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Handbook of Algal Technologies and Phytochemicals: Volume II Phycoremediation, Biofuels and Global Biomass Production [1 ed.]
 9780367178192, 9780429057892, 9780429603655, 9780429598135, 9780429609176

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Dr. G.A. Ravishankar is a Professor of Biotechnology, serving as Vice-President in Research and Development, Life Science division, Dayananda Sagar Institute, Bengaluru, Karnataka, India. Previously, Dr. Ravishankar worked as Chief Scientist & Head, Plant Cell Biotechnology Department, Council of Scientific and Industrial Research-Central Food Technological Research Institute, Government of India, Mysore, Karnataka, India. He has 35 years’ research experience in reputed national research laboratories. He has received international and national fellowships from various scientific boards, namely FAFST, FNASc, FNAAS, FBS, FSAB, FISAB, FAMI, FIAFoST (Canada), FIFST(UK), FIFT(USA). He is involved in various research fields such as Plant Biotechnology and Biochemistry, Plant secondary metabolites, Algal Biotechnology, and Food Science and Technology. His has more than 300 research papers in peer reviewed journals, review articles in peer reviewed journals, proceedings of symposia and seminar in India and abroad, and more than 40 chapters in books, 55 filed patents, 14 technologies developed, four books, and over 250 research papers presented in symposia in India and abroad, as well as over 200 invited lectures delivered in India and abroad. His papers have received citation by peer groups, with H index-53, and i-10 index of 196 with total citations of over 11,600 in Google Scholar. Dr. Ravishankar is actively contributing as editor and reviewer for various reputed international publishers.


Dr. Rangarao Ambati is a Research Assistant Professor in Food Science and Technology Programme, Department of Science and Technology, Beijing Normal University-Hong Kong Baptist University United International College, Zhuhai, Guangdong, China. Dr. Ranga Rao has completed a Ph.D in Biotechnology from the internationally recognized laboratory Central Food Technological Research Institute (CFTRI) which is a part of Laboratory of Council of Scientific and Industrial Research (CSIR), Government of India, India. Dr. Ranga Rao worked as Research Scientist, Postdoctoral Research Associate, Senior Research Fellow at various International Universities in USA, Malaysia, China and India. He is involved in group through his expertise in biotechnology; biochemistry, and food science and technology. Dr. Rangarao has published several research papers in the leading international journals including book chapters. He is also serving as editorial board member, guest editor, and reviewer for reputed international journals. He attended international and national conferences/symposia, traveling to the USA, Canada, China, Malaysia, Indonesia, and Muscat. His research citation was 880 with H-Index (11), and I-index (11) as per Google Scholar. He received research grants, travel grant awards, and scientific awards internationally and nationally. He has contributed research work to biological sciences with research publications in international and national journals. He received Fellow of the Society of Applied Biotechnology (FSAB-India); Young Scientist Award-World Food Congress by International Union of Food Science and Technology (IUFoST, Canada); TWAS-Young Affiliate by Regional office East and South-East Asia and the Pacific (ROESEAP), Chinese Academy of Sciences, China; Young Biotechnologist Award by Society of Applied Biotechnology (India), and Junior Scientist of the Year Award by National Environmental Science Academy, New Delhi, India.

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Handbook of Algal Technologies and Phytochemicals Volume II: Phycoremediation, Biofuels and Global Biomass Production

Handbook of Algal Technologies and Phytochemicals Volume II: Phycoremediation, Biofuels and Global Biomass Production

Edited by

Gokare A. Ravishankar and Ambati Ranga Rao

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2020 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper International Standard Book Number-13: 978-0-367-17819-2 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Dedication

E. Wolfgang Becker (1939–2017) We wish to offer our humble dedicaton of this volume to Dr. E. Wolfgang Becker, who was a doyen of Algal technology. During the 1970s, Dr. Becker was keen to find alternate sources of protein through algae-based foods to alleviate protein malnutrition in humans, which matched with the interests of the Central Food Technological Research Institute (CFTRI) at Mysore, India. This led to Dr. Becker’s decision to initiate algal biomass production at Mysore under the support of the Federal Ministry of Economic Cooperation, West Germany, and the Government of India from 1973 to 1976. He, along with Dr. L.V. Venkataraman, was able to lay a firm foundation for the mass production of microalgae for food applications, which is recognized globally. E. Wolfgang Becker was born in Wriezen, Brandenburg/Oderbruch, Germany on April 11, 1939. He obtained his education in SchleswigHolstein, West Germany, and later served in

the military from 1959 to 1960 as lieutenant. He earned his bachelor’s degree with a biology major in 1965, from the University of Hamburg, Germany, and master’s in biology in 1967, from the University of Tubingen, Germany. He earned his PhD in 1972 from the Institute for Plant Physiology, University of Tubingen, Germany, working under the guidance of Dr. H. Metzner, on the topic physiological analysis of photosynthesis of algae under extreme temperature conditions. From 1973 to 1976, Dr. Becker established the algal production unit at CFTRI under the Indo-German Algal Project. The studies focused initially on the cultivation of Chlorella and Scenedesmus. Realizing the difficulty in harvesting unicellular form for low-cost cultivation, the emphasis shifted to Spirulina— a multicellular cyanophycean form of high nutritional value. This laid a strong foundation for micro-algal cultivation technology. Dr. Becker was of the belief that technology should be

adaptable by rural populations to produce quality biomass for human consumption. He and his team developed several recipes for polarized algae-based foods. Integrated technology for use of poultry waste and biogas digester effluent to produce algae, and utilization of the algal biomass for poulty feed and fish feed, was developed to produce food and bioenergy. Strong scientific study of algal biomass production technology laid a firm foundation for the industrial production of microalgae at a global level. He evaluated the quality of Spirulina produced in India, Thailand, and Peru, which formed the guidelines for worldwide adoption of the low-cost production technology. After returning to Germany, Dr. Becker continued his association with the University of Tubingen, working on algae-based bioremediation for pollution abatement of heavy metals. He was actively involved in biomedical research, working on projects on drug-induced

allergic reactions in humans. He published nearly 90 research papers, and 5 books. Dr. Becker’s interest in algae, and his love for India and Indian food, brought him to Mysore almost every alternate year. His visits had lasting influence on the research and development at CFTRI for several decades to follow. Dr. Becker untiringly continued his interest in algal studies and was sought after by international scientific bodies and various countries for his expert advice. One of the editors—GAR—was associated with him closely and served as a member of an international delegation to China for quality assessment of Spirulina cultivation. Dr. E. Wolfgang Becker will be remembered for his contributions to algal cultivation technology worldwide. He was an ever smiling, friendly, and kindhearted person. His associates remember him as an excellent host! He breathed his last on April 1, 2017.

Contents Preface.........................................................................................................................................................................xi Acknowledgments.................................................................................................................................................... xiii Editors........................................................................................................................................................................xv Contributors.............................................................................................................................................................xvii

Section I  Phycoremediation Applications Chapter 1 Wastewater Phycoremediation by Microalgae for Sustainable Bioproduct Production...........................3 Najeeha Mohd. Apandi, Radin Maya Saphira Radin Mohamed, Adel Ali Saeed Al-Gheethi, and Amir Hashim Mohd. Kassim Chapter 2 Green Technology Applications for Algal Bloom Control.................................................................... 13 Mostafa M. El-Sheekh, Mohamed M. Abdeldaim, Samiha M. Gharib, and HalaY. El- Ksassas Chapter 3 Natural Algal Photobioreactors for Sustainable Wastewater Treatment................................................23 D. M. Mahapatra, N. V. Joshi, G. S. Murthy, and T. V. Ramachandra

Section II  Algal Biofuels Chapter 4 Opportunities and Challenges in Seaweeds as Feed Stock for Biofuel Production ..............................39 Mohammad Javad Hessami, Ambati Ranga Rao, and Gokare A. Ravishankar Chapter 5 Biodiesel Production from Microalgal Biomass: Challenges and Perspectives ................................... 51 Srijoni Banerjee and Debabrata Das Chapter 6 Carbon Dioxide Sequestration by Microalgae.......................................................................................63 G.V. Swarnalatha, A. Shekh, P.V. Sijil, C.K. Madhubalaji, V.S. Chauhan, and R. Sarada Chapter 7 Modulation of Lipid Biosynthesis by Stress in Diatoms.......................................................................77 Bing Huang, Virginie Mimouni, Annick Morant-Manceau, Justine Marchand, Lionel Ulmann, and Benoît Schoefs Chapter 8 Microalgal Biomass, Lipids, and Fatty Acids Production through Open or Closed Cultivation Systems: Challenges and Future Perspectives....................................................................................... 91 Ambati Ranga Rao and Gokare A. Ravishankar Chapter 9 Microalgae for Sustainable Fuel Technology: Coupling Photobioreactor and Bioelectrochemical System for Microalgae Cultivation and Hydrogen Generation............................................................ 101 Surajbhan Sevda, Dipak A. Jadhav, S.P. Jeevan Kumar, and T.R. Sreekrishnan

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Section III  Other Products of Economic Value Chapter 10 Seaweed as Source of Plant Growth Promoters and Bio-Fertilizers: An Overview........................... 111 Sananda Mondal and Debasish Panda Chapter 11 Algae: A Nutraceutical Supplement in Aquaculture........................................................................... 123 Helena M. Amaro, I. Sousa-Pinto, and A. Catarina Guedes Chapter 12 Techno-Economic Analysis of Multiple Scenarios for the Production of Microalgal Chemicals and Polymers....................................................................................................................................... 133 Giannis Penloglou and Costas Kiparissides Chapter 13 Kappaphycus Farming for Socio-Economic Development of Coastal People in India....................... 145 P.V. Subba Rao and C. Periyasamy Chapter 14 Diversity and Utilization of Marine Cyanobacteria............................................................................ 155 N. Thajuddin and G. Subramanian

Section IV  Mass Production of Microalgae Chapter 15 Open Cultivation Systems and Closed Photobioreactors for Microalgal Cultivation and Biomass Production........................................................................................................................................... 179 C.K. Madhubalaji, Ajam Shekh, P.V. Sijil, Sandeep Mudliar, Vikas Singh Chauhan, R. Sarada, Ambati Ranga Rao, and Gokare A. Ravishankar Chapter 16 Bioprocessing of Microalgae for the Production of Value Compounds..............................................203 Giorgos Markou, Christina Ν. Economou, and Imene Chentir Chapter 17 Production of Low-Cost EPA-Enriched Biomass with a Focus on the Filamentous Algal Strain Tribonema spp................................................................................................................ 211 Xuemei Bai, Hong Wu, Yu Chen, Hui ling Wang, Lin Wang, Jinfeng Geng, Zhongzhen Cai, Jinyang Zhang, Qing Li, Jie Teng, Qian Feng, Jiantao Luo, Fangwei Liu, Min Xu, and Zhenqi Zhu Chapter 18 New Strategies for the Design and Control of Raceway Reactors to Optimize Microalgae Production........................................................................................................................ 221 Marta Barceló Villalobos, Francisco Gabriel Acién Fernández, José Luis Guzmán, Jose María Fernández Sevilla, and Manuel Berenguel Chapter 19 Sustainable Water and Nutrient Management in Algal Biomass Production Systems........................ 231 Daniel P. Geller, Keshav C. Das, Gary L. Hawkins, Brian H. Kiepper, and Manjinder Singh

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Chapter 20 Technologies for Separation and Drying of Algal Biomass for Varied Applications.......................... 241 Júlio Cesar de Carvalho, Eduardo Bittencourt Sydney, Paulo Cesar de Souza Kirnev, Adriane Bianchi Pedroni Medeiros, and Carlos Ricardo Soccol

Section V  Production of Algal Biomass and Products Worldwide Chapter 21 Micro- and Macroalgae Production in Thailand for Food, Feed and Other Applications: Current Trends and Future Challenges................................................................................................ 253 Apiradee Hongsthong, Ratana Chaiklahan and Boosya Bunnag Chapter 22 Global Microalgal-Based Products for Industrial Applications.......................................................... 267 Ambati Ranga Rao and Gokare A. Ravishankar Chapter 23 Macro and Micro Algal Impact on Marine Ecosystem: A Global Perspective................................... 279 Sarban Sengupta and Ruma Pal Index........................................................................................................................................................................287

Preface Algae are the most significant group of autotrophic organisms which support life forms in aquatic, semi aquatic and terrestrial environments. They are also the earliest organisms on this planet which shaped the ecosystem, making it fit for an enormous number of diverse life forms including humans. Ironically, on one hand their biodiversity has still not been fully explored by mankind, but on the other hand we are unfortunately losing them from our ecosystem due to human activities. Their destruction is bound to create imbalances in our planet causing irreversible damage to the ecosystem and food chain. Human ingenuity to utilize the macroalgae and the seaweeds has been well known for several centuries. Whereas the advancement of science and technology has laid emphasis on the identification of micro- and macroalgal forms, their cultivation and utilization for various intended uses, their utility as sources of food, and as phytochemical factories, is beginning to open up immense opportunities for industrial production. They also have enormous abilities for environmental cleanup of polluted ecosystems. The shift in consumer preferences for nature derived, natural and nature identical molecules has provided a new impetus into research and development to explore the potential of micro- and macroalgae for food, health, energy and environmental needs. Realizing the need to provide a focus on this important group of organisms with unlimited potential for multifaceted utilities and industrial applications, we embarked on bringing out two comprehensive volumes for the benefit of researchers, industrialists, entrepreneurs and consumers. The contents of the two volumes offer an overview of the organisms, their distribution in various parts of the globe, the methods to culture them, scale-up technologies, downstream processing, biological activities, regulatory considerations, utilization of biomass in processed foods, feed, health and pharma products and other novel applications including fuel and environmental aspects. These aspects have been described in the two volumes and the following points provide focus at glance.

VOLUME I This volume relates to the use of micro- and macroalgae in food, health and nutraceutical applications.

Applications of algae for polysaccharides such as alginate, agar and most recent trends in utility of fucoidan in food, nutraceuticals and health care products have been highlighted. Utility of Spirulina and Chlorella which are the earliest cultivated microalgal forms has been discussed. Spirulina has been termed “super food” by WHO and is expected to rule the microalgal market in future as well. Antimicrobials from seaweeds are finding utility in various applications in food preservation, cosmetics and health formulations. The emerging opportunities of volatiles of algae add newer dimensions to the potentials of micro- and macroalgae. The extraction methods using ultrasound technology, supercritical carbon dioxide and subcritical water affords recovery of metabolite without the loss of biological activities during downstream processing. Applications of seaweed for treatment of diabetes are interesting as they open up newer option of usage as nutraceutical or functional food. For the vegetarians, the sources of B12 being limited, here is a detailed report from a novel seaweed source, with a possibility of utilization in vegan foods. Carotenoids such as β carotene and astaxanthin which are industrially produced pigments from microalgae Dunaliella and Haematococcus have been presented in detail with newer perspectives of future utility as nutraceuticals and cosmeceuticals. Fucoxanthin from seaweeds has been dealt with extensively, bringing to focus the health applications. Thus, the emerging applications of carotenoids from algal sources in the treatment of diseases such as cancer, ulcer and many more, provide future directions. A wide range of utilities of micro- and macroalgae with special reference to marine forms has been discussed with examples of human trials on the basis of several leads obtained through drug discovery programs leading to the evaluation of bio-efficacies. Their utility in the health foods and nutraceuticals segments is beginning to expand. The market share of algae-based products has been dealt in a few of the chapters providing global perspectives in bio-business. The future lies in the exploitation of omics tools to elucidate genetic regulation of the phytochemicals of microalgae for augmented production; also, to produce the molecules of interest in newer hosts, which is expected to open up production of novel products and processes.

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VOLUME II This volume relates to the use of micro- and macroalgae in bioremediation, biofuels and global biomass production for commercial applications. Phycoremediation is receiving attention due to its potential to clean up heavy metals, pollutants and atmospheric carbon dioxide, acting as a sequestration agent and in treatment strategies. The eco-friendly nature and efficiency of the process has been discussed. Also, the utilization of algal biomass in open cultivation, or closed cultivation through photobioreactor systems, for the treatment of contaminants is highlighted. Algae-based biofuels are being explored globally as a source of alternate fuels. However, the challenges to overcome low productivity are still a big bottle neck. However, emerging strategies to enhance the yields have been discussed. Integration of algal biofuel production with wastewater treatment, carbon dioxide sequestration and genetic regulation of lipid production pathways has been recommended as a sustainable production strategy. Photobioreactor applications for biofuels would provide adoption of technology independent of environmental conditions and also usage of flue gases in effective manner. The bio-refinery approach to produce multiple products is envisaged to provide economically viable technologies. Application of seaweed as a source of fertilizer, health foods and cosmetics, coupled to their cultivation methodologies for the production of value-added biomass for gainful employment is promising. Detailed treatise on the cyanobacterial diversity and potential for future applications has thrown open innumerable industrial possibilities. The scale up technologies are constantly being innovated for open pond production or closed cultivation in photobioreactors. The current research on the design of reactors for mass cultivation and downstream processing strategies for varied applications have been presented in a few chapters. Global perspectives of algal biomass production with a few of the major industries across the globe has been highlighted. Case studies with reference to industries in Thailand and Indonesia have been detailed.

Preface

The overall objective of these two volumes is to provide up-to-date information and projected future possibilities based on the advancing research and innovations in the diversified facets of phycotechnologies. The chapters are written by experts from 28 countries. This collective effort from scientists from all over the world represents global perspectives on the topics, which are discussed in a focused manner to bring up to date information, and is largely based on the authors’ own experience in working with various systems. The organization of the chapters in each of the volumes is under the following heads: Algal Constituents for Food, Health and Disease Applications (Vol I: Chapters 1 through 22) Algal Genomics and Metabolomics (Vol I: Chapter 23 through 25) Phycoremediation Applications (Vol II: Chapters 1 through 3) Algal Biofuels (Vol II: Chapters 4 through 9) Other Products of Economic Value (Vol II: Chapters 10 through 14) Mass Production of Microalgae (Vol II: Chapters 15 through 20) Production of Algal Biomass and Products Worldwide (Vol II: Chapters 21 through 23) These two volumes will be a treasure trove of information to students and researchers of plant sciences, biological sciences, agricultural sciences, foods and nutrition sciences, health sciences and environmental sciences. However, its application value will impact professionals such as agricultural scientists, food experts, biotechnologists, ecologists, environmentalists and biomass specialists. Its global relevance and outreach have economic implications in industries dealing with foods, nutraceuticals, pharmaceuticals, cosmeceuticals, bioenergy, health care products and bioenergy. Gokare A. Ravishankar Ambati Ranga Rao

Acknowledgments At the outset the editors are extremely thankful to the contributors for their dedication in providing the material in a comprehensive manner for the benefit of readers across the globe. Their kind cooperation in every facet of publication is gratefully acknowledged. The quality of these volumes is attributable to the contributors’ commitment to share their knowledge and experience with all those interested in the topic pertaining to basic and applied aspects of phycotechnologies. We are grateful to Alice Oven, Lara Spieker, Jennifer Blaise and their team at CRC Press for their diligent efforts in publishing these volumes in an elegant manner. We are thankful to our families who have extended wholehearted support and encouraged us to take up this task, even though it involved a lot of time away from them. G.A.R. thanks his wife Shyla, son Prashanth, daughter-in-law Vasudha, and daughter Apoorva. A.R.R. thanks his wife Deepika, daughter Jesvisree, parents Venkateswaralu and Tulasidevi, brothers, sisters-in-law, sisters and brothers-in-law. G.A.R. is thankful to Dr. Premachandra Sagar, Vice Chairman, Dayananda Sagar Institutions, Bengaluru, for granting permission to take this additional responsibility. A.R.R. is thankful to Dr. L. Rathaiah, Chairman; Mr. L. Sri Krishnadevarayalu, Vice-Chairman; Prof. Dr. K. Ramamurthy Naidu, Chancellor; Dr. M.Y.S. Prasad, Vice-Chancellor; Dr. Madhusudhan Rao, Director, Engineering and Management; Dean Academics, Dean R&D; and Head, Biotechnology Department of Vignan’s Foundation for Science, Technology and Research University for providing facility and support to fulfill this new assignment. We thank the institutions of the Government of India, Department of Biotechnology, Department of Science and Technology and Indian Council of Medical

Research, for the grant of financial support to our studies on algal biotechnology done at CSIR-Central Food Technological Research Institute (CFTRI), Mysuru, India. We thank the staff and students of the Plant Cell Biotechnology Department and collaborating departments at CSIR-CFTRI for the research done by our team, especially, the late Dr. L.V. Venkararaman, Dr. R. Sarada, Dr. M. Mahadevaswamy, Dr. KSMS Raghava Rao, Dr. V. Baskaran, Dr. Shylaja Dharmesh, T.R. Shamala, Dr. K. Udaya Sankar and many non-technical staff, including K. Shivanna, C.V. Venkatesh, H.S. Jaya in many of our algal projects. Also, thanks are due to a number of research students and associates of G.A.R. for their dedication to research and authorship in a large number of publications. G.A.R. is proud to have associated with doyens of Algal Biotechnology, the late Dr. L.V. Venkataraman (LVV) of CFTRI, Mysore and the late Dr. E. Wolfgang Becker of Eberhard-Karls-Universitat, Tubingen, Germany, who started the Algal Projects at CFTRI, with the support of GTZ (Gesellschaft für Technische Zusammenarbeit), Germany. This laboratory at CFTRI gained prominence as world renowned center for algal biotechnology research. G.A.R. is also thankful to the Department of Biotechnology Government of India for the National Technology Day Award conferred on him and LVV on May 11, 2003 for commercialization of Spirulina Technology in India. A.R.R. is thankful to IUFoST (Canada) and TWASCAS for being honored as a young affiliate for his work on Astaxanthin from Haematococcus. Gokare A. Ravishankar Ambati Ranga Rao

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Editors Gokare A. Ravishankar is presently the vice president of research and development (R&D) in life sciences and biotechnology at Dayananda Sagar Institutions, Bengaluru, India; he is a professor of biotechnology. Earlier, he had a distinguished research career of more than 30 years working at the Central Food Technological Research Institute (CFTRI), Mysuru, India, and in the institutions of government of India. He served as chairman of the board of studies in biotechnology at the Visvesvaraya Technological University, Belgavi, India, and as academic council member of Dayananda Sagar University. He has also been a member of the boards of eight universities. He is an internationally recognized expert in the areas of plant biotechnology, algal biotechnology, food biotechnology and postharvest technologies, plant secondary metabolites, functional foods, herbal products, genetic engineering, and biofuels, and served as visiting professor to universities in Japan, Korea, Taiwan, and Russia. Dr. Ravishankar earned a master’s and PhD degrees from Maharaja Sayajirao University of Baroda, India. He mentored more than 40 PhD students, 62 master’s students, 7 postdocs, and 8 international guest scientists; he has authored more than 260 peer-reviewed research papers in international and national journals, 48 reviews, 55 patents in India and abroad, edited 3 books, with an h-index of 59. He has presented more than 200 lectures in various scientific meetings in India and abroad, including visits to more than 25 countries. Dr. Ravishankar has received several coveted honors and awards: Young Scientist Award (Botany) by the then prime minister of India in 1992; National Technology Day Award of Government of India in 2003; Laljee Godhoo Smarak Nidhi Award for food biotechnology R&D of industrial relevance; the prestigious Professor V. Subramanyan Food-Industrial Achievement Award; Professor S.S. Katiyar Endowment Lecture Award in New Biology by Indian Science Congress; Professor Vyas Memorial Award of Association of Microbiologists of India; Professor V.N. Raja Rao Endowment Lecture Award in Applied Botany, University of Madras, India; Lifetime Achievement Award by the Society of Applied Biotechnologists; Dr. Diwaker Patel Memorial Award by Anand Agricultural University, India; Prof C.S. Paulose Memorial Oration Award by Society for Biotechnologists of India.

