, which catalyze the hydrolysis of organic P compounds and liberate orthophosphate in the surrounding mucilage available for cell uptake. N. palea is able to supplement C fixation with heterotrophy in high irradiances when organic substrate concentration is high (Tuchman et al. 2006). N. palea has been also observed within the mucilaginous colonies of the planktonic coccoid cyanobacterium Microcystis aeruginosa (Kütz.) Kütz. (Flower 1982; Morales et al. 2015; Round 1984). It is reasonable to hypothesize that mechanisms for organic substrate uptake and metabolisms are activated in N. palea when inhabiting algal mucilage under ambient inorganic nutrient limitation. According to Round (1984), the association of Nitzschia with mucilage producing algae, such as marine planktonic Phaeocystis, and mucilaginous

Diatom Symbioses with Other Photoautotroph 237 tube-forming diatoms (e. g., Cymbella, Berkeleya, Amphipleura, Navicula) is found frequently and may be linked with heterotrophy tendencies of Nitzschia. It has been demonstrated, that many algae growing under suboptimal light conditions are capable of utilizing diverse range of organic C sources (see Tuchman et al. 2006 and references therein). Indeed, these organic and inorganic compounds, available for heterotrophic metabolizing by diatoms in low nutrient conditions, could be actually excreted by other algae and cyanobacteria. Algal nutrient interactions are not considered in the bioassessment studies, which may affect the precision of the water quality assessment. For instance, Snoeijs and Murasi (2004) reported symbiotic relationship for diatoms inhabiting the mucilaginous colonies of N2-fixing cyanobacterium Rivularia atra Roth in Baltic Sea. Authors suggested, that diatoms probably profit by protection against grazing and physical disturbances, using cyanobacterial mucilage as a substratum for motility, and as source of inorganic and organic nutrients excreted from Rivularia cells. The members of Rivulariaceae are able to fix atmospheric N during periods of high inorganic P supply, and to form long colorless multicellular hairs under P-limited conditions, which are the sites of phosphomonoesterase activity for utilizing organic phosphates (Whitton & Mateo 2012). Obviously, the nutrient exchange and appropriate light conditions are beneficial for diatoms inhabiting algal mucilage, but whether or not this is the case for the host is not clear. Cholnoky (1929), Snoeijs and Murasi (2004), and Morales et al. (2015) believed that these diatom associations are symbiotic but temporary and optional for both diatoms and the host. Endophytic and more specialized associations between diatoms and seaweeds in marine environments may provide nutritional and protective benefits for diatoms. For example, Navicula endophytica Hasle had been described from and typically observed in the intercellular substance of receptacles and tips of brown seaweeds Ascophyllum nodosum (L.) Le Jolis, Fucus vesiculosus L., and F. serratus L. (Hasle 1968; Taasen 1972). The otherwise epiphytic diatom Cocconeis scutellum, sometimes is recorded with N. endophytica in the host (Hasle 1968; Taasen 1972). Navicula dumontiae Baardseth and Taasen grows endophytically in the mucilage of the red alga Dumontia incrassata (O. F. Müll.) J.V. Lamour. (Baardseth & Taasen 1973), but has also been found in sediments (Witkowski et al. 2000; Álvarez-Blanco & Blanco 2014). Gyrosigma coelophilum Ocamoto and Nagumo inhabits internodes of the red marine alga Coelarthrum opuntia (Endl.) Børgesen (Ocamoto et al. 2003). Pseudogomphonema sp. has been recorded inside the cystocarps and scattered throughout the whole blade in the cortical and meddular layers of marine red algae Neoabbottiella araneosa (Perest.) S. C. Lindstr. and N. decipiens Klochkova and Pisareva (Klochkova et  al. 2014). Partial heterotrophy is suspected for both Gyrosigma coelophilum and Pseudogomphonema sp., which receive low light intensity (Klochkova et al. 2014; Ocamoto et al. 2003). The exact nature of the relationship between endophytic diatoms and their hosts is unknown. However, the endophytic diatoms listed above posses well developed pigmented chloroplasts and silica-rich frustules without signs of structural reduction. Their cells divide inside the host, but it is unclear whether they re-colonize the hosts within every host generation and keep their autonomy or are vertically transmitted to the next host generation. It seems that multiple re-colonization of the host is the case in N. dumontiae, because the diatom is often recorded free-living outside the host. The mechanism of establishing the relationship, penetrating the seaweed surface and

238 Diatoms: Fundamentals and Applications transmission from one host thallus to another is unknown. It seems that vertical transmission may be possible for N. endophytica and Pseudogomphonema sp., which inhabit seaweed reproductive structures. Diatom cells could be distributed along with the spores into the new developing plants, and thus relationship may become obligatory for diatoms. The abundant endophytic growth of Pseudogomphonema sp. was reported to be potentially negative for Neoabbottiella, but not beyond causing morphological changes (i.e., warping of the host thalli) and not to the level of parasitism (Klochkova et al. 2014).

10.5

Diatom Endosymbionts in Dinoflagellates

Dinoflagellates in the kingdom Chromalveolata host a wide variety of endosymbionts. The tertiary endosymbiosis between a dinoflagellate host and diatom endosymbiont resulted in a group of dinoflagellates known as “dinotoms” (Imanian & Keeling 2014). Dinotom dinoflagellates have unique nuclear and mitochondrial redundancies derived from two evolutionarily distinct eukaryotic lineages in tertiary endosymbiotic events. The diverse dinoflagellate genus Symbiodinium hosts endosymbionts from at least five different eukaryotic phyla including diatoms (Stat et  al. 2006). The inclusion of diatom endosymbionts occurs in many different dinoflagellate taxa. In most examples the endosymbiotic diatom has been reduced and modified into a functional C-fixing plastid with pigments typical of diatoms rather than dinoflagellates. Inagaki et al. 2000 showed that the diatom endosymbionts inside two species of dinoflagellates (Peridinium balticum (now Durinskia dybowskii (Wołosz.) S. Carty and Peridinium foliaceum (now Kryptoperidinium foliaceum (F. Stein) Lindem.) were similar, indicating that the endosymbiont was obtained prior to the divergence of these Peridinium “sister species”. These “sister species” require further analyses since they have been transferred to different genera. Furthermore, the endosymbiont was a benthic species of a pennate diatom (Chesnick et al. 1997). The authors discuss the habitat juxtaposition of the planktonic dinoflagellate and the benthic diatom, specifically the establishment of a symbiosis between a planktonic flagellated dinoflagellate and a bottom-dwelling diatom. They suggest that environmentally associated life strategy stages of the host and symbiont may have been vital to the formation of diatom/dinoflagellate association. The sanddwelling dinoflagellate from Palau, Galeidinium rugatum M. Tamura et T. Horig., was also shown to contain a diatom endosymbiont that appears to be closely related to the pennate diatom, Cylindrotheca sp., which is typically a benthic diatom species (Tamura et al. 2005a). More recent phylogenetic and chloroplast genome analyses reconfirmed the pennate diatom ancestry for these endosymbionts suggesting close relationships with Phaeodactylum tricornutum Bohlin (Imanian et al. 2010) and Nitzschia (Takano et al. 2008). This further suggests that benthic endosymbionts may be more available to dinoflagellates that forage for prey in benthic habitats. Nutrients are often the key limiting factors to primary production in freshwater ecosystems and in tropical seas, where diatom symbioses are frequently observed. It seems that the nutrient limitation is a primary driving factor for diatoms, establishing intracellular and extracellular relationships with other photoauthotrophs for nutrition benefits throughout variable metabolic pathways. The diatom symbioses range from

Diatom Symbioses with Other Photoautotroph 239 temporary co-existence, permitting heterotrophic assimilation of nutrient compounds produced by other algae, to permanent obligatory accrual of intracellular N2-fixing cyanobacteria, and transfer of fixed N products to diatom host cell. Therefore, the establishment of a symbiotic association between diatoms and other photoauthotrophs might be considered an ecological adaptation to life in the oligotrophic habitats.

Acknowledgements Part of the original research on streams in California (by the author Rosalina Stancheva) was funded by the Surface Water Ambient Monitoring Program of the California State Water Resources Control Board. We thank Dr. Mary Power and two anonymous reviewers for suggestions that enhanced the manuscript.

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11 Diatom Sexual Reproduction and Life Cycles Aloisie Poulíčková1,* and David G Mann2,3 1

Department of Botany, Faculty of Science, Palacký University, Olomouc, Czech Republic 2 Royal Botanic Garden, Edinburgh, UK 3 Institute for Food and Agricultural Research Technology (IRTA), Sant Carles de la Ràpita, Catalunya, Spain

Abstract Following a reminder of the general plan of the life cycle in diatoms (which are diplontic organisms that mostly exhibit either oogamy or morphological isogamy) and a short historical account of the discovery of its special features (size-related sexuality, size restitution via an auxospore), we review recent research on the morphological and cytological processes of sexual reproduction in diatoms, mating systems, the environmental factors that induce auxosporulation, and spawning, updating the long 2004 review by V.A. Chepurnov et al. We also emphasize that auxosporulation is not always accompanied by sexual reproduction and that there may be many exceptions to the ‘normal‘ (i.e., oogamous) life cycle among centric diatoms, which are much less well investigated in this respect than pennates. The structure and formation of the wall structures (incunabula and perizonium) that contain and support the auxospore and control its development are discussed. Keywords: Auxospore, gametogenesis, incunabula, life-cycle, mating system, morphogenesis, perizonium, sexual induction, sexual reproduction, spawning

11.1

Introduction

Most diatoms have a life cycle in which there is an alternation between long periods (months, years) of vegetative growth and development involving mitotic cell division, and a short period (hours, days, rarely c. 1 month) in which sexual reproduction takes place. This in itself is not remarkable, since many multicellular plants and animals do the same. It is also not particularly strange that this alternation is coupled to changes in the size of the organism. In plants and animals, and in the brown algae (close relatives of diatoms), this is expected. What is odd is that, whereas in most ‘higher’ organisms, maturity comes only after a period of enlargement and differentiation and the outcome of sexual reproduction is the formation of a small, embryonic individual. In diatoms sexually mature individuals are among the smallest for the species and larger *Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (245–272) © 2019 Scrivener Publishing LLC

245

246 Diatoms: Fundamentals and Applications individuals of the same species are generally impotent (e.g., Chepurnov et  al. 2004; Drebes 1977; Geitler 1932; Round et al. 1990). The short sexual phase of diatoms is in fact tied to restoration of large size: size restitution is necessary because of the gradual reduction in cell dimensions that occurs during the long vegetative phase caused by the way diatom cells divide (new cell wall elements are formed within the confines of the existing wall), coupled with the physical properties of the cell wall (each valve or girdle band can bend but not stretch). Hence the formation and development of auxospores – the specially constructed cells of diatoms that expand to re-establish maximum size – is a rejuvenation of the cell lineage; it is usually also the stage when meiosis and sexual recombination occur (Mann, Crawford, & Round 2017; Round et al. 1990). Given the unique and counter-intuitive features of diatom life cycles (which can be detected even in fossil diatoms: see Beraldi-Campesi et al. 2015; Jewson & Harwood 2017), with the saw-tooth pattern of slow size reduction and rapid size restitution linked to sex, it is perhaps not surprising that it was a long time before the life cycle was fully understood. Pairing of cells and conjugation (fusion) of their contents were first documented by, Thwaites (1847a, b, 1848), with further observations in the next 10 years by Smith (1856), Griffith (1855) and Carter (1856), but the interpretation of the process was unclear. The products of conjugation were first thought to be spore-producing structures and Smith (1856) suggested that each enlarged cell divided up rapidly to form many small cells like those that had paired to form the ‘sporangium’ and he illustrated this in his plate C; his drawings are accurate, but they illustrate stages in the feeding of amoeboid protists, rather than diatom reproduction. It was not until 1869 that a clear account was provided of the mechanism of cell division in diatoms (MacDonald 1869; Pfitzer 1869; see also Crawford 1980), which leads (in most cases) to inexorable decline in cell size, hence showing the significance of the size restoration phase (Pfitzer 1871). Later, alternation of ploidy level, from diploid throughout the vegetative phase to haploid in the gametes, was confirmed by demonstration of meiosis during gametogenesis in Surirella by Surirella by Karsten (1912), and benthic diatoms were surveyed extensively over many years and shown to be morphologically isogamous, though sometimes behaviourally anisogamous (summarized by Geitler 1973; Geitler 1932). Finally, more than 100 years after Thwaites’ initial observations of sexuality in diatoms, von Stosch (1950; see also, for example, von Stosch & Drebes 1964; von Stosch et al. 1973) showed that some diatoms, especially planktonic species, do not conjugate in the same way as the benthic diatoms studied by Thwaites and others, but instead are oogamous. Even after this time, however, some (e.g., Margalef 1969) have questioned whether the size reduction–restitution cycle is as characteristic of diatoms as textbooks imply. There have been several reviews of sexual reproduction, auxospore formation, and life cycle dynamics in diatoms (Geitler 1973; Chepurnov et  al. 2004; Drebes 1977; Edlund & Stoermer 1997; Geitler 1932; Mann 2011; Round et  al. 1990) and more recently a paper (Kaczmarska et al. 2013) has been published that seeks to explain and standardize the terminology applied to these stages. We will not attempt to repeat or rephrase these accounts, since most of them are easily accessible and much of the information they contain is still valid. In the present chapter, we will restrict ourselves to reviewing some recent discoveries (especially, though not exclusively, since the review by Chepurnov et al. 2004) and making some general points that have perhaps received less attention than they deserve. We do not discuss the importance of the sexual phase

Diatom Sexual Reproduction and Life Cycles 247 in relation to population structure, which has begun to attract more attention in the last 15 years (e.g., Adams et al. 2009; Casteleyn et al. 2010; Chen & Rynearson 2016; Evans et al. 2009; Godhe et al. 2014; Harnstrom et al. 2011; Vanormelingen et al. 2015) and is critical for understanding speciation and the maintenance of diversity in diatoms.

11.2

Centric Diatoms

11.2.1 Life Cycle and Reproduction Traditionally, diatoms have been divided by taxonomists into two groups – centrics, with pattern (on the valves) organized radially about a circular or elongate ring (the ‘annulus’), and pennates, with pattern organized bilaterally about a rib-like ‘sternum’; the two groups were often recognized as orders (e.g., by Karsten 1928). This separation does not reflect phylogeny, in the sense that the centric diatoms are not monophyletic (e.g., Medlin, Kooistra, & Schmid 2000; Theriot et al. 2015), but it was and remains a convenient division, since it is based on easily visible features of valve pattern, has a broad relationship to habitat (most centrics are planktonic, most pennates are benthic), corresponds on the whole to a division based on the internal organization of the cell (most centrics have many small chloroplasts, most pennates have one, two or a few large chloroplasts), and, most important in the present context, reflects a fundamental difference in sexual reproduction: centrics exhibit oogamy, whereas pennates are isogamous (or very rarely anisogamous) and never produce sperm. In fact, flagella and centrioles have never been shown to exist at any stage of the life cycle in pennate diatoms and were presumably lost early in their evolution. However, while it seems to be true that oogamous auxosporulation is restricted to centric diatoms, this does not mean that all centric diatoms exhibit oogamy, nor that the characteristics of gametogenesis and auxospore development are uniform throughout, nor that a species or cell lineage that can restore size through oogamy will always do so. It is clear, for example, that some centric species are able to enlarge by auxospores produced non-oogamously, or through vegetative enlargement (vegetative enlargement is expansion of a cell after it has jettisoned its cell wall, without meiosis or fertilization and without the formation of the special protective and shaping structures, e.g. scales, perizonium, that are normally associated with auxosporulation). Furthermore, in at least one lineage that appears nested among other centric diatom lineages in current molecular phylogenies, oogamy has been replaced by a kind of nonflagellate isogamy highly reminiscent of what occurs in some araphid pennate diatoms (Davidovich et al. 2017). Non-oogamous auxosporulation and vegetative enlargement may be present instead of or as well as oogamy in a life cycle. Examples of asexually or autogamously produced auxospores are found in Coscinodiscus wailesii (Nagai et al. 1995), Thalassiosira angulata (Mills & Kaczmarska 2006) and Paralia guyana (Kaczmarska & Ehrman 2015); vegetative enlargement has been documented in Melosira moniliformis var. octogona (Drebes 1977), Coscinodiscus alboranii, (von Stosch 1985), Skeletonema spp. (Gallagher 1983), several species of Chaetoceros (von Stosch et al. 1973), and Ditylum brightwellii (von Stosch 1965; Koester et al. 2007). Von Stosch

248 Diatoms: Fundamentals and Applications (1965, 1986) noted that some populations of D. brightwellii seemed to restore size only or mostly via vegetative enlargement (e.g., in the German Bight of the North Sea and in Narragansett Bay, Rhode Island, USA), but that other populations were predominantly sexual (e.g., in Port Phillip Bay, SE Australia). However, interpretation is difficult because of the possibility that observations of natural populations of D. brightwellii refer to different cryptic species, growing either separately or together (Koester et al. 2010; Sharpe et al. 2012). Furthermore, although Koester et al. (2007) showed that individual clones of D. brightwellii from Maine, USA, could perform both oogamous auxosporulation and vegetative enlargement in culture, it is not yet known what relative contributions these two modes of enlargement make in natural populations. Mixed strategies of cell size restitution have also been documented recently in Skeletonema marinoi (Godhe et al. 2014). Another aspect of centric diatom life cycles that remains surprisingly unclear is the extent to which the cycle exhibits ‘cardinal points’, i.e., characteristic ranges of size in a species outside which particular sexual, physiological or developmental characteristics are not expressed. The concept of cardinal points is embedded in our current understanding of the diatom life cycle, largely as a result of the work of Geitler on pennate diatoms (especially Geitler 1932) and von Stosch in centric diatoms (see in particular, von Stosch 1951, 1956; von Stosch & Drebes 1964). Examples of cardinal points in pennate diatoms are the maximum size of cell produced through expansion of the auxospores (this maximum is a range), the maximum and minimum cell sizes at which cells can be sexualized and produce gametes (these cardinal points are threshold values) and the minimum size for viability (also a threshold value; the minimum viable size may or may not be the same as the minimum size for sexualization). Examples of cardinal points in centrics are the maximum cell size achieved following auxosporulation; the maximum and minimum size for production of egg cells; the maximum and minimum size for production of sperm; and the minimum viable cell size. To take one example, in Stephanopyxis turris the maximum cell diameter is close to 100 μm, the maximum size of cell capable of oogenesis something over 40 μm, the minimum size capable of gametogenesis 16 μm, and the minimum viable size 10 μm; the thresholds for spermatogenesis and oogenesis are the same, though the relative frequency of oogenesis is greater in larger cells (von Stosch & Drebes 1964). In Trieres (formerly Odontella) mobiliensis, the maxima and minima for spermatogenesis and oogenesis differ (von Stosch, 1956). Other centric species that are understood rather well with respect to ‘cardinal points’ include Melosira varians, Biddulphia rhombus, Odontella granulata and Chaetoceros didymum (von Stosch 1951, 1956; von Stosch et al. 1973), a diatom identified as Coscinodiscus pavillardii (Findlay 1969; the identification of this material is perhaps questionable: the micrographs illustrate a drum-shaped species resembling Coscinodiscus wailesii), and Skeletonema marinoi (Godhe et al. 2014). However, the existence of cardinal points has been established for very few species of centric diatoms (despite the relative ease with which centrics can be maintained in culture because of their homothallic nature) and in fact there are several reasons for doubting that the concept is as useful or applicable in centrics as in pennates. One reason is the demonstration of two- or three-step auxosporulation in several distantly related lineages of centrics. This phenomenon was included in the review by Chepurnov et al. (2004) but is still probably unknown by many phycologists. The most recently reported

Diatom Sexual Reproduction and Life Cycles 249 example is in Thalassiosira punctigera (Chepurnov et al. 2006), in which auxosporulation can take place in at least two steps. In culture, small cells 26–41 μm in diameter gave rise oogamously to initial cells 83–124 μm in diameter. After some divisions, the enlarged cells (measuring 92–120 μm) were themselves able to act as oogonia, producing auxospores that swelled to diameters of 132–153 μm. Moreover, Chepurnov et al. speculated that the upper size limit of 183 μm for T. punctigera reported by Hasle (1983) might indicate that a third step of expansion takes place in some circumstances. Similar phenomena have been reported in Melosira nummuloides (Schreiber 1931), M. moniliformis (Kustenko 1978), Coscinodiscus janischii (Roshchin 1994) and C. granii (Roshchin 1994). In all these cases, although there may be a lower limit to the size of cell that can reproduce sexually and a maximum size produced by the largest auxospores, it seems that the smallest cells cannot themselves give rise directly to the largest cells, but only to intermediate-sized cells, in contravention of the classic model of the diatom life cycle. Possibly the unexpectedly variable patterns of size change reported to occur in cultures of Thalassiosira weissflogii by Armbrust and Chisholm (1992) could also reflect the occurrence of multistep auxosporulation and the lack of true ‘cardinal points’. Inspection of molecular phylogenies (e.g., Theriot et al. 2015, fig. S1) indicates that the diatoms known to exhibit multi-step auxosporulation are not restricted to a single lineage (the Thalassiosirales, Coscinodiscales and Melosirales are not sister groups), showing that it cannot be assumed that either this type of life cycle, or the stricter ‘orthodox’ type of life cycle reported by von Stosch and colleagues (von Stosch 1951, 1956; von Stosch & Drebes 1964; von Stosch et al. 1973), is the prevalent one. Chepurnov et al. (2006) reported a further interesting phenomenon in Thalassiosira weissflogii, which may give hints as to how the isogamy of pennate diatoms (and that of the anomalous centric diatom Ardissonea: Davidovich et al. 2017b) may have arisen. This was the observation that the egg cells of T. weissflogii are capable of amoeboid movement: adjacent oogonia were seen to interact, the egg cell of one pushing inside the frustule of the other. A possibly similar observation was made by Findlay (1969) in Coscinodiscus pavillardii. Overall, then, the results of recent and older research suggest that the life cycles of centric diatoms may be more flexible and varied than was thought. One lesson to take from the observations described above is that no assumptions should be made a priori about the course of the life cycle in a particular centric species or population. Demonstration of a wide range of sizes in populations of a particular species may indicate that a size reduction–restitution cycle takes place (though it could also reflect the presence of different species with different but constant sizes, since size reduction is not inevitable in all diatoms: see Chepurnov et al. 2004), but it does not say anything about how restitution takes place. Oogamy is one possibility, but others are non-oogamous auxosporulation and vegetative enlargement. Furthermore, while formation of sperm is a prerequisite for oogamy in centric diatoms, the formation of sperm within a population does not mean that auxosporulation will take place oogamously, since examples are known where sperm are produced but auxospores are formed via autogamy (see Drebes 1977, p. 262; Chepurnov et  al. 2004). A rigorous demonstration of oogamy must include direct observation of spermatogenesis and oogenesis occurring simultaneously and either (1) observations of egg and sperm nuclei fusing (e.g., as done by von Stosch et al. 1973 in Chaetoceros didymum); or (2) detection via genetic evidence

250 Diatoms: Fundamentals and Applications that nuclear fusion has taken place (as done by Godhe et al. 2014, in Skeletonema marinoi). Oogamy is in fact extremely difficult to demonstrate conclusively; this is true even in culture but even more so in natural populations. On the other hand, it is also important to recognize that the absence of any observations of sexual reproduction in a species, either in culture or in natural populations, does not mean that it does not occur: Moore et al. (2017) have recently reported the induction of oogonia and sperm in Thalassiosira pseudonana, a species that previously seemed to be asexual (Chepurnov, Chaerle, Vanhoutte, & Mann 2012; Chepurnov et al. 2008), and there is now also genetic evidence for sexual lineages in this species, as well as for the recent evolution of an obligately asexual one that has apparently become globally distributed within a few hundred years (Koester et al. 2018).

11.2.2

Gametogenesis and Gamete Structure

In the last 12 years there have been few new reports about gametogenesis and gamete structure in centric diatoms. Spermatogenesis in diatoms has classically been classified into two categories according to whether the whole of the spermatocyte protoplast, including the chloroplasts, is apportioned among the four sperm produces after meiosis – this is hologenous spermatogenesis – or whether much of the spermatocyte cell, including the chloroplasts, are left behind in a ‘residual body’ – this is merogenous spermatogenesis. A variant type of merogeny, in which exclusion of the chloroplasts is left until the final stages of sperm formation has recently been documented by Samanta et al. (2018). The two classical patterns of spermatogenesis have been described and illustrated by Drebes (1977) and Round et al. (1990) and a recent paper by Idei et al. (2012) illustrates merogeny for Actinocyclus. It has sometimes been claimed (e.g., by Medlin & Kaczmarska 2004) that the occurrence of hologenous vs merogenous spermatogenesis has phylogenetic significance, each characterizing major lineages of centric diatoms, but this appears not to be the case: the two can occur even within the same genus (reviewed by Idei et al. 2012; Jensen et al. 2003; Mizuno 2008). In addition, it is evident that the binary classification into hologenous and merogenous spermatogenesis, as described in previous reviews (Drebes 1977; Round et al. 1990) is something of an oversimplification, since some diatoms exhibit what Idei et al. (2012) refer to as ‘plasmodial spermatogenesis’. In this, repeated mitotic divisions within an undivided spermatogonangium leads to the formation of a multinucleate plasmodium; each diploid nucleus undergoes meiosis within the plasmodium and the sperm are then budded off, leaving the plasmodium as a large anucleate mass. This type of spermatogenesis has recently been documented in some detail in Hydrosera by Hydrosera by Idei et al. (2015), but there are brief earlier reports of the same phenomenon in Pleurosira laevis, Aulacodiscus argus and Guinardia flaccida (Heath & Darley 1972; von Stosch & Drebes, 1964; Hoppenrath et al. 2009).The parallels with classical merogenous spermatogenesis are obvious, both types resulting in sperm that lack chloroplasts, and presumably therefore, in maternal inheritance of the chloroplast genome. As with hologeny and classical merogeny, the taxa exhibiting plasmodial spermatogenesis are not all closely related (Theriot et al. 2015, fig. S1); the selective advantages of plasmodial spermatogenesis, relative to ‘normal’ merogeny, are unknown.