Dr. Ravishankar is honored as a fellow of several national organizations in India, viz. National Academy of Sciences; National Academy of Agricultural Sciences; Association of Microbiologists of India; Society of Agricultural Biochemists; Society for Applied Biotechnology; Indian Botanical Society; and the Association of Food Scientists and Technologists of India. He has held honorary positions of President of the Society of Biological Chemists, Mysore Chapter, and President of Association of Microbiologists of India, Mysore and Bangalore Chapters. Several premier international bodies have also honored him with fellowships, viz., the International Academy of Food Science and Technology (Canada); the Institute of Food Technologists (USA); the Institute of Food Science and Technology (UK); and the Certified Food Scientists of the United States. Dr. Ravishankar is a very active member of the task forces of several organizations of the government of India and has served as an expert on the selection committees for the appointment of professors, scientists, and research students in universities, as well as R&D institutions. He has also served as advisor and resource at international conferences, seminars, workshops, and short courses; he has convened national and international seminars in biology, biotechnology, and food science and technology. He is an associate editor and reviewer of a large number of reputed research journals. Ambati Ranga Rao is a scientist and assistant professor in the Department of Biotechnology at Vignan’s Foundation for Science, Technology, and Research (Deemed to be University), Andhra Pradesh, India. Dr. Ambati earned bachelor’s and master’s degrees from Acharya Nagarjuna University, Andhra Pradesh, India, and a PhD degree from the University of Mysore, India. He started his research career in 2004 as a research assistant at the Department of Plant Cell Biotechnology, Council of Scientific and Industrial Research (CSIR) Central Food Technological Research Institute (CFTRI), Mysuru, India, under the supervision of Dr. G. A. Ravishankar and Dr. R. Sarada. He was awarded Senior Research Fellow of Indian Council of Medical Research (ICMR), New Delhi, in the year 2007. His PhD work at CFTRI focused on the production of astaxanthin from cultured green alga xv

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Haematococcus pluvialis, and its biological activities. He worked extensively on process optimization of algal biomass production; mass culture of various algal species in raceway ponds and photobioreactors; downstream processing of algal metabolites and evaluation of their possible nutraceutical applications in in vitro and in vivo models. Further, Dr. Ambati worked as lead scientist in Algal Technologies, Carot Labs Pvt Ltd, India; postdoctoral research associate in Laboratory of Algal Research and Biotechnology, Arizona State University, under the supervision of Prof. Milton Sommerfeld and Qiang Hu; visiting assistant professor in Food Science and Technology Programme, Beijing Normal University– Hong Kong Baptist University, United International College, China, under the supervision of Prof. Bo Lei; visiting senior research fellow in the Institute of Ocean and Earth Sciences, University of Malaya, Malaysia, under the guidance of Prof. Phang Siew Moi. He is the author

Editors

of more than 30 peer-reviewed publications, 36 international/national conference papers (including invited talks), 5 reviews, and 9 chapters in books. His research citations exceed 1,600, with h-Index (12), and i-index (14) as per Google Scholar. He has attended international and national conferences/symposia in the United States, Canada, Brazil, China, Malaysia, Indonesia, and Oman. Based on his research accomplishments, he is honored TWAS-Young Affiliate (2014) by Regional Office of East South-East Asia and the Pacific, Chinese Academy of Sciences (CAS), China; Carl Storm International Diversity Fellowship Award (2010) by Gordon Research Conferences. Dr. Ambati is a fellow of the Society for Applied Biotechnology (2013), India. He has received research grants and travel grant fellowships as both international and national awards, under young scientist schemes. He also serves as editorial board member and reviewer for reputed international and national journals.

Contributors Mohamed M. Abdeldaim Faculty of Veterinary Medicine Suez Canal University Ismailia, Egypt Adel Ali Saeed Al-Gheethi Micropollutant Research Centre Faculty of Civil and Environmental Engineering Universiti Tun Hussein Onn Malaysia Johor, Malaysia Helena M. Amaro Interdisciplinary Centre of Marine and Environmental Research (CIIMAR) University of Porto Matosinhos, Portugal

A. Catarina Guedes Interdisciplinary Centre of Marine and Environmental Research (CIIMAR) University of Porto Matosinhos, Portugal Ratana Chaiklahan Pilot Plant Development and Training Institute King Mongkut’s University of Technology Thonburi Bangkok, Thailand Vikas Singh Chauhan Plant Cell Biotechnology Department CSIR, Central Food Technological Research Institute Mysuru, India and

Najeeha Mohd. Apandi Micropollutant Research Centre Faculty of Civil and Environmental Engineering Universiti Tun Hussein Onn Malaysia Johor, Malaysia Xuemei Bai ENN Group Economic and Technology Development Zone Hebei, China Srijoni Banerjee Advanced Technology Development Center Indian Institute of Technology Kharagpur Khargpur, India

Academy of Scientific and Innovative Research Ghaziabad, India Yu Chen ENN Group Economic and Technology Development Zone Hebei, China Imene Chentir Department of Biotechnology Nature and Life Sciences Faculty Blida 1 University Blida, Algeria and

Manuel Berenguel Department of Informatics University of Almería Almería, Spain

Laboratory of Enzyme Engineering and Microbiology National School of Engineering of Sfax University of Sfax Sfax, Tunisia

Boosya Bunnag School of Bioresources and Technology King Mongkut’s University of Technology Thonburi Bangkok, Thailand

Debabrata Das Department of Biotechnology Indian Institute of Technology Kharagpur Kharagpur, India

Zhongzhen Cai ENN Group Economic and Technology Development Zone Hebei, China

Keshav C. Das College of Engineering University of Georgia Athens, Georgia

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Julio Cesar de Carvalho Bioprocess Engineering and Biotechnology Department Federal University of Paraná State Curitiba, Brazil Paulo Cesar de Souza Kirnev Bioprocess Engineering and Biotechnology Department Federal University of Paraná State Curitiba, Brazil

Contributors

Gary L. Hawkins J. Phil Campbell REC Watkinsville, Georgia Mohammad Javad Hessami Institute of Biological Sciences University of Malaya Kuala Lumpur, Malaysia and

Christina Ν. Economou Department of Chemical Engineering University of Patras Patras, Greece

Department of Biotechnology Faculty of Advanced Sciences and Technologies University of Isfahan Isfahan, Iran

HalaY. El-Ksassas National Institute of Oceanography and Fisheries Alexandria, Egypt

Apiradee Hongsthong Biochemical Engineering and Pilot Plant Research and Development Unit National Center for Genetic Engineering and Biotechnology National Science and Technology Development Agency King Mongkut’s University of Technology Thonburi Bangkok, Thailand

Mostafa M. El-Sheekh Botany Department Faculty of Science Tanta University Tanta, Egypt Qian Feng ENN Group Economic and Technology Development Zone Hebei, China Francisco Gabriel Acién Fernández Department of Chemical Engineering University of Almería Almería, Spain Daniel P. Geller College of Engineering University of Georgia Athens, Georgia Jinfeng Geng ENN Group Economic and Technology Development Zone Hebei, China Samiha M. Gharib National Institute of Oceanography and Fisheries Alexandria, Egypt Jose Luis Guzmán Department of Informatics University of Almería Almería, Spain

Bing Huang Metabolism, Bioengineering of Microalgal Molecules and Applications Mer Molécules Sante Le Mans University Le Mans, France Dipak A. Jadhav Department of Agricultural Engineering Maharashtra Institute of Technology Maharashtra, India S.P. Jeevan Kumar Department of Seed Biotechnology ICAR, Indian Institute of Seed Science Mau, India N.V. Joshi Energy and Wetlands Research Group Center for Ecological Sciences Indian Institute of Science Bangalore, India Amir Hashim Mohd. Kassim Micropollutant Research Centre Faculty of Civil and Environmental Engineering Universiti Tun Hussein Onn Malaysia Johor, Malaysia

xix

Contributors

Brian H. Kiepper 309 Poultry Science Building University of Georgia Athens, Georgia

Giorgos Markou Institute of Technology of Agricultural Products Hellenic Agricultural Organization-Demeter Lykovrysi, Greece

Costas Kiparissides Chemical Process and Energy Resources Institute (CPERI) Centre for Research and Technology Hellas (CERTH) Department of Chemical Engineering Aristotle University of Thessaloniki (AUTH) Thessaloniki, Greece

Adriane Bianchi Pedroni Medeiros Bioprocess Engineering and Biotechnology Department Federal University of Paraná State Curitiba, Brazil

Qing Li ENN Group Economic and Technology Development Zone Hebei, China

Virginie Mimouni Metabolism, Bioengineering of Microalgal Molecules and Applications Mer Molécules Santé Le Mans University Le Mans, France

Fangwei Liu ENN Group Economic and Technology Development Zone Hebei, China

Sananda Mondal Institute of Agriculture Visva-Bharati Central University West Bengal, India

Jiantao Luo ENN Group Economic and Technology Development Zone Hebei, China

Annick Morant-Manceau Metabolism, Bioengineering of Microalgal Molecules and Applications Mer Molécules Sante Le Mans University Le Mans, France

C.K. Madhubalaji Plant Cell Biotechnology Department CSIR, Central Food Technological Research Institute Mysuru, India and Academy of Scientific and Innovative Research Ghaziabad, India D.M. Mahapatra Energy and Wetlands Research Group Center for Ecological Sciences Indian Institute of Science Bangalore, India and Biological and Ecological Engineering Oregon State University Corvallis, Oregon Justine Marchand Metabolism, Bioengineering of Microalgal Molecules and Applications Mer Molécules Sante Le Mans University Le Mans, France

Sandeep Mudliar Plant Cell Biotechnology Department CSIR, Central Food Technological Research Institute Mysuru, India and Academy of Scientific and Innovative Research Ghaziabad, India G.S. Murthy Biological and Ecological Engineering Oregon State University Corvallis, Oregon Ruma Pal Phycology Laboratory Department of Botany University of Calcutta Kolkata, India Debasish Panda Institute of Agriculture Visva-Bharati Central University West Bengal, India

xx

Contributors

Giannis Penloglou Chemical Process and Energy Resources Institute (CPERI) Centre for Research and Technology Hellas (CERTH) Thessaloniki, Greece

Jose María Fernández Sevilla Department of Chemical Engineering University of Almería Almería, Spain

C. Periyasamy Department of Botany Pasumpon Muthuramalinga Thevar College Manonmanium Sundaranar University Tamil Nadu, India

Ajam Shekh Plant Cell Biotechnology Department CSIR, Central Food Technological Research Institute Mysuru, India

Radin Maya Saphira Radin Mohamed Micropollutant Research Centre Faculty of Civil and Environmental Engineering Universiti Tun Hussein Onn Malaysia Johor, Malaysia

and

T.V. Ramachandra Energy and Wetlands Research Group Center for Ecological Sciences Center for Sustainable Technologies Indian Institute of Science Bangalore, India

Academy of Scientific and Innovative Research Ghaziabad, India P.V. Sijil Plant Cell Biotechnology Department CSIR, Central Food Technological Research Institute Mysuru, India and Academy of Scientific and Innovative Research Ghaziabad, India

Sarada Ravi Plant Cell Biotechnology Department CSIR, Central Food Technological Research Institute Mysuru, India

Manjinder Singh 2012 Orchard Walk Watkinsville, Georgia

and

Carlos Ricardo Soccol Bioprocess Engineering and Biotechnology Department Federal University of Paraná State Curitiba, Brazil

Academy of Scientific and Innovative Research Ghaziabad, India Benoit Schoefs Metabolism, Bioengineering of Microalgal Molecules and Applications Mer Molécules Sante Le Mans University Le Mans, France Sarban Sengupta Phycology Laboratory Department of Botany University of Calcutta Kolkata, India Surajbhan Sevda Department of Biosciences and Biotechnology Indian Institute of Technology Guwahati Assam, India

I. Sousa Pinto Interdisciplinary Centre of Marine and Environmental Research (CIIMAR) University of Porto Matosinhos, Portugal and Faculty of Sciences of University of Porto Porto, Portugal T.R. Sreekrishnan Department of Biochemical Engineering and Biotechnology Indian Institute of Technology Delhi New Delhi, India

xxi

Contributors

P.V. Subba Rao Aquaculture Foundation of India Tamil Nadu, India G. Subramanian Professor and Head (Retired) Department of Microbiology Founder Director, National Facility for Marine Cyanobacteria Bharathidasan University Tiruchirappalli, India G.V. Swarnalatha Plant Cell Biotechnology Department CSIR, Central Food Technological Research Institute Mysuru, India and Department of Biochemistry Rayalaseema University Kurnool, India Eduardo Bittencourt Sydney Bioprocess Engineering and Biotechnology Department Federal University of Paraná State Curitiba, Brazil Jie Teng ENN Group Economic and Technology Development Zone Hebei, China N. Thajuddin Department of Microbiology School of Life Sciences Bharathidasan University Tiruchirappalli, India Lionel Ulmann Metabolism, Bioengineering of Microalgal Molecules and Applications Mer Molécules Santé Le Mans University Le Mans, France

Marta Barceló Villalobos Department of Informatics University of Almería Almería, Spain Hui Ling Wang ENN Group Economic and Technology Development Zone Hebei, China Lin Wang ENN Group Economic and Technology Development Zone Hebei, China Hong Wu ENN Group Economic and Technology Development Zone Hebei, China Min Xu ENN Group Economic and Technology Development Zone Hebei, China Jinyang Zhang ENN Group Economic and Technology Development Zone Hebei, China Zhenqi Zhu ENN Group Economic and Technology Development Zone Hebei, China

Section I Phycoremediation Applications

1

Wastewater Phycoremediation by Microalgae for Sustainable Bioproduct Production Najeeha Mohd. Apandi, Radin Maya Saphira Radin Mohamed, Adel Ali Saeed Al-Gheethi, and Amir Hashim Mohd. Kassim

CONTENTS Abbreviations...............................................................................................................................................................4 Introduction..................................................................................................................................................................4 Conventional Wastewater Treatment............................................................................................................................4 Phycoremediation and Wastewater Treatment.............................................................................................................6 Heavy Metal Removal via Phycoremediation..............................................................................................................6 Mechanism of Nutrient Removal Using Algae............................................................................................................7 Factors Affecting Microalgae Culture..........................................................................................................................8 Microalgae Cultivation System....................................................................................................................................9 Microalgal Biomass as a Sustainable Bioproduct........................................................................................................9 Conclusion................................................................................................................................................................. 10 Acknowledgements.................................................................................................................................................... 10 References.................................................................................................................................................................. 10 BOX 1.1  SALIENT FEATURES The rapid and ever-increasing world population along with the advancement in science and technology has increased the utilization of resources. This has created challenges in wastewater processing. The discharge of rich wastewater into rivers and aquatic environments without proper treatment contributes to eutrophication and deterioration in water quality. Most of the current wastewater treatment practices are not economically viable as they incur high operation costs and maintenance costs. In addition, these practices also produce an extensive volume of sludge. Through the phycoremediation method, sludge can be used for biomass production. The use of bacteria or other natural microorganisms such as microalgae for biological treatment is cost effective, accessible and requires low maintenance. The bioremediation of wastewater using microalgae also overcomes problems related to the physical and chemical treatment of wastewater. Compared to other conventional treatment methods, phycoremediation is a simple and practical wastewater treatment technique that uses algae.

The use of microalgae has been commercialized in wastewater treatment applications. Mass production of strains such as Scenedesmus almeriesnsis, Botrycoccus sp., Chlorella sp. and Dundiella sp. are being used for animal feeds, fertilizers, pharmaceuticals, nutraceuticals, cosmetics, aquaculture and pollution control. Moreover, biomass yield can be used as food supplements, bioenergy resources and pharmaceutical products. Microalgae have been previously suggested as one of the alternative methods for nutrient removal from wastewater. Nutrient removal has been shown to be more efficient when algae strains with special attributes are used. Recently, bio-treatment using microalgae has increased in popularity because of its photosynthetic capabilities which converts solar energy into useful biomasses and removes nutrients such as nitrogen and phosphorus that cause eutrophication. Microalgae systems can treat human sewage, livestock wastes, agro-industrial wastes, industrial wastes, municipal wastewater and domestic wastewater. The removal or remediation of nutrients by microalgae takes place through one of two

3

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Handbook of Algal Technologies and Phytochemicals

pathways. The primary components for microalgae growth are nutrients (nitrogen and phosphorus), carbon and other micronutrients (sodium, magnesium, potassium and iron). Microalgal biomass cultured from wastewater phycoremediation has varied applications such as food, source of energy, pharmaceuticals and pollution control. Of all these established applications and treatments using microalgae, previous studies have revealed that fish feed ingredients might lead to the reduction of biological aquatic diversity. Phycoremediation and wastewater treatment, heavy metal removal, mechanism of nutrient removal, factors affecting microalgae culture, microalgae cultivation systems and microalgal biomass as a sustainable bioproduct are discussed in this chapter.

ABBREVIATIONS BOD COD NH TP TN TKN PBR

Biochemical oxygen demand Chemical oxygen demand Ammonia Total phosphorus Total nitrogen Total kjeldahl nitrogen Photo-bioreactor

INTRODUCTION The escalating global population has resulted in the overproduction of wastewater which is normally discharged into water bodies. These wastes have contributed to environmental crises all over the world. Wastewater characteristics vary depending on living conditions and technologies used to produce wastewater. Hence, it is quite a challenge to provide any information regarding the quality and quantity of biomass production. The sustainability of phycoremediation for the elimination of pollution in wastewater has been widely published. Most of the current wastewater treatment practices are not economically viable as they incur high operation costs and maintenance costs. In addition, these practices also produce an extensive volume of sludge. In current wastewater treatment systems, it is important to decrease or minimize sludge production which otherwise would cause environmental hazards. Besides, most chemical methods increase pH levels, conductivity and dissolved matter in wastewater (Renuka et al. 2015). In this respect, bioremediation using algae is a better option for the dewatering and disposal of sludge.