Diatom Sexual Reproduction and Life Cycles 251 Sperm ultrastructure has recently been documented in two diatoms, Thalassiosira lacustris and Melosira moniliformis var. octogona by Idei et al. (2013), adding to the four – Lithodesmium undulatum, Pleurosira laevis, Coscinodiscus wailesii and Chaetoceros laciniosus – studied previously (Heath & Darley 1972; Jensen et al. 2003; Manton & von Stosch 1966). The number of studies is still very small, but several features noted earlier still seem to be general to diatoms, such as the absence of the central pair of microtubules in the flagellar axoneme (i.e., a 9 + 0 structure), the presence of nine doublets rather than triplets in the basal body, and the absence of a true flagellar root system. In contrast, the Bolidophyceae–Parmales, which comprise the sister group of the diatoms, have normal 9 + 2 flagella. However, whether 9 + 0 is common to all extant diatoms is still unknown: studies of Corethron and Leptocylindrus might be instructive, given their basal position in some molecular phylogenies (Theriot et al. 2015, fig. S1).

11.2.3 Spawning The function of sperm, of course, is to swim to and fertilize an egg cell. This process has been observed several times in the laboratory but in nature the dynamics and spatial patterns of egg and sperm formation and dispersal are a mystery. Oogamous centric diatoms are essentially ‘broadcast spawners’: the sperm and egg cells (whether set free from the oogonium or not) are free in the water column or film, where fertilization takes place. This might seem a risky strategy, since sperm and eggs could be quickly ‘diluted’ by turbulent movements much more rapid than the sperm can swim. In the case of benthic species, such as Melosira or Hydrosera, gametes may perhaps be concentrated temporarily and their contact facilitated by flows as suggested for invertebrate sperm and eggs by Crimaldi and Zimmer (2014), but this does not seem as likely for oogamous planktonic centrics like Thalassiosira weissflogii or Lithodesmium undulatum. Tight coupling of sexual reproduction to high cell densities, such as those present at the end of a bloom, may be part of the answer, as may be the ability to restore size through means other than sexual reproduction (discussed in relation to Skeletonema marinoi by Godhe et al. 2014). Coupling of sexual reproduction to bloom formation could be achieved by quorum sensing, positive feedback between cell density and sexualization mediated by pheromones (Godhe et al. 2014), or detection of external factors usually correlated with peak abundances (e.g., low levels of limiting nutrient: Jewson et al. 2008, indicate that low P may be important in the case of Aulacoseira skvortowii in Lake Baikal). Recently Moore et al. (2017) have suggested that high levels of ammonium could be involved in some cases, marking when a bloom has become sufficiently dense to support high numbers of grazers, the ammonium being released as the grazers feed (“We propose that ammonium released by grazers at bloom climax may be a principal ecological trigger for sexual morphologies in centric diatoms”: ibid., line 305). However, it has to be said that there is as yet rather little information about when sexual reproduction of centric diatoms occurs in nature. Crawford (1995) documented a mass sexual event in the marine planktonic diatoms Corethron and there are a few detailed studies of freshwater phytoplankton (e.g., Jewson & Granin 2015; Jewson 1992a, b) but such examples are rare, probably because the chances of detecting sexual events by microscopy are almost negligible without intensive sampling campaigns maintained for months or years. Metatranscriptomic approaches should lead to significant advances in

252 Diatoms: Fundamentals and Applications understanding reproduction in natural populations, for example by using markers of sexual reproduction such as the transcripts of the sig genes that code for elements of flagellar mastigonemes (Armbrust 1999; Yamagishi et al. 2009). It will be particularly interesting to see whether there are major differences in spawning behaviour between centric diatoms inhabiting the plankton and those living in the benthos, where it might be expected that the spatial and temporal dynamics of gamete formation, release and dispersal will be inherently more conducive to reproductive success.

11.3

Pennate Diatom Life Cycles and Reproduction

In the vast majority of pennates the gametes are morphologically similar. In the wider context of eukaryote sexual reproduction, the evolution of isogamy from oogamy, as must have occurred during the evolution of the pennates given their late origin from among centric diatom lineages, is surprising (Mann 1993). However, the isogamy found in most pennate diatoms is preceded by gametangial copulation and is connected with their benthic way of life. Pennate diatoms do not broadcast their gametes and thus stand in sharp contrast to centrics. Raphid diatoms in particular (but also at least some araphids: e.g. see Hopkins 1969; Sato & Medlin 2006) are motile and can occur in high densities in periphyton and epipelon, which permits a reproduction method based on the formation of small numbers of relatively large isogametes of limited motility (Mann 1993). Outstanding advances have been made in the last decade in our understanding of the biochemistry, genetic control and mechanisms of sexual reproduction in pennate diatoms, particularly the raphid diatom Seminavis (e.g., Bondoc et al. 2016; Gillard et al. 2013; Moeys et al. 2016; Patil et al. 2015; Vanstechelman et al. 2013; Basu et al. 2018). Sexualization and mate attraction have been demonstrated to be mediated by pheromones (Frankel et al. 2014; Gillard et al. 2013; Lembke et al. 2018; Moeys et al. 2016; Sato et al. 2011; see also the review by Venuleo et al. 2017), which can modify patterns of movement of mating cells by chemotaxis and chemokinesis (Bondoc et al. 2016). Gamete movement may also be guided by pheromones in pennates (Sato et al. 2011), though in other cases search seems to be through random walks (Edgar et al. 2014). In addition, some totally unexpected types of gamete movement and behaviour have been discovered in araphid pennates (Davidovich et al. 2012b; Edgar et al. 2014; Sato et al. 2011). Whereas previously the only known types of gamete movement and activity during fertilization in pennate diatoms were swelling and amoeboid movements (e.g., Geitler 1932; Mann & Stickle 1991, 1995), the ‘male’ gametes of some araphid pennates have now been shown to possess extensible projections that are involved in generating steady or jerky translocational or rotational movements (Davidovich et al. 2012b; Kaczmarska et al. 2017; Podunay et al. 2014; Sato et al. 2011). The ultrastructure of these projections has not been studied; they apparently contain microtubules but do not seem to be homologous to flagella (Sato et al. 2011). At least one group of centric diatoms – the extremely elongate genus Ardissonea – produces nonflagellate male gametes with similar projections (Davidovich et al. 2017a). Exploration of the genetic basis of sexual reproduction in pennate diatoms began with attempts to identify the mating type locus in the heterothallic diatom Seminavis

Diatom Sexual Reproduction and Life Cycles 253 robusta (Vanstechelman et  al. 2013) and searches for genes likely to be involved in meiosis in the genomes of three species (Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries, as well as the centric diatom Thalassiosira pseudonana) and the transcriptomes of two more (Seminavis robusta and Pseudonitzschia multistriata) (Patil et al. 2015). Undoubtedly, these studies are just the beginning of a new phase in our exploration of sexuality and auxosporulation in diatoms (e.g., see Basu et al. 2018). Genomic/transcriptomic evidence has already suggested that there may be significant differences between diatom meiosis and ‘classical’ meiosis (as determined for higher plants and opisthokonts), including possible differences in the mechanism of crossing over (Patil et al. 2015). They also hint that the ‘model’ diatom P. tricornutum may possess a sexual cycle, despite the absence until now (see Chepurnov et al. 2008) of any indication that it undergoes size-reduction and auxosporulation. The enormous variation in sexual reproduction and auxosporulation of pennate diatoms has been well known for more than 150 years. Geitler (1932) was already able to list over 90 records, classifying them into four main categories (derived from Hustedt 1930) and a number of subcategories according to characteristics such as the number of gametes produced per gametangium and the number of auxospores produced per mother cell. Subsequently, he developed a more detailed classification (Geitler 1973), dividing the main categories on the basis of gamete behaviour, the presence/absence of copulation structures, pairing methods, and auxospore orientation. However, although particular groups of related species or genera are sometimes characterized by a particular type of sexual reproduction, thus supporting or at least not contradicting classifications based on morphology and molecular evidence, it is not easy to find characters that continue to give a convincing phylogenetic signal at higher taxonomic levels. One example given by Mann (1993) was of Neidium and Biremis, which exhibit similar gametogenesis, plasmogamy and auxospore development; a close relationship between these genera has since been supported by molecular systematic data (Witkowski et al. 2014). One problem that sometimes makes it difficult to determine the evolution of sexual reproduction is that, because of taxonomic changes, sometimes we cannot be sure exactly which records of sexual reproduction belong to which species (e.g., Poulíčková & Mann 2006; Poulíčková et al. 2010), pointing to the importance of voucher material (e.g., Mann 2015). Although there has been rapid progress during the last two decades in our understanding of sexual reproduction in pennate diatoms, there is still no information for most diatom genera. Furthermore, a remarkable change has occurred in the sources of data on sexual reproduction. The vast majority of observations of pennate diatom reproduction made up till the 1990s were made on natural populations (e.g., Geitler 1973; Mann & Stickle 1995; Mann 1989), despite the long refractory periods in pennate diatom life cycles when cells cannot reproduce sexually because they are above the critical size threshold (e.g., Mann 2011). This dependence on natural populations (even by Geitler, who was largely responsible for initiating laboratory studies of sexual reproduction, for his 1932 treatise) greatly delayed the discovery that many pennate diatoms are heterothallic, but it had the advantage that the results of many more interclonal matings were surveyed than can normally be achieved in the laboratory and it gave some preliminary information about the seasonality of reproduction (though these data have yet to be reviewed and synthesized). Since the 1990s, especially

254 Diatoms: Fundamentals and Applications following the seminal work of (Roshchin 1994; see Chepurnov et al. 2004), data have come predominantly from laboratory experiments, which have allowed mating systems to be investigated and have provided information on the triggers of sexualization (e.g., Mouget et al. 2009) and also on the mechanisms through which cells stimulate and attract each other (Bondoc et al. 2016; Gillard et al., 2013; Moeys et al. 2016; Sato et  al. 2011), though not yet the ways in which cells recognize each other, which we suspect is the critical phase for speciation. The ability to attract cells of related species but not recognize them as ‘same’ is implied by observations such as those of Sellaphora capitata and S. blackfordensis by Mann et al. (1999) or Neidium ampliatum demes by Mann and Chepurnov (2005). Pennate diatoms have long been considered to be predominantly allogamous (biparental) sexual organisms (e.g., Chepurnov et al. 2004; Poulíčková et al. 2007) and this is probably true. However, some older generalizations have proved less reliable. For example, historically, all diatoms were thought to be homothallic, but whereas it does still appear that homothally is the rule in centric diatoms (the only known exception is Ardissonea: Davidovich et al. 2017), many pennates are proving to be heterothallic. The first records of heterothally were in three araphid pennates, Rhabdonema adriaticum (von Stosch 1958; Rozumek 1968), Grammatophora marina (von Stosch and Drebes, 1964) and Subsilicea fragilarioides (von Stosch and Reimann, 1970) and these were the only examples known up until Drebes’ (1977) review. Since then, however, examples of heterothallic diatoms have multiplied. In heterothallic pennate diatoms, sexual reproduction takes place between clones belonging to different mating types (Figure 11.1a–f). Where there is a difference in gamete behaviour between the mating types (“physiological” or “behavioural” anisogamy), it may be appropriate to talk about “male” vs “female”, or “+” and “–“ (Kaczmarska et  al. 2013), but there is sometimes no visible or behavioural difference between the gametangia and gametes produced by the different mating types (e.g., in Amphora: Mann and Poulíčková 2010). Recently, heterothally has been demonstrated in a wide range of raphid genera including Pseudo-nitzschia (e.g., Amato et al. 2005; Chepurnov et al. 2005; Scalco et al. 2016), Fragilariopsis (Fuchs et al. 2013), Cylindrotheca (Vanormelingen et al. 2013b), Neidium (Mann & Chepurnov 2005), Pinnularia (Poulíčková et  al. 2007), Haslea (2012a; Davidovich et al. 2009) and Schizostauron (Davidovich et al. 2017), adding to those already known by the time of Chepurnov et al.’s (2004) review. There have also been additional reports of heterothally in araphid pennates, such as Tabularia (Davidovich et  al. 2010), Ulnaria (Podunay et  al. 2014) and Plagiogrammaceae (Kaczmarska et al. 2017). Some species are not obligatorily heterothallic, since they exhibit some (usually rather limited) degree of intraclonal auxosporulation (e.g., Davidovich et al. 2010, and see below). Mating systems can vary between closely related species (e.g., within the same genus). Heterothallic and homothallic behaviour have been demonstrated in different species of Sellaphora and Pseudo-nitzschia (see Poulíčková et al. 2015, and QuijanoScheggia et al. 2009a, respectively), and both allomictic and automictic behaviour in different species of Neidium, Sellaphora, and Nitzschia (Mann et al. 2013; Poulíčková et al. 2015; Poulíčková 2008a; Rovira et al. 2015). Moreover, complex controls on mating behaviour are evident in Achnanthes longipes, where clones can be ‘monoecious’, ‘bisexual’ and ‘unisexual’ (Chepurnov & Mann 1997, 1999, 2000).

Diatom Sexual Reproduction and Life Cycles 255

Figure 11.1 Biparental reproduction in pennate diatoms: a–f Heterothallic reproduction in Pinnularia cf. gibba: a – sexualized cells in meiotic prophase (notice that cells belong to different clonal cultures differing in size), b – gametogenesis, c – gamete rearangement, d – early zygotes after gamete fusion, e – zygotes, f- expanding auxospores; g–j Homothallic reprodution in Luticola poulickovae Levkov, Metzeltin et Pavlov. g – pairing cells belonging to the same clonal culture, h – four gametes (two per gametangium), each with one chloroplast, i – two zygotes (each with two chloroplasts), j – expanding auxospores. Scale bar = 10 μm.

In Sellaphora and Nitzschia, it appears that automictic (uniparental) reproduction has evolved repeatedly from biparental ancestors (Geitler 1953; Poulíčková et al. 2015; Rovira et al. 2015) and, although we still do not have enough information (and what is available has not been subject to formal analysis), we believe that the wide distribution and inherently greater developmental complexity of heterothally is consistent with this state being primitive in pennate diatoms and that homothally and the different types of uniparental reproduction are derived. In both heterothallic and homothallic diatoms, it has been found that reproductive isolation is sometimes decoupled from morphological divergence. Habitual intraclonal reproduction has been observed in some species of the genus Navicula, for example the homothallic Navicula cryptocephala and N. lothargeitleri, which are reproductively isolated and form genetically differentiated sister clades but are almost indistinguishable on the basis of LM frustule morphology (Poulíčková & Mann 2006;

256 Diatoms: Fundamentals and Applications Poulíčková et al. 2016). A further related and morphologically similar species is the automictic N. trivialis (Poulíčková et al. 2010), in which each cell lineage is by definition reproductively isolated and comprises a separate ‘microspecies’. Microspecies (due to paedogamous automixis: see below) have been recorded also in Nitzschia sect. Lanceolatae (Rovira et al. 2015; Trobajo et al. 2006) and N. inconspicua has been found to comprise a complex of several clades, some of which are less closely related to each other than to morphologically very different diatoms (N. amphibia and Denticula kuetzingii) that have long been regarded as separate species (Rovira et al. 2015). Among heterothallic diatoms, evidence for reproductively isolated, cryptic or pseudocryptic species continues to accumulate, e.g. in Sellaphora (Vanormelingen et al. 2013a) and Pseudo-nitzschia (Quijano-Scheggia et al. 2009b). Incomplete reproductive isolation among sympatric Seminavis lineages has recently been analysed by De Decker et al. (2018). Sexual reproduction of homothallic clones is usually induced within a few days after inoculation into new medium (Idei et al. 2013) and can be so vigorous and rapid that the process may be missed if inspection of clones after isolation is not very frequent (e.g., Luticola: Poulíčková 2008b; Figure 11.1g–j). In theory, of course, the effects of such intraclonal reproduction can be detected, even if sexual reproduction itself is missed, by changes in cell size. Indeed, the proportion of small, sexually competent cells in a culture often declines rapidly due to vigorous intraclonal auxosporulation and there can be almost total replacement by enlarged initial and post-initial cells within a month or even a week (Poulíčková 2008b, and the authors’ unpublished observations of Pinnularia nodosa, Neidium cf. ampliatum, Nitzschia sp. and Sellaphora bisexualis). However, once the culture includes only newly enlarged cells, there may be no further sexual reproduction for months or years, until there are again sufficient cells below the sexual threshold. Hence it can be easy to overlook homothallic and other forms of intraclonal sexual reproduction. In the case of Luticola, vigorous rapid homothallic reproduction is perhaps understandable given its natural habitat – it grows aerophytically in caves (Poulíčková & Hašler 2007) – which is only intermittently wet and may provide only limited and sporadic opportunities to meet extraclonal sexual partners. However, homothallic species, e.g., S. bisexualis, do also occur in more mesic environments. In Diploneis pupula, intraclonal sexual reproduction was observed but some clones did not respond to replenishment of medium and remained vegetative (Idei et  al. 2013). In cases like this the possibility exists that the mating system is fundamentally heterothallic but with some capacity for intraclonal reproduction and such behaviour has been reported in Nitzschia, Tabularia, Fragilaria, Haslea and Ulnaria ( Davidovich, Kaczmarska, & Ehrman 2006; 2012a; Podunay et al. 2014; Roshchin 1994), and occurs also in S. blackfordensis (V.A. Chepurnov and D.G. Mann, unpublished observations). Roshchin (1994) interpreted this behaviour as an alternation of generations of both types of sexuality (monoecious, dioecious), but Davidovich et  al. (2006, 2010) reported intraclonal reproduction only within male clones of heterothallic Tabularia and Nitzschia, and proposed the term “facultative andromixis” for it (Davidovich et al., 2006; Kaczmarska et al. 2013). Possibly, facultative andromixis may represent an adaptive advantage for pioneering populations inhabiting new habitats, or a “last chance” for sexuality in populations in which heterothallic reproduction has not been initiated

Diatom Sexual Reproduction and Life Cycles 257 and which are consequently nearing the size threshold at which viabilty and/or sexual potency is lost (Davidovich et al. 2010). As with centric diatoms, the existence of a size reduction–restitution cycle does not imply that a population is sexual, with mating and sexual reproduction between cells. Auxospores can also be formed automictically (i.e., fusion takes place between gametes produced by a single cell) or apomictically. In both cases auxosporulation is ‘uniparental’: typically a single unpaired cell gives rise to a single auxospore, although pseudocopulation –involving pairing between cells that afterwards developed apomictically – has been recorded in Cocconeis (Geitler 1982). Uniparental reproduction has previously been considered infrequent (Chepurnov et al. 2004), but several new cases have been published quite recently within a variety of genera, including Achnanthes, Eunotia, Muelleria, Navicula, Neidium, Nitzschia and Pinnularia (Edlund & Spaulding 2006; Mann et al. 2013; Poulíčková et al. 2010; Poulíčková & Mann 2008; Poulíčková et al. 2015; Poulíčková 2008a; Rovira et al. 2015; Sabbe et al. 2004; Trobajo et al. 2006; Vanormelingen, Chepurnov, Mann, Sabbe, & Vyverman 2008). The principles of uniparental reproduction have been explained several times (e.g., Chepurnov et al. 2004). Briefly, two main forms of uniparental auxosporulation are known – apomixis and automixis. In apomixis, meiosis is absent or highly modified (‘pseudomeiosis’) and there is no sexual fusion of nuclei, whereas during automixis, a normal meiosis takes place, followed by fusion between two of the meiotic products. There are two variants of automixis (see Kaczmarska et al. 2013). In paedogamous automixis (Figure 11.2a– h), the unpaired parent cell forms two gametes after meiosis I, which fuse to form a diploid zygote (Mann et  al. 2013; Poulíčková 2008a). In autogamous automixis (Figure  11.2i–n), there is no cytokinesis after meiosis I and two haploid nuclei fuse within the undivided cell to create an auxospore. Not surprisingly, paedogamy has never been reported in centric diatoms, since sperm and egg cells are never formed in the same gametangium. In all cases known up to now in pennates, paedogamous reproduction occurs in genera where related biparentally reproducing species produce two gametes per gametangium (e.g., in Neidium and Nitzschia: Geitler 1970; Mann et al. 2013; Poulíčková & Mann 2008). In contrast, autogamy occurs in several species of Sellaphora, a genus in which most species are biparental and produce just one gamete per gametangium (Poulíčková et al. 2015). Ancestral character reconstructions suggest that Sellaphora is primitively biparental and that the uniparental auxosporulation evolved quite recently and probably several times (Poulíčková et al. 2015). Similarly, paedogamy has probably evolved at least twice in Nitzschia (Mann et al. 2013). Overall, automixis still seems to be uncommon in pennate diatoms, relative to biparental sexual reproduction, and occurs sporadically in unrelated lineages whose other members are allogamous (Mann et al. 2013; Trobajo et al. 2006). The form taken – paedogamy or autogamy – seems to depend to a large extent on the kind of behaviour present in the allogamous ancestor.

11.4

Auxospore Development and Structure

Besides all the peculiarities of the life cycle and sexuality mentioned above, one aspect of diatom auxosporulation has received less attention than it deserves, namely auxospore

258 Diatoms: Fundamentals and Applications

Figure 11.2 Uniparental reproduction in pennate diatoms: a–e Paedogamy in Nitzschia sp. a – gametes each with two chloroplasts, b – zygote with four chloroplasts, c–e – expanding auxospores; f–h Autogamy in Navicula trivialis. f – sexualized cell after meiosis II (with four nuclei), g – zygote , h- expanding auxospore with two unfused nuclei; i–n Autogamy in Pinnularia nodosa. i – sexualized cell in meiotic prophase, j – zygote (pathway I), k – pseudogametes (pathway II – pseudogametes never fuse: one or both die), l–n expanding auxospore. Scale bar = 10 μm.

development. This is the phase of the life cycle in which shape is generated de novo (e.g., Mann 1994) and understanding it is key to understanding how the huge diversity of diatom morphologies have arisen. However, although SEM images of auxospores first appeared in the literature 35 years ago (Mann 1982), and TEM and high quality LM images even earlier (von Stosch 1962, 1982), comparative studies of the ultrastructure of zygotes and auxospores are a relatively new field of interest.

Diatom Sexual Reproduction and Life Cycles 259

11.4.1 Incunabula It appears that the first event after fertilization is usually the formation by the young zygote of a polysaccharide layer (the primary wall), which may also contain silica scales. The scales are often flat,±round structures with an annulus resembling simplified centric valves, but sometimes scales are produced bearing dichotomously branching spines (von Stosch 1982; Samanta et al. 2017). It has been known for some time that auxospore development can involve the formation of either (1) scales alone (for example, see the recent account of Actinocyclus by Actinocyclus by Idei et al. 2012), or (2) scales together with a perizonium (sensu Kaczmarska et al. 2013) of shaping bands. It is possible that some auxospores have entirely organic walls, though as far as we know, this has never been proved: even in the delicate auxospores of Cylindrotheca, perizonial bands are present (Vanormelingen et al. 2013b). Elements formed before the growth of the auxospore are termed ‘incunabula’ (from the Latin for ‘swaddling clothes’ or ‘cradle’): in the words of Kaczmarska et al. (2013), “the primary zygote wall and parts that are subsequently added to the zygote wall (secondary elements) prior to its expansion as an auxospore compose the ‘incunabula’”. However, in Actinocyclus scales are present even around the egg cell (Idei et al. 2012), though many more are secreted to cover the zygote and expanding auxospore. Here the distinction between egg wall, incunabula and auxospore wall is unclear. In other diatoms, however, there is a clearer differentiation. For example, in Sellaphora species and Nitzschia inconspicua the incunabula contain round scales and this scale case is completed before auxospore expansion, whereas the auxospore wall is fortified by a full complement of transverse and longitudinal perizonial bands (Mann et al. 2011, 2013, and see below). In some Pinnularia and Nitzschia species, the incunabular elements include fine unpatterned strips, which are lightly silicified (Poulíčková & Mann 2008; Poulíčková et al. 2007; Trobajo et al. 2006). The strips are oriented approximately transverse to the long axis of the auxospore but criss-cross to some extent, sometimes resembling a “ball of wool” (Trobajo et al. 2006). The incunabula of the raphid diatom Neidium and its allies are particularly strongly silicified. In the best-known case (in a species of Neidium: Mann & Poulíčková 2009) there is a well-differentiated system of six incunabular elements – two polar caps and four lateral plates. The caps overlap the plates and might therefore be expected to be formed first, but development seems to take place more or less simultaneously and takes place while the zygotes are still enclosed within the gametangia. The ultrastructure of caps suggests that they are probably derived from fused scales. Later, the incunabula are split into two halves by the growth of the auxospore, each half comprising a cap and two of the lateral plates; these cover the ends of the auxospore and appear to ensure that it maintains a constant diameter as it expands. Similar incunabula seem to be present in Muelleria and Scoliopleura (Edlund & Spaulding 2006) and Biremis (Witkowski et al. 2014). The primary wall and incunabula are also split in two in other pennates, though it seems that the halves are not always as equal as in Neidium (e.g., Mann et al. 2011). The details of the incunabula and their development have rarely been established. Isodiametric scales overlying the perizonial bands have been reported several times in araphid pennates including Rhabdonema, Gephyria, Grammatophora, Pseudostriatella, and Tabularia (von Stosch, 1982; Sato et al. 2004, b, c, 2008a). Reports of such scales in

260 Diatoms: Fundamentals and Applications raphid diatoms have been infrequent but include Pseudo-nitzschia (Kaczmarska et al. 2000), Sellaphora (Mann et al. 2011) and Diploneis (Idei et al. 2013). The fact that scaly and strip incunabula can both be found in phylogenetically closely related groups, such as Sellaphora (scaly incunabula) and Pinnularia (strip incunabula; for the relationship between Sellaphora and Pinnularia, see Medlin & Kaczmarska 2004; Evans et al. 2008), and even within a single genus (e.g., Nitzschia: Mann et al. 2013; Trobajo et al. 2006), indicates that there has been convergent evolution of incunabula. This is further demonstrated by the occurrence of strip-like incunabula similar to Nitzschia and Pinnularia in the centric diatoms Hydrosera (Idei et al. 2015) and Ardissonea (Kaczmarska et al. 2018). Overall, it seems from the wide distribution of scaly incunabula in diatoms that this is the ancestral type (Mann et al. 2011); such coverings played an important part in the model of early diatom evolution put forward by Round and Crawford (1981) and invite comparison with the scale-case of the sister group of diatoms, the Parmales (Mann & Marchant 1989; Yamada et al. 2016).