Through the phycoremediation method, sludge can be used for biomass production. Other than that, chemicals are not necessary for effluent treatment using algae technology. The use of bacteria or other natural microorganisms such as microalgae for biological treatment is cost effective, accessible and requires low maintenance (Jais et al. 2017; Kobayashi et al. 2013). Moreover, to overcome problems related to the physical and chemical treatment of wastewater, the bioremediation of wastewater using microalgae is both applicable and energy efficient (Rawat et al. 2011). Compared to other conventional treatment methods, phycoremediation is a simple and practical wastewater treatment technique that uses algae (Abdel-Raouf et al. 2012). Phycoremediation can be classified as a sustainable and cost-effective method which benefits both the economy and the environment (Ajayan et al. 2015; Apandi et al. 2018b). Remarkably, phycoremediation not only removes nutrients but also other organic pollutants and heavy metals depending on the selected microalgae, type of wastewater and culture condition. Therefore, an excessive amount of nutrients is a pragmatic feature for wastewater as they can be recycled to yield biomass. Nasir et al. (2015) found that 90% of nutrients from aquaculture wastewater and biomass (bio-products) ranging between 15 to 20% (v/v) can be removed through wastewater treatment using microalgae (Chlorella sp.). Yaakob et al. (2014) suggested that microalgae can potentially restore the chemical components or meet the rising demand for natural supplements and animal feed. The use of microalgae has been commercialized in wastewater treatment applications. Mass production of strains such as Scenedesmus almeriesnsis, Botrycoccu ssp, Chlorella sp. and Dundiella sp. are being used for animal feeds, fertilizers, pharmaceuticals, nutraceuticals, cosmetics, aquaculture and pollution control (Darmaki et al. 2012; Vizcaino et al. 2014; Gani et al. 2017). Moreover, biomass yield can be used as food supplements, bioenergy resources, pharmaceutical products and so on (Gani et al. 2017; Spolaore et al. 2006). The current chapter aims to summarize research studies available on the potential uses of microalgae biomass produced from the phycoremediation of wastewater.

CONVENTIONAL WASTEWATER TREATMENT In a wastewater treatment system, the elimination or reduction of unnecessary contaminants such as biochemical oxygen demand (BOD), toxins, bacteria, suspended solids, nutrients and coliform is necessary and must therefore comply with international standards. Wastewater treatment consists of physical, chemical and

5

Wastewater Phycoremediation by Microalgae

biological processes. The process applied in conventional wastewater treatments is summarized in Figure 1.1. The preliminary step in wastewater processing removes large solid materials and large inorganic particulate contents of wastewater (Abdel-Raouf et al. 2012). The secondary treatment (biological treatment) is performed after the primary treatment, which is also known as the physical-chemical treatment. Sedimentation and floatation clear away the settling solids after the removal of coarse materials. During this treatment process, up to 70% of organic and inorganic solids are removed. AbdelRaouf et al. (2012) and Karia (2013) stated that 50–60% of suspended solids can be eliminated if the sedimentation tank is perfectly designed and is able to reduce up to 40% of BOD in the form of settleable solids. Secondary treatment is defined as a biological treatment process where the aerobic degradation of organic matter occurs. This secondary treatment is considered environmentally compatible and inexpensive. This stage can be defined as the activated sludge process where the primary effluent flows into an aeration tank and air diffusers are used to mix it with microorganisms through mechanical agitation (Abdel-Raouf et al. 2012). During this process, the breaking down or assimilation of chemicals present in wastewater is done by the microorganisms. (Rawat et al. 2011). The major mechanism of this assimilation process is the adsorption of colloidal and soluble organic matter by microalgae. According to Abdel-Raouf et al. (2012), several researchers have mentioned that the biological oxidation system can eliminate about 80–90% of organic solids.

The tertiary treatment process is the final stage where organic ions, dissolved solids and remaining suspended solids are removed using physical, chemical and biological processes. Tertiary treatment is 8 to 16 times more expensive than primary treatment (Abdel-Raouf et al. 2012). At this stage, certain nutrients such as phosphorus and nitrogen are removed using application systems such as membrane filtration, granular filtration, solar and UV light, chlorine, ozone and ultraviolet light. These applications are extremely expensive and may contribute to secondary pollution (Berbeka et al. 2012). For these reasons, numerous studies have focused on the removal of nitrogen and phosphorus through biological processes and different combinations of anaerobic, aerobic and sequencing batch reactors (Sombatsompop et al. 2011). Therefore, alternative methods for nutrient removal and metal precipitation through biological processes including the use of microalgae have been reviewed and practiced by many researchers. During the tertiary treatment stage, the role of microalgae is critical as it has the ability to uptake nitrogen even if it is in starvation mode. “Activated algae” is an intense algae culture which reduces land and space requirements for treatment systems (Olguín 2003). Martin et al. (1985) reported that microalgae have the ability to eliminate nitrogen and phosphorus in a very short period of time. The authors also stated that microalgae perform better under colder environmental conditions. However, more comprehensive studies are required to examine if the composition of other species plays a role. Moreover, NH3 or NH3 precipitation can also occur

FIGURE 1.1  Schematic of conventional wastewater treatment processes.

6

Handbook of Algal Technologies and Phytochemicals

through phycoremediation due to increased pH levels during photosynthesis (Rawat et al. 2011).

PHYCOREMEDIATION AND WASTEWATER TREATMENT Microalgae have been previously suggested as one of the alternative methods for nutrient removal from wastewater. Wastewater treatment using microalgae appears to be more reliable as it can be used for both biomass growth and removal of biological wastewater contaminants (Makareviciene et al. 2013). Nutrient removal has been shown to be more efficient when algae strains with special attributes are used. Such special attributes include tolerance towards extreme temperatures, chemical composition with predominance of high value-added products, quick sedimentation behavior and a capacity for growing mixotrophically (Apandi et al. 2018b; Gani et al. 2017). Recently, bio-treatment using microalgae has increased in popularity because of its photosynthetic capabilities which convert solar energy into useful biomasses and remove nutrients such as nitrogen and phosphorus that cause eutrophication. Microalgae systems can treat

human sewage, livestock wastes, agro-industrial wastes, industrial wastes, municipal wastewater and domestic wastewater (Abdel-Raouf et al. 2012; Maizatul et al. 2017). Algal systems have traditionally been employed as a tertiary process. They have been proposed as a potential secondary treatment system because of their ability to remove heavy metals, toxic compounds and organic ions. The tertiary process is usually more costly because it involves the removal of pollutants. Therefore, the treatment of wastewater using microalgae has been suggested due to the reasonable cost of the overall process. Recent studies on wastewater treatment using microalgae are presented in Table 1.1. Many microalgae species including Dunaliella, Pithophora, Scenedesmus obliquus and many others can be used for wastewater treatment.

HEAVY METAL REMOVAL VIA PHYCOREMEDIATION The most commonly found heavy metals in wastewater are Cadmium (Cd), Chromium (Cr), Lead (Pb), Mercury (Hg) and Zinc (Zn) which can cause poisoning to humans and aquatic life. Poisoning can occur through

TABLE 1.1 Summary of the Microalgae Use in Wastewater Treatment Source of Wastewater

Type of Treatment

Type of Microalgae

Industrial wastewater (brewing, soy sauce pulp and paper, dairy and poultry)

Biological treatment method via microalgae cultivation

Dunaliella

Brewery effluent

C. vulgaris

Brewery effluent

Biological treatment method via microalgae cultivation Filtration system

Cafeteria wastewater

Phycoremediation

Scenedesmus sp.

Wet market wastewater

Phycoremediation

Scenedesmus sp.

Scenedesmus obliquus

Parameters Studied BOD COD Rate of removal of ammonium and orthophosphate BOD removal COD removal BOD removal COD removal COD reduction Total nitrogen reduction Total carbon reduction Nutrient removal Element removal

Nutrient removal

Treatment Efficiency

Researcher

53 % 89 % 99 %

Sara et al. (2012)

18 % 13 % 27 % 15 % 57.5 % 20.8 % 56.9% TN (90.78%) PO4−³ (35.90%) TOC (73.36%) Fe (88.2%) Cu (60%) Zn (76.63%) TP (85%) TN (90%) TOC (65%)

Raposo et al. (2010)

Mata et al. (2010)

Mohamed et al. (2015)

Apandi et al. (2018b)

7

Wastewater Phycoremediation by Microalgae

drinking water or food chains since heavy metals tend to accumulate in the food chain. Heavy metals discharged from wastewater into rivers, lakes and the sea might lead the contamination of fishes and vegetables due to their high solubility in aquatic environments (Azimi et al. 2017). On the other hand, heavy metals can remain in the human body for a long time since they are not biodegradable. For example, too much Zn may cause health related diseases such as skin irritations, vomiting and stomach cramps (Azimi et al. 2017). Thus, heavy metal removal using microalgae is efficient, safe and cost effective (Ajayan et al. 2015). The summary of heavy metal removal by previous researchers is presented in Table 1.2. More than 50% of heavy metals can be removed using Scenedesmus sp. Ajayan et al. (2015) reported that Scenedesmus sp. removed 70%, 85%, 62% and 78% of Cr, Cu, Pb and Zn, respectively, in 50% of tannery wastewater. In 100% of tannery wastewater, Scenedesmus sp. also successfully reduced the concentration of Cr, Cu, Pb and Zn by about 57%, 79%, 48% and 65%, respectively, after a 12-day treatment. For industrial wastewater, Onalo et al. (2014) used Botryococcus sp. to reduce 94% of Cr and 45% of Cu. In a different study, Hadiyanto et al. (2014) examined two strains of immobilized microalgae, namely, Chlorella vulgaris and Spirulina plantesis, for the

removal of copper and chromium from textile wastewater. Their findings showed that the use of immobilized algae cells is very effective for removing heavy metals. In summary, most of the microalgae species stated in Table 1.2 possess different capabilities in removing heavy metals from wastewater. Microalgae can be used for the removal of phosphorus, nitrogen and heavy metals. The application of Scenedesmus sp. for the remediation of wet market wastewater also has been proven (Apandi et al. 2018). Moreover, the combination of wastewater and microalgae motivates research for biomass production. Thus, this study serves to add knowledge to the field of phycoremediation.

MECHANISM OF NUTRIENT REMOVAL USING ALGAE The removal or remediation of nutrients by microalgae takes place through one of the two pathways shown in Figure 1.2. The remediation mechanism is achieved through the metabolic pathways of algae cells for nutrient uptake or assimilation during photosynthesis for biomass growth (Whitton et al. 2015). The primary components for microalgae growth are nutrients (nitrogen and phosphorus), carbon and other micronutrients (sodium, magnesium, potassium and iron). However,

TABLE 1.2 Compilation of Microalgae Used for the Bioremediation of Heavy Metals Type of Microalgae

Source of Wastewater

Heavy Metal

Removal Efficiency (%)

Scenedesmus sp.

50% tannery wastewater

Scenedesmus sp.

100% tannery wastewater

Scenedesmus sp.

Food stall wastewater

Scenedesmus sp.

Wet market wastewater

Botryococcus sp.

Industrial wastewater

Chlorellavulgaris

Textile wastewater

Chromium (Cr) Copper (Cu) Lead (Pb) Zinc (Zn) Chromium (Cr) Copper (Cu) Lead (Pb) Zinc (Zn) Ferum (Fe) Copper (Cu) Zinc (Zn) Ferum (Fe) Zinc (Zn) Chromium (Cr) Chromium (Cr) Copper (Cu) Arsenic (As) Cadmium (Cd) Copper (Cu) Chromium (Cr)

70 85 62 78 57 79 48 65 88.2 60 75.61 92.47 92.40 91.5 94 45 9 2 89 90

References Ajayan et al. (2015)

Ajayan et al.(2015)

Latiffi et al. (2015)

Apandi et al. 2018a

Onalo et al. (2014)

Hadiyanto et al. (2014)

8

Handbook of Algal Technologies and Phytochemicals

FIGURE 1.2  Schematic diagram of a microalga cell summarizing the biochemical pathways of nitrogen and phosphorus remediation.

nitrogen and phosphorus are the major nutrients that are measured and required for algae growth. The assimilation of nitrogen is an essential constituent of biological substances when inorganic nitrogen such as nitrite (NO2−), nitrate (NO3−) and ammonium (NH4+) are converted into organic nitrogen across the cell membrane. Oxidized nitrogen subsequently reduces NO3−, followed by the incorporation of NH4− and assimilation into amino acids for the formation of protein (Apandi et al. 2018b). The biological reduction of nitrogen via glutamine and glutamate synthase within the intracellular fluid is shown in Figure 1.2. Phosphorus is one of the major nutrients and exists in the form of H2PO − 4 and HPO24−. It is consolidated into an organic compound through the phosphorylation process across the cell membrane. Cell growth and metabolism require a certain amount of photon energy from a light source. Thus, during phosphorylation, ATP (adenosine triphosphate) and ADP (adenosine diphosphate) allow the microalgae growth mechanism to assimilate and store phosphorus within algae cells as acid-insoluble polyphosphate granules (Gani et al. 2016). Phosphorus removal from wastewater via precipitation resulting

from chemical addition or elevated pH levels (Powell et al. 2009) in high rate algal ponds (HRAP) incurs high operation costs. Therefore, biological nutrient removal has more advantages than chemical precipitation due to its ability to produce energy-rich biomass after phosphorus uptake (Powell et al. 2009). Xin et al. (2010) demonstrated that the efficiency of Scenedesmus sp. decreases significantly when the nitrogen:phosphorus ratio exceeds 12:1. Whitton et al. (2015) stated that the specific uptake rate for nitrogen and phosphorus can be increased when the internal content is initially reduced or when the external concentration is limited. This condition represents a time of stress where nitrogen and phosphorus assimilation is necessary for continuous metabolic processes and algae growth.

FACTORS AFFECTING MICROALGAE CULTURE Microalgae have been widely cultured for many years and one of the significant findings is that microalgae can be used for the manufacture of value-added products (Spolaore et al. 2006). Microalgae already have

9

Wastewater Phycoremediation by Microalgae

well-established applications for biofuel, biodiesel, animal feed and fish feed supplements due to their natural source of pigments, antioxidants and other bioactive compounds which have functional properties in addition to their basic nutritional value (Chen et al. 2013). In addition, microalgae have also garnered the attention of researchers worldwide due to their high growth rate, high yield, biochemical compounds and potential bioremediation applications (Mohamed et al. 2017). In this regard, many researchers have been investigating factors that influence the survivability of microalgae in the environment (Hena et al. 2015; Wahidin et al. 2013). Several researchers have studied and reported the influence of pH, light intensity, nutrients, temperature and photoperiods on algal growth and production as shown in Table 1.3. The optimum photoperiod for microalgae culturing depends on the microalgae species. Table 1.3 reveals that the most favorable light exposure for microalgae growth is between 12 to 18 hours which allows the assessment of light penetration during photosynthesis development.

MICROALGAE CULTIVATION SYSTEM Microalgae can basically be cultivated in open systems and closed systems. The open system can be done on a large scale at a low cost. However, the risk of contamination by other microorganisms is high. The closed system in the form of tubes, plates or columns allows for accurate process control (light availability, nutrients, CO2 and O2 concentrations, temperature, mixing), a high degree of protection against contamination and high volumetric

productivity. The choice of a certain type of reactor depends on the location, available space, medium, cost and desired product. Microalgae cultivation depends on several factors such as volumetric productivity (VP) per unit reactor volume (g/L/d), area volumetric productivity (AP) per unit of ground area occupied by the reactor (g/m2d) and illuminated surface productivity (ISP) per unit of reactor illuminated surface area (g/m2d) (Richmond 2008; Özçimen et al. 2012). The selection of a system depends on current needs for an economical and sustainable technology, as well as the costs involved.

MICROALGAL BIOMASS AS A SUSTAINABLE BIOPRODUCT Microalgal biomass cultured from wastewater phycoremediation has varied applications such as food, source of energy, pharmaceuticals and pollution control as shown by previous researchers in Table 1.4. The engineering of largescale systems and algae harvesting methods are important for different algae cultures to produce high value products (Abdel-Raouf et al. 2012; Pahazri et al. 2016). Of all these established applications and treatments using microalgae, previous studies have revealed that fish feed ingredients might lead to the reduction of biological aquatic diversity (Apandi et al. 2017). Thus, new trends aim to use bio-products such as microalgae as fish feed since these organisms have high nutritional value. These nutrients include fats and carbohydrates which are able to provide energy, proteins which furnish amino acids, vitamins which serve as cofactors for enzymes,

TABLE 1.3 Summary of Optimum Conditions for Different Microalgae Species Factors

Microalgae Species

Optimal Condition

pH

Scenedesmus sp. Scenedesmus sp. Scenedesmus obliquus

7–8 7 7

Temperature

Scenedesmus sp. Scenedesmus sp. Scenedesmus sp. Nannochloropsis sp. Ankistrodesmusfalcatus Chlorella vulgaris Ankistrodesmusfalcatus Scenedesmus sp. Nannochloropsis sp.

25°C 20°C 27.5°C 12:12 12:12 16:8 60 µmol m–2s–1 4000 lux 100 µmol m–2s–1

Photoperiod

Light intensity

Nutrient/ Media BBM BG-11 Olive-mill wastewater BBM BG-11 Urea Seawater BG-11 Zehnder BG-11 BBM Seawater

Biomass Productivity/ Growth Rates n.d 0.38 gL–1d–1 n.d 5.5 × 106 cellmL–1 313.3 g P–1 0.0284 hr–1 6.5 × 107 cell mL–1 7.9 mg L–1 d–1 2.05 g L–1 d–1 7.9 mg L–1 d–1 2.28 × 106 cell mL–1 6.5 × 107 cell mL–1

References Latiffi et al. (2017) Difusa et al. (2015) Hodaifa et al. (2009) Latiffi et al. (2017) Li et al. (2011) Cassidy (2011) Wahidin et al. (2013) George et al. (2014) Amini Khoeyi et al. (2012) George et al. (2014) Latiffi et al. (2017) Wahidin et al. (2012)

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Handbook of Algal Technologies and Phytochemicals

TABLE 1.4 The Potential of Microalgae Biomass as Sustainable Bioproducts Microalgae Species

Protein %

Carbohydrates %

Lipids %

Scenedesmus sp.

41.7

7

23.2

Arthospiraplantesis

39.5

37.3

19.7

Chlorella sp.

NA

NA

21

Chlorella sp.

61.4

NA

9.2

Scenedesmus sp.

50.4

NA

10.3

Chlorella sorokiniana Scenedesmus sp. Chlorella sp.

33.94

NA

26.83

51.17 46.78

10 11.32

12.29 14.83

Culture Medium

Production Potential/ Application

Wet market wastewater Poultry litter leachate Piggery wastewater Nile tilapia effluent Nile tilapia effluent Cattle manure NA NA

Fish feed, pharmaceuticals Feed supplement

Markou et al. (2016)

Biodiesel

Kuo et al. (2015)

Aquaculture feed

Guerrero-Cabrera et al. (2014)

Aquaculture feed

Guerrero-Cabrera et al. (2014)

Animal feed

Kobayashi et al. (2013)

Fish feed Fish feed

Badwy et al. (2008) Badwy et al. (2008)

ions required for water balance and nerve and muscle function and selected elements that are incorporated into certain molecules synthesized by cells (Brune et al. 2009). Furthermore, microalgae are used as natural food for fishes. So far, the limitations of their utilization lie in the selection of microalgae species and biomass production. Nevertheless, the use of microalgae for a more sustainable future should be examined further for the benefit of future generations.

CONCLUSION The current review is motivated by the increasing potential of microalgae biomass and the implementation of environmentally friendly tools in treating wastewater. There is great potential in bringing microalgae to the next level in the field of biotechnological applications. Some researchers have highlighted that culture conditions may influence microalgae growth and biomass productivity. Another advantage of using microalgae in biotechnology is the reduction in biomass production costs through wastewater phycoremediation. Finally, the use of microalgae for phycoremediation shows promise for the removal of contaminants, chemicals and heavy metals from contaminated wastewater.

ACKNOWLEDGEMENTS The authors are grateful to the Ministry of Science, Technology and Innovation (MOSTI) for supporting this research under the E-Science Fund (02-01-13-SF0135) vot S029.