11.4.2 Perizonium In ‘radial centrics’ belonging to different lineages, such as Ellerbeckia (Schmid & Crawford 2001), Actinocyclus (Idei et al. 2012), Paralia (Kaczmarska & Ehrman 2015), and Thalassiosira (Schmid 1984), flat round scales are the only silica structures added to the auxospore wall. However, during auxospore expansion some centric diatoms (the ‘polar’ centrics, with elongate, polygonal or multipolar valves) and the pennates create complex systems of silica bands and hoops, referred to as the ‘perizonium’. Its functions appear to be to create and control anisometric expansion of the auxospore. The perizonium of pennate diatoms normally consists of two series of bands, transverse and longitudinal (von Stosch 1962; Mann 1982). The perizonium of some centric diatoms (previously referred to as the ‘properizonium’, e.g. by von Stosch 1982: see Kaczmarska et al. 2013) is a little less clearly differentiated, while the highly elongate auxospores of the anomalous centric diatom Ardissonea are supported by a curious system of ‘scaly bands’ (Kaczmarska et al. 2018), whose morphology and exact arrangement are difficult to discern because of their delicacy. The transverse perizonium is produced sequentially by the expanding auxospore as transverse bands (primary and secondary) are added at its tips (e.g., Mann 1994). The timing of longitudinal perizonium formation is still unclear in most cases, since it is formed internal to the transverse perizonium and is therefore hidden in whole mounts of developing auxospores in SEM, and almost invisible in LM; it is only in species without transverse perizonial bands (see Toyoda, Williams, Tanaka, & Nagumo 2006; Mather et al. 2014; Toyoda, Idei, Nagumo, & Tanaka 2005) that the longitudinal perizonium is exposed. Remarkably, there have as yet been no thin section studies of auxospore development. However, in studies of Fragilariopsis kerguelensis using the fluorochrome PDMPO, Fuchs et al. (2013) showed that, as might have been expected from its position, the longitudinal perizonium is added only after the transverse perizonium is complete. It is also because of the internal position of the longitudinal perizonial bands (but often also because of their delicacy) that, whereas the transverse perizonium has been

Diatom Sexual Reproduction and Life Cycles 261 known for c. 150 years (e.g., Lüders 1862) and its nature reviewed several times (von Stosch, 1962; Chepurnov et  al. 2004; Kaczmarska et  al. 2013; Mann 1994; Round et  al. 1990), very little is known about the detailed morphology of the longitudinal perizonium. It was discovered by von Stosch (1962) and full accounts of its structure are available only for Rhabdonema (von Stosch, 1962), Rhoicosphenia (Mann 1982), Achnanthes (Idei & Mizuno 1996; Toyoda et al., 2005, 2006), Nitzschia (Mann et al. 2013), Diploneis (Idei et al. 2013) and Tabularia (Mather et al. 2014). In four of these (Rhabdonema, Rhoicosphenia, Nitzschia and Diploneis), despite their wide phylogenetic separation, the longitudinal perizonium has the same basic structure and comprises five bands – a median primary band flanked on each side by two secondary bands (discussed by Idei et  al. 2013). However, in the two known cases where the longitudinal perizonium is unaccompanied by a transverse perizonium (Achnanthes and Tabularia: Mather et al. 2014; Toyoda et al., 2005, 2006), the number of bands is higher. Despite the planar appearance of its bands, the longitudinal perizonium can be a highly three-dimensional structure (Idei et al. 2013). Its function is unknown; Idei et al. (2013) speculate that “possibly it helps in the dehiscence of the auxospore to allow the initial cell to escape by providing a tongue-like brace against which the initial cell can press to open the transverse series and hence effect its escape” from the auxospore.

11.5

Induction of Sexual Reproduction

Sexual reproduction is required in diatoms for size restoration after the long period of vegetative division. One precondition for successful sexual reproduction in pennate diatoms is the occurrence of cells below the critical size threshold (Chepurnov et al. 2004) and another is the availability of compatible sexual partners. However, since populations of individual diatom species often consist mainly of relatively small cells that are below the critical size threshold (see Mann 2011), it is clear that the preconditions will often be met, providing that population densities are high enough, implying that external factors may be highly influential in controlling the occurrence of sexual reproduction. However, the nature of these factors and how they operate in particular populations are still very poorly understood; this situation is similar also in other algal groups, for instance the Zygnematophyceae (Zwirn et al. 2013). Light, temperature, and nutrients have all been shown to be influential in different species, and biotic factors (e.g., the presence of particular bacteria) have also been shown to have an effect in particular cases. The earlier literature has been reviewed by Drebes (1977) and Chepurnov et al. (2004). In the laboratory, low light levels and a short photoperiod favoured auxosporulation in Haslea ostrearia (Mouget et al. 2009), corresponding to conditions in winter and early spring at the site from which clones were isolated. Davidovich et al. (2018) have shown that summer temperatures also inhibit sexual reproduction in Haslea ostrearia and H. karadagensis, but allow rapid vegetative growth. Unfortunately, as with almost all other diatom species, there seem to be no data on Haslea sexual reproduction in the natural environment. Light conditions have also been found to be important for auxosporulation in Pseudo-nitzschia multiseries (Hiltz et al. 2000), Rhabdonema adriaticum (Rozumek 1968) and Cocconeis scutellum (Mizuno & Okuda 1985). However,

262 Diatoms: Fundamentals and Applications auxosporulation was apparently unaffected by light in other diatoms (Davidovich & Chepurnov 1993; Davidovich 2002). Continuous light has been shown to suppress sexual reproduction in Haslea and the centric diatom Chaetoceros (Furnas 1985; Mouget et al. 2009; Roshchin 1976), but to favour auxosporulation in other centrics (Cyclotella and Coscinodiscus: Schulz & Trainor 1968; Roshchin 1972, 1976). Photoperiod seems to be important for sexualization of some diatoms (Vaulot & Chisholm 1987), perhaps through synchronization of the cell cycle (Mouget et  al. 2009). Two pathways of autogamy occurred in Pinnularia nodosa and their relative frequency seemed to be influenced by light conditions (Poulíčková & Mann 2008). In an early publication (Baatz 1941), light quality (wavelength) was reported to have an effect of auxosporulation in Chaetoceros (Baatz 1941) and red light seems to be necessary for sexualization in Haslea ostrearia (Mouget et al. 2009). In responding to light quality, diatoms resemble other algae, both those that are relatively closely related and those belonging to different supergroups; examples are gamete release in the brown alga Silvetia compressa (Pearson et al. 2004), zoid production in Scenedesmus (Cepák & Přibyl 2006), and sexualization in Zygnematophyceae (Zwirn et al. 2013). In other algae, nutrient levels have been shown to be important for eliciting a sexual response, e.g. sexual induction in Zygnematophyceae and zoid production in Klebsormidium and Scenedesmus (Cepák & Přibyl 2006; Lokhorst 1996; Zwirn et al. 2013). As already mentioned (under “Spawning”), some centric diatom species also respond to nutrient levels – to high levels of ammonium in Thalassiosira (Moore et al. 2017), replenishment of nitrate in Stephanodiscus sp. (Jewson 1992b), and apparently to low levels of phosphorus in Aulacoseira skovortzowii (Jewson et al. 2008), though in A. subarctica Jewson (1992a) has suggested that the cue for sexual reproduction is a check to growth imposed by low light levels. On the other hand, Chepurnov et al. (2004) stated that “as yet, no case is known in pennate diatoms where sexual reproduction is triggered by severe nutrient (e.g., N or P) depletion” and the papers published on pennate mating systems since 2004 generally record that sexualization occurred in exponentially growing cultures. A particularly detailed study of sexualization and reproductive success has been made by Scalco et  al. (2014) in a marine planktonic species (Pseudo-nitzschia multistriata) that exhibits pronounced seasonal variation in abundance and might therefore be expected, perhaps, to respond to variations in nutrient levels. They recorded that the principal requirements for successful auxosporulation, besides the presence of compatible cells at the right stage in the life cycle, were unlimited growth (successful reproduction dropped off rapidly towards the end of the exponential phase), high cell density (ensuring frequent cell–cell encounters), and absence of a vigorous mixing regime.

Acknowledgments The main portion of the research on diatom reproduction of Aloisie Poulíčková was supported by projects GACR 206/07/0115 from the Czech Republic, Internal grant agency of Palacký University Prf-2018–001 and EU Framework 6 SYNTHESYS award (GB-TAF-643). David Mann is grateful to the Marine and Continental Waters Program, IRTA, Sant Carles de La Ràpita, for providing facilities. The Royal Botanic

Diatom Sexual Reproduction and Life Cycles 263 Garden Edinburgh (RBGE) is supported by the Scottish Government’s Rural and Environmental Science and Analytical Services Division.

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Diatom Sexual Reproduction and Life Cycles 269 Pfitzer, E. (1871). Untersuchungen über Bau und Entwickelung der Bacillariaceen (Diatomaceen). In Botanische Abhandlungen . 1. pp. 1–189. Hanstein (ed). Podunay, Y.A., Davidovich, O.I., Davidovich, N.A. (2014). Mating system and two types of gametogenesis in the fresh water diatom Ulnaria ulna. 24. pp. 3–18. Algologiya: Bacillariophyta. Poulíčková, A., Veselá, J., Neustupa, J., Škaloud, P. (2010). Pseudocryptic diversity versus cosmopolitanism in diatoms: a case study on Navicula cryptocephala Kütz. (Bacillariophyceae) and morphologically similar taxa, Protist, 161(3), 353–369. Poulíčková, A., Mann, D.G. (2008). Autogamous auxosporulation in Pinnularia nodosa (Bacillariophyceae), J. Phycol., 44(2), 350–363. Poulíčková, A. (2008a). Pedogamy in Neidium (Bacillariophyceae, Folia Microbiol. (Praha)., 21, 125–129. Poulíčková, A. (2008b). Morphology, cytology and sexual reproduction in the aerophytic cave diatom Luticola dismutica (Bacillariophyceae), Preslia, 80, 87–99. Poulíčková, A., Hašler, P. (2007). Aerophytic diatoms from caves in central Moravia (Czech Republic), Preslia, 79, 185–204. Poulíčková, A., Mann, D.G. (2006). Sexual reproduction in Navicula cryptocephala (Bacillariophyceae), J. Phycol., 42(4), 872–886. Poulíčková, A., Mayama, S., Chepurnov, V.A., Mann, D.G. (2007). Heterothallic auxosporulation, incunabula and perizonium in Pinnularia (Bacillariophyceae), Eur. J. Phycol., 42(4), 367–390. Poulíčková, A., Neustupa, J., Hašler, P., Tomanec, O., Cox, E.J. (2016). A new species, Navicula lothargeitleri sp. nov., within the Navicula cryptocephala complex (Bacillariophyceae, Phytotaxa, 273(1), 23–33. Poulíčková, A., Sato, S., Evans, K.M., Chepurnov, V.A., Mann, D.G. (2015). Repeated evolution of uniparental reproduction in Sellaphora (Bacillariophyceae, Eur. J. Phycol., 50(1), 62–79. Quijano-Scheggia, S., Garcés, E., Andree, K., Fortuño, J.M., Camp, J. (2009a). Homothallic auxosporulation in Pseudo-Nitzschia brasiliana (Bacillariophyta), J. Phycol., 45(1), 100–107. Quijano-Scheggia, S.I., Garcés, E., Lundholm, N., Moestrup, Øjvind., Andree, K., Camp, J. (2009b). Morphology, physiology, molecular phylogeny and sexual compatibility of the cryptic Pseudo-nitzschia delicatissima complex (Bacillariophyta), including the description of P. arenysensis sp. nov, Phycologia, 48(6), 492–509. Roshchin, A.M. (1972). Vliyanie uslovij osveshcheniya na obrazovanie auksospor i skorost deleniya kletok Coscinodiscus granii Gough, Fiziologiya Rastenij, 19, 180–185. Roshchin, A.M. (1976). Vliyanie uslovij osveshcheniya na vegetativnoe razmnozhenie kletok i polovoe vosppoizvedenie dvuh vidov tsentricheskih diatomovyh vodoposlej, Fiziologiya Rastenij, 23, 715–719. Roshchin, A.M. (1994). Zhiznennye tsikly diatomovyh vodoroslej. Kiev: Naukova Dumka. Round, F.E., Crawford, R.M. (1981). The Lines of Evolution of the Bacillariophyta. I. Origin, Proceedings of the Royal Society B: Biological Sciences, 211(1183), 237, B 211–260. Round, F.E., Crawford, R.M., Mann, D.G. (1990). The diatoms. Biology and morphology of the genera. Cambridge: Cambridge University Press. Rovira, L., Trobajo, R., Sato, S., Ibáñez, C., Mann, D.G. (2015). Genetic and Physiological Diversity in the Diatom Nitzschia inconspicua, J. Eukaryot. Microbiol., 62(6), 815–832. Rozumek, K.E. (1968). Der Einfluß der Umweltfaktoren Licht und Temperatur auf die Ausbildung der Sexualstadien bei der pennaten Diatomee Rhabdonema adriaticum Kütz, Beiträge zur Biologie der Pflanzen, 44, 365–388. Sabbe, K., Chepurnov, V.A., Vyverman, W., Mann, D.G. (2004). Apomixis in Achnanthes (Bacillariophyceae); development of a model system for diatom reproductive biology, Eur. J. Phycol., 39(3), 327–341.

270 Diatoms: Fundamentals and Applications Samanta, B., Heffell, Q., Ehrman, J.M., Kaczmarska, I. (2018). Spermatogenesis in the bipolar centric diatom Plagiogrammopsis vanheurckii (Mediophyceae, Phycologia, 57(3), 354–359. Samanta, B., Kinney, M.E., Heffell, Q., Ehrman, J.M., Kaczmarska, I. (2017). Gametogenesis and auxospore development in the bipolar centric diatom Brockmanniella brockmannii (family Cymatosiraceae, Protist, 168(5), 527–545. Sato, S., Beakes, G., Idei, M., Nagumo, T., Mann, D.G. (2011). Novel sex cells and evidence for sex pheromones in diatoms, PLoS ONE, 6(10), e26923. Sato, S., Mann, D.G., Nagumo, T., Tanaka, J., Tadano, T., Medlin, L.K. (2008a). Auxospore fine structure and variation in modes of cell size changes in Grammatophora marina (Bacillariophyta), Phycologia, 47(1), 12–27. Sato, S., Mann, D.G., Matsumoto, S., Medlin, L.K. (2008b). Pseudostriatella (Bacillariophyta): a description of a new araphid diatom genus based on observations of frustule and auxospore structure and 18S rDNA phylogeny, Phycologia, 47(4), 371–391. Sato, S., Kuriyama, K., Tadano, T., Medlin, L.K. (2008c). Auxospore fine structure in a marine araphid diatom Tabularia parva (Bacillariophyta), Diatom Research, 23(2), 423–433. Sato, S., Medlin, L.K. (2006). Motility of non-raphid diatoms, Diatom Research, 21(2), 473–477. Sato, S., Nagumo, T., Tanaka, J. (2004). Auxospore formation and the morphology of the initial cell of the marine araphid diatom Gephyria media (Bacillariophyceae), J. Phycol., 40(4), 684–691. Scalco, E., Amato, A., Ferrante, M.I., Montresor, M. (2016). The sexual phase of the diatom Pseudo-nitzschia multistriata: cytological and time-lapse cinematography characterization, Protoplasma, 253(6), 1421–1431. Schmid, A.-M.M. (1984). Wall morphogenesis in Thalassiosira eccentrica: comparison of auxospore formation and the effect of MT-inhibitors. In Proceedings of the Seventh International Diatom Symposium. pp. 47–70. D.G. Mann (ed). Koenigstein: Koeltz Scientific Books. Scalco, E., Stec, K., Iudicone, D., Ferrante, M.I., Montresor, M. (2014). The dynamics of sexual phase in the marine diatom Pseudo-nitzschia multistriata (Bacillariophyceae), J. Phycol., 50(5), 817–828. Schmid, A.-M.M., Crawford, R.M. (2001). Ellerbeckia arenaria (Bacillariophyceae): formation of auxospores and initial cells, Eur. J. Phycol., 36(4), 307–320. Schreiber, E. (1931). Über Reinkulturversuche und experimentelle Auxosporenbildung bei Melosira nummuloides, Archiv für Protistenkunde, 73, 331–344. Schultz, M.E., Trainor, F.R. (1968). Production of male gametes and auxospores in the centric diatoms Cyclotella meneghiniana and C. cryptica, J. Phycol., 4(2), 85–88. Sharpe, S.C., Koester, J.A., Loebl, M., Cockshutt, A.M., Campbell, D.A., Irwin, A.J., et al. (2012). Influence of cell size and DNA content on growth rate and photosystem II function in cryptic species of Ditylum brightwellii, PLoS ONE, 7(12), e52916. Smith, W. (1856). Synopsis of British Diatomaceae, vol. 2. J. London: van Voorst. Stosch, H.A.V. (1951). Entwicklungsgeschichtliche Untersuchungen an zentrischen Diatomeen I. Die Auxosporenbildung von Melosira varians, Archiv für Mikrobiologie, 16, 101–135. Stosch, H.A.V. (1956). Entwicklungsgeschichtliche Untersuchungen an zentrischen Diatomeen II. Geschlechtszellenreifung, Befruchtung und Auxosporenbildung einiger grundbewohnender Biddulphiaceen der Nordsee, Archiv für Mikrobiologie, 23, 327–365. Stosch, H.A.V. (1958). Kann die oogame Araphidee Rhabdonema adriaticum als Bindeglied zwischen den beiden grossen Diatomeengruppen angesehen werden? Ber. Dtsch. Bot. Ges., 71, 241–249. Stosch, H.A.V. (1962). Über das Perizonium der Diatomeen, Vorträge aus dem Gesamtgebiet der Botanik, 1, 43–52.

Diatom Sexual Reproduction and Life Cycles 271 Stosch, H.A.V., Drebes, G. (1964). Entwicklungsgeschichtliche Untersuchungen an zentrischen Diatomeen, IV. Die Planktondiatomee Stephanopyxis turris – ihre Behandlung und Entwicklungsgeschichte. Helgoländer wissenschaftliche Meereuntersuchungen, 11, 209–257. Stosch, H.A.V. (1982). On auxospore envelopes in diatoms, Bacillaria, 5, 127–156. Stosch, H.A.V. (1985). Some marine diatoms from the Australian region, especially from Port Phillip Bay and tropical north-eastern Australia, Brunonia, 8, 293–348. Stosch, H.A.V. (1986). Some marine diatoms from the Australian region, especially from Port Phillip Bay and tropical north-eastern Australia. II Survey of the genus Palmeria and of the family Lithodesmiaceae including the new genus Lithodesmioides, Brunonia, 9, 29–87. Stosch, H.A.V. (1966). Manipulierung der Zellgrösse von Diatomeen im Experiment 1, Phycologia, 5(1), 21–44. Stosch, H.Avon., Reimann, B.E.F. (1970). Subsilicea fragilarioides gen. et spec. nov., eine Diatomee (Fragilariaceae) mit vorwiegend organischer Membran, Nova Hedwigia, Beiheft, 31, 1–36. Stosch, H.A., Theil, G., Kowallik, K.V. (1973). Entwicklungsgeschichtliche Untersuchungen an zentrischen Diatomeen, Helgolander. Wiss. Meeresunters., 25(2-3), 384–445. Theriot, E.C., Ashworth, M.P., Nakov, T., Ruck, E., Jansen, R.K. (2015). Dissecting signal and noise in diatom chloroplast protein encoding genes with phylogenetic information profiling, Mol. Phylogenet. Evol., 89, 28–36. Thwaites, G.H.K. (1847a). On conjugation in the Diatomaceae, Annals and Magazine of Natural History, 20, 9–11. Thwaites, G.H.K. (1847b). On conjugation in the Diatomaceae, Annals and Magazine of Natural History, 20, 343–344. Thwaites, G.H.K. (1848). XVI.— Further observations on the Diatomaceæ; with descriptions of new genera and species, Annals and Magazine of Natural History, 1(1), 161–172. Toyoda, K., Idei, M., Nagumo, T., Tanaka, J. (2005). Fine-structure of the vegetative frustule, perizonium and initial valve of Achnanthes yaquinensis (Bacillariophyta), Eur. J. Phycol., 40(3), 269–279. Toyoda, K., Williams, D.M., Tanaka, J., Nagumo, T. (2006). Morphological investigations of the frustule, perizonium and initial valves of the freshwater diatom Achnanthes crenulata Grunow (Bacillariophyceae, Phycological Res., 54(3), 173–182. Trobajo, R., Mann, D.G., Chepurnov, V.A., Clavero, E., Cox, E.J. (2006). Taxonomy, life cycle, and auxosporulation of Nitzschia fonticola (Bacillariophyta), J. Phycol., 42(6), 1353–1372. Vanormelingen, P., Chepurnov, V.A., Mann, D.G., Sabbe, K., Vyverman, W. (2008). Genetic divergence and reproductive barriers among morphologically heterogeneous sympatric clones of Eunotia bilunaris sensu lato (Bacillariophyta), Protist, 159(1), 73–90. Vanormelingen, P., Evans, K.M., Chepurnov, V.A., Vyverman, W., Mann, D.G. (2013a). Molecular species discovery in the diatom Sellaphora and its congruence with mating trials, Fottea, 13(2), 133–148. Vanormelingen, P., Evans, K.M., Mann, D.G., Lance, S., Debeer, A.E., D'Hondt, S, D’Hondt, S., et al. (2015). Genotypic diversity and differentiation among populations of two benthic freshwater diatoms as revealed by microsatellites, Mol. Ecol., 24(17), 4433–4448. Vanormelingen, P., Vanelslander, B., Sato, S., Gillard, J., Trobajo, R., Sabbe, K., et  al. (2013b). Heterothallic sexual reproduction in the model diatom Cylindrotheca, Eur. J. Phycol., 48(1), 93–105. Vanstechelman, I., Sabbe, K., Vyverman, W., Vanormelingen, P., Vuylsteke, M. (2013). Linkage mapping identifies the sex determining region as a single locus in the Pennate diatom Seminavis robusta, PLoS ONE, 8(3), e60132. Vaulot, D., Chisholm, S.W. (1987). Flow cytometric analysis of spermatogenesis in the diatom Thalassiosira weissflogii (Bacillariophyceae), J. Phycol., 23(1), 132–137.

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12 Ecophysiology, Cell Biology and Ultrastructure of a Benthic Diatom Isolated in the Arctic Ulf Karsten1,*, Rhena Schumann1 and Andreas Holzinger2 1

Institute of Biological Sciences, Applied Ecology and Phycology, University of Rostock, Rostock, Germany 2 Department of Botany, Functional Plant Biology, University of Innsbruck, Innsbruck, Austria

Abstract Benthic diatoms are common and abundant components of soft bottom microphytobenthic communities in the shallow water coastal zones of the Arctic region. This phototrophic community plays an ecological key role as high primary producer, as major food source for benthic suspension or deposit feeders, as filter for oxygen and other elemental fluxes at the sediment/water interface and as stabilizer of sediment surfaces by the excretion of extracellular polymeric substances. Consequently, benthic diatoms represent a key component in the functioning of trophic webs in many Arctic coastal regions. The Arctic exhibits strong seasonally fluctuating environmental conditions which have strong implications for primary production and seasonal growth of benthic diatoms. Particularly temperature and the polar night act as stressors, and hence their effects on the physiology and cell biology of Navicula directa isolated from a subtidal rock of Kongsfjorden, Svalbard was studied in detail. All data indicate that this representative and abundant species is well adapted to the harsh environmental conditions in the Arctic. Keywords: Arctic, cell activity, cell viability, chloroplast, dark survival, growth, mitochondria, polar night, respiration, storage lipids, survival, temperature, viability dyes

*Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (273–288) © 2019 Scrivener Publishing LLC

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274 Diatoms: Fundamentals and Applications

12.1

Introduction

Diatoms play an ecological key role in the oceans, not only in the planktonic realm but also in the benthic habitat particularly on soft bottoms. Here, they contribute as pennate forms in high abundance to microphytobenthic communities, which often cover extensive sediment areas. These unicellular photoautotrophs excrete sticky extracellular polymeric substances (EPS), thereby stabilizing the sediment against erodibility under hydrodynamic forces (de Brouwer, Wolfstein, Ruddy, Jones, & Stal 2005). In addition, benthic diatoms modify the surface properties of sediments, and hence affect the horizontal exchange of oxygen and nutrients across the sediment-water interface (Petersen, Rysgaard, Nielsen, & Revsbech 1994). Benthic and pelagic diatoms are responsible for 25% of global carbon fixation (Smetacek 1999) and contribute to 45–50% to the total marine primary production (Mann 1999; Yool & Tyrrell 2003). There are many reports on the generally high primary production of benthic diatoms worldwide (Cahoon 1999), resulting in up to 50% contribution to the total production in different estuaries and tidal flats (Underwood & Kromkamp 1999). The microalgae in these rather temperate habitats, however, exhibit large variations of cell numbers at the sediment surface during periods of emersion as a result of vertical migration within the sediment matrix (MacIntyre, Geider, & Miller 1996; Paterson & Hagerthey 2001). Studies on microphytobenthic communities in the Arctic are still rare, but the very few point to high rates of primary production which can be compared with those in temperate regions (Glud, Woelfel, Karsten, Kühl, & Rysgaard 2009; Woelfel, Eggert, & Karsten 2014; Glud, Kühl, Wenzhöfer, & Rysgaard 2002; Sevilgen, de Beer, Al-Handal, Brey, & Polerecky 2014; Woelfel et al. 2010). Consequently, benthic diatom assemblages represent, although still badly studied, a key component in the functioning of trophic webs on soft substrates in Arctic coastal regions. Although higher trophic levels in the Arctic are well described (e.g., Hop et al. 2002), information on the ecophysiology and cell biology of benthic diatoms is still scarce. Therefore, we aim to fill this gap by investigating ecophysiological and cell biological traits of an Arctic diatom.

12.2

Environmental Settings in the Arctic

Low temperatures, seasonally fluctuating solar radiation, and long periods of ice and snow cover are the key environmental factors controlling primary production in the Arctic (Hop et al. 2002). Benthic diatoms experience only short periods with sufficient light over the course of the year with the polar day ranging from mid-April till end of August and twilight conditions prevailing in spring and autumn (Svendsen et al. 2002). The polar night lasts for about four months from end October to mid of February, and the annual solar radiation at 80°N is about one third less compared to mid latitudes. Additionally, these long periods of darkness can be further extended, for example, in the inner parts of protected bays and fjords by sea ice formation during the winter, with a varying break-up of the sea ice between spring and early summer (e.g., Svendsen et al. 2002). If the ice is also covered by snow, solar radiation can be diminished to less than 2% of the surface value. Consequently, phototrophic benthic communities may

Ecophysiology, Cell Biology and Ultrastructure 275 be exposed up to about 10 months of darkness or very low light conditions (Chapman & Lindley 1980; Dunton 1990). Such extreme fluctuating light conditions in Arctic waters have strong implications for primary production and seasonal growth of benthic diatoms. Although solar radiation exhibits extreme seasonal qualitative and quantitative variability in the Arctic, the annual patterns (e.g., of day lengths) are unaffected by global change. In contrast, Arctic water masses have generally warmed by 0.7 °C to 1.2 °C per decade since 1981 (Serreze & Francis 2006), with unknown consequences for benthic diatoms and other marine organisms (Hegseth & Tverberg 2013). The mean surface temperatures in the Arctic Ocean can geographically vary depending on regular warm water intrusions from the Northern Atlantic (e.g., by the West Spitzbergen Current). In the Fram Strait (78 ° 50’ N), for example, water masses on the west coast of Spitzbergen (9 °E) exhibit 5 °C while along the east coast of Greenland (8 °W) −2 °C were recorded based on measurements made between September 2002 to August 2003 (Schauer et al. 2008). The signs of continuing warming of the Arctic Ocean are numerous, and are reflected in raised surface air temperatures, a retreat and thinning sea ice cover, a longer melt season and increasing river discharges (Bintanja & van der Linden 2013; Overland, Wang, Walsh, & Stroeve 2014). Locally, the annual mean temperature from June 2011 until June 2012 was 2.4 ± 2.1 °C at a shallow (5 m water depth) coastal sandy sediment site in Kongsfjorden (Svalbard), which exhibits a diverse and dense microphytobenthic community (Sevilgen et al. 2014). However, during summer, water temperatures can temporarily increase to >8 °C at this station (Sevilgen pers. comm.). In contrast, the temperatures in deeper waters > 5 m in Kongsfjorden were relatively constant, ranging from 1 °C to 5 °C from June through August in 2011 (Woelfel et al. 2014).

12.3

Growth as Function of Temperature

From an ecophysiological view, growth response patterns represent the most relevant process to describe the performance of benthic diatoms under a gradient of temperatures because they optimally integrate all positive and negative environmental effects on the cell and hence reflect their acclimation potential (Gustavs, Schumann, Eggert, & Karsten 2009). Benthic diatoms in the Arctic are preferentially growing in shallow waters on top of sediments and hard substrata at low, but relatively constant temperatures. The first ecophysiological study on growth of Arctic benthic diatoms were undertaken on two Fragilaria species, isolated as epiphytes on seaweeds collected in Kongsfjorden (Svalbard). Both taxa showed optimum growth rates at 12–14 °C, grew still well but with reduced rates at 0 °C and did not survive 20 °C (Karsten, Schumann, Rothe, Jung, & Medlin 2006). In contrast, Navicula directa (W.Smith) Ralfs, which was isolated from sublittoral rocks in Kongsfjorden, grew between 1 °C and 15 °C with very similar rates, and thus exhibited a broad growth optimum (Figure 12.1). However, exposure to 20 °C led to high mortality (Figure 12.1). More recently, Schlie & Karsten (2017) evaluated eight benthic diatom species isolated from the Adventfjorden (Svalbard) concerning their temperature requirements for growth under controlled conditions between 1 °C and 20 °C. Five out of 8

276 Diatoms: Fundamentals and Applications

Growth rate ( d−1)

0.9

0.6 a a

a

a

0.3

0 0

5

10 15 Temperature (°C)

20

25

Figure 12.1 The effect of increasing temperatures on the growth rate of the benthic diatom Navicula directa isolated from subtidal rocks of Kongsfjorden, Svalbard. Cells were grown in sterilized Baltic seawater enriched with sea salt (Sel marin hw professional, Wiegandt GmbH, Krefeld, Germany), vitamins and silicate resulting in a salinity of 33 PSU, and kept at 30 μmol photons m-2 s-1 under a 16:8 hr light:dark cycle. Data represent mean values ± SD (n = 4) and were fitted according to Blanchard, Guarini, Richard, Gros and Mornet (1996).

of the investigated species grew between 1 °C and 20 °C, but with different rates, while growth of 2 isolates was inhibited at 1 °C and of 1 strain at 20 °C. In addition, Schlie & Karsten (2017) reported that only Grammonema rostockensis Stachura-Suchoples and Nitzschia sp. aff. dubiiformis Hustedt exhibited partly low-temperature requirement for growth, while all other examined benthic diatoms showed higher growth potential at elevated temperatures. Consequently, the general response patterns for most taxa clearly showed a preference for 6–15 °C, pointing to rather eurythermal and psychrotolerant growth behaviour of Arctic benthic diatoms (Schlie & Karsten 2017). For the Arctic diatom Navicula directa, increased growth rates were observed between 0.5 °C and 4.5 °C (Torstenssson et al. 2012), but higher temperatures have not been tested. In contrast to Arctic diatoms, the characteristic features of Antarctic benthic diatoms are very low-temperature requirements for growth in conjunction with an often narrow temperature tolerance. For Antarctic isolates of Gyrosigma subsalinum Peragallo and Odontella litigiosa (van Heurck) Hoban optimum growth rates were measured at 0 °C, while both stenothermal benthic diatoms exhibited upper survival temperatures between 6 °C and 8 °C (Longhi, Schloss, & Wiencke 2003). The enhanced temperatures led to high mortality during long-term exposure (several weeks), while short periods were tolerated. Similarly, the Antarctic Nitzschia lecointei van Heurck optimally grew between 2 °C and 5 °C, while slightly enhanced temperatures of 8 °C were accompanied by a strong growth inhibition (Torstensson, Hedblom, Andersson, Andersson, & Wulff 2013). An explanation for these striking differences in the temperature requirements for growth in Arctic and Antarctic benthic diatoms is related to the cold water history of both polar regions (Zacher, Rautenberger, Hanelt, Wulff, & Wiencke 2009). Antarctica is considered to have a much longer cold water history of about 23 Mio years (Sabbe, Verleyen, Hodgson, Vanhoutte, & Vyverman 2003), compared to the ‘young’ geological

Ecophysiology, Cell Biology and Ultrastructure 277 cold water history of the Arctic (c. 2 Mio years). These striking differences in both cold water systems have supported the development of many marine endemic organisms in Antarctica, most of which are sensitive to even slightly enhanced temperatures > 0–5 °C (e.g., Gómez et al. 2009; Longhi et al. 2003).

12.4

Growth After Long-Term Dark Incubation

The physiological state in which polar diatoms survive the polar night and the underlying biochemical and cell biological mechanisms are still poorly understood (Schaub, Wagner, Graeve, & Karsten 2017). In these microalgae different mechanisms have been documented for long term dark survival (McMinn & Martin 2013). Those include the utilization of stored energy products (Schaub et al. 2017), a reduction of metabolic activity (Palmisano & Sullivan 1982), formation of resting stages (Durbin 1978; McQuoid & Hobson 1996), and/or a facultative heterotrophic lifestyle (Tuchman, Schollett, Rier,& Geddes 2006). The utilization of energy storage products, such as lipids (triacylglycerol) was recently shown to provide energy for the cellular maintenance metabolism in Arctic benthic diatoms during long periods of darkness (Schaub et al. 2017). Experiments on the dark survival potential were also undertaken with an isolate of the benthic diatom Navicula directa from Spitsbergen. This species was kept for up to 5 months in darkness mimicking the polar night, and subsamples were re-irradiated after 2, 3 and 5 months by continuous low photon fluence rates of 10 μmol photons m-2 s-1. After 2 and 3 months dark incubation, Navicula directa exhibited high growth rates in the light (μ = 0.41–0.46 d-1), while after 5 months treatment the growth rate strongly decreased to μ = 0.17 d-1 (Figure 12.2). In parallel, however, the longer the dark incubation time the longer the lag-phase before optimum growth could be established again (Figure 12.3).

Growth rate ( d−1)

0.6

a

a

0.4

b

0.2

0 1

2

3

5

Time (months)

Figure 12.2 Dark survival potential of the benthic diatom Navicula directa isolated from a sublittoral rock of Kongsfjorden, Spitsbergen. Cells were kept at 5 °C for 2, 3 and 5 months in darkness followed by re-irradiation with low photon fluence rates (10 μmol photons m-2 s-1). Growth rates daily were measured as increase in chlorophyll fluorescence according to Gustavs et al. (2009). Cells were grown in sterilized Baltic seawater enriched with sea salt (Sel marin hw professional, Wiegandt GmbH, Krefeld, Germany), vitamins and silicate resulting in a salinity of 33 PSU. Data represent mean values ± SD (n = 3).

278 Diatoms: Fundamentals and Applications 40 c Lag phase (d)

30 b

20 a 10

0 1

2 3 Time (months)

5

Figure 12.3 Lag-phase for regaining growth in the benthic diatom Navicula directa isolated from a sublittoral rock of Kongsfjorden, Spitsbergen. Cells were kept at 5 °C for 2, 3 and 5 months in darkness followed by re-irradiation with low photon fluence rates (10 μmol photons m-2 s-1). Growth rates were daily measured as increase in chlorophyll fluorescence according to Gustavs et al. (2009). Cells were grown in sterilized Baltic seawater enriched with sea salt (Sel marin hw professional, Wiegandt GmbH, Krefeld, Germany), vitamins and silicate resulting in a salinity of 33 PSU. Data represent mean values ± SD (n = 3).

After 2, 3 and 5 months darkness the lag-phase strongly increased to 12, 17 and 27 days, respectively, and hence after 5 months simulating the polar night it took almost 1 months under re-irradiation before Navicula directa grew (Figure 12.3). A high dark survival potential of polar benthic diatoms has been reported earlier (Karsten, Schlie, Woelfel, & Becker 2012; Schlie, Woelfel, Ruediger, Schumann, & Karsten 2011; Wulff, Roleda, Zacher, & Wiencke 2008). It seems that particularly high latitude diatoms have the capability to withstand long periods of darkness, which may be beneficial when considering the fluctuating and variable radiation conditions in the Arctic and Antarctica. The photosynthetic apparatus of dark-incubated temperate pelagic diatoms seems to be impaired already after few weeks as reflected by a very long recovery phase after re-irradiation. In contrast, light harvesting for photosynthesis and growth can fast resume in their Antarctic pendants after the polar night (Peters 1996). Other benthic diatom species from Arctic waters such as Fragilaria striatula showed a 30–40% reduction in chloroplast lengths after 3 months of dark incubation at 5 °C, indicating the recruitment of energy for a maintenance metabolism through decomposition of organelle components (Karsten et al. 2012). Since the dark survival mechanisms of Arctic benthic diatoms are generally poorly understood, a comprehensive and precise evaluation has to be carried out, such as on the remobilization of storage lipids (Schaub et al. 2017). A new bacterial-like mechanism explaining how benthic diatoms might survive long periods of darkness was recently suggested by Kamp, de Beer, Nitsch, Lavik and Stief (2011). These authors reported a strong correlation between the dark survival potential and the ability to intracellularly accumulate NO3-. The stored NO3- is dissimilatorily reduced to ammonium in darkness, i.e., the diatoms seem to be capable to respire nitrate to keep their maintenance metabolism.

Ecophysiology, Cell Biology and Ultrastructure 279

Percentage of intact cells (%)

100 80 60 40 20

m on th s 5

m on th s 2

th on m 1

St at ph as e

Lo g

ph

as e

0

Figure 12.4 Percentage of intact cells (n = 400) in the benthic diatom Navicula directa isolated from a sublittoral rock of Kongsfjorden, Spitsbergen. Cells were kept at 5 °C for 1, 2 and 5 months in darkness followed by staining with the viability dye SYTOX (Veldhuis et al. 2001).

12.5

Cell Biological Traits After Long-Term Dark Incubation

To better understand cell biological features contributing to the dark survival mechanisms in the Arctic benthic diatom Navicula directa, cell viability and activity were evaluated using various fluorescent dyes. Diatoms with intact cell membranes (plasmalemma) were distinguished from those with permeabilized membranes, using the nucleic acid stain SYTOX according the protocol of Veldhuis, Kraay and Timmermans (2001). SYTOX can only pass through compromised or damaged membranes, and stains the nucleus leading to enhanced fluorescence under blue light excitation. Cell activity of Navicula directa was determined by intracellular esterase activity using 5-chloromethylfluorescein diacetate (CellTrackerTM Green CMFDA) as an artificial substrate according to Freese, Karsten and Schumann (2006). CMFDA is hydrolyzed enzymatically to the green fluorescing 5-chloromethylfluorescein (CMF), which is bound by its chloromethyl group to the intracellular protein pool. Diatoms that are positively stained with CMFDA must have at least two different enzyme classes: a hydrolase (esterase) and a transferase (glutathione-S-transferase). Such cells were interpreted as (hydrolytically) active diatoms. In addition, the fluorogenic CTC (5-Cyano-2,3-ditolyltetrazolium chloride) is reduced by an active electron transport system. Hence, CTC is widely used to detect respiring microorganisms (Freese et al. 2006). Application of the SYTOX stain clearly indicated that >95% of all Navicula directa cells exhibited intact membranes, even after 5 months of darkness, since only a minor fraction of nuclei exhibited fluorescence (Figure  12.4). Membrane integrity, as an important defense barrier against all environmental influences, is considered as the prerequisite for the cell’s further prosperity, and hence it was reported to be the least stress-sensitive trait of microorganisms (Freese et al. 2006). The data indicate that no cell biological damage occured during long-term darkness in Navicula directa.

280 Diatoms: Fundamentals and Applications

Percentage of active cells (%)

100 80 60 40 20

m on th s 5

m on th s 2

th on m 1

St at ph as e

Lo g

ph

as e

0

Figure 12.5 Percentage of active cells (n = 400) in the benthic diatom Navicula directa isolated from a sublittoral rock of Kongsfjorden, Spitsbergen. Cells were kept at 5 °C for 2 and 5 months in darkness followed by staining with a dye for intracellular esterase activity using 5-chloromethylfluorescein diacetate (CellTrackerTM Green CMFDA) as an artificial substrate according to Freese et al. (2006). The asterisk indicates no measurements.

In strong contrast to cell viability, the esterase activity in Navicula directa strongly decreased from 80–90% in the control to 50% decrease in chloroplast volume (unpublished results). Consequently, the recruitment of energy for a maintenance metabolism through decomposition of chloroplast components seems to be a key mechanism in Arctic benthic diatoms to survive the polar night.

12.6

Ultrastructural Traits

Freshly harvested Navicula directa cells were fixed for transmission electron microscopy by standard chemical fixation (2.5% Glutaraldehyde, 1% OsO4) according to Holzinger, Roleda and Lütz (2009). Cross sections in the central region revealed a central nucleus (Figure  12.9a). Large areas occupied by lipid bodies were visible. The chloroplasts showed intact thylakoid membranes and each a pyrenoid. In longitudinal sections

Ecophysiology, Cell Biology and Ultrastructure 283

CW P ChI L V

P

N ChI

P L

ChI (a)

(b)

L

L

P

N

M (c)

G

(d)

M

ChI

Figure 12.9 Transmission electron micrographs of Navicula directa (a) cross section in the central part, nucleus, a massive lipid body and the parietal chloroplasts with pyprenoid, (b) longitudinal section with chloroplasts and pyrenoid and central vacule and lipid body, (c) detail in the central parts with golgi bodies, nucleus and mitochondrium, (d) detail near the chloroplast with pyrenoid transversed by thylakoid membranes (arrow), elongated mitochondrium. Bars a,b 2 μm; c, d 500 nm.

the extensive lipid bodies, again parietal chloroplasts with a pyrenoid were visible (Figure 12.9b). When viewing the ultrastructure in higher magnification, Golgi bodies and endoplasmic reticulum surrounding the nucleus were obvious (Figure 12.9c), accompanied by numerous mitochondria, occasionally up to 3 μm long (Figure 12.9d). The pyrenoids were up to 1.8 μm in diameter and contained only few thylakoid membranes (Figure 12.9d).

12.7

Conclusions

In the present study several aspects of physiological activity and ultrastructure of an Arctic isolate of Navicula directa were investigated. It can be concluded that this species has a broad ecological amplitude, concerning the temperature requirements for growth (between 1 °C and 15 °C almost equal growth rates) and tolerates long term exposure to darkness (up to 5 months), which is naturally occurring during the polar night. An

284 Diatoms: Fundamentals and Applications active electron transport system, as well as intact membranes were observed even after prolonged dark incubation, and only hydrolytic activities decreased during this treatment. However, after transfer to light, the lag-phase took 27 d after the longest dark exposure (5 months), suggesting that substantial repair was needed. From the ultrastructural observations, it can be concluded that Navicula directa cells contain large mitochondria and accumulate reserves in form of storage lipids, which will be used for energy production together with chloroplast degradation during darkness. These traits enable Navicula directa to survive the harsh environmental conditions at high latitudes, and it is likely, that the observed warming of the Arctic waters will not readily replace this species.

Acknowledgements We thank Mandy Rickler, University of Rostock for providing some of the ecophysiological and cell biological data. Moreover, we thank Doris Kofler, University of Innsbruck, for help in silica scale preparation by H2O2 digestion. The field-work has been performed at the Ny-Ålesund InternationalArctic Environmental Research and Monitoring Facility. The authors thank the crew and divers at the AWIPEV-base in Ny Ålesund for technical support. Ulf Karsten greatly appreciates financial support by the Deutsche Forschungsgemeinschaft (Projects KA899/12-1/2/3 and KA 899/15-1/2) in the frame of Priority Program 1158 Antarctic Research.

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13 Ecology of Freshwater Diatoms – Current Trends and Applications Aloisie Poulíčková1,* and Kalina Manoylov2 1

Department of Botany, Faculty of Sciences,, Palacký University in Olomouc, Czech Republic 2 Department of Biological & Environmental Sciences, Georgia College and State University, Milledgeville, GA, USA

Abstract Freshwater systems are considered vulnerable due to the relative scarcity of resources and the conflicting needs between humans and aquatic organisms, where microbial aquatic organisms remain of least concern. This review summarizes directions of the very active research in diatom ecology in the last few decades, focusing on diatom distribution and conditions influencing abundance. The influence of climate vs local environmental predictors is beginning to grow as an area of research, while understanding diatom biogeography and global ecology has been concentrated for extreme environments. The role of traditional taxonomic resolution for freshwater ecosystem biomonitoring remains relevant, but new molecular methodologies show great promise. Ecological guilds, eco-morphological functional groups, and phylogenetic signals present new understanding in the complex ecology of diatom assemblages. Keywords: Diatoms, distribution, dispersal, diversity, assemblages, classification, metacommunities, biomonitoring

13.1

Introduction

The ecology of microbial communities remains mostly unexplored, as we often apply and test ecological principles developed for groups with larger size, longer life span, and smaller community diversity. The assumption often is that the most abundant species respond to the current conditions as best competitors, while low abundance taxa are present at random and are outcompeted by the dominant taxa. In low nutrient conditions, the expectation is to document low diversity communities with specialized low nutrient taxa (Manoylov et  al. 2013), but diatoms (as all algae) will proliferate in luxurious nutrient conditions. Therefore, in high nutrient conditions, community indices like biodiversity, richness, and abundance need to be carefully considered. When nutrients are available in luxurious concentrations, many taxa *Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (289–310) © 2019 Scrivener Publishing LLC

289

290 Diatoms: Fundamentals and Applications will increase in abundance and as a result, will improve evenness (Manoylov et al. 2013). How high diversity can be a negative indicator of water quality has not been addressed to this day. At very high nutrient conditions, few taxa are assumed to grow faster than their competitors and dominate the community. Very rarely a single diatom dominates freshwater communities. The challenge for algal ecologists remains the understanding of species identity, community interactions and population density. The ecology of freshwater diatoms can be approached from many directions, as they occur in all aquatic habitats. Freshwater diatoms perform a great variety of ecological functions. As widespread primary producers, they significantly contribute to the production of oxygen (20–25% of world primary production; Werner 1977), and are particularly important to nutrient cycling, energy flow, and higher trophic levels’ supply of energy (Falkowski, Fenchel, & Delong 2008; Soininen et al. 2016). The number of species is estimated within a range of 30,000 to 100,000 (Mann & Vanormelingen 2013), which compared with 12,000 described species (Guiry 2012) means that the majority of species await their description. Diatoms are abundant in all waters except hot and hypersaline habitats, being the most ubiquitous group within the microalgae (Zimmermann et  al. 2014). They live freely in the plankton of lentic waters or attached to the surfaces in benthos of both lotic and lentic ecosystems (Round 1981). The most important freshwater diatom communities with selected representatives are shown in Figure 13.1. Benthic diatom ecology is much less understood than planktic diatom ecology. The “golden era” of plankton research was connected particularly with lake areas all over the world (Poulíčková, Duchoslav & Dokulil 2004; Reynolds et al. 2012; Talling 2012; Rühland et  al. 2014; 2015). As the main interest shifted in recent years from local to global changes, the current research is mostly „indirectly based on plankton“, primarily based on paleolimnological reconstructions of lake sediments (De Laender et al. 2012; Reavie et al. 2017). Benthic diatoms have been largely ignored by ecologists due to difficulties with sampling, particularly quantitative (Round et  al. 1990). Great progress in this field has been achieved recently (Poulíčková et al. 2014; Kelly et al. 2016; Virtanen & Soininen 2016). In Arctic lakes, where temperatures are increasing most rapidly, diatom communities changed significantly due to ice cover dynamics and vegetative habitat availability (Griffiths, Michelutti, Sugar, Douglas, & Smol 2017). In the Anthropocene, remote lakes continue to shift towards new ecological paradigms (Hobbs et al. 2010). The last decade has been exciting for diatom community research, as no prior analogs exist to the changes observed and predicted for lakes in colder climates. Diatoms are predicted to increase in biomass and as food source, due to anthropogenic nutrient deposition and overall temperature increase (Rühland et al. 2015). Diatoms with high surface to volume ratio stay buoyant, strongly influence energy transfer through the food web, and contribute to increased carbon uptake. Abiotic drivers affected the size structure of planktonic communities and warmer climate favours small-sized diatom cells (Winder et al. 2009). In high latitude lakes, there has been an expansion of pelagic, small diatom species representative of the genus Cyclotella probably due to a longer ice-free season and increased stratification (Saros & Anderson 2015). How those trends are related to structural, functional, and trophic level changes is unclear, and has prompted a series of monitoring initiatives in large temperate lakes (Warner & Lesht 2015; Reavie et al. 2016; Beall et al. 2016).

Ecology of Freshwater Diatoms – Current Trends and Applications 291 Tychoplankton

Euplankton 6

13

5

1

7

12

Epiphyton - Endophyton

Meroplankton

3

4

11 29

2

8

9 31

14

30

Planktic Freshwater diatom assemblages

Epilithon

10

27 32

Epizoon - Endozoon

28 15

16

19

17

18

Bentic 22

25 23 24

21

26

20 Epipelon - Endopelon

Epipsammon - Endopsammon

Figure 13.1 Freshwater diatom assemblages with schematic drawings (orig. Štěpánka Michalíková – Poulíčková) representing the most typical genera. Schematic illustration sizes are not proportional, but highlights the general contrast between small planktonic and mostly robust benthic diatoms. Plankton consist of euplanktonic diatoms adapted to life in water column (1-Asterionella,2-Cyclotella, 3-Stephanodiscus, 4-Nitzschia acicularis, 5-Aulacoseira) and tychoplanktic or meroplanktic diatoms living a part of life cycle in close connection with sediment (shallow ponds or littoral of deeper lakes; 6-Fragilaria, 7-Tabellaria). Epiphyton (on the surface of algae and macrophytes) and endophyton (within mucilage envelopes of other algae): low profile diatoms (8-Cocconeis, 9-Achnanthidium), high profile diatoms (10-Gomphonema,11- Cymbella) and motile (12-Navicula, 13-Nitzschia, 14-Pinnularia). Epizoon (on the surface of animals) represented by 15-Nitzschia. Epipelon (on the surface of fine sediments) and endopelon (within the fine sediments) are represented by motile species (16-Sellaphora,17- Neidium, 18-Caloneis, 19-Cymatopleura, 20-Amphora, 21-Pinnularia, 22-Planothidium. Epipsammon (on the surface of sand grains) and endopsammon (within sandy sediments) are represented by 22-Planothidium, 23-Campylodiscus, 24-Surirella, 25-Opephora, 26-Nitzschia. Epilithic diatoms are attached to stones: low profile (27-Achnanthidium, 28-Meridion, 29-Diatoma), high profile 30-Gomphonema, 31Didymosphenia, 32- Cymbella and motile (12,13,14).

Diatoms show strong responses to a wide range of ecological factors, and their sensitivity to changes in pollution, nutrient availability, salinity, and acidity make them excellent bioindicators (Eloranta & Soininen 2002; Lavoie et al. 2014; Stevenson 2014) Moreover, in contrast to direct chemical analyses, diatoms reflect long-term changes in water chemistry (Smucker & Vis 2011; Stevenson 2014) due to integrating ecological conditions across temporal and spatial scales (Virtanen & Soininen 2016). Water quality has been identified as a very important issue related with human health. In this paper we address the role of diatom analyses to assess and predict the state of aquatic ecosystem services and goods. Anthropogenic contaminations in aquatic environments with acids, metals, and agricultural runoff have been increasing due to demand for coal generated energy, industry, and the need for more food production with human

292 Diatoms: Fundamentals and Applications population growth, respectively. Here we synthesize current knowledge regarding diatom diversity, distribution and dispersal, and community structure. Measures of ecosystem processes, such as nutrient uptake rates, extracellular enzyme activities, and metabolism are increasingly being used as assessment tools, but remain in their infancy relative to traditional community structure-based approaches. The continued development, testing, and implementation of functional measures and their use alongside diatom community structure metrics will further advance bioassessment, will inform management decisions, and quantify progress toward restoration goals.