Reference Apandi et al. (2017)

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Berbeka, K., Czajkowski, M., & Markowska, A. Municipal wastewater treatment in Poland–efficiency, costs and returns to scale. Water Science and Technology. 2012. 66(2): 394–401. Brune, D. E., Lundquist, T. J., & Benemann, J. R. Microalgal biomass for greenhouse gas reductions: Potential for replacement of fossil fuels and animal feeds. Journal of Environmental Engineering. 2009. 135(11): 1136–1144. Cassidy, K. O. Evaluating algal growth at different temperatures. Master thesis. University of Kentucky. 2011. Chen, L., Wang, C., Wang, W., & Wei, J. Optimal conditions of different flocculation methods for harvesting Scenedesmus sp. Cultivated in an open-pond system. Bioresource Technology. 2013. 133: 9–15. Darmaki, A. Al., Talebi, L. G., Al-Rajhi, S., S., & Tahir Al-Barwani, Z. A. Cultivation and characterization of microalgae for wastewater treatment. In: Proceedings of the World Congress on Engineering, July 4–6, 2012. Vol. 1. 2012. WCE, London, U.K. Difusa, A., Talukdar, J., Kalita, M. C., Mohanty, K., & Goud, V. V. Effect of light intensity and pH condition on the growth, biomass and lipid content of microalgae Scenedesmus species. Biofuels. 2015. 6(1–2): 37–44. Gani, P., Sunar, N. M., Matias-Peralta, H., Jamaian, S. S., & Latiff, A. A. A. Effects of different culture conditions on the phycoremediation efficiency of domestic wastewater. Journal of Environmental Chemical Engineering. 2016. 4(4): 4744–4753. Gani, P., Sunar, N. M., Matias-Peralta, H., Mohamed, R. M. S. R., Latiff, A. A. A., & Parjo, U. K. Extraction of hydrocarbons from freshwater green microalgae (Botryococcus sp.) biomass after phycoremediation of domestic wastewater. International Journal of Phytoremediation. 2017. 19(7): 679–685. George, B., Pancha, I., Desai, C., Chokshi, K., Paliwal, C., Ghosh, T., & Mishra, S. Effects of different media composition, light intensity and photoperiod on morphology and physiology of freshwater microalgae Ankistrodesmusfalcatus–A potential strain for biofuel production. Bioresource Technology. 2014. 171: 367–374. Guerrero-Cabrera, L., Rueda, J. A., García-Lozano, H., & Navarro, A. K. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent. Bioresource Technology. 2014. 161: 455–460. Hadiyanto, A. B. P., Buchori, L., & Budiyati, C. S. Biosorption of heavy metal Cu2+ and Cr2+ in textile wastewater by using immobilized algae. Research Journal of Applied Sciences, Engineering and Technology. 2014. 7(17): 3539–3543. Jais, N. M., Mohamed, R. M. S. R., Al-Gheethi, A. A., & Hashim, M. K. A. The dual roles of phycoremediation of wet market wastewater for nutrients and heavy metals removal and microalgae biomass production. Clean Technologies and Environmental Policy. 2017. 19(1): 37–52. Karia, G. L., & Christian, R. A. Wastewater Treatment: Concepts and Design Approach. 2013. PHI Learning Pvt. Ltd.

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Kobayashi, N., Noel, E. A., Barnes, A., Watson, A., Rosenberg, J. N., Erickson, G., & Oyler, G. A. Bioresource Technology Characterization of three Chlorella sorokiniana strains in anaerobic digested effluent from cattle manure. Bioresource Technology. 2013. 150: 377–386. Kuo, C. M., Chen, T. Y., Lin, T. H., Kao, C. Y., Lai, J. T., Chang, J. S., & Lin, C. S. Cultivation of Chlorella sp. GD using piggery wastewater for biomass and lipid production. Bioresource Technology. 2015. 194: 326–333. Latiffi, A., Atikah, N., Mohamed, R., Saphira, R. M., Apandi, Mohd., Kassim, N. M., & Hashim, A. Application of phycoremediation using microalgae Scenedesmus sp. as wastewater treatment in removal of heavy metals from food stall wastewater. Applied Mechanics and Materials. 2015. 773: 1168–1172. Latiffi, A., Atikah, N., Mohamed, R., Saphira, R. M., Apandi, N. M., & Tajuddin, R. M. Experimental assessment on effects of growth rates microalgae Scenedesmus sp. in different conditions of pH, temperature, light intensity and photoperiod. Key Engineering Materials. 2017. 744: 546–551. Li, X., Hu, H. Y., & Zhang, Y. P. Growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. under different cultivation temperature. Bioresource Technology. 2011. 102(3): 3098–3102. Maizatul, A. Y., Radin Mohamed, R. M. S., Al-Gheethi, A. A., & Hashim, M. K. A. An overview of the utilisation of microalgae biomass derived from nutrient recycling of wet market wastewater and slaughterhouse wastewater. International Aquatic Research. 2017. 9(3): 177–193. Makareviciene, V., Skorupskaite, V., & Andruleviciute, V. Biodiesel fuel from microalgae-promising alternative fuel for the future: A review. Reviews in Environmental Science and Bio/Technology. 2013. 12(2): 119–130. Markou, G., Iconomou, D., & Muylaert, K. Applying raw poultry litter leachate for the cultivation of Arthrospiraplatensis and Chlorella vulgaris. Algal Research. 2016. 13: 79–84. Martin, C., De la Noüe, J., & Picard, G. Intensive cultivation of freshwater microalgae on aerated pig manure. Biomass. 1985. 7(4): 245–259. Mata, T. M., Martins, A. A., & Caetano, N. S. Microalgae for biodiesel production and other applications: A review. Renewable and Sustainable Energy Reviews. 2010. 14(1): 217–232. Mohamed, R., Saphira, R. M., Maniam, H., Apandi, N., Al-Gheethi, A. A. S., Kassim, M., & Hashim, A. Microalgae biomass recovery grown in wet market wastewater via flocculation method using Moringaoleifera. Key Engineering Materials. 2017. 744: 542–545. Mohamed, R., Saphira, R. M., Mohd Apandi, N., Matias Peralta, H. M., Kassim, M., & Hashim, A. Removal of nutrients from cafeteria wastewater using varying concentrations of microalga Scenedesmus sp. Presented at the International Conference on Environmental Forensics. iENFORCE 2015. August 19–20, 2015. Putrajaya Marriot Hotel, Malaysia.. Nasir, N. M., Bakar, N. S. A., Lananan, F., Abdul Hamid, S. H., Lam, S. S., & Jusoh, A. Treatment of African catfish, Clariasgariepinus wastewater utilizing phytoremediation

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of microalgae, Chlorella sp. with Aspergillusniger bio-harvesting. Bioresource Technology. 2015. 190: 492–498. Olguín, E. J. Phycoremediation: Key issues for cost-effective nutrient removal processes. Biotechnology Advances. 2003. 22(1–2): 81–91. Onalo, J. I., Peralta, H. M. M., & Sunar, N. M. Growth of freshwater microalga, Botryococcus sp. in heavy metal contaminated industrial wastewater. Journal of Science and Technology. 2014. 6(2). Özçimen, D., Gülyurt, M. Ö., & İnan, B. Algal biorefinery for biodiesel production. In: Biodiesel-Feedstocks, Production and Applications. 2012. InTech. Pahazri, N. F., Mohamed, R., Al-Gheethi, A. A., & Kassim, A. H. M. Production and harvesting of microalgae biomass from wastewater: A critical review. Environmental Technology Reviews. 2016. 5(1): 39–56. Powell, N., Shilton, A., Chisti, Y., & Pratt, S. Towards a luxury uptake process via microalgae–defining the polyphosphate dynamics. Water Research. 2009. 43(17): 4207–4213. Raposo, M. F. D. J., Oliveira, S. E., Castro, P. M., Bandarra, N. M., & Morais, R. M. On the utilization of microalgae for brewery effluent treatment and possible applications of the produced biomass. Journal of the Institute of Brewing. 2010. 116(3): 285–292. Rawat, I., Ranjith Kumar, R., Mutanda, T., & Bux, F. Dual role of microalgae: Phycoremediation of domestic wastewater and biomass production for sustainable biofuels production. Applied Energy. 2011. 88(10): 3411–3424. Renuka, N., Sood, A., Prasanna, R., & Ahluwalia, A. S. Phycoremediation of wastewaters: A synergistic approach using microalgae for bioremediation and biomass generation. International Journal of Environmental Science and Technology. 2015. 12(4): 1443–1460. Richmond, A. (Ed.) Handbook of Microalgal Culture: Biotechnology and Applied Phycology. 2008. John Wiley & Sons.

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Sara, A. R., Raut, N., Fatma, A. Q., Qasmi, M., & Al Saadi, A. Treatments of industrials wastewater by using microalgae. In: International Conference on Environmental, Biomedical and Biotechnology IPCBEE. 41. 2012. Sombatsompop, K., Songpim, A., Reabroi, S., & Inkongngam, P. A comparative study of sequencing batch reactor and movingbed sequencing batch reactor for piggery wastewater treatment. Maejo International Journal of Science and Technology. 2011. 5(2): 191. Spolaore, P., Joannis-Cassan, C., Duran, E., & Isambert, A. Commercial applications of microalgae. Journal of Bioscience and Bioengineering. 2006. 101(2): 87–96. Vizcaíno, A. J., López, G., Sáez, M. I., Jiménez, J. A., Barros, A., Hidalgo, L., Camacho-Rodríguez, J., Martínez, T. F., Cerón-García, M. C., & Alarcón, F. J. Effects of the microalga Scenedesmus almeriensis as fishmeal alternative in diets for gilthead sea bream, Sparusaurata, juveniles. Aquaculture. 2014. 431: 34–43. Wahidin, S., Idris, A., & Shaleh, S. R. M. The influence of light intensity and photoperiod on the growth and lipid content of microalgae Nannochloropsis sp. Bioresource Technology. 2013. 129: 7–11. Whitton, R., Ometto, F., Pidou, M., Jarvis, P., Villa, R., & Jefferson, B. Microalgae for municipal wastewater nutrient remediation: Mechanisms, reactors and outlook for tertiary treatment. Environmental Technology Reviews. 2015. 4(1): 133–148. Xin, L., Hong-Ying, H., Ke, G., & Ying-Xue, S. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp. Bioresource Technology. 2010. 101(14): 5494–500. Yaakob, Z., Ali, E., Zainal, A., Mohamad, M., & Takriff, M. S. An overview: Biomolecules from microalgae for animal feed and aquaculture. Journal of Biological Research (Thessalon). 2014. 21(1): 6.

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Green Technology Applications for Algal Bloom Control Mostafa M. El-Sheekh, Mohamed M. Abdeldaim, Samiha M. Gharib, and HalaY. El- Ksassas

CONTENTS Introduction���������������������������������������������������������������������������������������������������������������������������������������������������������������13 Green Technology Applications for Algal Bloom Control���������������������������������������������������������������������������������������14 Biological Controls (Bio-Remediation)���������������������������������������������������������������������������������������������������������������14 Bio-Remediation of Wetlands by Planting (BWP)�������������������������������������������������������������������������������������������14 Plant Cultivation ����������������������������������������������������������������������������������������������������������������������������������������������14 Activating Pressure of Grazing������������������������������������������������������������������������������������������������������������������������14 Bio-Remediation����������������������������������������������������������������������������������������������������������������������������������������������14 Controlling of HABs by Bacteria���������������������������������������������������������������������������������������������������������������������14 Physical Controls��������������������������������������������������������������������������������������������������������������������������������������������������14 Air Circulation�������������������������������������������������������������������������������������������������������������������������������������������������14 Mechanical Circulation �����������������������������������������������������������������������������������������������������������������������������������15 Hypolimnetic Oxygenation������������������������������������������������������������������������������������������������������������������������������15 Surface Skimming��������������������������������������������������������������������������������������������������������������������������������������������15 Ultrasound��������������������������������������������������������������������������������������������������������������������������������������������������������15 Chemical Controls������������������������������������������������������������������������������������������������������������������������������������������������15 Algaecides��������������������������������������������������������������������������������������������������������������������������������������������������������15 Hydrogen Peroxide (H2O2)�������������������������������������������������������������������������������������������������������������������������������15 Barley Straw�����������������������������������������������������������������������������������������������������������������������������������������������������15 Mandarin Skin Extract�������������������������������������������������������������������������������������������������������������������������������������15 Case Studies: Harmful Algal Blooms in Egyptian Waters����������������������������������������������������������������������������������������15 Conclusion����������������������������������������������������������������������������������������������������������������������������������������������������������������19 References����������������������������������������������������������������������������������������������������������������������������������������������������������������� 19

INTRODUCTION Water bodies worldwide are step by step eutrophicated as a result of human obstruction in various ways like pesticide utilization, industrialization and so forth. The eutrophicated water is the great habitat for the rich development of algal blooms. In late decades, harmful algal blooms (HABs) represent a great hazard to health of citizens, water organisms, economical fisheries, water quality of lakes, water reservoirs and along the coast of the sea (Lee and Kim 2008; Stoecker et al. 2008; Kim et al. 2010). During the previous decade, there has been increasing attention to reduce the algae bloom, although the progress of field applications is still insufficient (Anderson 1997). Different investigations have been done to clarify

the variables in charge of bloom development and their toxin creation. It is difficult to discover the factors in charge of algal generation. Some algal species can create assorted types of toxins. Of the most common harmful algae are species within the dinoflagellates, diatoms and cyanophytes. Around the seas of the world, the planktonic algae are the basic food for filter-feeding bivalve (mollusks, mussels, shellfish, scallops) and in addition the hatchlings of economic crustaceans and various fishes. In many cases, the overwhelming of the microalgae reached up to several million cells per liter, which is considered useful for aquaculture and mariculture. Be that as it may, in a few circumstances algal blooms can have a negative impact, causing serious financial misfortune in aquaculture, fisheries and tourism 13

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operations and having major ecological and human wellbeing impacts. Among the 5,000 species of surviving marine phytoplankton, approximately 300 species can occur in such high numbers that they clearly stain the surface of the ocean (so-called ‘red tides’) (Figure 2.1).

GREEN TECHNOLOGY APPLICATIONS FOR ALGAL BLOOM CONTROL Biological Controls (Bio-Remediation) Bio-Remediation of Wetlands by Planting (BWP) This includes new wetlands and also the plants flourishing as floating mats on the water surface. The plant roots give enough surface zones to filter and reserve excess nutrients. BWP furthermore supports biofilm forms that diminish cyanobacteria densities. Covered surface zone limits light penetration and limits opportunity for algae growth. Plant Cultivation Cultivating the areas with different kinds of plants near surface waters in order to keep a balance between the water and point/non-point wellsprings of contamination. Blocking of supplements and different contaminations from entering surface waters. Providing shade from daylight, which reduces higher temperatures that can cause HABs (Kim et al. 2014). Activating Pressure of Grazing Several methods can be applied in order to activate the pressure of grazing by the zooplankton, bottom fauna and the other herbivore organisms to feed upon cyanobacteria, in this manner restricting the expansion of cyanobacteria populations. Strategies include: the expulsion of fish that feast upon zooplankton and other benthic fauna

Handbook of Algal Technologies and Phytochemicals

or the acquaintance of predators with these fish and the improvement of specialties to empower the development of beneficial creatures (Lampert et al. 1986). Bio-Remediation The dense blooming of phytoplankton in the hypertrophic waters can be done in vast fields using the stock filter-feeding silver carp (Hypophthalmichthys molitrix) which so far improve the quality of water (Ma et al. 2010; Xiao et al. 2010). Experiments have demonstrated that filter-feeding fish can decrease phytoplankton biomass to a specific degree, in spite of the fact that the last proficiency relies upon the attributes of the given condition. Notwithstanding, the utilization of such biomanipulation may prompt distinctive impacts relying on the community structure of the first plankton compositions (i.e., zooplankton and phytoplankton), the species and the density of the stocking fish and the water temperature (Xiao et al. 2010). Although a few experiments have demonstrated that stocking silver carp neglects to reduce phytoplankton biomass within the presence of extensive herbivorous cladocerans (Xiao et al. 2010; Zhao et al. 2013), a key explanation behind this was the diminishment of pressure on phytoplankton by zooplankton because of fish predation (Zhao et al. 2013). Controlling of HABs by Bacteria Early, González et al. (2000) were interested in studying the effect of Roseobacter lineage culture to prevent the algal bloom of dinoflagellates. But during application of the treatment, it is important to search about the harmful algal blooms (HAB) especially in freshwater environments used humanely (Buchan et al. 2014). In freshwater environments, few workers have used microorganisms involving bacteria for controlling harmful algal blooms. Early, Lee et al. (2010) made an experiment on Rhodococcus, a Gram-positive bacterium that prevails in hypertrophic body waters, and illustrated cyanobactericidal effect of bacterial filtrate. Furthermore, treatment of cyanobacterial blooms by heterotrophic bacteria must be preceded by or carried out together with sufficient nutrient cut off from the freshwater environment.

Physical Controls

FIGURE 2.1  Red tide recorded in Nozha Hydrodrome, Nov. 2002 (A) (Shakweer and Gharib 2005); and Western Harbor, 2013 (B) (Gharib and Dorgham 2006).

Air Circulation Aerators direct air all through the water section to disturb stratification. Air may be drawn through a diffuser close to the base of the water body, bringing about the produced plumes that rise to the surface that make vertical circulation cells as they spread out from the aerator.

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Green Technology Applications for Algal Bloom Control

This strategy restricts the availability of nitrogen and phosphorus and disturbs the behavior of blue green algae to migrate vertically and reduces the upper hand of cyanobacteria by keeping up suitable levels of dissolved O2 (Klumb et al. 2004; Skinner et al. 2014). Mechanical Circulation This method operates by pumping of water from the surface layer downwards or by moving water up from the bottom. Mechanical mixers interfere with stratification of the water column, catching conditions perfect for HABs to happen. This technique constrains the accessibility of both nitrogen and phosphorus to the upper layers and changes the behavior of cyanobacteria to migrate vertically and reduces the upper hand of blue greens by keeping up high levels of dissolved O2 (Cooke et al. 1993). Hypolimnetic Oxygenation This is a strategy to expand O2 concentrations in the hyper layer. Mechanisms incorporate submerged O2 chambers, direct O2 infusion. High O2 delivery rates lower the potential for sediment to release nitrogen and phosphorus (Beutel 2006). Surface Skimming Cyanobacterial sprouts frequently frame surface scums, particularly in the final stages of a bloom. Oil slick skimmers have been utilized to expel blue greens from these surface scums. Oftentimes this technique is coupled with the implementation of some coagulant or flocculent. Helpful technique for sprouts that are in later stages and have framed surface scums (Carmichael 2008). Ultrasound An ultrasound gadget is utilized to control HABs by emanating ultrasonic floods of a specific frequency with the end goal that the cellular structure of cyanobacteria is decimated by cracking interior gas vesicles utilized for lightness control. This strategy has been successfully actualized in ponds and other small water bodies (Klemenčič and Griessler-Bulc 2010).

Chemical Controls Algaecides Algaecides are synthetic compounds connected to a water body to eliminate cyanobacteria, and include Potassium permanganate; Coppersulphate; Chlorine; Alum; Lime; Ferric salts; Clay (Coagulation and Flocculation); Ozone and Hydrogen peroxide (H2O2) (McCoy and Martin 1977).

Hydrogen Peroxide (H2O2) Consideration of the cyanobacterial and cyanotoxin removal methods studied in the literature suggests that hydrogen peroxide (H2O2) may be appropriate for diminishing cyanobacterial and cyanotoxin concentrations and might be more fruitful when combined with other alleviation methods (Cooper et al. 1994; Bauzá et al. 2014). These algaecides cause blue greens to settle down far from the top layer of water body. Flok, which is an aluminum hydroxide precipitate, is formed when alum is applied to the water body. As the flok moves down, it expels phosphorus and particulates (including algae) from the water column. The flok settles on the sediment forming a layer that acts as a barrier to phosphorus. Phosphorus, discharged from the silt, combines with the alum and isn’t discharged into the water to fuel algae blooms. Barley Straw According to Roelke et al. (2007), disintegrating barley straw discharges inhibitory exacerbates that hinder algae growth development. Barley straw bundles are softened, separated and placed in a light net conveyed around the border of the water body to encourage the vital chemical responses and natural procedures that prevent algae growth. This is a low-cost method for preventing HABs. Mandarin Skin Extract Chen et al. (2004) discovered that algal development was essentially hindered on using mandarin skin extract (0.1% w/v). At the point when its concentration expanded to 0.5% w/v, no algal development was recognized.

CASE STUDIES: HARMFUL ALGAL BLOOMS IN EGYPTIAN WATERS Egyptian waters are subject to significant amounts of many sources of nutrients that can stimulate harmful algal blooms; these untreated sources include cabbages, some groundwater inflow, atmosphere deposition, animal wastes, as well as agricultural and other fertilizer run off. In Egyptian waters, prior emotional events of harmful algae growth happened on a few occasions because of planktonic blooms of algae. Reports on the harmful algal blooms were initiated by Halim since 1956 (Halim 1960) on studying the Egyptian Mediterranean Sea water. He described the new genus and new species of dinoflagellates Alexandrium minutum. From that point forward, a considerable number of investigations happened along the Egyptian drift. Amid (2007) and Ismael (2014) recorded

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Handbook of Algal Technologies and Phytochemicals

a substantial green tide of Micromonas pusilla joined by a bloom of Peridinium quinquecorne along the coast of Alexandria. Hassan (1972) and Ismael (1993) recorded that P. quinquecorne was confined toward the Eastern Harbor (Table 2.1). Labib (2000) also reported the three dinoflagellates, Scrippsiella trochoidea, Gymnodinium catenatum and Prorocentrum triestinum, that caused water discoloration during 1997 and 1998 in Mex Bay, as well as in the Eastern Harbor of Alexandria, Egypt. Moreover, 14 Oscillatoriales species were reported from the Western Alexandria coast with the perennial existence of Oscillatoria brevis (Shams El Din and Abdel Halim 2008) (Figure 2.2). Likewise, six cyanobacteria species were recorded along the shore of Alexandria in 2005 after an episode of mass mortality of the bottom angling fish, Siganus rivulatus, in the Eastern Harbor of Alexandria (Ismael 2012). Freshwater assets in Egypt are currently debilitated by the presence and increment of unsafe algal blooms. The

HABs are once in a while a direct result of anthropogenic contamination entering water bodies, for example, halfway treated nutrient rich effluents and the draining of fertilizers and animal wastes. Many researchers have discussed the harmful algal blooms phenomena, and many attempts have been made to reduce their effect or to control their impact (Dorgham 2011). The river Nile that is the main source of drinking water in Egypt is continuously subjected to nutrient loading coupled with year-round warm weather which favors the growth of cyanobacteria. Harmful cyanobacterial sprouts are perceived as a quickly extending worldwide issue that debilitates human and biological community wellbeing. The genera most responsible for the massive blooms are Gomphosphaeria, Microcystis and Oscillatoria. These genera can deliver cyanotoxins that are discharged into the water section (Mohamed and Carmichael 2000). Microcystis is the most across the board and habitually experienced living being. It can

TABLE 2.1 Main Harmful Algal Blooms (HABs) Events in Egypt Time

Location

HAB Species

Sept. 1990 May 1987 April 1992 Oct. 1994 Sept. 1998 July 1999 May 2000 Sept. 2000 Oct. 2000 Sept. 1998 Oct. 1998 Aug. 1999 May 1999 Oct. 2000 21 Aug. 2004 7 Aug. 2004 13 Aug. 2004 Feb. 1990 Oct. 1990 July 1999 July 1999 Feb. 1987 Nov. 2002 April 1992 June 1992 2012 2013 2013

Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Eastern Harbor Western Harbor Western Harbor Western Harbor Western Harbor Nozha Hydrodrome Nozha Hydrodrome El-Max Bay El-Max Bay Alexandria shoreline Alexandria shoreline Western Harbor

Anabaena sp. Alexandrium minutum Prorocentrum triestinum Alexandrium minutum Chattonella antiqua Chattonella antiqua Chattonella antiqua Chattonella antiqua Chattonella antiqua Gymmodinium mikimotoi Gymmodinium mikimotoi Gymmodinium mikimotoi Gymmodinium mikimotoi Gymmodinium mikimotoi Chattonella antiqua Gymnodinium catenatum Gymnodinium mikimotoi Prorocentrum cordatum Pseudo-nitzschiadelicatissima Alexandrium minutum Prorocentrum triestinum Anabaenopsis circularis Microcystis aerugicnosa Prorocentrum triestinum Scrippsiella trochoidea Peridinium quinquecorne Oscillatoria spp. Eutreptiella sp.

Cell × 106 21.1 15 75 24 0.54 0.85 1.14 0.65 0.31 0.5 0.13 0.93 1.8 0.42 1.26 2.73 0.16 2 5 10.6 6.8 6.8 3.2 63.5 6.11 9.9 0.13 17

Source Zaghloul (1996) Zaghloul (1995) Zaghloul (1995) Halim and Labib (1996) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail (2001) Mikhail et al. (2005) Mikhail et al. (2005) Mikhail et al. (2005) Zaghloul (1996) Zaghloul (1996) Gharib and Dorgham (2006) Gharib and Dorgham (2006) Gharib (1991) Shakweer and Gharib (2005) Mikhail (1997) Mikhail (1997) Ismael (2014) Ismael et al. (2014) Heneash et al. (2015)

Green Technology Applications for Algal Bloom Control

17

FIGURE 2.2  Species recorded from the Eastern Harbor and Abu Qir area. (A) Oscillatoria nigroviridis, (B) Oscillatoria spp., (C) Planktothrix c.f. agardhii, (D) O. acutissima, (E and F) O. limosa, (G and H) Lyngba sp. and (I) large mat of O. acutissima in the Eastern Harbor (the platform of oceanography department) (Ismael 2012).

alter the pristine nature of water and cause numerous issues, for example, event of smelly scent, fish kill, decay of recreational esteem and clogging of filters for water supply frameworks. It can likewise deliver the microcystin toxin (Carmichael 1992), which is harmful to fishes, amphibian invertebrate creatures, domestic creatures and humans. Abdel Mohsen and El Gammal (2008) found that the addition of potassium fertilizer (potassium sulfate) was suitable for inhibited Microcystisaeruginosa growth in waters and safe for fish (Figure 2.3). Due to their unique properties, nanomaterials have been playing an important role in controlling harmful algae. Recently, lab work carried out by El-Sheekh and El-Kassas (2014) reported the anti-algal activity of in vivo biosynthesized silver nanoparticles (Ag-NPs) by different microalgae species that belong to different groups, namely, Spirulina platensis, Chlorella vulgaris and Scenedesmus obliquus. Silver-NPs greatly inhibited the growth of Microcystis aeruginosa which is known to produce hepatotoxins that affect aquaculture and humans. Moreover, El-Kassas and Ghobrial (2017) concluded the complete green remediation of the bloom forming blue green alga named Oscillatoria simplicissima, using Ag-NPs biosynthesized by the three marine plant species; they added that it might have been due to some bioactive compound carried over from the biological materials and not only to silver alone. Biosynthesized FeO3-NPs have been characterized, and results revealed they too can be used to separate Ag-NPs and silver for complete remediation and further applications. Additionally, El-Kassas and Okbah (2017) reported that Copper-NPs synthesized by Corallina officinalis

have excellent phytotoxic effect on the harmful alga, namely, Lyngbya majuscula, which is considered as a standout amongst the most widely recognized types of Lyngbya worldwide in a measurement subordinate way. Copper-NPs synthesized by Corallina mediterranea extract created direct consequences for L. majuscula dry weight. The outcomes additionally demonstrated that there were sharp declines in chlorophyll a content in L. majuscule with the increase in Cu-NPs concentrations derived from either C. officinalis or C. mediterranea. The heterotrophic Diphylleia rotans can assume a vital part in the decrease of Microcystis biomass and microcystin toxins and consequently could be utilized as a safe bioagent for the biocontrol of harmful algal sprouts in oceanic conditions (Mohamed and Al-Shehri 2013). Furthermore, numerous bacterial strains as Trichoderma citrinoviride have been accounted for as conceivable specialists for repressing and controlling destructive cyanobacterial Microcystis aeruginosa blooms (Mohamed et al. 2014). Alum application in fish earthen lakes at the Central Laboratory for Aquacultural Research, Abbassa, Egypt, uncovered that alum successfully diminished blue green algal biomass and diminished the water turbidity of tilapia earthen lakes with no negative impact on fish growth (Dawah et al. 2015). Applying ferric chloride and ferric sulfate as coagulants to waters of Nile surface water at El Maadi, Greater Cairo Urban Region, treated with the combined pre-oxidation system viably lessened algal numbers by 60% and better the total organic carbon diminishment and leftover aluminum in the treated water (El-Dars et al. 2015).

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Handbook of Algal Technologies and Phytochemicals

FIGURE 2.3  Farm visit and clinicopathological pictures of affected Nile Tilapia. The farm visit revealed (A) an earthen pond with dark green water, some dead fish on the water surface and huge quantities of waterweeds and water hyacinth and (B) the presence of dead Nile Tilapia on the pond bunds. Closer examination found (C) large-size dead fish with detached scales and external hemorrhages, (D) moribund fish exhibiting abdominal distension and vent inflammation and (E) gill filaments showing a marbling appearance. Panel (F) shows wet mount broth culture with Gram-positive cocci arranged in a chai (black arrow) (Gram’s stain × 1,000) (Abu-Elala et al. 2016).

In laboratory study, Mohamed (2001) described the efficiency of a mixture of alum (75 mg L−1) and lime (100 mg L−1), instead of the usual use of alum only, to precipitate and coagulate all phytoplankton without release of any substances into the water and precipitated phosphate from the Nile river water used as a source of drinking water in Egypt. Nam et al. (2016) reported that the use of biomanipulation of massive nuisance algal blooms using herbivorous Daphnia similoides in a eutrophic agricultural reservoir has long been considered an ecosystem-friendly approach for the management of algal blooms. The Suez Canal, one of the main canals in Egypt, has become eutrophic, and filamentous algae are the most common aquatic weed problem in the canal. Abou El Ella et al. (2007) controlled the algae by utilizing rice straw. The decay of rice straw in water delivers and discharges many compounds. Hydrogen peroxide might be viewed as the principle substance in controlling algae. Aquatic creatures, for example, mollusks, snails and other filter-feeding shellfish, prominently affect nutrient expulsion from eutrophic water bodies (Wang et al. 2012).

The common treatment of stocking filter-feeding silver carp (Hypophthalmichthys molitrix) in eutrophic water bodies has been for the most part associated with control of over the top phytoplankton levels and improvement in the nature of water bodies (Xiao et al. 2010; Ma et al. 2010). Silver carp has a long life expectancy in common water bodies (6–10 years, even 20 years in a few examples (Ma et al. 2010)). Regularly supplied in water reservoirs in developing nations, silver carp is an omnivorous filter-feeder that can filter particles > 10 mm, including zooplankton and phytoplankton (Xiao et al. 2010). Filter-feeding fish, for example, silver carp, have appeared to choose zooplankton on the premise of prey escape capacity; for example, cladocerans are more defenseless than copepods to fish predation because they have lower escape ability (Zhao et al. 2013). A case of aquatic creature utilization to enhance water quality is the presentation of Asiatic shellfish (Corbicula fluminea) into the Potomac River, United States of America. This was done in the mid-1980s, when chlorophyll a concentration in the Potomac River had all the earmarks of being emphatically drained, at levels of under 1 g/L (Li et al. 2010). The Asiatic shellfish can

19

Green Technology Applications for Algal Bloom Control

likewise advance supplement recovery. Therefore, the species imposes simultaneous top-down and bottom-up effects on the ecosystem (Xiao et al. 2010). Trials have demonstrated that filter-feeding fish can lessen phytoplankton biomass to a specific degree, despite the fact that the last proficiency relies upon the attributes of the given ecosystem. In any case, the utilization of such biomanipulation may prompt distinctive impacts relying upon the composition of the underlying plankton community (i.e., zooplankton and phytoplankton), the species and stocking density of fish and the water temperature (Xiao et al. 2010). Silver carp use to control algal biomass remains questionable. For example, a few examinations have demonstrated that stocking silver carp neglects to diminish phytoplankton biomass within the sight of expansive herbivorous cladocerans (Xiao et al. 2010; Zhao et al. 2013). A key explanation behind this was the lessening of touching weight on phytoplankton by zooplankton because of fish predation (Zhao et al. 2013).

CONCLUSION In conclusion green technology applications for algal bloom control involved the following: A) Advanced techniques for detecting harmful algal blooms such as remote sensing techniques, quality control/quality assurance and low-pressure (high-vacuum) scanning electron microscopy and gene probes and other biomarkers. B) Techniques and applications for bloom control such as naturally derived compounds using barley straw, Batangas mandarin skin and dwarf banana peel in addition to clay flocculation. Other green technological techniques such as macro-algae, bacteria, LG sonic solutions and green synthesized nanomaterials and aquatic animals are also reviewed. C) Some of case studies for control of eutrophication in lakes and reservoirs as well as control of marine eutrophication with a reference to microalgal biomass systems for bioremediation and fuel production are investigated in different countries and have also been reviewed. D) Causes and results of river eutrophication and the strategies for protection of rivers from eutrophication in addition to river eutrophication control techniques including microbial dosing, catchment sensitive farming, bio-film, bioceramics, bio-film carrier (filamentous bamboo) and biocord would offer solutions.

REFERENCES Abou El Ella, S. M., Hosny, M. M., Bakry, M. F. Growth inhibition of bloom-forming using rice straw in water courses (case study). Eleventh International Water Technology Conference, IWTC11 Sharm El-Sheikh, Egypt, 2007. Abu-Elala, N. M., Abd-Elsalam, R. M., Marouf, S., Abdelaziz, M., Moustafa, M. Eutrophication, ammonia intoxication, and infectious diseases: Interdisciplinary factors of mass mortalities in cultured Nile tilapia. J Aquat Anim Health. 2016. 28(3): 187–198. Anderson, D. M. Turning back the harmful red tides. Nature. 1997. 388(6642): 513–514. Bauzá, L., Aguilera, A., Echenique, R., Andrinolo, D., Giannuzzi, L. Application of hydrogen peroxide to the control of eutrophic lake systems in laboratory assays. Toxins (Basel). 2014. 6(9): 2657–2675. Beutel, M. W. Inhibition of ammonia release from anoxic profundal sediments in lakes using hypolimnetic oxygenation. Ecol Eng. 2006. 28(3): 271–279. Buchan, A., LeCleir, G. R., Gulvik, C. A., González, J. M. Master recyclers: Features and functions of bacteria associated with phytoplankton blooms. Nat Rev Microbiol. 2014. 12(10): 686–698. Carmichael, W. A world overview – One hundred twenty seven years of research on toxic cyanobacteria, where do we go from here. In: Hudnell, K.(ed) Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. Springer. 2008. 105–125. Carmichael, W. W. Cyanobacteria secondary metabolites. The cyanotoxins. J Appl Bacteriol. 1992. 72(6): 445–459. Chen, J., Liu, Z., Ren, G., Li, P., Jiang, Y. Control of Microcystis aeruginosaTH01109 with Batangas mandarin skin and dwarf banana peel: Technical note. Water SA. 2004. 30(2): 279–282. Cooke, G. D., Welch, E. B., Peterson, S. A., Newroth, P. R. Artificial circulation. In: Cooke G. D., Welch, E. B., Peterson, S. A., Newroth, P. R. (eds) Restoration and Management of Lakes and Reservoirs. Lewis Publishers, London. 1993. 419–449. Cooper, W. J., Shao, C. W., Lean, D. R. S., Gordon, A. S., Scullym, F. E. Factors affecting the distribution of H2O2 in surface waters. In: Baker L.A. (ed)Environmental Chemistry of Lakes and Reservoirs. American Chemical Society, Washington, DC. 1994. 237: 391–422. Dawah, A., Soliman, A., Abomohra, A., Battah, M., Anees, D. Influence of alum on cyanobacterial blooms and water quality of earthen fish ponds. Environ Sci Pollut Res. 2015. 22(21): 16502–16513. Dorgham, M. M. Eutrophication problem in Egypt. In: Ansari, A.A., Sarvajeet, S.G., Lanza, G.R., Rast, W. (eds) Eutrophication: Causes, Consequences and Control. Springer, Netherlands. 2011. El Gammal, M. Potassium fertilizer inhibits the Growth of Cyanobacteria (Microcystisaeruginosa) in Fishpond. In: 8th International Symposium on Tilapia in Aquaculture. 2008.

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El-Dars, F. M. S. E., Abdel Rahman, M. A. M., Salem, O. A. M., Abdel-Aal, E. A. Algal control and enhanced removal in drinking waters in Cairo, Egypt. J Water Health. 2015. 13: 4. El-Kassas, H. Y., Ghobrial, M. G. Biosynthesis of metal nanoparticles using three marine plant species: Antialgal efficiencies against—Oscillatoriasimplicissima. Environ Sci Pollut Res. 2017. 24(8): 7837–7849. El-Kassas, H. Y., Okbah, M. A. E. A. Phytotoxic effects of seaweed mediated copper nanoparticles against the harmful alga: Lyngbyamajuscula. J Genet Eng Biotechnol. 2017. 15(1): 41–48. El-Sheekh, M. M., El-Kassas, H. Y. Application of biosynthesized silver nanoparticles against a cancer promoter cyanobacterium, Microcystisaeruginosa. Asian Pac J Cancer Prev. 2014. 15(15): 6773–6779. Gharib, S. M. Study of the biological productivity of the Nozha Hydrodrome as a model of artificially fertilized fish farm. Ph.D. Thesis, El-Mansoura Univ. Fac. Sci. 453 pp. 1991. Gharib, S. M., Dorgham, M. M. Eutrophication stress on phytoplankton Community in the Western Harbour of Alexandria, Egypt. IJOO. 2006. 1(2): 261–273. Gonzalez, L. E., Bashan, Y. Increased growth of the microalga Chlorella vulgaris when coimmobilized and cocultured in alginate beads with the plant-growth-promoting bacterium Azospirillumbrasilense. Appl Environ Microbiol. 2000. 66(4): 1527–1531. Halim, Y. Alexandriumminutum n. gen., n. sp. Dino flagella provocant des “eaux rouges”. Vie Milieu. 1960. 11(1): 102–105. Halim, Y., Labib, W. First recorded toxic Alexandriumminutum Halimbloom. The Intergovernmental Oceanographic Commission of UNESCO. Harmful Algae News. 1996. No. 14. Hassan, A. K. Systematic and Ecological Study of the Dinoflagellates in the Area of Alexandria. M.Sc. Thesis, Faculty of Science, Alexandria University. 316 pp. 1972. Heneash, A. M. M., Tadrose, H. R. Z., Hussein, M. M. A., Hamdona, S. K., Abdel-Aziz, N., Gharib, S. M. Potential effects of abiotic factors on the abundance and distribution of the plankton in the Western Harbour, southeastern Mediterranean Sea, Egypt. Oceanologia. 2015. 57(1): 61–70. Ismael, A. A. The ecological distribution of planktonic dinoflagellates along the Coastal water of Alexandria. M.Sc., Alexandria Univ., Unpublished manuscript. 119 pp. 1993. Ismael, A. A. Benthic bloom of cyanobacteria associated with fish mortality in Alexandria waters. Egypt J Aquat Res. 2012. 38(4): 241–247. Ismael, A. A. Coastal engineering and Harmful Algal Blooms along Alexandria coast, Egypt. Egypt J Aquat Res. 2014. 40(2): 125–131. Ismael, A. A. H., Mohamed, E. A., El-Sheekh, M. M., Hegazy, W. Ecological distribution of harmful epiphytic Oscillatoriales in Alexandria coast, Egypt, with special reference to DNA identification. J Coast Life Med. 2014. 2(4): 274–280.

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Kim, D.-K., Zhang, W., Watson, S., Arhonditsis, G. B. A commentary on the modelling of the causal linkages among nutrient loading, harmful algal blooms, and hypoxia patterns in Lake Erie. J Gr Lakes Res. 2014. 40(3): 117–129. Kim, Y. M., Wu, Y., Duong, T. U., Ghodake, G. S., Kim, S. W., Jin, E. S., Cho, H. Thiazolidinediones as a novel class of algicides against red tide harmful algal species. Appl Biochem Biotechnol. 2010. 162(8): 2273–2283. Klemenčič, A. K., Griessler-Bulc, T. The efficiency of ultrasound on algal control in A closed loop water treatment system for cyprinid fish farms. Fresenius Environ Bull. 2010. 19 – No 5a. Klumb, R. A., Bunch, K. L., Mills, E. L., Rudstam, L. G., Brown, G., Knauf, C., Burton, R., Arrhenius, F. Establishment of a metalimnetic oxygen refuge for zooplankton in a productive Lake Ontario embayment. Ecol Appl. 2004. 14(1): 113–131. Labib, W. Dinoflagellate “brown tides” in Alexandria, Egypt Waters During 1997–1998. Pak J Marine Sci. 2000 9(1&2): 33–49. Lampert, W., Fleckner, W., Rai, H., Taylor, B. E. Phytoplankton control by grazing zooplankton: A study on the spring clear-water phase. Limnol Oceanogr. 1986. 31(3): 478–490. Lee, M. O., Kim, J. K. Characteristics of algal blooms in the southern coastal waters of Korea. Mar Environ Res. 2008. 65(2): 128–147. Lee, Y. K., Ahn, C. Y., Kim, H. S., Oh, H. M. Cyanobactericidal effect of Rhodococcus sp. isolated from eutrophic lake on Microcystis sp. Biotechnol Lett. 2010. 32(11): 1673–1678. Li, X. N., Song, H. L., Li, W., Lu, X. W., Nishimura, O. An integrated ecological floating bed employing plant, freshwater clam and biofilm carrier for purification of eutrophic water. Ecol Eng. 2010. 36(4): 382–390. Ma, H., Cui, F., Liu, Z., Fan, Z., He, W., Yin, P. Effect of filter-feeding fish silver carp on phytoplankton species and size distribution in surface water: A field study in water works. J Environ Sci. 2010. 22(2): 161–167. McCoy Jr., L. F., Martin, D. F. The influence of Gomphosphaeriaaponina on the growth of Gymnodiniumbreve and the effects of aponin on the icthyotoxicity of Gymnodiniumbreve. Chem Biol Interact. 1977. 17(1): 17–24. Mikhail, S. K. Ecological Studies of Phytoplankton in Mex Bay. Ph.D. Thesis, Fac. Sci. Alexandria Univ. 266 pp. 1997. Mikhail, S. K. Toxic red tide species are on rise in Alexandria waters (Egypt). In: The Intergovernmental Oceanographic Commission of UNESCO Harmful Algae News. 2001. No. 22. Mikhail, S. K., Oakbah, M. A., Labib, W. Toxic phytoplankton species linked to massive invertebrate and fish mortality in the Eastern Harbour of Alexandria. The Intergovernmental Oceanographic Commission of UNESCO. Harmful Algae News. 2005. No. 29. Mohamed, Z. Alum and lime-alum removal of toxic and nontoxic phytoplankton from the Nile River water: Laboratory study. Water Resour Manage. 2001. 15: 213–221.