13.2

Diatom Distribution

Although diatoms have been investigated for almost 300 years (first certain record is known from 1703; Round et al. 1990), their diversity, distribution, and biogeography remain poorly known (Mann & Vanormelingen 2013). The traditional view suggests that microorganisms including diatoms have unlimited dispersal potential (Finlay 2002) lacking any biogeography. Unlimited dispersal has been expected as a result of microbes’ small body size and astronomic population sizes (Finlay & Clarke 1999; Passy 2012). Paradoxically, some macroorganisms (e.g., bryophytes) were found to correspond more closely to the “everything is everywhere” pattern than diatoms (Hájek et al. 2011) most likely due to the microscopic size of bryophyte propagules. Similar inverse relationships of body size to abundance and distribution has been recorded for other small organisms (protists and multicellular forms) across aquatic and terrestrial environments (Passy 2012). More specifically, species with wide niches, high abundances, and broad distribution exhibit optimized body sizes (small in diatoms, but intermediate in fish; Passy 2012); This type of distribution would be governed by local environmental conditions (Van der Gucht et al. 2007). In contrast, several studies demonstrated a spatial pattern in microbial distribution and biogeographical patterns largely similar to those known for macroorganisms (Martiny et  al. 2006; Vyverman et  al. 2007; Verleyen et  al. 2009; Astroga et al. 2012). Finlay et  al. (2004) proposed that rapid and ubiquitous dispersal in protists leads to low proportion of endemism. Although we have no clear example of narrow endemism within diatoms, new species described from Lakes Prespa, Ohrid, Baikal or the Hawaii islands (Levkov, Krstic, Metzeltin, & Nakov 2007; Kulikovskiy, Lange-Bertalot, Metzeltin & Witkowski 2012; Kociolek et al. 2016) have not been documented outside of their original locations to date. In contrast, geographically structured populations have been documented (based on microsatellite data) in cases of widespread diatoms, such as Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle, Sellaphora capitata Mann and McDonald, and Eunotia bilunaris (Ehrenberg) Schaarschmidt (respectively, Vanormelingen, Chepurnov, Mann, Cousin, & Vyverman 2007; Evans & Mann 2009; Casteleyn et al. 2010).

13.3

Diatom Dispersal Ability

Freshwater habitats mostly represent discrete sites („islands“) surrounded by terrestrial habitats (Mac Arthur and Wilson, 1967, Incagnone et al. 2014). Size of the water body

Ecology of Freshwater Diatoms – Current Trends and Applications 293 is a key variable affecting diatom species richness of benthic assemblages and small island effect is a characteristic feature of their species-area relationship (Bolgovics et al. 2016). Despite the lack of connectivity between the sites, microalgae lacking the capability of active dispersal may passively achieve wide distribution through agents such as wind, water flow, animal vectors, and human activities (Kristiansen 2008; Padisák et al. 2015). All potential dispersal vectors have been reviewed by Kristiansen (1996) and few papers appeared just recently dealing with air-dust, ballast-water and introduction of non-native fish (Sharma & Singh 2010; Boltovskoy et al. 2011; Villac, Kaczmarska, & Ehrman 2013; Görgényi et al. 2016). Despite the progress in all aspects of phycology we still can advocate for Round s statement that we have no real evidence on diatom transport mechanisms (Round 1981). In general, periphytic taxa (attached) seem to possess lower dispersal ability than phytoplankton species (Wetzel et al. 2012). Dispersal ability is crucial for understanding changes in biodiversity, invasions and proper conservation (Wilk-Wozniak and Najberek 2013) and is primarily connected with the ability of algae to survive different types of stress (e.g., desiccation). In the case of living diatom cells, short distance dispersal over a few meters should be possible. Common longdistance dispersal of freshwater diatoms is not expected, due to their low tolerance for desiccation and temperature stress (Souffreau et al. 2010; Souffreau et al. 2013). Higher dispersal potential can be expected in terrestrial diatoms, particularly soil-inhabiting diatoms such as Pinnularia borealis Ehrenberg and Hantzschia amphioxys (Ehrenberg) Grunow, which show higher tolerances to temperature extremes and desiccation (Souffreau et al. 2010; Souffreau et al. 2013). Although this advantage ranks P. borealis and H. amphioxys among the hot candidates for cosmopolitan distribution, both species were found to be genetically highly differentiated, suggesting cryptic diversity within these aerophytic diatom complexes (Souffreau et al. 2013). Some microalgae are expected to be able to survive on the wet surface of animals without specialized protective structures, or in form of dormant stages (spores, akinetes, cysts; Padisák et al. 2004 & Villac et al. 2013). Stoyneva (2016) noticed that over 90% of the species associated with long transportation by birds have mucilaginous sheaths. Zoochory has been regarded long-ago as important mechanism of algal transport (Figuerola & Green 2002; Cellamare, Leitão, Coste, Dutartre & Haury 2010 and Stayneva 2016). The capability of algae to resist digestion within the alimentary tracts of fish has been tested in filter-feeding Asian carps, which are frequently introduced into eutrophic lakes and many rivers throughout the world. With the massive invasion of the Asian carp into the Mississippi River delta and its tributaries, the change in phytoplankton communities and survival of pennate planktonic diatoms were documented (Pongruktham et al. 2010).Viable cells of diatoms, cyanobacteria, desmids and green algae have been found in cultured hindgut samples (Görgényi et al. 2016). However, this fact on its own does not guarantee successful diatom colonization of new biotopes. Diatoms are limited by their reproductive behavior, namely by cell diminution (within large portions of the populations) during their vegetative division and size restitution via auxosporulation, which is usually accompanied by sexual reproduction. Thus, in most diatoms, sex is not facultative, as in other algae (Mann 1999). As the majority of diatoms is heterothalic (Chepurnov et al. 2004; Poulíčková et al. 2007; Vanormelingen et  al. 2013), representatives of both (compatible) sex partners need to survive the transport, which is quite unlikely. This seems to be clear applying experiences from

294 Diatoms: Fundamentals and Applications laboratory experiments on sexual reproduction, because isolation of many clonal cultures in sexual size range is necessary to obtain both sex partners (in heterothalic diatoms) (Chepurnov et al. 2004; Poulíčková et al. 2007). Consequently, how are microalgae performing as invasive species? Biological invasions are caused by non-native species that can alter dominant species in the community (Mack et al. 2000). The most famous example of an invasive diatom is Didymosphaenia geminata (Lyngbye) M. Schmidt (Didymo). Didymo is a benthic freshwater diatom, native to rivers of the Circumboreal Region and the Northern Hemisphere (Blanco & Ector 2009). Historically, it was described a rare diatom of rivers with cold water and moderate flow, and was not considered as an invasive species until the last few decades (Coste & Ector 2000), when it began to form massive biomass. Currently, it is successfully invading phosphorus poor rivers in North America, Europe, Asia, as well as freshwater localities of the Southern Hemisphere (New Zealand, Argentina, Chile, Spain (reviewed by Jaramillo et al. 2015; Montecino et al. 2016; Sanmiguel et al. 2016). Recently, Didymo is cited in over fifty countries and affects all continents except Antarctica (Sanmiguel et al. 2016). Is it possible that it was present everywhere, but overlooked? Recent study support the hypothesis that its ecological niche is expanding (Sanmiguel et al. 2016). Unfortunately, the study of genetic structure of Didymo populations is in the stage of molecular methods optimization (Jaramillo et al. 2015), so we have little information about genetic diversity of its populations, but some progress is being made (Keller, Hilderbrand, Shank, & Potapova 2017).

13.4

Functional Classification in Diatom Ecology

The composition and dynamics of benthic diatom assemblages depend on taxa colonization ability, cell size and biovolume, nutrient demands, and sensitivity to disturbances (Ács & Kiss 1993; Stenger-Kovács et al. 2013; B-Béres et al. 2014; Kókai et al. 2015). Functional classification in diatom ecology is becoming increasingly important for a deeper understanding of the dynamics and functioning of diatom assemblages. While such classifications of planktic algae and cyanobacteria have a long tradition (Salmaso et al. 2014), benthic diatom functional groups have not received attention until quite recently (Passy 2007; Berthon et al. 2011; Rimet & Bouchez 2012a; b; Kókai et al. 2015). From functional approaches developed to predict phytoplankton species distribution along environmental gradients, the most widespread has been an approach sensu Reynolds et al. (2002) & Padisák et al. (2009); however, proper classification of a single species into corresponding functional groups requires in-depth knowledge of their autecology (Padisák et al. 2009). The most recent functional classification is based on species traits, i.e. physiological, morphological and phenological features (Litchman & Klausmeier 2008; Rimet & Bouchez 2012a). Despite missing information for many species, the functional trait approach is frequently applied (Vogt et al. 2010 & Weiher et al. 2011; Žutinic at al. 2014; Cellamare et al. 2016). In the case of benthic diatoms, several were established functional groups guilds: low profile, high profile, motile guilds (Passy 2007). Low profile guild species are adapted to an unfavorable position in the biofilm due to small body size and horizontal growth (Figure 13.2b, g, i-k). Species in high-profile and motile guilds

Ecology of Freshwater Diatoms – Current Trends and Applications 295

Figure 13.2 Representatives of freshwater diatom assemblages documented with LM images (BF, DIC, Zeiss AxioImager), diatom images from aquatic habitats in the Czech Republic. Planktic genera: (a) - Fragilaria, (c) - Acanthoceras, (d) - Asterionella, (e) – Aulacoseira. Bentic genera, low profile guild: (b) - Meridion, (g) - Diatoma, (i,j)- Achnanthidium attached by short mucilaginous stalks (arrow) to green filamentous alga Oedogonium (characteristic rings marked by asterics), (k) Cocconeis attached by valve face, (l) - Fragilaria attached by mucilagenous pad. Bentic genera, high profile guild: (f)- Melosira, (h) - Gomphonema with long branched stalks. Bentic genera, motile guild: (m) Nitzschia, (n) Navicula. Scale bars 10 μm.

296 Diatoms: Fundamentals and Applications can secure more beneficial positions using their specialized habitats e.g. long stalks (Figure  13.2h) or motility (Figure  13.2m, n). Here we illustrate a few additional growth patterns of the low profile guild that could allow them to be elevated as epiphytes: short stalk epiphytic (Figure 13.2j), adnate epiphyte (Figure 13.2k), and pin cushion epiphyte with a mucilage pad (Figure 13.2l). There is still much unknown in understanding ecological guild classifications as diatoms tend to change strategies. For example, in a culture experiment, Euinotia bilunaris (Figure 13.3c,d) was either motile or formed tufted colonies (Vanormelingen et  al. 2007). In a competition experiment using genetically identical populations (commercially obtained cultures were used) of Achnanthidium minutissimum (Kützing) Czarnecki (Figure  13.2i), populations grown under low light and high nutrient conditions produced short pads and stalks, and attached to available vertical substrates (Manoylov 2009). The presence of the stalk forming Cymbella cistula (Ehr.) Kirchner (member of the high profile guild) facilitated growth of A. minutissimum (member of the low profile guild). Regardles of initial density, A. minutissimum populations grown under variable light and nutrient conditions, produced waving mucilagenous strands that were attached to the substrate. The frustules within those globular strands were with either loose or clumpted arrangement (Manoylov 2009). Besides previously mentioned classification into three guilds (Passy 2007), planktonic guild (Figures 13.2a,c–e and 13.3a,b,e) has been added by Rimet and Bouchez (2012a), as planktonic species are frequently present in biofilms due to sedimentation. Another type of classification into five size classes has been suggested by Berthon et al. (2011). Classification based on SEM morphological characteristics will bring even finer scale classifications when striae patterns, undulations, size of raphe slit, punctae occlusions, and other structures get related to specific function in diatom communities (Figure  13.3). Finally B-Beres et al. (2016) combined both classifications into eco-morphological functional classification and obtained flexible and fine-scale tools for application of dynamic assessment approaches. Both diatom species and guild composition varied in response to local environment and spatial and climatic variables, but guild distributions are driven primarily by the environment and are less dependent on historic factors (Soininen et al. 2016). Thus, guild composition is more suitable for global environmental changes assessment (eutrophication, acidification, global warming).

13.5

Spatial Ecology and Metacommunities

Increasingly, attention is paid to spatial ecology of microorganisms (Potapova & Charles 2002) and global factors affecting freshwater diatoms such as climate (reviewed by Soininen 2012). Long-term environmental information archives, represented by lake sediments with their subrecent and fossil records (particularly diatoms), can be used for paleolimnological reconstructions of both local and global influences within lakes as well as within catchments (Battarbee 2000; Williamson et al. 2009). The accuracy of lake ecosystem assessments depend strongly on our understanding of key diatom species ecology. Cyclotella sensu lato should serve an early indicator of global changes, but we need to keep in mind that the most apparent factor, like

Ecology of Freshwater Diatoms – Current Trends and Applications 297

Figure 13.3 Representatives of freshwater diatom assemblages documented with SEM images (Joel SEM (JSM-IT100) Georgia College and State University or at at the Center for Advanced Microscopy at Michigan State University, images from aquatic habitats in the States of Georgia and Florida, US). Planktic genera: (a) - Thalassiosira lacustris (Grunow) Hasle undulate valve face with numerous simple pore openings of subcentral fultoportulae, (b) – Staurosira venter (Ehrenberg) Cleve and Möller, frustule with numerous copulae without perforations, hollow spines positioned between striae on the valve mantle, (e) - Pseudostaurosira brevistriata (Grunow) Williams and Round internal valve view, irregular third row of round areolae with mostly eroded but visible complex vela; Benthic genera, low profile guild: (c, d) - Eunotia bilunaris, (c)- external view at the pole with hooked raphe on the valve face and raphe shortened on mantle, (d)- internal pole view with well-developed helictoglossa, a rimoportula in close proximity, and a smooth area interrupting the striae; Benthic genera, high profile guild: (g)Amphora sp. internal view, dorsal striae are composed of several small areolae (5-7) and ventral striae are composed of two areolae (near the apices by three areolae), proximal raphe endings are simple without central helictoglossae. Benthic genera, motile guild: (f)- Aneumastus rostratus (Hustedt) Lange-Bertalot with three different shapes of pores and different types of occlusions, (h)- Surirella amphioxys W. Smith isopolar valve, narrow-lanceolate with depressed hyaline axial area in the center of the valve. Scale bars: h 20 μm, a f 10 μm; b, e, g 2 μm and c-d 1 μm.

298 Diatoms: Fundamentals and Applications temperature, is not the only driver of changes in their abundance and species composition (Saros & Anderson 2015; Wang et al. 2016). Environmental effects are expected to follow some kind of hierarchy: large-scale patterns influence, fine-scale habitat features which influence organisms within their environment. This is particularly apparent in river ecosystems (Burcher et  al. 2007; Atkinson & Cooper 2016). Pajunen et  al. (2016) focused on climate influence on diatom distribution. Their results suggest that large-scale climatic factors influence significantly influenced the diatom distribution, while overriding the local variables at relatively small, regional scales. It supports some previous studies on other groups of microorganisms (Hillebrand et al. 2001; Martiny et al. 2006; Passy 2009); however, this influence seems to be species-specific (Pajunen et al. 2016). Spatial structure of biodiversity predictions based on metacommunity theory recently emerged as subdiscipline of ecology – metacommunity ecology (Liu et al. 2013; Heino et al. 2017). Ecologists use four main theoretical types of metacommunity organization (Leibold et al. 2004; Cottenie 2005): 1 – patch dynamics, 2 – species sorting, 3 – mass effect and 4 – neutrality (Bottin et al. 2016). Metacommunities depend strongly on the physical structure of the landscape and the configuration of the available habitat. In the case of freshwater organisms, metacommunities as discrete local communities connected by dispersal can be represented by dendritic ecological networks, such as river basins (Grant et al. 2007; Brown et al. 2011; Liu et al. 2013; Heino et al. 2017). Stream metacommunities are structured by a combination of local (environmental filtering) and regional (dispersal) processes, the latter being more important for instance in mountain rivers (Dong et al. 2016). Overland distances among sites, previously used for spatial relationship models on river organisms, seem to be poor predictors (Liu et al. 2013). Benthic diatom metacommunities appear to be confined to the boundaries of the river network, and dispersal via the river is more important than through the air or via animal vectors (Liu et al. 2013; Dong et al. 2016). Similarly, stream corridors are the primary dispersal pathway in high mountains due to dispersal limitation among streams by mountain barriers (Dong et al. 2016). Differences can be found in the spatial distribution of guilds (low profile vs high profile and motile), the latter being more frequent in the lower zones of the river network (Passy 2007; Liu et al. 2013), due to differences in current velocities. Such results highlight that trait-based approaches in metacommunity ecology can play an important role (Wetzel et al. 2012; De Bie et al. 2012; Liu et al. 2013). River basin identity can be a slightly better predictor of community structure than local environmental variables, which was proved in the case of organisms surveyed in streams (diatoms, bryophytes and zooplankton), not in case of organisms surveyed in lakes (insects, macrophytes and fish). Although, both influences (environmental and basin effects) lead to high beta diversity in all organismal groups and habitats under study (Heino et al. 2017). Different patterns should be caused by differences in dispersal limitation between lake and stream organisms or by their body/propagule size (Finkel et al. 2009; Lavoie et al. 2010; De Bie et al. 2012; Hájek et al. 2014).

Ecology of Freshwater Diatoms – Current Trends and Applications 299

13.6

Aquatic Ecosystems Biomonitoring

Species composition of benthic diatoms has been related to environmental conditions in streams to develop diatom indicators for assessment of biological condition (Stevenson 2014). Loss of biodiversity due to anthropogenic pollution is considered to be the most significant ecological crisis of our time. Freshwater systems are particularly vulnerable due to the anthropogenic overuse that alters ecosystem functions (Dudgeon et  al. 2006). Ecosystem functioning models for lotic systems have begun to take in account the importance of autotrophic processes in driving river production. The diversity of aquatic microorganisms in large river systems is likely critical for understanding how rivers provide services such as oxygen availability, nutrient abatement, and food production (Abell et al. 2008). Besides the changes in diversity and species composition, pollution can lead to teratology in diatoms. What components kill-off some diatom species and not others? Based on the unique way diatoms divide asexually, an alteration in morphology that should kill an individual is expected to occur during morphogenesis. As complementary valves within frustules are synthesised at different times and under different conditions, often only one valve has an altered morphology. Are all toxicants likely to induce similar deformities? Lavoie et al. (2017) found species specific sensitivity to metal(s) (Leguay et al. 2016) and organic compound(s) (Morin et  al. 2010) affecting disproportionally araphid, eunotioid, and asymmetric biraphid diatoms (Mc Farland et al. 1997; Lavoie et al., 2017). Finally, non-taxonomic parameters, i.e. number and size of lipid bodies and ratios of carotenoids to chlorophyll a, were altered without changes in frustule outline under contamination with Cd and Zn (Pandey & Bergey 2016). Species traits are usually calculated using weighted averages methods to define species environmental optima, tolerances, and regression (Schönfelder, Gelbrecht, Schönfelder & Steinberg 2002; Poulíčková et al. 2004). Taxa are defined as either sensitive (if disappear under certain conditions) or tolerant (where increase in stressor concentrations is not changing relative abundance of taxa). In the US, state and federal programs have incorporated diatoms in addition to analyses of fish and macroinvertebrates, while European programs are based on phytoplankton, macrophytes and phytobenthos (Kelly et al. 2016). Most indices were created to assess ecosystem health reflecting general water quality and regional climate (e.g., Lavoie et al. 2006a). There are also many studies reporting the response of diatom to metal contamination (Pandey & Bergey 2016), acid mine drainage (Smucker et al. 2014) and organic contaminants (e.g., herbicides and pesticides, Debenest et al. 2008; 2010). Standard protocols often call for enumerating a certain number of valves (400 EU, 600 NAWQA, 800 State of Montana, 1000 State of Kentucky) and identification to the lowest possible taxonomic level. Species-specific ecological roles of common taxa have been explored (Potapova and Charles 2012), but there are many more rare taxa that require more work. Nutrient concentrations and other anthropogenic stressors were used in stream classifications representing levels of impairment in US streams (Hausmann et al. 2016). Diatom assemblages from the same streams were used in the Biological Condition Gradient (BCG) classification. Diatom-based BCG levels correlated most strongly with total phosphorus and the percentage of forest cover.

300 Diatoms: Fundamentals and Applications In a recent study, Young et al. (2016) addressed the regulatory properties of diatom Rubisco and found that the activation status of Rubisco differs in response to environmental conditions (e.g., temperature, light, CO2, and nutrients). They suggested species-specific physiology that is largely unknown. The impact of resource use efficiency of diatoms synchronized with basic biological functions like photosynthesis will require further research. Recognizing the ecological value of abundance, Lavoie et al. (2009) removed 40% of the documented diatom species, or all taxa with abundance of less than 2% and found no difference in the ecological signal. Excluding a greater number of taxa still allows for the distinction between impacted and reference sites (the goal of most bioassessment projects), although subtle changes in the ecological status are lost (Poulíčková et al. 2017). They presented ordinations based on presence/absence or genus-level identification that resulted in a loss of information on subtle changes, but gross separations between impacted and reference sites, in different project, were still possible. Indeed, applied ecologists requiring cost-effective biomonitoring methods, tend to replace species-based approach by higher taxa or trait-based approach (Jones 2008; Mandelik, Roll, & Fleischer 2010; Mellin et al. 2011; Bevilacqua et al. 2012; Bevilacqua, Claudet, & Terlizzi 2013; Soininen et  al. 2016), ecological guilds, life forms and history, or survival strategies (Kokai et al. 2015). The use of higher taxa (genera, families) as surrogates for species is frequently discussed across terrestrial, freshwater and marine environments and is based on concept of taxonomic sufficiency. Taxonomic sufficiency has been tested also in diatoms, indicating that genus level resolution seems sufficiently robust to describe the main gradients in datasets (Raunio & Soininen 2007; Rimet & Bouchez 2012b; Chen et  al. 2016; Poulíčková et  al. 2017). Thus, recent trends in diatom research seems to follow two contradictory directions: avoidance of time consuming fine taxonomy leading to species surrogacy (sensu Terlizzi et al. 2003) versus cryptic diversity recognition based on molecular methods and barcoding (Zimmermann et al. 2014), finally allowing for a „taxonomy free“ approach (Apothéloz-Perret-Gentil et al. 2017). However, species within the big genera differ in ecological demands (Heino & Soininen 2007) and cryptic species within species complexes are in some cases ecologically differentiated (Poulíčková et al. 2008; Poulíčková et al. 2017), thus ignoring fine taxonomy in bioassessment will potentially result in the loss of fine scale ecological information. Retention of greater ecological information (fine taxonomic resolution) has been recommended particularly in case of easy recognizable good indicator species (Bevilacqua et al. 2012; Poulíčková et al. 2017). The first molecular approaches in water quality monitoring were mainly based on species-specific oligonucleotide sequences (Nguyen et al. 2011; Manoylov 2014). Some of the more current ideas incorporate the use of Microarray (phylochip) analysis for expected community and to identify freshwater pathogens that can be of risk for human health (Baudart et  al. 2017). These molecular methods allow for analysing diatom communities without cultivation, benefiting biodiversity and bioassessment studies. Using environmental DNA (eDNA) after detection of Didymo with morphological analyses allowed the understanding that the blooming population had genes from multiple global origins (Keller et  al. 2017). Rapid detection

Ecology of Freshwater Diatoms – Current Trends and Applications 301 of the presence of this potential invader can be achieved by exploring and testing phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow for the accurate discrimination of diatoms at the species level (Cimarelli et al. 2015). Small-scale prototype DNA chip can recognize and identify multiple diatom species simultaneously. These methodologies, when perfected, will contribute to a deeper understanding of diatom communities and quicker data returns for timely management decisions.

13.7

Conclusions

In the future understanding functional diversity in freshwater ecosystems is important, but will remain under explored and requires long-term investment and commitment. We should strive to obtain a better understanding of the interactions and feedbacks among taxonomic, genetic and functional diversity of algal and more specifically diatom assemblages. Integrating so many parameters (environmental, taxonomic, genetic, and ecological) into a biological model will be challenging. Both existing and novel modeling methods should be tested, validated, and optimized as necessary.

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14 Diatoms from Hot Springs of the Kamchatka Peninsula (Russia) Tatiana V. Nikulina1,*, Elena G. Kalitina2, Natalya A. Kharitonova2, Georgy A. Chelnokov2, Elena A. Vakh3 and Olga V. Grishchenko1 1

Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of Russian Academy of Science, Vladivostok , Russia 2 Far Eastern Institute of Geology, Far Eastern Branch of Russian Academy of Science, Vladivostok, Russia 3 Far Eastern Federal University, Vladivostok, Russia

Abstract The diatom flora of 14 hot springs belonging to four geothermal fields (Malkinsky, Nachikinsky, Paratunsky, and Mutnovsky) in the south-eastern Kamchatka, were surveyed in summer-autumn period of 2012, 2013, and 2015. The diatom flora is represented by 170 species (177 intraspecies taxa) from the Classes Coscinodiscophyceae, Fragilariophyceae, and Bacillariophyceae. Most common were 25 species found in periphyton communities and in floating algobacterial mats. The main dominants in the thermas were diatoms Pinnularia acidojaponica, Achnanthidium exiguum, Caloneis bacillum, Nitzschia amphibia, Rhopalodia acuminata, and Gomphonema parvulum. The algal communities of thermal springs are mainly composed of algae of cold waters that have adapted to high temperatures. As a rule, in acidic water with the chemical type: SO4-HCO3-Na-Ca and at temperature higher than 55 °C, species composition of diatom communities is significantly reduced. In the diatom flora, those that are benthic, indifferent to salinity, alkaliphilic, oligo- and beta-mesosaprobic, and widely distributed species are dominant. Keywords: Kamchatka Peninsula, thermal water, hot springs, diatom flora, algobacterial mat, bacterial flora, the chemical type of water

14.1

Introduction

The Kamchatka Peninsula is a unique region located in far eastern Russia, lying between the Sea of Okhotsk on the west and the Pacific Ocean and Bering Sea on the east. Kamchatka is extremely active geologically and has numerous volcanoes, geysers, and thermal springs. Kamchatka is located in the Kuril-Kamchatka volcanic arc zone, part of the Pacific Ring of Fire.

*Corresponding author: [email protected], [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (311–334) © 2019 Scrivener Publishing LLC

311

312 Diatoms: Fundamentals and Applications The natural outflows of volcanic regions are hot springs, which are numerous on the Kamchatka Peninsula, have a wide range of temperature, pH, chemical composition, and are represented by different types. About 75 % of Russia's thermal water resources are located in Kamchatka (Chudaev et al. 2000). In 1936, B.I. Piip composed a map with 64 largest thermal springs of Kamchatka (Piip 1937). At the present time, more than 150 major springs (115 thermal groups and 37 cold mineral sources) are known on the Kamchatka Peninsula (Goleva 1972). Data on algae and cyanobacteria (Cyanophyta, Cyanoprokaryota, Cyanobacteria, blue-green algae) from the Kamchatka Peninsula were first recorded at late 19th century. The most complete studies of diatom flora from hot springs were published by S. Schmidt Schmidt (1885), Gutwinski (1891), A.A. Elenkin (1914), and J.B. Petersen (1946). For example, Gutwinski recorded 19 taxa of diatoms from Banny (Banna) hot springs. Сomprehensive algological material from large rivers, lakes, and hot springs of Kamchatka was collected during the Kamchatka Complex Expedition (“F.P. Ryabushinsky’s Expedition”) conducted by the Russian Geographical Society in 1908– 1910. According to A.A. Elenkin, the diatom flora of reservoirs, rivers, and springs, visited by the Expedition, contained 115 species and varieties, of which 23 taxa were found in warm and hot springs (Elenkin 1914). Petersen examined over a dozen of hot springs, including Nachikinsky hot springs (Natchika), Paratunka, Karimshinsky (Karymchina), Sopka Goryachaya (Garaschie Gara), Banny (Bannaja), Nizhne-Kireunsky, and Maliy Klyuch (Malenki Klutchik), Khodutkinsky (Schadutka), in basins of Savan River and Opala River, and others. The total diatom flora of the examined sources included 132 species, varieties, and forms. Among those, 27 diatom taxa were among the dominant and most frequent occurrences, including Achnanthes exigua, Nitzschia amphibia, Rhopalodia gibberula, Eunotia exigua, Cocconeis placentula, Pinnularia subcapitata var. hilseana, Navicula minima, Fragilaria pinnata, forms of Fragilaria construens, Fragilaria vaucheriae, Diploneis pseudovalis, Amphora normanii, etc. (Petersen 1946). Information on more recent studies of diatom flora of the peninsula has been presented in a few publications. The paper by N.I. Golovenkina (1981) was dedicated to the diatoms of the thermal springs of the Uzon volcano caldera. The author examined three hot springs; the diatom flora of which was represented by 124 species and varieties. Pinnularia braunii var. amphicephala (A. Mayer) Hustedt was a most common species; as well as several other species with high abundance (P. microstauron (Ehrenberg) Cleve, P. interrupta f. minor Petersen, Gomphonema angustatum var. productum Grunow, G. parvulum var. micropus (Kützing) Cleve, and Nitzschia palea (Kützing) W. Smith). Also, the diatom communities of five hot springs (Kipelye, Tumrok, Kireunsky, and ones located in the basins of Savan River and Khodutka River) were studied by Japanese and Russian scientists (Yoshitake, Fukushima & Lepskaya 2008). A total of 39 taxa of diatoms were recorded, of which Achnanthes exigua Grunow, Amphora veneta Kützing, Caloneis bacillum (Grunow) Cleve, Neidium ampliatum (Ehrenberg) Krammer, Nitzschia amphibia Grunow, N. frustulum (Kützing) Grunow, and Pinnularia marchica I. Schönfelder were found as dominants. Finally, data on the diatom flora of the Malkinsky, Nachikinsky, and Verkhne-Paratunsky hot springs were described by T.V. Nikulina et al. (2015, 2016) and E.G. Kalitina et al. (2015).

Diatoms From Hot Springs of the Kamchatka Peninsula 313 In this paper we present the results of a survey on diatom species composition from 14 hot springs at four geothermal fields (Malkinsky, Nachikinsky, VerkhneParatunsky, and Mutnovsky) in the south of Kamchatka Peninsula (Russian Far East). Diatom species prevailing by abundance in periphyton and floating algobacterial mat communities have been studied and eco-geographic characteristic (connection to habitat, salinity, pH, saprobity, and geographical distribution) for diatom flora have been analyzed.

14.2

Materials and Methods

Samples of algobacterial mats were collected according to standard methods (Wasser et al. 1989). All samples were cleaned by the method of Swift (1967), and processed onto permanent microscope slides. The algological material (not cleaned valves) fixed with 4% formalin (Wasser et al. 1989) was used for taking photographs of diatoms from the genus Pinnularia. Light microscope observations were made with a Axioskop 40» (Zeiss, objective 40х/0,65 and 100х/1,25 oil) and «Alphaphot-2 YS-2» (Nikon, objective 40х/0,65 и 100х/1,25 oil). Diatom photos were taken at the Center of Collective Use (Prof. V.P. Bulgakov, Chief) at the Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far East Branch of Russian Academy of Sciences (FSCEATB FEB RAS). Permanent slides are housed at the FSCEATB FEB RAS, Vladivostok, Russia. To estimate the frequency of taxa occurrence at the stations, we used the six point Korde scale (1956): 1 – solitary (1–5 cells in the slide); 2 – rare (10–15 cells in the slide); 3 – not infrequent (25–30 cells in the slide); 4 – frequent (1 cell in each row of the cover glass at magnification with immersion); 5 – very frequent (several cells under the same conditions); 6 – in bulk (several cells in each visual field under the same conditions). The taxa with Korde values of 6 and 5 were referred to as dominants and subdominants, respectively. All the algae with Korde values of 4 and lower were considered to be secondary. Literature data on the ecology and distribution of diatoms were used in description of the ecological and geographical characteristics of the diatom flora of algae (Barinova et al. 2006; Bukhtiyarova 1999; Sládeček 1986; Van Dam, Mertens, & Sinkeldam 1994).

14.3

Description of Sampling Sites

During a survey of four geothermal fields of south-eastern Kamchatka in August 2012, October 2013, August and September 2015, the personal of the Far Eastern Institute of Geology and Federal Scientific Center of the East Asia Terrestrial Biodiversity (FEB RAS) visited four geothermal fields including Malkinsky, Nachikinsky, VerkhnayaParatunka (Sopka Goryachaya and Karimshinsky hot springs), and Mutnovsky (Verkhne-Vilyuchinsky and Dachny hot springs – Activnaya group and Medvezhya group) (Figure 14.1, Table 14.1). Within these geothermal fields, algobacterial samples were collected from 14 hot springs. Diatoms were present in all selected samples.

314 Diatoms: Fundamentals and Applications

Figure 14.1 Map of sample sites of hot springs of Kamchatka Peninsula. Sampling place: I, II – hot springs of Malkinsky geothermal field; III, IV – hot springs of Nachikinsky geothermal field; VerkhnayaParatunka geothermal field: V – Verkhnaya-Paratunka hot spring, VI – Sopka Goryachaya hot spring, and VII – Karimshinsky hot spring; and Mutnovsky geothermal field: VIII–XII – Dachny hot springs of Activnaya group, XIII – Dachny hot spring of Medvezhya group, and XIV – Verkhne-Vilyuchinsky hot spring.

14.3.1 Malkinsky Geothermal Field Malkinsky thermal springs are located on the left bank in the low courses of Kluchovka River – a left tributary of Bystraya River (130 km from the Petropavlovsk-Kamchatsky). There are six groups/formations of thermal springs on a small area covered with gravel. Thermal water forms different size lakes, pools and also fills man-made holes. In October 2013 and August 2015, algobacterial mats were sampled from water surface in two small thermal lakes. The highest water temperature was 65.9 °C, however, sampling temperature varied from 28 °C to 55.0 °C, and pH from 3.5 to 9.0, accordingly (Table 14.1). Water in those pools belong to hydrocarbonate-chloride-sulfate sodium type (HCO3-Cl-SO4-Na) and hydrocarbonate-sulfate sodium type (HCO3-SO4-Na), with high content of H2SiO3 (>50 mg/l), and with TDS (mineralization) up to 0.7 g/l. Associated gases were represented mainly by N2 (about 98 % volume) (Kiryukhin, Kiryukhin, & Manukhin 2010; Kalitina, Nikulina, Kharitonova, & Vakh 2015; Nikulina, Kalitina, Vakh, & Kharitonova 2015). Thionic bacteria was dominant in water samples (1.0×103 cells/ml). Also, we found major development of saprophytic bacterial flora and denitrifying bacteria (2.1×102 cells/ml). Saprophytes were dominant in mats (1.5×102–6.1×102 cells/cm3). Thermophilic bacteria were mainly represented by Bacillus acidocaldarius, Sulfolobus acidocaldarius, and Thiobacillus ferooxidans (Kalitina et al. 2015; Nikulina et al. 2015). Three species of cyanobacteria (Nostoc linckia (Roth) Bornet ex Bornet et Flahault f. rivulare (Kützing) Elenkin, Mastigocladus laminosus Cohn, and Lyngbya aestuarii (Mertens) Liebman ex Gomont) along with green algae-filaments (Bulbochaete sp. ster., Oedogonium sp. ster., and Ulothrix sp.), and unicellular desmids (Cosmarium granatum

Mutnovsky (Dachny, Activnaya Group)

VIII

IX

X

XI

XII

August 2012

August 2012

August 2012

August 2015

August 2015

spring № 6

spring № 5

spring № 3

spring № 2

spring № 1

pool

periphyton

periphyton

periphyton

periphyton

periphyton

periphyton

periphyton

VII

spring

floating mat

VerkhneSeptember 2015 Paratunsky (Karimshinsky)

VI

pool

floating mat

floating mat

floating mat

floating mat

Biotope

periphyton

October 2013

VerkhneParatunsky (Goryachaya Sopka)

V

pool

pool

lake № 2

lake № 1

Stream, reservoir

September 2015

September 2015

IV

August 2015

VerkhneParatunsky

III

August 2015

October 2013

II

October 2013

Malkinsky

Nachikinsky

I

Date of sampling

Hot springs

Sampling sites number

98

89,5

-

[96.1]

-

75.3

34.5

39.5

78.3

54.9

42.5

42.5

65.9

Max T°С

Table 14.1 The main characteristics of hot springs at the sampling sites.

≈55.0

≈55.0

≈40.0

≈50.0

≈40.0

≈35.0-40.0

34.5

39.5

≈55.0

≈45.0

42.5

28.0

≈55.0

5.7

3.3

[acid]

[acid]

[acid]

8.1

7.9

8.2

8.4

8.4

7.9

9.0

3.5

Water T°С at sampling sites pH

55

Cl-SO4-Na Cl-SO4-Na

SO4-HCO3-Na-Ca

SO4-HCO3-Na-Ca

[SO4-HCO3-Na-Ca]

[SO4-HCO3-Na-Ca]

[SO4-HCO3-Na-Ca]

SO4-HCO3-Na-Ca

Cl-SO4-Ca-Na

31

37

32

23

43

15

32

50

71

HCO3-SO4-Na

Cl-SO4-Ca-Na

57

HCO3-Cl-SO4-Na

55

23

Chemical composition of water

Cl-SO4-Ca-Na

Diatom species, varieties and forms number

(Continued)

N52°31΄489" E158°11΄31"

-

-

-

N 52°48΄3971" E158°05΄3167"

N52°49'25.0" E158°9'48.0"

N 52°49 254 E158°09 483

N 53°07 15 E157°47 58

N 53°19 19 E157°32 18

Сoordinates

Diatoms From Hot Springs of the Kamchatka Peninsula 315

August 2012

August 2015

Mutnovsky (Dachny, Medvezhya Group)

Mutnovsky (VerkhneViluchinsky)

XIV

XIII

Sampling sites number

lake

spring № 4

Stream, reservoir

floating mat

periphyton

Biotope

Notes: in the square brackets – data from Chudaev et al. 2000; Chudaev 2003; “-“ – data absent.

Date of sampling

Hot springs

Table 14.1 Cont.

63

-

Max T°С

37.0

≈30.0

7.2

-

Water T°С at sampling sites pH

Diatom species, varieties and forms number 41

36

Chemical composition of water [SO4-HCO3-Na-Ca]

SO4-Cl-HCO3-Na

N52°39'15.7" E158°13'29.0"

-

Сoordinates

316 Diatoms: Fundamentals and Applications

Diatoms From Hot Springs of the Kamchatka Peninsula 317 Brébisson and Cosmoastrum punctulatum (Brébisson) Palamar-Mordvintseva) formed algobacterial mats.

14.3.2

Nachikinsky Geothermal Field

Nachikinsky thermal springs are located on the upper reaches of the Plotnikova River one of the left inflows of the Bystraya River (90 km N from Petropavlovsk-Kamchatsky). Thermal springs form a wetland terrace on the right river bank, and then water flows down into artificial pools. Algobacterial samples were collected in October 2013 and August 2015 from water surface in a small thermal pools. The maximum water temperature in the pool reached 54.9 οС. Sampling temperature ranged from 42.5 to 45.0 οС, and рН from 7.9 to 8.4 (Table 14.1). Geothermal water within Nachikinsky thermal field belonged to chloride–sulfate sodium type (Cl-SO4-Na), with high content of H2SiO3 (>50 mg/l), and, also, with TDS up to 1.2 g/l. Associated gases represented by N2 on 98vol. % (Kiryukhin et al. 2010; Kalitina et al. 2015; Nikulina et al. 2015). Dominant denitrifying (6.2×103 cells/ml) and thionic bacteria (4.5×102 cells/ml) were found in water, and mainly saprophytic bacterial flora (3.7×103–3.6×104 cells/ cm3) in mats. Bacteria of the genus Bacillus along with Thermus aquaticus, Thermothrix thiopara, and Bacillus thermocatenulatus were abundant in water and in mats. Cyanobacteria Leptolyngbya сf. thermarum (Woronichin) Anagnostidis et Komárek and L. angustissima (West & G.S.West) Anagnostidis et Komárek (Order Synechococcales) formed the bulk of the algobacterial mats. Other species of cyanobacteria belonging to orders Nostocales and Oscillatoriales (Mastigocladus laminosus, Nostoc linckia f. rivulare, Homoeothrix varians Geitler, Lyngbya aestuarii, Phormidium corium (C. Agardh) Gomont, and Ph. uncinatum (C. Agardh) Gomont ex Gomont), and green Oedogonium sp. ster. were found sporadically in mats.

14.3.3 Verkhnaya-Paratunka Geothermal Field On the Verkhnaya-Paratunka thermal field, the algobacterial mats were collected from water surface in a small pool near discharge of a geothermal well at Termalny village. Water temperature from the well discharge was nearly 78.3 οС, and at sampling sites in the pool – nearly 55 οС (Table 14.1). The chemical type of geothermal water was identified as chloride–sulfate–calcium-sodium type (Cl-SO4-Ca-Na), with high content of H2SiO3 (>50 mg/l), TDS at about 1.0 g/l, and alkaline рН (8.4). Here we found largest average number of bacteria from different ecological and trophic groups. Within those groups thionic (1.2×104 cells/ml), manganese reducing (2.1×102 cells/ml), and denitrifying (4.5×103 cells/ml) bacteria were dominant in water. Besides, in October 2013, we observed in water samples predominanting development of heterotrophic nitrifying bacteria (7.8×103 cells/ml). Heterotrophic nitrifying bacteria were numerous in mats (4.2×104 cells/cm3). Most abundant in both water and algobacterial mats were bacteria from the genera Pseudomonas and Arthrobacter, as well as Thermus thermophilus, Thiobacillus thioxidans, and Bacillus caldolyticus. In floating algobacterial mats we found Phormidium uncinatum in high quantity, whereas cyanobacteria Ph. autumnale (Agardh) Trevisan ex Gomont, Mastigocladus laminosus, Lyngbya aestuarii, Nostoc linckia f. rivulare, N. linckia f. spogiaeforme

318 Diatoms: Fundamentals and Applications (Agardh) Kützing, Chroococcus turgidus (Kützing) Nägeli, and Microcystis aeruginosa Kützing emend. Elenkin were lower frequency of occurrence.

14.3.3.1 Goryachaya Sopka Hot Spring The Goryachaya Sopka thermal springs belong to Verkhnaya-Paratunka thermal field. These hot springs are located on the north-east slope of the Goryachaya Sopka hill, 70–80 m above Paratunka River. Three springs flow from discharge area forming a small waterfall. The diatoms sampling was conducted in October 2013 and August 2015 from the waterfall spring. Water temperature in the waterfall at different seasons of sampling varied from 34.5 to 39.5 οС, and рН – from 7.9 to 8.2 (Table 14.1). The chemical type of geothermal water in Goryachaya Sopka thermal field was chloride–sulfate–calcium– sodium (Cl-SO4-Ca-Na), with high content of H2SiO3 (>50 mg/l), and with TDS at 1.0 g/l. Associated gases were represented mainly by N2 (97 vol.%) (Manukhin & Vorozheikina 1976; Kalitina et al. 2015; Nikulina et al. 2015). Bacterial flora in water samples was represented mostly by heterotrophic nitrifying (1.3×103 cells/ml), and in mats – by saprophytic (1.7×104 cells/cm3), thionic (6.3×103 cells/cm3), and ammonifying bacteria (1.0×103 cells/cm3). Three species: Dichothrix gypsophila (Kützing) Bornet et Flahault, Lyngbya aestuarii (Cyanobacteria), and Ulothrix sp. (Chlorophyta) were found on rocky surface outgrowths; the last two were abundant in periphyton community.

14.3.3.2 Karimshinsky Hot Spring Karymshinsky thermal springs are located in a valley off the left inflow of Karymshina River and belong to the Verkhnaya-Paratunka thermal fields. Thermal water was seeped through rocks forming the small springs. These springs drained into a manmade depression creating a pool where algobacterial mats were collected in September 2015. The maximum water temperature was about 75.3 οС, and in sampling sites ranged from 35 to 40 οС. Water from the pool belonged to sulfate–hydrocarbonate–sodium– calcium type (SO4-HCO3-Na-Ca), with high content of H2SiO3 (>50 mg/l), TDS up to 1.0 g/l, and alkaline рН=8.1 (Table 14.1). Saprophytic (1.0×103 cells/ml), denitrifying (1.2×103 cells/ml), and thionic bacterial organisms (2.5×103 cells/ml) were most common in thermal water, and saprophytic bacteria were abundant in mats (1.4×104 cells/cm3). Only one species, Lyngbya aestuarii, from the Division Cyanobacteria was found in algobacterial mats.

14.3.4 Mutnovsky Geothermal Field The Mutnovsky geothermal field is located within the East Kamchatka volcanic belt and represented by Mutnovsky, Goreliy, Vilyuchinsky, and Zhirovskoy volcanoes. Mutnovsky Volcano area was well studied by geologists and hydrogeologists for many years. There are three types of thermal springs in there area: 1 – crater fumaroles fields; 2 – thermal springs of northern slope of volcano (northern Mutnovsky hot springs and

Diatoms From Hot Springs of the Kamchatka Peninsula 319 Dachny hot springs); and, 3 – peripherical thermal fields including Vilyuchinsky thermal springs (Chudaev 2003).

14.3.4.1

Dachny Hot Springs

The Dachny hot springs comprise the largest area of the Mutnovsky geothermal field. Dachny hot springs is an active fumarole area with hot underground gases heating groundwater. 14.3.4.1.1 Activnaya Group In August 2015 periphyton samples from surface of stones and rocks were collected in five thermal streams in the area. The maximum water temperature in streams varied from 89.5 to 98 οС, whereas sampling sites temperature varied from 40 to 55 οС (Table 14.1). Water in streams was sulfate–bicarbonate-calcium-sodium type (SO4-HCO3Ca-Na), with рН=3.3–5.7. In water from the thermal spring with temperature 89.5 °C we found a low representation of bacteria. Among those the most abundant were autotrophic ironoxidizing (0.6×102 cells/ml), denitrifying (1.0×102 cells/ml), and thionic bacteria (1.5×103 cells/ml). Colorless sulfur bacteria (4.0×105 cells/cm3) from the genera Beggiatoa and Thiotrix were discovered in mats. In water from the stream with the highest temperature (98 οС) we identified autotrophic nitrifying (1.0×103 cells/ml), nitrogen-fixing (1.1×102 cells/ml), and thionic bacteria (0.6×102 cells/ml); saprophytes (6.2×103 cells/cm3) were found in mats. Several species of cyanobacteria: Leptolyngbya laminosa (Gomont ex Gomont) Anagnostidis et Komárek, L. granulifera (Copeland) Anagnostidis, Jaaginema geminatum (Schwabe ex Gomont) Anagnostidis et Komárek, Nostoc microscopicum Carmichael, N. paludosum Kützing ex Bornet et Flahault, Lyngbya major Meneghini ex Gomont, L. aestuarii, and Phormidium uncinatum were present in periphyton communities. 14.3.4.1.2 Medvezhya Group Periphyton samples were collected from stones in a stream that flows through a thermal seepage. Water temperature at the sampling place was about 30 οС. Water in hot spring of the Medvezhya group was sulfate–hydrocarbonate–sodium–calcium type (SO4-HCO3-Na-Ca) (Chudaev et al. 2000). In the periphyton, only one desmid Cosmarium undulatum Corda (Division Chlophyta) was recorded.

14.3.4.2 Verkhne-Vilyuchinsky Hot Spring Verkhne-Vilyuchinsky hot springs are located at the foot of the Vilyucha volcano, on the right bank of Vilyucha River. Algobacterial mats were collected in September 2015 from water surface in a thermal small lake. The chemical type of geothermal water was sulfate–chloride bicarbonate-sodium (SO4-Cl-HCO3-Na), with high content of H2SiO3 (>50 mg/l), and рН=7.2. The maximum water temperature was 63 οС, and at the sampling site – 37 οС (Table 14.1).

320 Diatoms: Fundamentals and Applications Water from the Verchne-Vilyuchinskiye hydrothermal sources was characterized by abundant development of denitrifying (9.5×102 cells/ml) and iron-oxidizing autotrophic bacteria (2.5×102 cells/ml). In mats saprophytes (1.2×104 cells/cm3), heterotrophic nitrifying (6.9×102 cells/cm3), and sulfate-reducing bacteria (8.6×102 cells/cm3) were observed. High abundance of two cyanobacterial species: Planktolyngbya cf. limnetica (Lemmermann) Komárková-Legnerová et Cronberg and Phormidium autumnale were found in floating mats. Other species identified from samples: Homoeothrix varians, Lyngbya aestuarii, Planktolyngbya circumcreta (G.S. West) Anagnostidis et Komárek, and Chroococcus turgidus (Kützing) Nägeli were rare.

14.4

Results

The diatom flora of 14 hot springs belonging to four geothermal fields (Malkinsky, Nachikinsky, Paratunsky, and Mutnovsky) in the south-eastern Kamchatka, were surveyed in summer-autumn period of 2012, 2013, and 2015. The diatom flora is represented by 170 species (177 intraspecies taxa) from the Classes Coscinodiscophyceae, Fragilariophyceae, and Bacillariophyceae (Table 14.2). It should be noted that not all of the diatoms identified by us were collected live. Only 30 % to 75 % of all diatom cells had undamaged valves and colored chromatophores, characteristics which usually confirm their live status. The class Bacillariophyceae is most diverse; it includes 141 intraspecies taxa (79.7 % of all diatoms). The genera Pinnularia (30 intraspecific taxa), Eunotia (11) and Nitzschia (15) contain the greatest number of species and varieties and are classified as leading. In addition to diatoms, 21 species of cyanobacteria belonging to the Orders Chroococcales, Nostocales, Oscillatoriales, Pseudanabaenales, and Synechococcales, as well as filamentous green algae from the Classes Chlorophyceae and Ulvophyceae (Bulbochaete sp. ster., Oedogonium sp. ster., Ulothrix sp.), and desmids (Cosmarium granatum, C. undulatum, Cosmoastrum punctulatum) were identified.

14.4.1 Malkinsky Geothermal Field In October 2013, the diatom flora from one of the Malkinsky hot springs was characterized by a poor species composition and represented by 23 intraspecific taxa. Caloneis bacillum, which had an abundance rating of very frequent, two subdominant species of Navicymbula pusilla (previously identified by us as Cymbella aff. pusilla (Nikulina et al. 2015)), and Achnanthidium exiguum, whose abundance did not exceed frequent, are classified as prevalent. In August, 2015, 57 species and varieties were present in another from Malkinsky hot springs, and the species Caloneis bacillum was also included in the number of prevalent associated with Gomphonema parvulum and Nitzschia frustulum (Table 14.3).

14.4.2

Nachikinsky Geothermal Field

Algobacterial mats floating on the surface of water in formed by thermal springs pools in different years (October 2013 and August 2015) contained a significant number of intraspecific diatom taxa (55 and 72, correspondently). Combined list includs 89 species, varieties, and forms (Table  14.2). The complex of prevailing species in the hot springs communities was characterized by the presence of the dominant Rhopalodia

Diatoms From Hot Springs of the Kamchatka Peninsula 321 Table 14.2 Taxonomic Composition of Diatoms From Hot Springs of Kamchatka Peninsula.

Order

Family

Genus

Species

Variety and form

Percent (%)

Coscinodiscophyceae

2

2

2

3

3

4.4

Fragilariophyceae

2

2

8

13

17

24.6

Bacillariophyceae

6

11

21

49

49

71.0

Total

10

15

31

65

69

100

Coscinodiscophyceae

2

2

2

6

6

6.8

Fragilariophyceae

1

1

7

9

10

11.2

Bacillariophyceae

8

16

27

71

73

82.0

Total

11

19

36

86

89

100

Coscinodiscophyceae

3

3

3

4

4

4.6

Fragilariophyceae

2

2

9

14

17

19.3

Bacillariophyceae

8

16

26

66

67

76.1

Total

13

21

38

84

88

100

Coscinodiscophyceae

1

1

1

3

3

2.7

Fragilariophyceae

2

2

8

11

12

10.8

Bacillariophyceae

8

16

29

95

96

86.5

Total

11

20

38

109

111

100

Class Malkinsky

Nachikinsky

Verkhne-Paratunsky

Mutnovsky

Common diatom flora of four geothermal fields Coscinodiscophyceae

3

3

3

8

8

4.5

Fragilariophyceae

2

2

11

23

28

15.8

Bacillariophyceae

8

17

35

139

141

79.7

Total

13

22

49

170

177

100

Eunotia exigua (Brébisson) Rabenhorst

Family Eunotiaceae

Order Eunotiales

Class Bacillariophyceae

Meridion circulare (Greville) C. Agardh

D. mesodon (Ehrenberg) Kützing

Diatoma hiemale (Lyngbye) Heiberg

Family Fragilariaceae

Order Fragilariales

Class Fragilariophyceae

Aulacoseira alpigena (Grunow) Krammer

Family Aulacoseiraceae

Order Aulacoseirales

Class Coscinodiscophyceae

BACILLARIOPHYTA

Diatom taxa

-

-

-

-

-

2–4

3–4

1

1

2015

2013

-

II

I

Hot spring, date

-

-

1

1

-

2013

III

-

-

1

1

4–5

2015

IV

-

1–5

1–3

-

-

2015

V

1

-

1

-

-

2013

VI

-

2–3

1

-

-

2015

-

-

-

-

-

2015

VII

4

-

1

2–3

-

2012

VIII

3–4

-

-

1

-

2012

IX

Table 14.3 Species Composition of Dominants in Hot Springs of Kamchatka Peninsula.