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Mohamed, Z. A., Al-Shehri, A. M. Grazing on Microcystisaeruginosa and degradation of microcystins by the heterotrophic flagellate Diphylleiarotans. Ecotoxicol Environ Saf. 2013. 96: 48–52. Mohamed, Z. A., Carmichael, W. W. Seasonal variation in microcystin levels of river Nile water at Sohag City, Egypt. Ann Limnol - Int J Lim. 2000. 36(4): 227–234. Mohamed, Z. A., Hashem, M., Alamri, S. A. Growth inhibition of the cyanobacterium Microcystisaeruginosa and degradation of its microcystin toxins by the fungus Trichodermacitrinoviride. Toxicon. 2014. 86: 51–58. Nam, G. S., Lee, E. H., Chang, K. H. Application of an algal bloom control technique using large zooplankton predators in an eutrophic agricultural reservoir: A Preliminary Study. Irrig Drain. 2016. 65: 230–238. Roelke, D. L., Errera, R. M., Kiesling, R., Brooks, B. W., Grover, J. P., Schwierzke, L., Pinckney, J. L. Effects of nutrient enrichment on Prymnesiumparvum population dynamics and toxicity: Results from field experiments, Lake Possum Kingdom, USA. Aquat Microb Ecol. 2007. 46(2): 125–140. Shakweer, L. M., Gharib, S. M. Chemical and biological conditions during the harmful algal bloom in the NozhaHydrodrome water during autumn 2002. Proceedings of the 15th International Conference on “Environmental Protection Is a Must”, 3–5 May, 2005. Shams El Din, N., Abdel Halim, A. M. Changes in phytoplankton community structure at three touristic sites at western Alexandria Egypt. J Aquat Biol Fish. 2008. 12(4): 85–118.

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Shirota, A. Red tide problem and countermeasures. Int J Aquacul Fish Technol. 1989. 1: 195–223. Skinner, M. M., Moore, B. C., Swanson, M. E. Hypolimnetic oxygenation in Twin Lakes, WA. Part II: Feeding ecology of a mixed cold- and warmwater fish community. Lake Reserv Manage. 2014. 30(3): 240–249. Stoecker, D. K., Adolf, J. E., Place, A. R., Glibert, P. M., Meritt, D. W. Effects of the dinoflagellates Karlodiniumveneficum and Prorocentrum minimum on early life history stages of the eastern oyster (Crassostreavirginica). Mar Biol. 2008. 154(1): 81–90. Wang, J., Liu, X. D., Lu, J. Urban river pollution control and remediation. Proc Environ Sci. 2012. 13: 1856–1862. Xiao, L., Ouyang, H., Li, H., Chen, M., Lin, Q., Han, B. P. Enclosure study on phytoplankton response to stocking of silver carp (Hypophthalmichthys molitrix) in a eutrophic tropical reservoir in South China. Int Rev Hydrobiol. 2010. 95(45): 428–439. Zaghloul, F. A. Comparative study of phytoplankton production, composition and diversity index in the eutrophic Eastern Harbour of Alexandria, Egypt. Bull High Inst Public Health. 1995. 25(3): 665–678. Zaghloul, F. A. Further studies of the assessment of eutrophication in Alexandria Harbours, Egypt. Bull Fac Sci Alex. 1996. 36(1): 281–294. Zhao, S. Y., Sun, Y. P., Lin, Q. Q., Han, B. P. Effects of silver carp (Hypophthalmichthysmolitrix) and nutrients on the plankton community of a deep, tropical reservoir: An enclosure experiment. Freshw Biol. 2013. 58(1): 100–113.

3

Natural Algal Photobioreactors for Sustainable Wastewater Treatment D. M. Mahapatra, N. V. Joshi, G. S. Murthy, and T. V. Ramachandra

CONTENTS Introduction................................................................................................................................................................24 Diversity of Pond Systems.........................................................................................................................................25 Algal Ponds (Oxidative/High Rate)...................................................................................................................25 Anaerobic Ponds................................................................................................................................................26 Facultative Ponds...............................................................................................................................................26 Maturation/Polishing Ponds..............................................................................................................................26 Pond Architecture and Design...................................................................................................................................26 Areal Loading Rate Model................................................................................................................................26 Gloyna and Hermann Model.............................................................................................................................27 First Order Kinetics—Plug Flow Model...........................................................................................................28 Marais and Shaw: Complete Mix Model...........................................................................................................28 Wehner–Wilhelm Model and Thrimurthi Application.......................................................................................28 Wastewater Characteristics, Algal Ponds, Nutrients, Pollutants and Pathogen Removal..........................................29 Algae’s Role in Bioremediation and Potential Scope for a Biorefinery....................................................................30 Economic Feasibility of Pond Systems.....................................................................................................................30 Conclusion................................................................................................................................................................. 32 Acknowledgment....................................................................................................................................................... 32 Bibliography.............................................................................................................................................................. 32

BOX 3.1  PARAMETERS AFFECTING THE OPERATIONAL CHARACTERISTICS OF PONDS 1. LIGHT • Helps in photosynthesis and dissolved oxygen production. • Especially wavelength from 400 to 700 nm (photosynthetic active radiation, PAR). • Influences phototrophs and pond dynamics (Curtis et al., 1994). • Moreover, the photoperiods light to dark hours also influences the oxygen saturation and availability of oxygen during the night. • Presence of suspended material interferes with the light penetration and can create anoxic/ anaerobic zones. • Sometimes natural scattering of light due to components present in water helps in better distribution of light (USEPA 2011).

2. TEMPERATURE • Pond system are dependent directly on microclimatic conditions that govern the temperature. • As the rate of the various chemical reactions and metabolism are directly dependent on temperature, the temperature governs a number of key processes that are critical to the treatments in the pond systems as nitrification/denitrification (Kirby et al, 2009). • Production of methane in anaerobic ponds (Sukias and Craggs, 2011). • Removal of a variety of contaminants as heavy metals, xenobiotics, COD, etc., are also dependent on temperature of the ponds (Halpern et al., 2009). • Especially during the seasonal transitions, temperature plays a major role in deciding the pond performance as well as physicochemical and biological integrity of such systems. • Increased pond depth and high variations in the temperatures across seasons results in

23

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Handbook of Algal Technologies and Phytochemicals

stratification (warm upper layers and cold bottom water across the water column) that hinders mixing and resuspension of particulate organic matter or suspended solids (Finney and Middlebrooks, 1980). Such stratification restricts sunlight penetration as well as dissolved oxygen equilibration and thus creates anaerobic conditions (Marais, 1966). Such thermal stratifications are disadvantageous for both the algal and the heterotrophic population, bringing down the overall treatment performance of the ponds (USEPA, 2011). 3. WIND • The wind helps in mixing the warm and cool layers and aids in prevention of stationary zones and anaerobic conditions (Marais, 1966; USEPA, 2011). Such wind conditions help in equilibration of the conditions and avoid stratification, malodor generation and treatment inefficiency due to water short-circuiting (USEPA, 2011). • The wind speed, direction and seasonal pattern have been reported to influence the bacterial removal, although the pond geometry, architecture positions of inflows and outflows and position type of baffles also critically affect the bacterial removal. • It has been observed that, in facultative ponds, the winds help in vertical mixing that distributes the algal cells, in the absence of which, algal forms a thick layer on the top of the ponds and alters the pond dynamics and critically reduces the performance and the quality of the pond effluent (Mara and Pearson, 1998). 4. POND ARCHITECTURE • The pond architecture plays a very important role in obtaining a high-quality effluent. Various pond types, with different dimensions and depths, have shown strikingly different performances. • Favorable microclimatic conditions coupled with the pond architecture can result in high treatments and overall performance (Pearson et al., 1995). • A series of ponds with low dimensions and shallow depths has often resulted in good effluent quality with better treatments. Compared to

horizontal pond orientation in terms of flow the vertical flow designed ponds have shown a high N removal with better effluent quality (Hamdan and Mara, 2011). • The number and dimension of baffles used also decides the treatment efficiency. In this context, studies conducted on optimization of treatment through selection of baffle size and number showed high performance with four number of baffles and the performance is unaffected by the dimension of the baffles (Abbas et al., 2006). Due to poor mixing, the lowest performances were observed with no baffles. 5. PROCESS PARAMETERS • Various process parameters such as feed to microbe (f/m) ratio, organic loading rate, strength of influents, hydraulic retention time (HRT), pH and redox potential are crucial in the treatment in pond processes. • HRT is a function of influents wastewater characteristics, and the organic loads can vary from two to three days (Green et al, 1995) to 24–30 days (Faleschini et al., 2012). • pH has been one of the most important process parameters in the pond treatment processes. In most of the natural treatment process in ponds, the pH typically rises due to excess of hydroxide ions as a consequence of algal photosynthesis. • In many of the studies the pH has been regulated although at smaller scales (Bhatnagar et al., 2010). • Similarly, other parameters also have profound effects on the treatment aspects of ponds. For instance, flow rate alterations (Faleschini et al., 2012), pond volume (Taddesse et al., 2004), area (Jamwal et al., 2009).

INTRODUCTION Traditional ponds, lakes and lagoons as natural algal photobioreactors are the most attractive and economic options for wastewater treatment and help in remediation functioning as stabilization ponds, facultative lagoons, oxidation ponds, high rate algal ponds, etc. These water bodies offer great advantages compared to the mechanically driven energy intensive systems provided that the nutrient inflow does not surpass the carrying capacity of remediation. In tropical climates traditionally, such algal ponds have been used for water storage and nutrient

Natural Algal Photobioreactors for Sustainable Wastewater Treatment

assimilation resulting in pond amelioration. Due to high water residence time, these ponds act as both reservoir and in-situ water treatment units by the virtue of their physico-chemical and biological characteristics and have offered enormous ecosystem services. These aquatic ecosystems are immensely resilient and self-sufficient due to highly functional ecosystems with potential abiotic and biotic components encompassing key microbial communities such as phages, bacteria, protozoans, fungi and beneficial algae (Ramachandra et al., 2014, 2015). The key processes in the systems involve algal bacterial symbiotic interaction with high rate oxygen production in the algal zone. These processes can simultaneously occur in a single pond unit or can occur in parallel through a cascading pond system. A typical algal pond system used in our earlier wetland studies comprise of a) anaerobic zone, b) facultative zone and c) aerobic zone (Ramachandra et al., 2014). Pond systems are stabilized by a balance in influx nutrients and organic matter, dissolved autochthonous minerals, dissolved oxygen, potentially growing algal communities, light, temperature, wind related turbulence and mixing (Konig, 1984; Konig et al., 1987; Hosetti and Frost, 1995; AmengualMorro et al., 2012). These systems have been very robust and self-sustaining due to their stable trophic interaction that is a key characteristic of aquatic ecosystems. The present-day treatment ponds also referred as natural photo-bioreactors and similar ecosystems have the prospect of potentially being used for treatment of various kinds of wastewaters. Today’s engineered treatment ponds systems can be variably operated by altering the organic and nutrient loads into the system; that potentially creates different redox environments thus varying pond microflora and stepwise removing nutrients and treating wastewater (Mahapatra and Ramachandra, 2013). These ponds have been used for treatment of high strength wastewater as anaerobic lagoons, for treatment of moderate strength municipal wastewater (black and grey water) as facultative lagoons or polishing ponds for partially treated municipal wastewaters or low strength wastewaters (Ramachandra et al., 2012a, b, 2018). Various studies have used such pond systems that are more oxidative in nature (Hosetti and Frost, 1995), anaerobic (Kayombo et al., 2010) and high rate advanced algal ponds (Craggs et al., 2012). The most important and striking characteristics of the ponds are their simplicity in design albeit having a complex, interactive relationship with the synergistic environment and drastically reducing the dependencies on advanced and mechanically operated forced heterotrophy-based systems (Mahapatra, 2015). These are mostly suited for developing tropical nations, and many such economies

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have been using the treated effluents from these pond systems for irrigation as in Bangalore (Ramachandra et al., 2018) and for aquaculture as in east Kolkata wetlands (Ghosh, 2018). This is also followed internationally as in Australia, South-east Asia and Latin America where such systems have been initiated since the 1980s. Such pond systems are vital in Third World countries, and most of the research emphasis has been laid by the pioneering organizations, capitalizing various projects under resource recovery initiatives by the United Nation Development Program (UNDP) and the Pan America Health Organization (PAHO) (Shuval et al., 1986). The pond system has been followed globally and can be seen operational in Mediterranean and southern European nations, especially in suburbs comprising of facultative and maturation ponds as in Algeria, Egypt, Greece, Morocco and Tunisia (Mara and Pearson, 1998). Due to simple operation, these systems are easy to maintain (Hosetti and Frost, 1998) and require minimal monitoring to ensure the effluents meet the regulatory standards. Lagoon systems, whether single or multi celled, have their own advantages and treat the wastewater in a cascading fashion. Traditionally lagoons were used to handle high strength wastewater, especially manure wastewaters, and have been more frequently used for livestock and agricultural systems. The algal ponds as natural photobioreactors offer several advantages such as a) simple design with the ease of construction, maintenance and operation; b) low cost; c) pathogen removal; d) sludge treatment; e) handling varied strength wastewaters; f) inactivating helminths and nematode eggs; g) efficient BOD removal with a great deal of advantage for small and rural communities. Despite these advantages there are many opportunities to improve such systems to minimize the land footprint and maximize the algal solids recovery at the downstream (Mara et al., 1992).

DIVERSITY OF POND SYSTEMS Although there are many variations in pond design and operations, the following sections present the details of four major prevalent pond systems.

Algal Ponds (Oxidative/High Rate) They are also known as advanced oxidative systems (AOS) or high rate algal ponds (HRAP) and are able to consistently maintain a highly aerobic environment across the depth of the water column due to rapid algal photosynthesis. Such systems are usually shallow, 30–45 cm in design (USEPA, 2011), which ensures proper dissolved

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oxygen production and equilibration across the depth of the water column. The daytime photosynthesis provides surplus dissolved oxygen, and nighttime DO is maintained through a combination of residual DO left from the daytime photosynthesis with wind turbulence. These high rate algal systems are good at BOD removal, have a reasonably large land footprint with higher ecosystem goods (fish, fodder, etc.) and have potential to support livelihood of the local population. The usual detention time for these ponds are two to six days, with a BOD loading of 12–25 g/KL/day and performing at a BOD removal efficiency of 95% (USEPA, 2011).

Anaerobic Ponds These systems function without the involvement of dissolved oxygen in a highly reducing environment. Under a strict methanogenic environment, at a relatively high organic loading, the process yields methane and carbon dioxide (Chanakya et al., 2012). The water residence time for such ponds is 1–1.5 days with a depth of 2–5 m. These anaerobic ponds are highly efficient at relatively warm temperatures (min 15–20°C) and a pH of ~6.2 (Kayombo et al., 2010). However, these ponds have high organic loading rate of 300 g/m2/day (Quiroga, 2011). Such activities have been possible in tropical climate with optimal efficiency and thus are highly variable based on nature and type of climate in the treatment region (USEPA, 2011). The main principle of these types of deep pond systems is rapid sedimentation and consequent organics degradation providing a clarified effluent. This type of deep pond system also ensures relatively high microorganism removal, as microbes are mostly attached to the solids that fall off into the bottom, and the freely suspended microbes are subsequently turned over into algal communities with decrease in the organic nutrients and predation. The anaerobic pond systems have been usually designed at the start of the ponding treatments unit to capture solids and effectively clarify the water for further treatments (Mahapatra and Ramachandra, 2013; Martinez et al., 2014).

Facultative Ponds Facultative system usually provides all dynamic environments such as (a) an aerobic environment in the top predominated by algal communities heterotrophic bacteria; (b) an anaerobic environment at the pond bottom and (c) the middle depth regions that has transient aerobic to anoxic nature, which allows both aerobic and anaerobic bacteria to thrive (Veeresh et al., 2010, 2010). The facultative pond systems are usually a part of the

Handbook of Algal Technologies and Phytochemicals

treatment chain initiated with an aerobic pond and ending up with polishing ponds at the downstream regions. The organic loads handled by these systems are typically around 10–40 g BOD/m2/day with BOD removal efficiency of 95%. Such pond systems usually have a relatively high hydraulic retention period (7–14 days) with a depth profile 1–2 m and are substantially treated by the characteristic algal communities such as flagellated motile algae as Chlamydomonas, Euglena, etc. (Mahapatra et al., 2013b).

Maturation/Polishing Ponds Maturation ponds are close to facultative ponds, which depend on algae as the main treatment module and remove low BOD and target removal of pathogens, fecal coliforms and nutrients (Kayombo et al., 2010). Such ponds are shallower with a depth of 1–1.5 m and are mostly aerobic in nature at the surface with accumulation of matured sludge. The pond system is either managed by a single unit in case of a low strength wastewater or by a series or combination of such ponding units. Successful cases are treatment plants in Mysore (Mahapatra et al., 2013b) with the pond system augmented by extended aeration and parallel systems. The pond system in series is ideal during summer (low organic loadings) (USEPA, 2011). However parallel systems can be operated during the winter, to maintain a higher biological activity (Mara and Pearson, 1998). The pond characteristics (% removal efficiency, land requirement and costing) are shown in Table 3.1, and effluents from the ponds are depicted in Figure 3.1.

POND ARCHITECTURE AND DESIGN Ponds function as completely mixed reactors and/or as plug flow reactors. However, the mass transport is sometimes more critical and often decides the fate of treatment through flow characteristics such as shortcircuiting and formation of stationary stagnant zones. The major mass transport mechanisms in these pond systems are a) diffusion, b) advection, c) sedimentation and d) interception. The relative effectiveness of the mechanisms with variability in all these processes are based on organic loads and pond design and architecture (Pena and Mara, 2003).

Areal Loading Rate Model The areal loading rates are the most crucial, designed to match up with organic loadings for optimal treatment and are governed by a number of factors such as a) type

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Natural Algal Photobioreactors for Sustainable Wastewater Treatment

TABLE 3.1 Pond Characteristics (Adapted From Sperling, 2007) Pond Characteristics Generic Parameters

Specific Parameters

Removal Efficiency (%)

BOD COD SS Ammonia Nitrogen Phosphorus Coliforms Area (m2/inhab.) Power (W/inhab.) Construction O&M

Requirements Costs (USD/inhab)

Facultative

Anaerobic– Facultative

Facultative Aerated

Compl.-mix Aerated– sedim.

75–85 65–80 70–80 < 50 < 60 < 35 90–99 2.0–4.0 ≈0 15–30 0.8–1.5

75–85 65–80 70–80 < 50 < 60 < 35 90–99 1.2–3.0 ≈0 12–30 0.8–1.5

75–85 65–80 70–80 < 30 < 30 < 35 90–99 0.25–0.5 1.2–2.0 20–35 2.0–3.5

75–85 65–80 80–87 < 30 < 30 < 35 90–99 0.2–0.4 1.8–2.5 20–35 2.0–3.5

Anaerobic – Facultative– Maturation 80–85 70–83 73–83 50–65 50–65 > 50 99.9–99.9999 3.0–5.0 ≈0 20–40 1.0–2.0

FIGURE 3.1  Images indicating (a) anaerobic environment, (b) facultative zone (high euglenoids growth and (c) maturation ponds observed in algae-based treatment ponds.

and nature of organic components in the wastewater, b) volumetric loading, c) dissolved oxygen availability, d) areal BOD loading and e) capability of treatment platform (algal communities) (USEPA, 1983). However, the environmental and microclimatic conditions decide the BOD loading, being a function of microbial metabolism and temperature. At subzero temperatures in temperate cold belts at BOD loading of 11–25 g/m2/day the treatment requires a residence time of four to eight months (Gloyna, 1971; USEPA, 2011). Whereas in the tropics a higher BOD loading can be handled. Thus, areal loading rate directly depends on microclimatic conditions and thus requires additional understanding of the reaction mechanisms. In this context, various models are discussed in the subsequent sections.

Gloyna and Hermann Model To address the inaccuracies and limitations of the areal loading rates, Hermann and Gloyna have proposed a

model that estimates the reactor, i.e. pond, volume that continues to maintain a high BOD removal (η = 0.9) even at low temperature regimes (Marais, 1966). The volume of the reactor is estimated graphically when the minimum temperature and the loading factor (in g per capita per day) are known. This establishes a model that estimates the reactor volume and is provided in Equation 3.1.

(

)

V = 3.5 × 10 −5 QLa θ(35−T ) αf (3.1)

where, V = pond volume, (m3); Q = influent flow rate (l/d); La = ultimate BOD or COD, mg/L; θ = the temperature coefficient; T = pond temperature, (°C); α = algal toxicity factor (1: domestic and industrial wastes); f = sulfide oxygen demand (1 when SO4 concentration < 500 mg/L).