3–4

-

-

2

-

2012

X

5

-

-

-

-

2015

XI

1

-

1

1

-

2015

XII

3

-

-

3–4

-

2012

XIII

-

1

1

-

-

2015

XIV

B

B

B

B

P

B

i

hb

hb

hb

i

H

alf

χ

χ-ο

χ

χ

ο

S

k

k

a-a

a-a

k

G

(Continued)

acf

alf

i

i

pH

Ecological-geographical characteristics

322 Diatoms: Fundamentals and Applications

Planothidium frequentissimum (Lange-Bertalot) Round et Bukhtiyarova

Achnanthidium exiguum (Grunow) Czarnecki [=Achnanthes exigua Grunow]

Family Achnanthidiaceae

Order Achnanthales

Gomphonema parvulum (Kützing) Kützing

Family Gomphonemataceae

Navicymbula pusilla (Grunow) Krammer [=Cymbella aff. pusilla Grunow]

Encyonema gracile Ehrenberg

Family Cymbellaceae

Order Cymbellales

Diatom taxa

Table 14.3 Cont.

-

4

-

4

-

1–2

5–6

-

4

2015

2013

-

II

I

Hot spring, date

-

4–5

1

2

1

2013

III

4–5

2

1

2

-

2015

IV

-

2–3

1–4

1–2

1

2015

V

-

6

1

3

-

2013

VI

-

1–4

1–2

-

-

2015

-

6

-

1

-

2015

VII

-

-

1

-

-

2012

VIII

-

-

1

-

-

2012

IX

-

-

1

-

-

2012

X

1

1–3

1

1–2

-

2015

XI

-

1

1

-

-

2015

XII

-

1

1

-

-

2012

XIII

-

1–2

1–3

-

1

2015

XIV

B

B

B

B

B

B

i

hb

i

i

i

H

ο-α

β

β

-

β

S

k

k

b

k

a-a

G

(Continued)

alf

alf

alf

alf

i

pH

Ecological-geographical characteristics

Diatoms From Hot Springs of the Kamchatka Peninsula 323

Family Amphipleuraceae

-

P. acoricola Hustedt

2

-

Nitzschia amphibia Grunow

N. communis Rabenhorst

Family Bacillariaceae

Order Bacillariales

-

-

5

P. acidophila Hofmann et Krammer

Pinnularia acidojaponica Idei et H.Kobayasi [=P. marchica I.Schönfelder]

Caloneis bacillum (Grunov) Cleve

Family Pinnulariaceae

Frustulia saxonica Rabenhorst

-

-

P. lanceolatum (Brébisson ex Kützing) LangeBertalot

Order Naviculales

2015

2013

Diatom taxa

-

1

-

-

-

4–5

-

1

II

Hot spring, date

I

Table 14.3 Cont.

-

1

-

-

1–2

2

-

5

2013

III

-

1–2

-

-

-

2

-

1

2015

IV

-

1

-

-

-

2–5

-

1–2

2015

V

-

6

-

-

1

2

-

3

2013

VI

-

1–2

-

-

1

1–2

-

1

2015

-

-

-

-

1

3–4

-

-

2015

VII

-

-

-

-

6

-

4–5

-

2012

VIII

-

-

-

3–4

6

-

-

1

2012

IX

-

-

1

-

6

-

-

1

2012

X

-

2–3

4

-

5

1–4

5

2–3

2015

XI

-

-

4–5

5–6

1–2

2

1

2015

XII

-

-

-

-

6

-

1

2

2012

XIII

3

3

1–2

-

1–2

1

1

1

2015

XIV

B-P

B-P

B

B

B

B-P

B

B

B

-

i

i

-

-

i

hb

i

H

ο

ο-α

ο

-

-

ο

-

χ-β

S

k

k

-

-

-

k

-

k

G

(Continued)

alf

alf

-

-

-

alf

acf

alf

pH

Ecological-geographical characteristics

324 Diatoms: Fundamentals and Applications

-

3

-

1

1

-

1

-

-

1

2

2–3

4–5

1

1

4

2

3

2013

III

4–5

5–6

2–4

3–4

1–2

1–2

1–2

2015

IV

-

3

-

-

-

2

1–3

2015

V

1

1

-

-

1

1

3

2013

VI

-

1–2

-

-

-

1

1–4

2015

-

3

-

-

-

1

-

2015

VII

-

-

-

-

-

3

1

2012

VIII

-

-

-

-

-

-

-

2012

IX

-

-

-

-

-

1

-

2012

X

1

-

-

1

1

2–3

-

2015

XI

-

-

-

-

-

5–6

-

2015

XII

-

-

-

-

-

2

-

2012

XIII

-

1

-

1

-

1–2

-

2015

XIV

B-P

B

B

B

B

B

B

B

hl

mh

i

i

mh

i

hl

H

alf

-

alb

alb

alf

i

alb

pH

ο

-

β

β

β

α

ο

S

Ecological-geographical characteristics

k

-

k

k

k

k

k

G

Notes: I–XIV – station numbers, see in Table 14.1. To estimate the frequency of taxa occurrence at the stations, we used the six point scale: 1 – solitary (1–5 cells in the slide); 2 – rare (10–15 cells in the slide); 3 – not infrequent (25–30 cells in the slide); 4 – frequent (1 cell in each row of the cover glass at magnification with immersion); 5 – very frequent (several cells under the same conditions); 6 – in bulk (several cells in each visual field under the same conditions) (Korde 1956). B (biotope): P – planktonic, B-P − benthic-planktonic, B − benthic. H (relation to salinity): mh – mesohalobic, hl – halophilous, hb – halophobic, i – indifferent. pH (relation to рН of water): alf – alkaliphilous, alb – alkalibiontic, acf – acidophilous, i – indifferent. S (relation to saprobity of water): χ − xenosaprobous, χ-ο − xeno-oligosaprobous, χ-β − xeno-betamesosaprobous, ο − oligosaprobous, β − betamesosaprobous, ο-α – oligo-alphamesosaprobous, α − alphamesosaprobous. G (geographical distribution): a-a – arctic-alpine, b – boreal, k – cosmopolitan. “-“ − data absent.

Surirella ovalis Brébisson

Family Surirellaceae

Order Surirellales

Rhopalodia acuminata Krammer

E. adnata var. porcellus (Kützing) Ross

Epithemia adnata (Kützing) Brébisson

Family Rhopalodiaceae

Order Rhopalodiales

Tryblionella apiculata Gregory

-

3

N. frustulum (Kützing) Grunov

N. palea (Kützing) W. Smith

2015

2013

Diatom taxa

2–5

II

Hot spring, date

I

Table 14.3 Cont.

Diatoms From Hot Springs of the Kamchatka Peninsula 325

326 Diatoms: Fundamentals and Applications acuminata, except for it Planothidium lanceolatum, P. frequentissimum, Achnanthidium exiguum, Aulacoseira alpigena, and Surirella ovalis developed abundantly (Table 14.3).

14.4.3

Verkhnaya-Paratunka Geothermal Field

The total species composition of diatoms from algobacterial mats from two pools and of periphyton from a thermal stream (Verkhnaya-Paratunka, Goryachaya Sopka, and Karimshinsky hot springs) flowing along the rock and forming a waterfall in this location includes 88 intraspecific taxa from the Classes Coscinodiscophyceae, Fragilariophyceae, and Bacillariophyceae. In September 2015, 55 species and varieties were present in one of the pools of the Verkhnaya-Paratunka, with the following dominants: Caloneis bacillum and Meridion circulare (estimated abundance is very frequent) and the subdominant Gomphonema parvulum (estimated abundance is frequent) (Table 14.3).

14.4.3.1 Goryachaya Sopka Hot Spring During the autumn of 2013 and 2015, the species number in periphyton communities varied from 50 to 32, respectively. Although the different number of taxa in communities, their species composition was very similar. The dominant species Achnanthidium exiguum in combination with Nitzschia amphibia (estimated abundance is bulk) and Nitzschia frustulum (estimated abundance is frequent) were found. All other species were classified as secondary, and their frequency was estimated as solitary–frequent) (Table 14.3).

14.4.3.2 Karimshinsky Hot Spring Diatom flora of these hot springs formed poll is extremely low and includes only 15 species from the Class Bacillariophyceae, of which only two species had a high abundance – Achnanthidium exiguum (in bulk) and Caloneis bacillum (frequent).

14.4.4 Mutnovsky Geothermal Field The seven hot springs (Dachny and Verkhne-Vilyuchinsky hot springs) examined by us in this geothermal field had a wide temperature range, and the composition of diatoms of the periphyton communities includes a significant number of taxa. A total of 111 species and varieties of diatoms are recorded herein. Primarly, these species belong to the Class Bacillariophyceae (Table 14.2).

14.4.4.1

Dachny Hot Springs

14.4.4.1.1 Activnaya Group On stones and rocky substrates in the hot springs of the Activnaya Group, several prevalent species were identified: the only dominant Pinnularia acidojaponica (frequency of taxa occurrence is bulk) and subdominants – Eunotia exigua, Frustulia saxonica, Pinnularia acidophila, and P. acoricola (frequency of occurrence frequent–very frequent) (Table 14.3).

Diatoms From Hot Springs of the Kamchatka Peninsula 327 14.4.4.1.2 Medvezhya Group Periphyton communities in hot spring from of the Medvezhya group contained a moderate number of intraspecific taxa (41). The species Pinnularia acidojaponica, which had the highest abundance, was dominant and Diatoma hiemale was subdominant (Table 14.3).

14.4.4.2 Verkhne-Vilyuchinsky Hot Spring For Verkhne-Vilyuchinsky hot springs, 36 species were identified. There were no dominants, all species had low abundance (solitary or rare), and only the diatoms Nitzschia amphibian, Gomphonema parvulum, and Nitzschia communis were found with the abundance estimate of not infrequent (Table 14.3). The diatom flora of the investigated thermal springs was moderately diverse and abundant. Most common were 25 species found in periphyton communities and in floating algobacterial mats (Table 14.3). The main dominants in the thermas were diatoms Pinnularia acidojaponica, Achnanthidium exiguum, Caloneis bacillum, Nitzschia amphibia, Rhopalodia acuminata, and Gomphonema parvulum. They were observed in many studied water bodies with the highest abundance. The species Achnanthidium exiguum (=Achnanthes exigua), Gomphonema parvulum, Caloneis bacillum, and Nitzschia amphibia were also found in other hot springs of Kamchatka (Petersen 1946; Golovenkina 1981; Yoshitake et al. 2008). More than likely that the most common species Pinnularia acidojaponica was mistakenly identified and recorded in previous publications: as Pinnularia subcapitata var. hilseana (Petersen 1946); as Pinnularia braunii var. amphicephala (Golovenkina 1981), and as Pinnularia marchica (Yoshitake et al. 2008; Kalitina et al. 2015; Nikulina et al. 2015). Pinnularia acidojaponica was described in 2001 by Japanese researchers from the extreme habitats of Japan – springs with acid reaction water and rivers with high organic pollution of water (Idei & Mayama 2001). Later on this species was recorded from the periphyton algal communities of the in thermal springs of the on Kuril Islands and Sakhalin Islands (Nikulina 2010; Nikulina & Kociolek 2011). Specimens of this species found in samples from Dachny hot springs differ morphologically in small ways from ones described for typical habitats from Japan springs and rivers (Nikulina & Grishchenko 2017). In our opinion, P. acidojaponica is a true thermophilic species. There are known that the genus Pinnularia is characterized by single free-moving cells, chain-forming or short colonies (Lund, 1950; Van de Vijver, Gremmen, Beyens, & Le Cohu, 2004; Van de Vijver, Agius, Gibson, & Quesada, 2009). For species P. acidojaponica, we have noted uncharacteristic "colonies" formations, i.e.,columnar and rounded forms with different numbers of cells in them(Fig. 2A, B, Fig. 3) (Nikulina and Grishchenko, 2017). Kamchatka’s hot springs showed prevalence of saprophytic, thionic and denitrifying bacteria that indicate about their involvement in the destruction of organic matter and the processes of oxidation of nitrogen and sulfur compounds. Saprophytic bacteria prevailed in the waters of Karimshinsky hot springs; thionic bacteria prevailed in Verkhnaya-Paratunka, and denitrificants predominated in Nachikinsky hot springs. The algobacterial mats of Kamchatka thermal springs showed prevalence of saprophyte bacteria with their highest quantity in the mats of Nachikinsky hot springs

328 Diatoms: Fundamentals and Applications

10μm

10μm

(a)

(b)

Figure 14.2 Columnar form “colonies” of Pinnularia acidojaponica, (a) – from the valve and girdle view, (b) – from the apical ends of the frustules (from Nikulina & Grishchenko 2017).

10μm

Figure 14.3 Round form “colony” of Pinnularia acidojaponica (from Nikulina & Grishchenko 2017).

and Verkhnaya-Paratunka Geothermal Field (Karimshinsky, Goryachaya Sopka). All springs contain abundant heterotrophic bacteria Bacillus sp. Malkinsky springs contained thermophilic aerobic bacteria Sulfolobus sp. Mesophilic bacteria with predominant Pseudomonas sp. and Arthrobacter sp. were found in Verkhnaya-Paratunka hot springs. Colorless sulfur bacteria Beggiatoa sp. and Thiotrix sp. were discovered only in periphyton communities of Dachny (Activnaya Group) hot springs. As we have mentioned before, the main biomass of floating algobacterial mats and periphyton communities of solid substrates in hot springs were cyanobacteria and filamentous green algae. Between two and eight species of cyanobacteria developed in the communities of floating algobacterial mats, and up to four species vegetated in periphyton; but both types of communities were characterized by the presence of Lyngbya aestuarii, Mastigocladus laminosus, and Phormidium uncinatum. Only the communities of floating mats in pools and small water bodies contained Leptolyngbya сf. thermarum, L. angustissima (Nachikinsky hot springs), Planktolyngbya cf. limnetica, and P. circumcreta (Verkhne-Vilyuchinsky hot springs). The species Jaaginema geminatum, Leptolyngbya granulifera, and L. laminosa were found only in periphyton communities of Dachny (Activnaya Group) hot springs. Mass growth of these

Diatoms From Hot Springs of the Kamchatka Peninsula 329 Table 14.4 Distribution of Diatoms from Hot Springs of Kamchatka Peninsula into Ecological and Geographical Groups. Ecological-geographical group

Number of taxa

Percent (%)

141

79.7

planktonic

5

2.8

benthic-planktonic

30

16.9

epiphytic

1

0.6

No data

0

0

177

100

euhalob

0

0

mesohalob

8

4.5

halophil

17

9.6

indifferent

98

55.4

halophob

20

11.3

No data

34

19.2

Total

177

100

alkalibiont

14

7.9

alkaliphil

71

40.1

indifferent

33

18.6

acidobiont

0

0

acidophil

24

13.6

No data

35

19.8

Total

177

100

xenosaprobic (χ, χ-ο)

21

11.9

oligosaprobic (ο-χ, χ-β, ο, ο-β)

59

33.3

betamesosaprobic (β-ο, ο-α, β, β-α)

41

23.2

Biotope benthic

Total Relation to salinity of water

Relation to рН of water

Relation to saprobity of water

(Continued)

330 Diatoms: Fundamentals and Applications Table 14.4 Cont. Ecological-geographical group

Number of taxa

Percent (%)

alphamesosaprobic (α-β, β-ρ, α, α-ρ)

8

4.5

polysaprobic (ρ-α, ρ)

1

0.6

No data

47

26.5

Total

177

100

cosmopolitan

92

52.0

boreal

30

16.9

arctic-alpine

18

10.2

No data

37

20.9

Total

177

100

Geographical distribution

thermophilic species was observed in the hot springs temperature water range from 37.0–55.0 °C. However, no coexistence preferences were found between the diatom taxa and particular species of bacteria and cyanobacteria. General analysis of the diatom flora of 14 thermal springs from 4 geothermal fields in Kamchatka (Malkinsky, Nachikinsky, Verkhne-Paratunsky, and Mutnovsky) showed that most species are benthic (79.7 % of the total number of identified species). In relation to salinity most species would be classified as indifferent (55.4 %). In relation to the pH, the alkaliphil species were most common (40.1 %) (Table 14.4). In terms of geographical distribution, the 52.0 % of the species would be classified as widespread or cosmopolitan (Table 14.4). For the diatom flora of these 14 thermal springs, oligoand betamesosaprobionts were the most representative indicators of water saprobity (33.3 % and 23.2 %, respectively) (Table 14.4).

14.5

Summary

The diatom flora of 14 hot springs of the Malkinsky, Nachikinsky, Paratunsky, and Mutnovsky geothermal fields (the south-eastern Kamchatka) is represented by 177 species, varieties and forms. In the diatom flora, those that are benthic, indifferent to salinity, alkaliphilic, oligo- and beta-mesosaprobic, and widely distributed species are dominant. The algal communities of thermal springs are mainly composed of algae of cold waters that have adapted to high temperatures. As a rule, in acidic water with the chemical type: SO4-HCO3-Na-Ca and at temperature higher than 55 °C, species composition of diatom communities is significantly reduced. According to Elenkin (1914) and Petersen (1946), there are no true thermophiles in the thermal springs of

Diatoms From Hot Springs of the Kamchatka Peninsula 331 Kamchatka, but we found Pinnularia acidojaponica and P. acidophila, which, in our opinion, are true thermophiles and characteristic representatives of hot springs. In addition, in algal communities of hot springs of Kamchatka several diatoms (Achnanthidium exiguum, A. minutissima, Planothidium lanceolatum, Caloneis bacillum, Pinnularia acoricola, Nitzschia amphibia, N. frustulum, N. palea, Rhopalodia acuminata, Gomphonema parvulum, Eunotia exigua, Navicymbula pusilla, Navicula cincta) with different abundance have been identified and have been mentioned by many researchers from hot springs in different parts of the world (Stockner 1967; Stockner 1968; Ekins & Rushforth 1986; Kim 1999; Balashova 2011; Covarrubias, Cantoral-Uriza, Casas-Flores & Garcia-Meza 2016; and others). This fact confirms that these species are eurythermic and possess high adaptogenic properties that allow their survival in high-temperature habitats.

References Balashova, N.B. (2011). Diatoms of Issyk-Ata thermal springs (Kyrgyzstan Mountain, TianShan), Bulletin of St. Petersburg University 3(3), 15–25. Barinova, S.S., Medvedeva, L.A., Anissimova, O.V. (2006). Diversity of algal indicators in environmental assessment. Tel-Aviv: piles Studio. in Russian. Bukhtiyarova, L.N. (1999) Diatoms of Ukraine. Inland waters. Kyiv, Ukraine. Chudaev, O.V., Chudaeva, V.A., Karpov, G.A., Edmunds, W.M., Shand, P. (2000). Geochemistry of waters of the main geothermal arears of Kamchatka. Vladivostok: Dalnauka. Chudaev, O.V. (2003) Composition and origin of the recent hydrothermal systems of the Far East Russia. Vladivostok: Dalnauka. Covarrubias, Y., Cantoral-Uriza, E.A., Casas-Flores, J.S., García-Meza, J.V. (2016). Thermophile mats of microalgae growing on the woody structure of a cooling tower of a thermoelectric power plant in Central Mexico. Revista Mexicana de Biodiversidad, 87(2), 277–287. Ekins, L., Rushforth, S.R. (1986). Diatom flora of Cowboy Hot Spring, Mono County, California. Great Basin Nat., 46(3, 4), 612–624. Elenkin, A.A. (1914). Freshwater algae of Kamchatka. In: Kamchatka's expedition of F.P. Ryabushinsky. Botanical department. Spore plants of Kamchatka: 1) algae, 2) fungi, Publishing house of PP. Ryabushinsky, A.A Elenkin (ed.), pp. 3–405. Moscow: Publishing house of PP. Ryabushinsky. In Russian. Goleva, G.A. (ed). (1972). Hydrogeology of the USSR. Kamchatka, Kurile and Komandorskie Islands. Moskow: Nedra. Golovenkina, N.I. (1981). Diatoms from thermal springs in the caldera of the Uzon volcano from Kamchatka, The Diatoms. Proceedings of the Biological Research Institute, 30(132–148). Gutwinski, R. (1891). Algarume lacu Baykal et e peninsula Kamtschatka clariss. prof. Dr. B. Dybowski anno1877 reportatarum enumeratio et diatomacearum lacus Baykal cum iisdem tatricorum,italicorum atque franco-gallicorum lacuum comparatio, Nuova Notarisia, 2, 1–27,300–305, 357–366, 407–417. Idei, M., Mayama, S. (2001). Pinnularia acidojaponica M. Idei et H. Kobayasi sp. nov. and P. valdetolerans Mayama et H. Kobayasi sp. nov. – new diatom taxa from Japanese extreme environments. In: Lange-Bertalot-Festschrift: Studies on Diatoms. Dedicated to Prof. Dr. Dr. h.c. Horst Lange-Bertalot on the occassion of his 65th Birthday. pp. 265–277. J.R. Kociolek, A. Witkowski and P. Compère (eds). A.R.G. Gantner Verlag. K.G. P.

332 Diatoms: Fundamentals and Applications Kalitina, E.G., Nikulina, T.V., Kharitonova, N.A., Vakh, E.A. (2015). Materials for studying of the microorganisms diversity in the thermal springs of Kamchatka (Russia). In Modern problems of hydrogeology, engineering geology and hydrogeoecology of Eurasia. Materials of the Russian conference. pp. 510–513. A.Yu. Dmitriev (ed.). Tomsk: Tomsky Politechnical University. Kim, B.-H. (1999). Ecology of a cyanobacterial mat community in a Korean thermal wastewater stream, Aquatic Ecology, 33(4), 331–338. Kiryukhin, A.V., Kiryukhin, V.A., Manukhin, Y.F. (2010). The Hydrogeology of Volcanogenic Rocks. St. Petersburg: Nauka. Korde, N.V. (1956). Survey procedure of biological study of bottom sediments. In: The life of fresh waters of the USSR. 4(1). pp. 383–413. E.N Pavlovsky and V.I Zhadin (eds). Moskow, Leningrad: Publishing house of Academy Sciences of the USSR. Lund, J.W.G. (1950). Contributions to our knowledge of British Algae. XI. A new colonial Pinnularia (P. cardinaliculus Cl. emend), Hydrobiologia, 2(3), 281–284. Manukhin, Y.F., Vorozheikina, L.A. (1976). Hydrogeology of the Paratunsky hydrothermal system and conditions of its formation. In: Hydrothermal systems and thermal fields of Kamchatka, V.M Sugrobov (ed), pp. 143–178. Vladivostok: Far Eastern Scientific Center. Nikulina, T.V. (2010). Biodiversity of algae of hot springs from Kuril Islands (Russia), Algologia, 20(3), 334–356. In Russian. Nikulina, T.V., Kalitina, E.G., Vakh, E.A., Kharitonova, N.A. (2016). List of diatoms from three hot springs from Kamchatka – Malkinskiye, Nachikinskiye and Verhne-paratunskiye (Russia). In: Freshwater Life, Vol. 2, V.V Bogatov (ed), pp. 108–115. Vladivostok: Dalnauka. Nikulina, T.V., Kociolek, J.P. (2011). Diatoms from hot springs from Kuril and Sakhalin Islands (Far East, Russia). In: The Diatom World, J. Seckbach and J.P Kociolek (eds). pp. 333–363. Springer, New York, London. Nikulina, T.V., Kalitina, E.G., Vakh, E.A., Kharitonova, N.A. (2015). Bacteria and diatoms of Malkinskiye, Nachikinskiye and Verkhne-Paratunskiye hot springs (Kamchatka, Russia). In: Present state and methods of studying inland water ecosystems. Materials of the Russian scientific conference dedicated to the 100th anniversary of the birth of I.I. Kurenkov, S.L Rudakova (ed), pp. 104–110. KamchatNIRO, Petropavlovsk-Kamchatsky. Nikulina, T.V., Grishchenko, O.V. (2017). Diatom flora of Dachnye thermal springs (Kamchatka Peninsula, Russia). In Vladimir Ya. Levanidov’s Biennial Memorial Meetings, 7, E.A. Makarchenko (ed), pp. 185–193. Vladivostok: FNCEATB FEB RAS. Petersen, J.B. (1946). Algae collected by Eric Hultén on the Swedish Kamtschatka Expedition 1920–22, especially from hot springs, Det Kgl. Danske Vidensk. Selskab, Biol. Meddel, 20(1), 3–120. Piip, B.I. (1937). Thermal springs of Kamchatka. Kamchatskaya ser. Moskow, Leningrad: Acad. Sci. USSR. Schmidt, C. (1885). Hydrologische Untersuchungen In Die Thermalwasser Kamtschatka’s, 7(32, 18). St. Pétersbourg: Mémoire de l’Académie Impériale des Sciences Sládeček, V. (1986). Diatoms as indicators of organic pollution, Acta hydrochim. hydrobiol., 14(5), 555–566. Stockner, J.G. (1967). Observations of thermophilic algal communities in Mount Rainier and Yellowstone National Parks, Limnol. Oceanogr., 12(1), 13–17. Stockner, J.G. (1968). The ecology of a diatom community in a thermal stream, British Phycological Bulletin, 3(3), 501–514. Swift, E. (1967). Cleaning diatoms frustules with ultraviolet radiation and peroxide, Phycologia, 6(2/3), 161–163. Van Dam, H., Mertens, A., Sinkeldam, J. (1994). A coded checklist and ecological indicator values of freshwater diatoms from the Netherlands, Netherlands J. Aquat.Ecol., 1(28), 117–133.