28

Handbook of Algal Technologies and Phytochemicals

The equation provides a satisfactory estimate to compute the pond volume. However, the model is not applicable to all ponds, due its inaccuracies in predicting the pond depths and BOD removal efficiencies. The use of such models results in the treatment chains design with a number of ponds with smaller geometry connected in series (Finney and Middlebrooks, 1980).

First Order Kinetics—Plug Flow Model Along with the BOD removal efficiency, the reaction rate is very crucial in design of ponds. This model takes into consideration both BOD and the reaction rate (K′), where K′ is calculated from the BOD loading rate and temperature conditions in the pond. Figure 3.2 depicts BOD loading rate as a function of reaction rate. This model can be used to predict the effluent influent BOD concentration at 20°C and is provided in Equation 3.2. Ce = Co e − k ′t (3.2)



where, Ce = effluent BOD (mg/L); Co = influent BOD (mg/L); K′ = first-order reaction rate in plug flow (/d); t = hydraulic resident time in each pond, (d).

kT = k20 ′ (1.09)T − 20 (3.3)

where, reaction rate at minimum operating water kT =  temperature (/day); k′20 = reaction rate at 20°C (/day); T = minimum operating water temperature (°C).

BOD Loading Rate (kg/Ha/d)

This model considers both the first order kinetics and the completely mixed flow reactor conditions and thus has been used for designing the aerobic ponds (USEPA, 2011). Such a model applies to completely mixed systems, operational under equilibrium conditions, where there is slight difference in the influent and effluent concentrations. Moreover, the applicability of this model considers the efficiency of system increases with a series of ponds and where BOD does not settle as sludge (Marais, 1966; Finney and Middlebrooks, 1980). Ce = Co



1 (3.4) (1 + kc t n )n

where, Ce = effluent BOD (mg/L); Co = influent BOD (mg/L); kc = first-order reaction rate for complete-mix flow, (/day); tn = HRT in each cell (day); n = number of equal-sized pond cells in series. The temperature adjustments are provided by the following equation.

The plug flow model can be used and applied to other temperatures by applying Equation 3.3.

Marais and Shaw: Complete Mix Model

kT = k35 ′ (1.085)T −35 (3.5)



where, kT = reaction rate at minimum operating water temperature (/day); k′35 = reaction rate at 35°C (/day) = 1.2/d; T = minimum operating water temperature (°C).

Wehner–Wilhelm Model and Thrimurthi Application This model complements Marais and Shaw’s model and adds in the component of reaction kinetics (Thirumurthi, 1974; Finney and Middlebrooks, 1980).

120 100



80 60 40 20 0 0

0.03

0.06 0.09 k' (/d)

0.12

0.15

FIGURE 3.2  BOD loading rate as a function of K′ at 20°C (USEPA, 1983).

Ce = Co

4ae1/ 2 d (3.6) (1 + a)2 e a / 2 d − (1 − a)2 e − a / 2 d

where, Ce = effluent BOD (mg/L); C0 = influent BOD (mg/L); a = √ (1 + 4ktd ) ; d = Dt/L2; k = first order BOD removal coefficient, (/day); t = mean retention time, (days); d = dimensionless dispersion number; D = axial dispersion coefficient, (ft2/h).

Natural Algal Photobioreactors for Sustainable Wastewater Treatment

The temperature adjustments of parameters are given by the equation:

kT = k20 ′ (1.09)T − 20 (3.7)

where, kT = reaction rate at minimum operating water temperature (/day); k′20 = reaction rate at 20°C (/day) = 0.15/d; T = minimum operating water temperature (°C). In case of anaerobic ponds, the key parameter considered for the designs is the volumetric loading rate. Where the treatment efficiency can be determined by the operational parameters, the major ones are water temperature and hydraulic retention time (HRT). Anaerobic ponds designed at lower depths (0.9 to 1.2 m) operating year-round with an aerial BOD loading ranging from 20 to 100 kg/ha/d showed an average removal efficiency of 70% (Parker, 1970). Similarly for ponds with larger depths, 2.5–3 m, studies conducted by Eckenfelder (1961), Oswald et al. (1967), Cooper (1968) and Malina and Rios (1976) also showed removal efficiencies from 60 to 70% with a relatively high BOD areal loading rate, with retention times varying from 5 to 50 days (USEPA, 2011).

WASTEWATER CHARACTERISTICS, ALGAL PONDS, NUTRIENTS, POLLUTANTS AND PATHOGEN REMOVAL Wastewater comprises of organic matter (degraded/ partially degraded/inert), particulate/dissolved nutrients/minerals, heavy metals, xenobiotic compounds, pharmaceuticals and emerging contaminants. Various strategies have been employed for removal of nutrients through application of pond systems. For nitrogen (N), the main pond process involved is ammonia volatilization (Kayombo et al., 2010) that often occurs when there is a significant increase in the pond pH due to high photosynthesis. The other processes for N removal comprise of sludge sedimentation, nitrification, de-nitrification, ANAMMOX, etc. (USEPA, 2011; Mahapatra et al., 2013b). The algal ponds have been very efficient in the removal of N as observed in our earlier studies (Mahapatra et al., 2013b; Ramachandra et al., 2016, 2017a, b, c, 2018). Various design configurations have also aided in rapid nutrient removal as in case of the combined pond systems with wetlands that are usually called hybrid systems (Yeh et al., 2010; Ramachandra et al., 2016, 2017a, b, c, 2018). The high rate advanced algal facultative pond systems have also shown higher

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nutrient removal rates at relatively low HRT (Nurdogan and Oswald, 1995; Veenestra et al., 1995; Veeresh et al., 2010; Craggs et al., 2012). One of the major challenges in the treatment of wastewater is the heavy metals that have very dangerous impacts both on the environment and to human health (Ogunfowokan et al., 2008). Pond systems largely benefit in reducing the concentrations of heavy metals such as Cu, Cr and Ni from pulp and paper mill effluent (Achoka, 2002); Zn and Fe (Batty et al., 2008); Co and Cr (IV) from textile mill effluent (Mona et al., 2011); Pb from industrial waste (Banerjee and Sarker, 1997) and Al and Ni from acid mine drainage (Kalin and Chaves, 2003). Ponds have been also responsible of higher organic matter removal especially through the initial anaerobic zone or anaerobic pond systems. This has been also accomplished in facultative pond systems where the organic matter is removed in two stages, i.e. a first stage where the organic matter is broken down to soluble organics, carbon dioxide and dissolved mineral nutrients by the bacteria action, and during the second stage the dissolved organics, carbon dioxide and nutrients are taken up by the algal communities. This produces oxygen that is again taken up by the heterotrophic bacteria for decomposition of organic matter, and this cycle repeats. Whereas under high organic loading, in the anaerobic stage, hydrolysis, acidogenesis, acetogenesis and methanogenesis takes place, which converts the bulk of the organic matter to methane, carbon dioxide and water (Chanakya et al., 2012). Just after such processes, the predominance of heterotrophic bacteria and algae increases due to increased mineralization and availability of dissolved soluble nutrients. Various studies have reported treatment of poly aromatics such as PCBs and dissolved organic matter through pond processes (Musikavong and Wattanachira, 2007; Badawy et al., 2010). The pond systems have also demonstrated their utility in treatment of other xenobiotic compounds such as pesticides (Ahmad et al., 2004), pharmaceuticals (Spongberg et al., 2011) and emerging pollutants such as hormones like estrogen (Gomez et al., 2007). The wastewater also consists of a variety of pathogens comprising of coliforms, protozoans, helminths and phages that can cause numerous diseases and ailments, e.g., dysentery, gastroenteritis, cholera, typhoid, hepatitis (Parker, 1970; Mara and Pearson, 1998; Senderovich et al., 2008). The algal pond systems have been known for their effectiveness in the treatment of pathogens especially through the interplay between key parameters such as pH, light intensity, temperature and dissolved oxygen (Mara and Pearson, 1998). In the majority of pond systems, the sunlight inactivates pathogens such as E. coli, Cryptosporidium, Campylobacter, Salmonella,

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Ascaris eggs and phages (Gloyna, 1971; Gopo et al., 1997; Taner and Sukias, 2003; Sinton et al., 2007; Da Silva et al., 2008; Reino and Becares, 2008; Jamwal et al., 2009; Maiga et al., 2009).

ALGAE’S ROLE IN BIOREMEDIATION AND POTENTIAL SCOPE FOR A BIOREFINERY Algae are the major drivers for the effectiveness of pond processes, being potential assimilators of N, P and inorganic C forms such as CO2. Algal communities uptake these nutrients and temporarily immobilize them, thus aiding in the nutrient removal and recovery. Simultaneously, these algal communities produce oxygen through photosynthesis which is essential for heterotrophic bacteria for the degradation of organic matter (Mara, 2012). In a natural setup, the pond algal microflora primarily comprises of green algae such as Chlamydomonas, Chlorella and Euglenoides (Mahapatra, 2013b). Various studies with controlled setups have showed the effectiveness of Scenendesmus sp. (Garcia et al., 2000), Chlorella sp. and Oscillatoria sp. (Tharavathi and Hosetti, 2003). The algae-based facultative ponds have their own advantages in treatment and harbor beneficial motile flagellate algal communities, i.e., Euglena spp. and Chlamydomonas (Mahapatra, 2015) and non-motile algae such as Chlorella and Chlorococcum (Mahapatra et al., 2013b; Mahapatra and Ramachandra, 2013, 2014; Mahapatra et al., 2018). The typical architecture of facultative ponds and lagoons, especially in tropical waters, has multi-tier algal communities that help in efficient nutrient remediation and wastewater purification (Mahapatra et al., 2011a,b, c; Chanakya et al., 2012, 2013; Mahapatra, 2015; Mahapatra et al., 2017). The experiments conducted on algal cultivation with municipal wastewaters have demonstrated higher nutrient recovery and biomass productivity with high P concentrations in unialgal and polycultures especially with the mixotrophic community (Mahapatra et al., 2013a– f, 2014, 2017). Wastewater treatment systems involving facultative ponds have shown a higher techno-economic feasibility for algal single cell proteins (SCP), i.e., for euglenoids (Mahapatra et al., 2016) and as biofertilizer as Spirulina sp. (Mahapatra et al., 2018) with a lower environmental impact (Ramachandra et al., 2015a, b; Ramachandra and Mahapatra, 2015). A modular algal bioprocess (Figure 3.3) designed at the Indian Institute of Science (IISc), Bangalore, has been successfully working for nutrient capture and recovery at the Jakkur Lake in Bangalore City (Mahapatra, 2015; Ramachandra et al., 2018, 2014, 2016).

Handbook of Algal Technologies and Phytochemicals

According to studies conducted by Mara and Pearson (1998), algal members are from families such as Chlorophyceae (Chlamydomonas sp., Eudorina sp.); Cyanophyceae (Anabaena sp.); Euglenophyceae (Euglena spp., Phacus sp.); Bacillariophyceae (Navicula sp. and Cyclotella sp.). In spite of myriads of advantages like high productivity, short growth time, low land footprint and high value biomass production in algal pond systems, one of the major challenges is to promote those algal communities that have suitable properties of sedimentation/flocculation, which ensures a better-quality effluent with acceptable levels of suspended solids. Many studies have attempted to harvest the algal biomass from pond systems through flocculation or through neutralization of their negative surface charges through application of various chemicals such as alum/polymers (De-Bashan and Bashan, 2004). Such cell concentration strategies will ensure high harvestability and suitable bioproducts production during the wastewater treatment process that can be used suitably as biofuels in the form of lipids (Ramachandra et al., 2009; Craggs et al., 2011; Pitttman et al., 2011; Rawat et al., 2011; Chanakya et al., 2012); biomethanation to biogas (Shilton et al., 2008; Rawat et al., 2011; Mishra et al., 2017); biohydrogen production (Kumar et al., 2018; Prabakar et al., 2018) and in microbial fuel cells with present day innovation catalysis for improved electricity production (Kumar et al., 2018). A schematic algal biorefinery from ponds treating wastewater has been depicted in Figure 3.4. There are a number of biochemical (biomethanation) and thermochemical routes (liquefaction, pyrolysis, combustion and gasification) that can be used for processing algal biomass to energy rich products (Rawat et al., 2011).

ECONOMIC FEASIBILITY OF POND SYSTEMS Economic viability of treatment systems depends on the location and quantum aspects. Globally, there is great deal of difference in the human resource and commodity/infrastructure availability that sets the labor and the materials cost. The major cost components in the development of these pond systems are the infrastructure and the capital cost and operation and maintenance costs (O&M). The capital cost covers the land value, cost for acquiring the land with legal procedures, earth works, constructions, infrastructure and design/architecture cost (Mara et al., 1992; Mara, 2004), whereas the O&M costs involve the repair and maintenance with staff and recurring energy use costs. Analyses of the provisioning goods (fish, fodder, value added products from algae, etc.) from the pond ecosystem works out to be Rs (rupees) 10,500/hectare/day

Natural Algal Photobioreactors for Sustainable Wastewater Treatment

FIGURE 3.3  Integrated wetlands system for managing water and wastewater.

FIGURE 3.4  Pond systems to treat wastewater and algal bio-refinery.

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(Ramachandra et al., 2018). The cost incurred on the construction and O&M of such algal ponds can be recovered by harvesting and commercialization of the algal bioproducts and other goods (fish, fodder, etc.) from the pond system.

CONCLUSION Algal ponds offer a technically feasible, economically viable, socially acceptable and environmentally sustainable treatment option. They can be very efficiently used in small cities and communities mostly in tropical developing countries due to their simple design, extremely minimal maintenance and low cost. Such sustainable treatment practices using naturally predominant wastewater algae ensures higher availability of recyclable water and evades climate change with negligible GHG emissions and thus will ensure environmental sustainability. The present artificial lagoon system in various cities can be engineered and retrofitted with algal harvesting modules. Such augmentations will create an economically viable and technically feasible situation, which addresses nutrient remediation, wastewater treatment and bioproducts generation. Such systems that generate revenue (economically viable) are bound to sustain and be successful in the system. Integration of pond systems with the wastewater treatment processes on a larger scale requires understanding of the nutrient regimes and seasonal dynamics. A thorough understanding of the linkages of algal growth with wastewater strength and characteristics from different sources with various growth platforms, algal assemblages, at various microclimatic variabilities, nutrient uptake and mobilization trends and biochemical composition is vital for a complete wastewater treatment and subsequent harvest and utilization of biomass for diverse applications following the biorefinery approach. More studies on natural algal assemblages with heterotrophic–autotrophic interactions and their community characterization in dynamic environments will eventually lead to the novel design with the optimal wastewater treatment processes.

ACKNOWLEDGMENT We thank (i) the Science and Education Research Board (SERB), IUSSTF INDO-US Postdoctoral Fellowship (2016–2018), Government of India; Department of Biotechnology (DBT); (ii) NRDMS Division Ministry of Science and Technology (DST); (iii) ENVIS Division, The Ministry of Environment, Forests and Climate Change (MoEFCC), Government of India; (iv) Indian Institute of Science and Biological; and (v) Department

Handbook of Algal Technologies and Phytochemicals

of Biological and Ecological Engineering, Oregon State University for the financial and infrastructural support.

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Section II Algal Biofuels

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Opportunities and Challenges in Seaweeds as Feed Stock for Biofuel Production Mohammad Javad Hessami, Ambati Ranga Rao, and Gokare A. Ravishankar

CONTENTS Abbreviations������������������������������������������������������������������������������������������������������������������������������������������������������������39 Introduction���������������������������������������������������������������������������������������������������������������������������������������������������������������39 Seaweed Cultivation��������������������������������������������������������������������������������������������������������������������������������������������������40 Challenges for Seaweed Cultivation�������������������������������������������������������������������������������������������������������������������������40 Bioethanol from Seaweed�����������������������������������������������������������������������������������������������������������������������������������������41 Challenges for Saccharification of Macroalgal Biomass������������������������������������������������������������������������������������������41 Bioethanol Production from Seaweed Biomass��������������������������������������������������������������������������������������������������������43 Challenges of Fermenting Seaweed’s Sugars�����������������������������������������������������������������������������������������������������������44 Opportunities of the Macroalgal Biofuel������������������������������������������������������������������������������������������������������������������45 Conclusion����������������������������������������������������������������������������������������������������������������������������������������������������������������46 Acknowledgments�����������������������������������������������������������������������������������������������������������������������������������������������������46 Bibliography��������������������������������������������������������������������������������������������������������������������������������������������������������������46

ABBREVIATIONS GHG CO2 FAO MT FW Mwh Bpd DW MM3 RS FPU CBU

Greenhouse gases Carbon-dioxide Food and agriculture organization Metric tons Fresh weight Megawatt hour Barrels per day Dry weight Million cubic meters Reducing sugar Filter paper unit Cellobiase units

INTRODUCTION Woody biomass is considered as energy dense when compared to herbaceous ones, however a lot of efforts have also gone in to find the utility of non-woody biomass as a source of bioenergy (Lu et al. 2015). However, the fact of the lower growth rate of woody biomass compared to that of herbaceous plants is another influencing factor for use as feed stock. Climate change, shortage of freshwater, raising the global temperature and increasing the desertification

process have drawn attention to seaweeds as a source of sustainable biomass for various applications including that of biofuels. One advantage of seaweeds over landbased biomass is that they possess higher photon conversion efficiency (6–8% more photosynthetic activity than terrestrial plants), and they synthesize and accumulate massive amounts of carbohydrate from very abundant and cheap substrates (Gao and McKinley 1994, Aresta et al. 2005). Kelp forests that include Laminaria spp., which grows in sub-tidal areas, are amongst the most productive communities on earth, generating large amounts of organic carbon. In Nova Scotia, it is reported that kelp forests produce 1.75 kg organic carbon m−2 year−1; however, an average of 1.0 kg organic carbon m−2 year−1 is more typical of kelp beds (Bjerregaard et al. 2016), compared to the optimum yield for global net primary productivity of land-based crops of 0.47 kg organic carbon m−2 year−1(Hughes et al. 2012). Seaweed can be also be cultivated in non-productive land that is unsuitable for agriculture or in brackish, saline and wastewater that has few competing demands (Adams et al. 2009). Also, little impact is expected on the production of food and other products derived from terrestrial crops, unlike the use of corn or sugar-cane for bioenergy needs (Searchinger et al. 2008, Hughes et al. 2012). Another positive aspect of seaweed cultivation for biofuel is potential to reduce 39

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the generation of greenhouse gases (GHG) and to recycle CO2 emissions from flue gases from power plants and natural gas operations as indicated by preliminary life cycle assessments (Darzins et al. 2010). Terrestrial plants produce lignin, a complex polymer to provide strength and to protect plants of pathogen attack, but marine plants generally contain very low levels of lignin (Gressel 2008), and this advantageous feature can ease the process of carbohydrate conversion into reducing sugars. Another issue with first generation biofuel is its effect on global warming, where it is claimed that the final result of using land crop-based bioethanol, instead of producing a 20% saving, nearly doubles greenhouse emissions over 30 years and increases greenhouse gasses for the next 167 years (Searchinger et al. 2008). It is also pointed out that biofuels from switchgrass, if grown on US corn lands, increase emissions by 50%. This raises concerns about large corn-based biofuel and highlights the value of utilizing other sources to produce biofuels. Therefore, producing bioethanol from marine algae has recently attracted more attention. Considering advantages of seaweeds over land crop biomass, the opportunities and challenges of seaweeds as biofuels could be investigated (Hessami et al. 2018a). Though macroalgal biomass are basically suitable for wet biofuel conversion types, namely anaerobic digestion and alcoholic fermentation. In this chapter, we have focused on bioethanol production from seaweeds, its current scenario and future challenges and opportunities.

SEAWEED CULTIVATION The main concept of cultivation of seaweed is the open ocean farm using a framework structure that fixed to depth of open ocean to cultivate kelp (Macrocystis pyrifera) for fuel production (Bender 1980). In general, the method of seaweed faming depends on seaweed species with specific emphasis on species growth, reproduction and development. For example, free-floating species such as Ulvae sp. can be cultivated in ponds; a similar method is used for microalgal production, while the other seaweed species may need ropes, seabed or similar structures to attach on (Buschmann et al. 2005). But the land-based cultivation, harvesting, near shore and offshore cultivation are the four general approaches of seaweed cultivation. Offshore cultivation is under development and seaweed harvesting from the ocean provides as little as 6% of the whole seaweed market share. The highest share is generated by near shore cultivation (Burton et al. 2009, Buschmann et al. 2017). The landbased cultivation approach is applied for small scale production for specific markets. These methods have a

Handbook of Algal Technologies and Phytochemicals

variety of designs and constructions. Hence, mass cultivation of seaweed is conducted in different methods including seabeds (Gracilaria spp.), using nets (Porphyra spp., Gracilaria spp., Cladosiphon okamuranus, Ulva spp.) and the most common type which is on lines and ropes (S. japonica, Kappaphycus spp., Eucheuma spp., Hypnea musciformis and Gigartina atropurpurea). Nowadays, seaweeds are mass-cultivated in Asia. According to recent studies there is high potential for seaweed mass-production in Atlantic waters such as Ireland, France, Spain, Germany, UK and Canada (Black 1950, Kraan 2013, Buschmann et al. 2017). According to latest statistics, the global total seaweed yield is 30.14 MT (fresh weight), wherein both China (47%) and Indonesia (38.7%) are the dominant seaweeds producers, globally. Saccharina japonica (8.2 MT), Undaria pinnatifida (2 MT) and Pyropia spp (1.35 MT) belong to brown seaweeds that are mainly farmed for human food consumption. The red seaweed species are mainly cultivated for non-food purposes where Gracilaria spp. (4.1 MT) is cultivated for agar production, Kappaphycus alvarezii (1.5 MT) and the most important commercial seaweed species, Eucheuma spp. (10.5 MT), for the carrageenan industry (FAO 2016). In general, due to market demand, the production of seaweeds is increasing, however innovations in its farming, harvesting and processing are expected to maximize the productivity and production.