Diatoms From Hot Springs of the Kamchatka Peninsula 333 Van de Vijver, B., Gremmen, N., Beyens, L., Le Cohu, R. (2004). Pinnularia sofia Van de Vijver and Le Cohu spec. nov., a new spine-bearing, chain-forming Pinnularia species from the subantarctic region, Diatom Research, 19(1), 103–114. Van de Vijver, B., Agius, J.T., Gibson, J.A.E., Quesada, A. (2009). An unusual spine-bearing pinnularia species from the antarctic livingston island (South Shetland islands), Diatom Research, 24(2), 431–441. Wasser, S.P., Kondratieva, N.B., Masyuk, N.P., Palamar-Mordvintseva, G.M., Vetrova, Z.I., Kordyum, E.L., et al. (1989). In The reference book, S.P Wasser (ed). Kiev, Ukraine: Naukova dumka. Yoshitake, S., Fukushima, H., Lepskaya, E.V. (2008). The diatom flora of some hot springs in Kamchatka, Russia. Proceedings of the 19 International Diatom Symposium. pp. 151–168. Bristol: Biopress Ltd.

15 Biodiversity of High Mountain Lakes in Europe with Special Regards to Rila Mountains (Bulgaria) and Tatra Mountains (Poland) Nadja Ognjanova-Rumenova1,2,*, Agata Z. Wojtal3, Elwira Sienkiewicz4, Ivan Botev2 and Teodora Trichkova2 1

Geological Institute, Bulgarian Academy of Sciences, Sofia, Bulgaria 2 Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria 3 Institute of Nature Conservation, Polish Academy of Sciences, Kraków, Poland 4 Institute of Geological Sciences, Polish Academy of Sciences, Research Centre at Warsaw, Warsaw, Poland

Abstract This paper synthesises the knowledge on diatom biodiversity of two high-mountain regions in Europe: the Rila Mts. (Bulgaria) and the Tatra Mts. (Poland). The composition of the benthic diatom communities in the studied lakes showed considerable differences, both in regards to the number of species and their relative abundance. This must be seen in relation to the different geomorphological, hydrochemical and hydrological characteristics of the lakes and their catchments. There is a relationship between species diversity and the main gradients in the water chemical environments. The lake sediment continuum constitutes an excellent model for integrative studies. Current biodiversity informatics is an important contribution to the ongoing efforts to take the measurement of biodiversity. Keywords: Diatoms, biodiversity, mountain lakes, environmental factors, palaeolimnology, Rila Mountains, Tatra Mountains

15.1

Introduction

Global biodiversity clearly declined in recent years. The decline was greater in freshwater than in terrestrial or marine systems (Turak et al. 2017). Ecology of mountain lakes remains still poorly understood. They are characterised by low productivity and isolation. The majority of high-mountain lakes are relatively small, having cold, oligotrophic and dilute waters. Oligotrophic lakes are threatened by direct (nutrient enrichment, land-reclamation, etc.) and indirect (diffuse airborne pollution, climate change) impacts. Moreover, in contrast to lowland lakes mountains lakes often register climate changes directly (Catalan et al. 2009). High-mountain lakes are sensitive indicators of *Corresponding author: [email protected] Joseph Seckbach and Richard Gordon (eds.) Diatoms: Fundamentals and Applications, (335–354) © 2019 Scrivener Publishing LLC

335

336 Diatoms: Fundamentals and Applications past, present and future global change (Catalan et al. 2009, 2013; Catalan 2015). There are over 50 000 remote mountain lakes across Europe, occurring from Arctic to southern Iberian Peninsula (Kernan et al. 2009). All these lakes support unique aquatic plant and animal communities. The individual characteristics of the habitats dictating which diatoms live there are: altitude (the higher the habitat, the colder its water and the less organic remains enter it because there is less vegetation in the basin), mineral salts concentration in water (vary depending on the geology of the basin), and the size of lakes (vary from a few square metеrs to more than ten hectares in the case of the largest). Oligotrophic aquatic habitats have been neglected by ecologists and taxonomists including diatomists, whose attention has been more focused on aquatic environments affected by human impacts (eutrophication, acidiphication, etc.) and by their characteristic diatom species (e.g., Kociolek & Stoermer 2009; Cantonati & Lange-Bertalot 2010). These habitats are often located in National Parks, and this facilities the conservation of their ecological integrity. Oligotrophic lakes are perceived as important also as reference sites for monitoring systems and ecological restoration projects (Kociolek & Stoermer 2009). Lange-Bertalot and Steindorf (1996) proposed the first Red List for diatoms in the region of Central Europe, where they supposed that the strictly oligotraphenitic diatom taxa are endangered.

15.1.1

Factors Which Control the Diatom Distribution

The biodiversity and diatom composition in aquatic ecosystems are controlled by many regional and global environmental factors. The excellent material to estimate the changes in diatom assemblages occurring throughout the years are dated sediments collected from mountain lakes. The natural distribution of diatoms is mainly driven by climate and water chemistry (pH, dissolved oxygen, nutrients availability etc.). Due to isolated geographic location of mountain ecosystems the alterations in diatom assemblages occurred in these lakes are mainly caused by climate changes and environmental conditions (Battarbee 2005; Bigler et al. 2006). Usually remote, mountain lakes are located in pristine places, often in protected areas without intensive anthropogenic activity, forestry practices and agriculture pressure. However, in the 20th century the human-induced changes such as an increase of airborne pollution and climate warming after the Little Ice Age (LIA) caused a higher trophic level which affected the diatoms distribution (Battarbee 2005). Generally alpine lakes are located on the sensitive bedrock with low acid neutralizing capacity (ANC). As a result of poorly buffered rocks in the catchment, even small load of pollution delivered to the oligotrophic lakes may disrupt the environmental balance. The most of mountain ecosystems were oligotrophic from the onset of the lakes’ existing but approximately since the second half of the 20th century more fertile water conditions are observed. An increase of nutrients amount was connected with climate warming accelerated weathering of rocks and the tourism intensification caused enhanced of soil erosion and delivery of the base cations from the catchment to the lakes (Toro & Granados 2002). The rise of total phosphorus and nitrogen often is limiting factor for phytoplankton development i.e. the decrease of their biodiversity and species richness. Lower diversity of aquatic biota might be expected if species grow slowly in

Biodiversity of High Mountain Lakes in Europe 337 comparison with the rate of changes in lake environment which often occur in spring and autumn (Hinder et al. 1999). The distribution of diatom flora in mountain ecosystems also depends on lakes’ elevation. In Swiss Alps the lakes located at the highaltitude were dominated by small Fragilaria sensu lato such as Staurosirella pinnata (Ehrenb.) Williams et Round, Pseudostaurosira pseudoconstruens (Marc.) Williams et Round, P. brevistriata (Grun.) Williams et Round and Achnanthidium minutissimum (Kütz.) Czarnecki. In lakes located at lower elevation a higher frequency of planktonic Asterionella formosa Hassal, Cyclotella cyclopuncta Håkansson et Carter and Tabellaria flocculosa (Roth) Kütz. were observed (Bigler et al. 2006).

15.1.2

Biodiversity Assessment

There are various methods for the assessment of biodiversity like biodiversity indices, richness indices, evenness indices, ecosystem indices, biodiversity models for ecosystem and species ordination techniques (Sreekanth et al. 2015). One of the many indices estimating the diatom diversity ( ¯ ), evenness (E’) and species richness is the Shannon-Weaver index (Shannon & Weaver 1964). ShannonWeaver diversity index ( ¯ ) represents a combination of richness and abundance and it is calculated using two based logarithms to evaluate diatom biodiversity in the benthic and sediment diatom samples. The taxonomic richness (S), corresponding to the total number of taxa present, was also characterized for each lake. The diversity index was used to examine the general trend in diatom distribution and evenness is a measure of the similarity of different species. A high value of ¯ indicates diverse community and lower value of ¯ represent less diversity. If the value equals 0, the composition of community consists of one species. Among 16 lakes located in the Italian Alps, the range of the Shannon-Weaver index in littoral diatom assemblages was relatively wide ( ¯ =1.7–5.0) what was confirmed by high species richness (Tolotti 2001). An alpine, oligotrophic acidophilous taxa belonging to the genera Navicula, Eunotia, Pinnularia, Achnanthes s.l. and Cymbella s.l. dominated these lakes. Seasonal collecting of epilithic diatoms from Laguna de La Caldera (Sierra Nevada, Spain) showed that during 1996–1998 the Shannon-Weaver index did not fall below 1.0 with except for the two last sampling in 1996, when the lake was dominated by Fragilaria rumpens (Kütz.) Carlson ( ¯ =0.4–0.6), but other taxa were almost completely absent. In the next period the diatom assemblages established itself and the most frequent species was Achnanthidium minutissimum with accompanying Encyonema minutum (Hilse) Mann, Encyonopsis microcephala (Grun.) Krammer and Navicula cryptocephala Kütz. The values of diversity index also increased and varied between 1.0 and 2.2. Ordination methods are used to measure the association of species with different sites, environments and habitats. These multivariate techniques arrange sites along axes on the basis of data on species composition, that is to say powerful methods for analyze ecological niches. There are various methods of species ordination techniques: principal component analysis, canonical correspondence analysis, etc. The numerous methods and tools used in the diversity studies require lengthy calculations, estimations and derivations. A number of various software platforms are available, e.g. PAST, BiodiversityPro, CANOCO, etc. (Ter Braak et al. 2002).

338 Diatoms: Fundamentals and Applications

15.2

Recent Datom Biodiversity in High Mountain Lakes in Bulgaria and Poland

This study includes two integrated regional studies in two protected areas – the Tatra Mts., Poland and the Rila Mts., Bulgaria. Each region posses its own driving mechanisms, which are multi-faced and inter-related. The Tatra Mts. are the highest part of the 1300 km long Carpathian range. The Tatras area is protected as Polish National Park and is under a special protection as an International Biosphere Reserve (Kawecka 2012). The algal flora of this area was studied and recently the elaborations of diatoms from Tatra Mts. streams (Kawecka 2012) and springs (Wojtal 2013) were published. The numerous lakes were also studied (e.g., Kawecka & Galas 2003; Sienkiewicz & Gąsiorowski 2014), however any summarized study was not made. The Tatra Mts., were study lakes were selected are mainly built of magmatic rock (granitoids), and are inhabited by oligotraphentic diatoms. The Rila National Park covers the highest parts of the Rila Mts. (altitude of 1000–2925 m a.s.l.) and it is the largest National Park in Bulgaria. This area is remarkable by its 140 glacial lakes, which are close to the Central European alpine lakes regarding their age and evolution. The investigation of the diatom flora of the Rila glacial lakes is interesting from the ecological and floristic point of view because the Rila Mts. is one of the Southwestern European mountains with glacial lakes of an alpine type (Ognjanova-Rumenova 2012). The aim is to investigate and compare the diatom diversity of lakes, using benthic and sedimentary assemblages within and between two distinct protected regions of Europe – the Tatra and the Rila mountains.

15.2.1 The Rila Lakes, Bulgaria The Rila Mts., the highest mountain on the Balkan Peninsula (peak Mousala is 2925 m a.s.l.), are situated in Southwest Bulgaria. Compared to other mountains, the Rila Mts. are remarkable for their clearly outlined block-fault tectonic structure, high ridges rising above 2600–2800 m a. s. l. and widespread alpine and subalpine relief. Most characteristic hydrographic elements are the mountain lakes and the rivers that spring from them and flow radially to the Danube and the Aegean Sea (Zyapkov & Naidenow 2000). The total number of lakes in Rila is 140 (Ivanov et al. 1964). All these lakes are of glacial origin. They are considered to be between 13000 and 14000 years old, formed when the ice masses, covering the high altitudinal zones during Pleistocene began to thaw. Most numerous are lakes located at 2200 to 2500 m a.s.l. The temperature of the lake waters varies from 0.0 to 20.0 C. The pH values fluctuate from ~6.0 to 7.8, showing insignificant differences on the surface of the lakes and on their bottom (Ivanov et al. 1964; Zyapkov & Naidenow 2000). The first and basic records about the living assemblages and biological processes in the lake ecosystems of the Rila Mts. were provided by Valkanov (1932, 1938), Petkoff (1939), Vodenitcharov (1962), and Kawecka (1974, 1976). The recent benthic diatoms have been precise investigated according to the pan-European study of mountain lakes: the project EMERGE (European mountain lake ecosystems: regionalisation diagnostics & socio-economic evaluation) during 2001–2005. A lot of results obtained on diatoms, invertebrates and water chemistry were published (Ognjanova-Rumenova et al. 2006, 2009a, 2009b; 2011; Catalan et al. 2009; Curtis et al. 2005; Camarero et al. 2009a, 2009b; Ognjanova-Rumenova 2012). The latest investigations focus on some more specific questions regarding the Rila

Biodiversity of High Mountain Lakes in Europe 339 lakes, such as – phytoplankton structure (Beshkova et al. 2016), lake evolution and climate change (Lotter & Hofmann 2003). The diversity of recent epilithic diatoms from nine lakes, situated in three different cirques in the Rila Mts. was analyzed by Ognjanova-Rumenova et al. (2009a). A total of 166 diatom taxa belonging to 32 genera were recorded. The number of the diatom species in the investigated lakes ranged from 79 to 148. The minimum species number (79) was recorded in Lake Ledeno, one of the highest glacial lakes on the Balkan Peninsula (2709 m a.s.l.). The Shannon-Weaver Index for the nine lakes varied between 2.70–4.50. The most diverse diatom epilithic flora was observed in lakes Sulzata and Karakashevo, the lowest species richness was found in the slightly acidified lake Alekovo. The composition of the benthic diatom communities showed relationships between species diversity and the main chemical gradients (pH, conductivity, alkalinity and nutrients). The diatom community structures generally confirmed the status of the studied lakes as judged from their water chemistry. Two main groups of lakes were distinguished. The first group included mostly deep, non-acidified lakes, where the circumneutral and alkaliphilic benthic species belonged to genus Fragilaria s.l. prevailed. The second group included shallow, moderately acidified lakes, where acidophilous diatoms are the most abundant (Eunotia glacialis Meist., E. exigua (Breb. ex Kütz.) Rabenh. and Tabellaria flocculosa). There is a larger proportion of acidophilic planktonic species of genus Aulacoseira Thw. – A. pfaffiana (Reinsch) Krammer and A. alpigena (Grun.) Krammer.

15.2.2 The Tatra Lakes, Poland The Tatras are the highest mountains in Poland (Rysy peak, 2499 m. a.s.l.). They are located in the West Carpathians, with a total area in Poland - 785 km2. The Tatras are divided in two parts: High Tatra Mts. (East Tatras) and West Tatra Mts. The High Tatra Mts. area is built mainly from magmatic rocks (granitoids), highly resistant to erosion. The West Tatras area is composed of crystalline metamorphic rocks (gneiss and crystalline schists) or sedimentary carbonate rocks (mainly limestone). The first data on diatoms from lakes, streams and springs in Tatras were provided by Schumann (1867) and Gutwiński (1909). Next, many articles about recent diatoms and fossils from Tatra materials were published (e.g., Wasylik 1965; Starmach 1973; Kawecka 2012; Wojtal 2013; Sienkiewicz & Gąsiorowski 2016). The total number of lakes in Tatras is c.a. 200. In the Polish Tatras there are about 40 of them, the rest are located on the Slovak side. The Polish lakes were investigated by Marciniak (1982), Marciniak & Ciesla (1983) – Przedni Staw lake, Kawecka & Galas (2003) – lakes: Czarny Staw pod Rysami, Zadni Staw Polski, Czarny Staw Polski, Wielki Staw Polski, Zadni Staw Gąsienicowy, Zmarzły Staw Gąsienicowy, Długi Staw Gąsienicowy, Zielony Staw Gąsienicowy, Dwoisty Staw Gąsienicowy, and Czarny Staw Gąsienicowy, Sienkiewicz & Gąsiorowski (2014, 2016, 2017) – Smreczyński Staw, Toporowy Staw Niżni, Zielony Staw Gąsienicowy, Czarny Staw Gąsienicowy,Wielki Staw Polski, Przedni Staw Polski, Czarny Staw Polski, Czarny Staw pod Rysami and Morskie Oko. The status of high-mountain lakes was estimated, according to the EU Water Framework Directive, in projects AL:PE2 (ALpine LAkes: PAleolimnology and ECology), molar (MoUntain LaKe REsearch) and EMERGE (Kawecka & Galas 2003).

340 Diatoms: Fundamentals and Applications Their aims were measurement of the response of mountain lakes to acidification and recent species distribution assessment in relation to environmental gradients. The diversity of recent epilithic diatoms from the Polish and Slovak Tatra Mountain lakes were analyzed by Kawecka & Galas (2003) and Štefková (2006). Among ten Polish Tatra lakes the highest species diversities ( ¯ ) were observed in Zadni Staw Polski and Zielony Staw Gąsienicowy, while the lowest was in Czarny Staw pod Rysami. Generally the Shannon-Weaver index varied between 1.6 and 4.45 while evenness ranged 0.34–0.73. Lakes intermediately acidified were dominated by Psammothidium marginulatum (Grun.) Bukhtiyarova et Round, P. helveticum (Hust.) Bukhtiyarova et Round and P. helveticum var. minor (Flower et Jones) Bukhtiyarova et Round. In non-acidified lakes mainly occurred Achnanthidium minutissimum, Staurosirella pinnata, Fragilaria capucina Desm., Encyonema minutum and Achnanthes spp. The number of taxa in these lakes varied from 29 to 72 (Kawecka & Galas 2003). In the total of 34 Slovak Tatra lakes, 127 diatom species belonging to 26 genera were identified. The lowest species richness was found in acidified Starolesnianske pleso (seven taxa) but the highest number of diatoms (28-32) were in Veľké spišské pleso, Nižné Jamnícke pleso, Dračie pleso, Ľadové pleso, Vyšné Žabie bielovodské pleso and Prostredné spišské pleso. The most common taxa present in more than 20 lakes were: Achnanthidium minutissimum, Psammothidium helveticum, P. subatomoides (Hust.) Bukhtiyarova et Round, Encyonema minutum, Denticula tenuis Kütz., Staurosirella pinnata, Pinnularia microstauron (Ehrenb.) Cl. and Tabellaria flocculosa. The Shannon-Weaver index varied between 0.68 and 2.88. The highest species diversities were estimated in Batizovské pleso and Ľadové pleso, while the lowest in Starolesnianske pleso. The non-acidified lakes were mainly dominated by Achnanthidium minutissimum with lower frequency of Denticula tenuis, Encyonema minutum and Diadesmis gallica var. perpusilla (Grun.) Lange-Bertalot. In lakes with acidic water pH were found Psammothidium helveticum, P. marginulatum, Aulacoseira distans (Ehrenb.) Sim. and Tabellaria flocculosa (Štefková 2006). However, analysis of the epilithic diatoms shows the changes in seasonal biodiversity concerning relatively modern times, because the monitoring data usually are available for a few last decades. The changes in diatom distribution in wider context can be obtained by study of phytoplankton from the dated lake sediments.

15.3

Diatom Community Changes in High-Mountain Lakes in Bulgaria and Poland from Pre-Industrial Times to Present Day

The high mountain lakes could be considered as potential archives, documenting changes in air-pollution deposition or climate change through detailed studies of their preserved biological remains deposited within the sediment record (Clarke et al. 2005).

15.3.1 The Rila Mts. Sediment short cores (14–17 cm) were collected from nine lakes situated in three different cirques above the natural timberline in the Rila Mts. (Ognjanova-Rumenova et al. 2011). Diatoms were analyzed from the top (present day) and the bottom (pre-industrial)

Biodiversity of High Mountain Lakes in Europe 341 6.0 Top

Bottom

Shannon-weaver index

5.0 4.0 3.0 2.0 1.0

a

Le de no Al ek ov o Ka ra ka Go sh ev rn o o M ar i c Do hi no ln o M ar ich in o

ak

a izm

ek br Bu

Bl

ot o Ok

Su

lza

ta

0.0

Sediment samples

Figure 15.1 Histogram showing the Shannon-Weaver index of the top/bottom sediment samples for studied lakes in the Rila Mts. according to EMERGE project’2000.

sediment samples. This “top/bottom” comparison approach is commonly used in the palaeolimnological studies for presenting a “snap-shot” of the environmental changes and for the assessment of limnological conditions before the identified effects of industrialization, recent climate change and extensive catchment land-use changes (e.g., Clarke et al. 2005). In total 213 taxa, belonging to 36 genera were recorded. The total number of species found in the different cirques (Sedemte ezera, Musalenski and Marishki) ranged between 115 and 183. The lowest number (45 taxa) was recorded in Lake Ledeno (Musala cirque) – the second highest natural lake in Bulgaria. Alkalinity, pH, and conductivity summarize the main chemical characteristics of the studied lakes. Changes in the ratio planktonic to non-planktonic diatom abundance could be also related to changes in climate and to associated changes in nutrients, ice-cover and erosion caused by climate warming. The Shannon-Weaver index of the diatom communities in bottom samples (~c.a. 150 years before) varied between 3.25 and 5.44. The detected pH change over the last 150 years could explained the lower values of the Shannon-Weaver index and species richness in the diatom assemblages for all top sediments. (Figure 15.1) The increase in the planktonic diatoms in the lake could be affected by changes in the icecover regimes. Owing to the limited number of sites (9) and the limited gradients from which our lakes have been chosen (i.e., oligotrophic, bedrock type lakes), our conclusions for the changes in ice-cover regimes could be considered preliminary. Further monitoring is needed to evaluate the direction and size of past and future changes, and to investigate the effects of the increasing load of atmospheric pollutants. Chemical and biological sedimentary records of the high alpine lake “Bubreka”, Rila Mountains, Bulgaria, were used to reconstruct palaeoecological conditions and to determine the phases in the ontogeny of the lake (Ognjanova-Rumenova et  al. 2009b). Four

342 Diatoms: Fundamentals and Applications sediment cores were taken in the deepest area of the lake for a multy–pollutant, multi-proxy study to determine the temporal trends in pollution inputs and ecological change. One core was radiometrically dated by 210Pb and 137Cs and other cores correlated to it by lithostratigraphic means. They were analysed for percentage of dry weight (DW), loss on ignition (LOI), pigments, diatoms, chrysophyte cysts, cladoceran, chironomids and spheroidal carbonaceous particles (SCP). These precise studies show that high mountain lakes are good sites for the study of the evolution and effects of the long range transport of atmospheric pollutants from the industrial lowlands to mountain regions. In total, 171 diatom taxa were distinguished. The Shannon-Weaver index in the diatom communities ranged between 4.30 and 5.64. Four distinct successional phases can be described in the lake history during the last ~250 years, revealing complex interactions between anthropogenic impact and climate. Phase 1 (c.a. AD 1762–1834) is characterized by stable oligotrophical conditions. The diatom flora is dominated by epiphytic species, such as Psammothidium curtissimum (Carter) Aboal, Eunotia bilunaris (Ehrenb.) Schaarschmidt but also by Gomphonema parvulum (Kütz.) Kütz. The Shannon-Weaver index in the diatom assemblages during this period ranged between 4.30 and 5.21. During Phase 2 (c.a. AD 1834 −1928) there is a change from oligotrophic to mesotrophic conditions. The diatom flora is very rich and diverse, the Shannon-Weaver index ranged between 4.70 and 5.25. An increase in the abundance of Aulacoseira alpigena is also observed, but among the subdominant diatoms there are different Cymbella s.l. species. Phase 3 (c.a. AD 1928 - 1994) shows an increase in LOI due to the high proportion of organic material. The shorter ice cover period in the lake may have created conditions more suitable for the growth of phyto- and zooplankton and changed the overall primary production from benthos to plankton. There is a peak in the planktonic and tychoplanktonic diatoms (30.9%), mainly Aulacoseira valida (Grun.) Krammer. The Shannon-Weaver index ranged between 4.31 and 5.64. In Phase 4 (c.a. AD 1994 - 2000) there is increased LOI, increasing percentages of periphytic diatoms, less favorable conditions for cladocerans, and rheophilic chironomid assemblages which indicate rapid input of nutrients causing mesotrophication (Ognjanova-Rumenova et  al. 2009b). The centric planktonic species decrease markedly. The diatom flora shows an increase in periphytic species, such as Staurosira construens Ehrenb., S. venter (Ehrenb.) Cleve et Möller, Fragilaria arcus (Ehrenb.) Cl. and Diatoma mesodon (Ehrenb.) Kütz. The Shannon-Weaver index ranged between 4.99 and 5.32. The SCP record and comparisons with other European mountain lakes suggest there is a moderate impact from atmospheric deposition compared with similar lakes elsewhere in Europe.

15.3.2 Tatra Mts. The examples of alterations in the diatom flora diversity are three cores encompassing sediments accumulated between 240 and 520 years ago and collected from Toporowy Staw Niżni (TSN), Morskie Oko (MOK) and Czarny Staw Polski (CSP). Toporowy Staw Niżni (TSN) belongs to dystrophic water bodies while MOK and CSP are oligotrophic lakes. TSN is fishless whereas CSP was artificially stocked by brook trout. MOK is the only one lake located in the Polish part of the Tatra Mts. with natural fish population, which was additionally stocked in 1881 and in the 1960s. The ShannonWeaver index in the sediments of MOK ranged between 1.88 and 3.41 with an average value of 2.85. During the last 500 years the most common diatoms occurred in the lake

Biodiversity of High Mountain Lakes in Europe 343 were: Fragilaria nanana Lange-Bertalot, Achnanthidium minutissimum, A. caledonicum (Lange-Bertalot) Lange-Bertalot and Psammothidium levanderi. The lowest value of ¯ was at the beginning of the 1990s together with the lowest evenness (0.53) and number of species (34). From that time to the present, the lake was dominated by Aulacoseira subarctica (O.Müll.) Haworth and Fragilaria capucina, which reached a maximum frequency in the core. Both species prefer mesotrophic to eutrophic water and indicate a higher nutrient level. The reconstruction of diatom-inferred TP pointed to mesotrophic conditions in the lake during this time (Sienkiewicz & Gąsiorowski 2016). TSN is characterized by relatively high biodiversity during the last two centuries although this is dystrophic lake with water pH