CHALLENGES FOR SEAWEED CULTIVATION In case of mass-scale seaweed cultivation activities, various issues will emerge, including technical problems, environmental concerns and those that must be managed by alteration of routine activities. Growth of epiphytes, damage to grazers and disease, possible damage to natural marine environment, occupation of vast marine area and disruption to marine transportation and fishing activities are among the technical and social issues that have been studied widely, but one of biggest issues that may occur, once considering seaweed biomass as biofuel feedstock, is the huge required volume of biomass and subsequently the huge marine environment that must be allocated. It is assumed that if a country needs to provide a meaningful contribution to energy requirements, a country-scale activity is demanded, in which fundamental alteration will be required in societal attitudes to utilize the marine environment as well as regulatory changes in many sections. On the other hand, currently the inshore waters are highly under pressure, so for mass-cultivation of seaweeds at biofuel scale, very large amounts of continental waters should be used. To comprehend the vast size of offshore seas, it helps to mention that currently, 0.04%

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Opportunities and Challenges in Seaweeds as Feed Stock 

of these waters are using for aquaculture (Duarte et al. 2009). To estimate the capacity of biofuel (bioethanol) production from macroalgal biomass, the Republic of China has undertaken a vast study. Based on the latest statistics, China is the biggest fuel importer (11.6 million bpd) and also the main seaweed producer (47.9% of total global seaweed production) (FAO 2018). Under the most optimistic condition, the maximum bioethanol production from seaweeds is calculated (Figure 4.1.), where it is shown that maximum annual bioethanol production in China would cover only 0.23% of its fuel demand. What if we are targeting to supply 50% (62.5 × 1012 Mwh) of annual global energy use? In that case, considering the macroalgal average productivity at 1,000 dry tons/km2 (Sze 1993, Bjerregaard et al. 2016), the required marine environment to cultivate seaweeds would be 25 million km2, which is equal to 1.5% of the ocean surface area. The predicted annual seaweed biomass yield in that case would be 25 billion dry tons by which 2.5 billion tons of protein and 0.75 billion tons of algal oil would be produced (Bjerregaard et al. 2016). Lack of more mechanized technologies for offshore farming might be another challenge (Hughes et al. 2012) for biofuel-scale seaweed production. Another concern regarding mass-cultivation of seaweeds is halocarbon emission by seaweeds, however the released amount is far lower than natural halocarbon emission by the ocean itself (Nightingale et al. 1995, Keng et al. 2013). Eventually, with respect to the fact that carbohydrate is the main constituent of seaweeds biomass and they are highly dependent on atmospheric CO2 (Moreira and Pires 2016), there is uncertainty whether natural uptake and distribution of CO2 in deep waters would meet the demand in dense seaweed farms or if artificial techniques must be applied.

BIOETHANOL FROM SEAWEED Currently, bioethanol derived from sugarcane in Brazil is the only economically feasible biofuel that shows a significant net energy gain (Gressel 2008, Walker 2010, Turetta et al. 2017). Seaweeds (green, red and brown seaweeds) produce different types of carbohydrates leading to different degrees of saccharification process, which has a diverse influence on the fermentation method and yield, as well. Prior to fermentation, the biomass must be harvested and processed to ensure the quality of the carbohydrate has not been reduced. Thus, the algal biomass has to undergo a series of processes including saccharification, detoxification, fermentation, ethanol recovery and waste management.

CHALLENGES FOR SACCHARIFICATION OF MACROALGAL BIOMASS The carbohydrates need to undergo a process called saccharification by which the carbohydrate polymers cleave into the constituent monomers. Cellulose breaks to glucose, hemicellulose gives some different hexoses and pentose sugars such as xylose, arabinose and glucose. Various methods have been reported while the most common approaches are grouped into enzymatic and chemical hydrolysis (Taherzadeh and Karimi 2007a). In addition, there are other hydrolytical methods in which no chemicals or enzymes are applied. For instance, lignocelluloses may be hydrolyzed by gamma-ray or electronbeam irradiation or microwave irradiation. However, these processes are far from being commercially applied (Saini et al. 2015). Other saccharification approaches beside enzymatic or chemical treatments include electron-beam irradiation and gamma-ray microwave, which

FIGURE 4.1  The estimated bioethanol production from current native seaweed production in China. Source: www.ecns.cn/ business/2018/02-07/291780.shtml. MT, million tons, MM3, MM3, million cubic meters; FW, Fresh weight; DW, dry weight.

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Handbook of Algal Technologies and Phytochemicals

still require further development for commercial application (Taherzadeh 1999). Carbohydrates from seaweed are very different from land-crops and they also vary based on the seasonal changes and species (Kim et al. 2015). Through a survey of tropical seaweeds in Malaysia, Hessami (2017) reported that total carbohydrate content varied from 12.16 ± 2.11% to 71.22 ± 0.71% for Sargassum binderi and K. alvarezii, respectively, while even an higher amount (78.3 ± 11.5%) was reported in Papua, Indonesia (Meinita et al. 2012a). In addition, they contain very low amounts of lignin and hemicellulose; thus it is more amenable for enzymatic conversion to reducing sugars (Gressel 2008). Seaweeds contain unique carbohydrate composition: besides starch, cellulose, agar, carrageenan and alginate, they may also contain mannitol and laminarin, making them distinctively different from terrestrial biomass. Thus, it is important to apply appropriate methods to seaweed biomass and to select appropriate microorganisms that are pivotal for successful bioethanol fermentation (Tan and Lee 2014). Acid hydrolysis is the most common type of saccharification and is carried out by two methods, concentrated acid hydrolysis and dilute-acid hydrolysis (Taherzadeh and Karimi 2007a). The former type is conducted with a high concentration of acid (30–70%) and at a low temperature (30–40°C) with a very high yield of glucose production (90% of theoretical) (Taherzadeh and Karimi 2007a). Besides the high yield of this method, use of this method might be extremely dangerous due to a corrosive attribute of concentrated acid especially once temperature increases; it is also expensive as specialized acid resistant material must be used in reactors

with a high level of safety. Another hydrolysis type using diluted acids is more common in comparison with concentrated acid hydrolysis and can be used either as a pre-treatment or as the actual method of hydrolyzing biomass to fermentable sugars (Qureshi and Manderson 1995). Single stage hydrolysis in batch reactors has been widely applied for the kinetic study of the hydrolysis of biomass to ethanol production in pilot or laboratory scales (Taherzadeh and Karimi 2007a). The main drawback of single stage hydrolysis is degradation of parts of sugar that release from less resistant polymers into fermentation toxins (Larsson et al. 1999). It is recommended that dilute-acid hydrolysis is conducted in more than one stage to avoid degradation of sugars. A comparison of macroalgal saccharification yield using sulfuric acid is illustrated in Table 4.1. It is shown that the conditions for chemical treatment for converting macroalgal carbohydrates are highly diverse and might be dependent on substrates or the strategies that have been chosen by researchers. Regardless, the fact is that dilute acid hydrolysis is a common method applied to hydrolyze seaweed biomass, but this method has its drawbacks including degradation of sugar to fermentation inhibitors. In that case, detoxification using a different method such as over liming might be applied, by which along with toxins, some parts of reducing sugar contents would be discarded, whereas sugar loss of 12% is reported in detoxification of K. alvarezii hydrolysate (Hessami et al. 2018a). Hence, a safer method for feedstock hydrolysis is the enzymatic procedure. Enzymes are naturally found in certain plants and microorganisms which cause a chemical reaction to breakdown polymers. Cellulose as the most abundant polymer in

TABLE 4.1 Comparison of Chemical Saccharification Using Sulfuric Acid from Different Seaweed Species Seaweed spp. Laminaria hyperborea Undaria pinnatifida Saccharina japonica Gelidium amansii Gelidium amansii Kappaphycus alvarezii Palmaria palmata Kappaphycus alvarezii Gracilaria corticata Gracilaria manilaensis Kappaphycus alvarezii

Treatment Condition Acid Conc. /Time/ Temp

RS Conc (g/L)

RS Yield (g/g)

pH=2/60 min/65°C 0.7%/60 min/121°C 0.4% and Saccharification with Bacillus sp. 2.5%/150°C 1%/60 min/121°C 2%/15 min/130°C 4%/25 min/125°C 1%/5 min/140°C 1%/15 min/120°C 2.5%/45 min/120°C 2.5%/45 min/120°C

20 28.65 45.6 NA 43.5 4.4 NA 38.3 NA 55.77 61.28

NA NA NA 0.42 NA NA 0.16 0.31 0.13 0.34 0.29

Conc: concentration; temp: temperature; RS: reducing sugar; NA, not available.

Reference Horn et al. (2000) Cho et al. (2013) Jang et al. (2012) Park et al. (2012) Ra et al. (2013b) Meinita et al. (2012a) Mutripah et al. (2014) Ra et al. (2016) Sudhakar et al. (2016) Hessami et al. (2018a) Hessami et al. (2018a)

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Opportunities and Challenges in Seaweeds as Feed Stock 

the plant can be degraded to its monomer by the enzyme cellulase. To conduct enzymatic hydrolysis, the enzymes must obtain access to the molecules to be hydrolyzed and the crystalline structure of cellulose must be reduced to increase the access of enzyme to molecules. To obtain this condition, some kind of physical or chemical pretreatment process is applied (Badger 2002). Cellulase enzymes are highly specific catalysts which act under mild conditions (pH = 4.5–5.0 and temperature at 40 to 50°C). This allows for low corrosion of equipment, low energy consumption and also the low toxicity of the hydrolysates (Taherzadeh and Karimi 2007b). This process is performed by the synergistic action of at least three major classes of enzymes: endo-glucanases, exoglucanases and ß-glucosidases. These enzymes together are usually called cellulase or cellulolytic enzymes, and the result of cooperation of these enzymes is generating glucose from cellulose (Wyman 1996). This enzyme has been widely used in macroalgal saccharification studies, and a high cellulase efficiency (87%) is reported in saccharification of the red seaweed, Gracilaria verrucosa (Kumar et al. 2013). Seaweeds have different polysaccharides rather than cellulose and hemicellulose that are common in terrestrial crops. Hemicellulose is only found in some green seaweeds, Ulva (Ye et al. 2010) and Enteromorpha (Ray 2006), but unique polysaccharides such as carrageenan, alginate, agar, etc., are found in seaweeds (Barsanti and Gualtieri 2005, Michel et al. 2006). Therefore, special enzymes are required for seaweed enzymatic hydrolysis. Agar is a valuable phycocolloid extracted from the cell walls of the red seaweeds. The main source of agar is Rhodophyceae, which includes Gelidium, Gracilaria and Porphyra spp. The first bacterium with an agarolytic enzyme was isolated from seawater (Michel et al. 2006). A few microorganisms were found in seawater, coastal marine sediments or water column and reported to have same attributes (Stanier 1942). Moreover, some marine microorganisms with the potential for agarose production have been discovered from Pseudomonas, Alteromonas, Pseudoalteromonas, Vibrio, Alterococcus, Microbulbifer, Agarivorans, Thalassomonas and Saccharophagus (Michel et al. 2006). Carrageenan is another specific compound which is extracted from some species of the class Rhodophyceae, mainly Chondrus, Gigartina, Kappaphycus and Eucheuma (Necas and Bartosikova 2013). The main blocks of carrageenan are of D-galactose and 3,6-anhydro-galactose which are joined by α1→3 and β1→4 linkage. In comparison to agar-degrading bacteria, very few microorganisms have been reported to have hydrolytic activity to digest carrageenan. All these

bacteria were isolated in the marine environment and belong to the gamma proteobacteria, Flavobacteria or Sphingobacteria classes. Alginate was described as being composed of different blocks of G, M and MG (Aarstad et al. 2012) and is an unbranched polysaccharide polymer without repeating subunit structures and can be found widely in brown seaweeds and some bacteria including Azotobacter vinelandii and Pseudomonas aeruginosa (Hansen et al. 1984). Numerous bacteria are capable of producing alginase, but, unlike carrageenase, the majority of them are marine bacteria which are active in algal decomposing residues (von Riesen 1980). Among all hydrolytic enzymes, commercial production of amylase is widely developed, and also great investment in production of low-cost cellulase has been made, and currently, it is claimed that the company Novozymes is on the edge of low-cost production of cellulase for biofuel process. A comparison of saccharification process and reducing sugars yield is presented in Table 4.2. Seaweeds are rich in jell-like compounds and this attribute introduced them as the most hygroscopic biomaterials. Seaweeds may contain over 90% moisture, and this means that one unit of dry seaweed biomass may keep its solid state even after the addition of nine units of the water. However, in the process of saccharification, obtaining concentrated slurry of seaweed biomass is not easily feasible, and consequently, reduced sugar may very rarely reach 7% concentration (Table 4.2). Subsequently bioethanol content can hardly meet industrial requirements, 4–5% (v/v) (Fan et al. 2003, Lu et al. 2010).

BIOETHANOL PRODUCTION FROM SEAWEED BIOMASS Bioethanol can be extracted from a variety of feed stocks that possess fermentable sugars generally in a mixture of polysaccharides and free sugars. The microorganisms used for ethanoic production are divided into three categories which are mold, bacteria and yeast (Naik et al. 2010). The different sugar composition of seaweeds causes difficulty in the fermentation process by using one or a few strains of microbes in fermentation. The seaweed biomass must be ground at the first stage to small pieces and then transferred to saccharification. The saccharified solution can be concentrated by evaporation if low sugar content was obtained. The hydrolysate is then transferred to the fermentation reactors to produce ethanol. The fermented product is distilled and dehydrated to achieve a concentration of 99.9% (v/v) which is needed for fuel quality specifications. Also, the residues of fermentation can be utilized to produce heat and electricity (Roesijad et al. 2010).

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Handbook of Algal Technologies and Phytochemicals

TABLE 4.2 Comparison of Enzymatic Saccharification of Selected Seaweeds Seaweed spp.

Target Polymer

Ulva fasciata Ulva rigida

Cellulose Starch cellulose Cellulose, starch Cellulose, starch Starch Cellulose

Ulva pertusa Alaria crassifolia Saccharina japonica Nizimuddinia zanardini Laminaria japonica

Cellulose

Laminaria japonica, Caulerpa sp. Gracilaria salicornia

Alginate

Gelidium elegans Gracilaria verrucosa

Cellulose, starch Cellulose

Kappaphycus alvarezii Grascilaria manilaensis Kappaphycus alvarezii

Cellulose

Enzyme/Enzyme Conc.

RS (g/L)

Cellulase/2% (v/v) Amyloglucosidase α-amylase, cellulase Meicelase/5 g/L Meicelase/5 g/L Amylase 120 L Cellulase, β-glucosidase Cellobiase 55 CBU/g Cellulase 45 FPU/g Rapidase/ Viscozyme /dextrozyme Cellulase/0.5% (w/v)

Yield (g/g)

Reference

NA NA

0.2 0.19

Trivedi et al. (2013) Korzen et al. (2015)

43 67 20.6 NA

0.82 (glucan) 0.58 (glucan) 0.31 0.07

Yanagisawa et al. (2011) Yanagisawa et al. (2011) Jang et al. (2012) Yazdani et al. (2011)

34

0.24

Ge et al. (2011)

8.3

NA

Choi et al. (2009)

NA

Wang et al. (2011)

49 40

Cellulose Cellulose

Meicelase/5 (g/L) Cellulase/20 (FPU/g) β-glucosidase 60 (U/g) Cellulase 45 (FPU/g) Cellulase/10%

0.013 (wet biomass) 0.67 (glucan) 0.87 (cellulose)

90 105

0.76 (cellulose) 0.7

Hargreaves et al. (2013) Hessami et al. (2018b)

Cellulose

Cellulase/10%

120

0.76 (residues)

Hessami et al. (2018b)

Yanagisawa et al. (2011) Kumar et al. (2013)

Conc, concentration; Temp, temperature; NA, not available; RS, reducing sugar; FPU, filter paper unit; CBU, cellobiase units.

Seaweeds of Europe and East Asia have been much investigated for bioethanol production. In Europe brown seaweeds dominate in the cold climate, and in East and South-East Asia the red seaweeds are abundant. Among brown seaweed species, Laminaria spp. (Horn 2000, Horn et al. 2000, Cui et al. 2002, Lee and Lee 2010, Adams et al. 2011, Lee and Lee 2011, Tedesco et al. 2014), Undaria spp. (Cho et al. 2013), Saccharina spp. (Adams et al. 2009, Jang et al. 2012) are the most investigated seaweeds in bioethanol production, while in red seaweeds the most interest has been towards Kappaphycus spp. (Khambhaty et al. 2012, Meinita et al. 2012a, Hargreaves et al. 2013, Hessami et al. 2018a, Hessami et al. 2018b), Gelidium spp. (Wi et al. 2009, Jeong et al. 2011, Park et al. 2012, Meinita et al. 2013, Ra and Kim 2013, Cho and Kim 2014, Kim et al. 2015) and Gracilaria spp. (Amanullah et al. 2013, Kumar et al. 2013, Meinita et al. 2013, Ahmad 2014, Wu et al. 2014). It can be noticed that, among all types of seaweeds, red seaweeds are the most investigated in bioethanol production studies, where Kappaphycus spp., Euchemia spp., Gracilaria spp. and Gelidium spp. are dominant seaweed species in bioethanol studies. In general, two main

current methods of bioethanol production with these seaweed species are using seaweed’s whole plant and seaweed’s residues of carrageenan, agar or fiber industries. The seaweed hydrolysates are subjected to fermentation process with commercial fermentative yeasts (mostly S. cerevisiae), other specific yeasts (Pichia stipites, Kluyveromyces marxianus, Brettanomyces bruxellensis) (Lee and Lee 2012, Ra et al. 2013a, Moktaduzzaman et al. 2015, Takagi et al. 2015, Hessami 2017), isolated marine bacteria (Bacillus sp., Gracilibacillus sp.) (Tang et al. 2009, Jang et al. 2012) or genetically engineered microorganisms (Sphingomonas sp., E. coli) (Takeda et al. 2011, Wargacki et al. 2012). A summary of bioethanol production from some macro algal biomass is illustrated in Table 4.3.

CHALLENGES OF FERMENTING SEAWEED’S SUGARS Bioethanol from seaweed has its specific technical issues that have hindered it in commercial production. In seaweeds, specific carbohydrates and their corresponding reduced sugars are not suitable substrate for most

45

Opportunities and Challenges in Seaweeds as Feed Stock 

TABLE 4.3 Bioethanol Production from Macroalgal Biomass Seaweed spp.

Microorganism Used

Laminaria hyperborea Undaria pinnatifida Saccharina japonica

Pichia angophorae Pichia angophorae Pichia angophorae, Pichia stipites, S. cerevisiae, Pachysolen tannophilus S. cerevisiae Brettanomyces custersii Scheffersomyces stipitis S. cerevisiae S. cerevisiae Kluyveromyces marxianus S. cerevisiae S. cerevisiae S. cerevisiae

Saccharina latissima Gelidium amansii Gelidium amansii Kappaphycus alvarezii Palmaria palmata Kappaphycus alvarezii Gracilaria corticata Kappaphycus alvarezii Gracilaria manilaensis

EtOH Conc (%)

EtOH Yield (g/g RS)

Fermentation Yield (%)

NA 0.94 0.77

0.43 NA 0.33

84 27 NA

Horn et al. (2000) Cho et al. (2013) Jang et al. 201

0.45 2.7 2.0 0.16 NA 1.6 0.3 2.09 1.81

NA NA NA NA 0.012 0.42 0.10 0.34 0.32

NA 38 91 66 24 NA NA 71.4 67.9

Adams et al. 200 Park et al. (2012) Ra et al. (2013b) Meinita et al. (2012a) Mutripah et al. (2014) Ra et al. (2016) Sudhakar et al. (2016) Hessami et al. (2018a) Hessami et al. (2018a)

Reference

NA, not available; EtoH, ethanol, S. cerevisiae, Saccharomyces cerevisiae.

fermentative microorganisms. For instance, galactose, as one of the most abundant monosaccharides in red seaweeds, has a different metabolism pathway than glucose where it should pass Leloir pathway (Frey 1996) in order to become a fermentable sugar. For starters, D-galactose is transported into the cell by galactose permease (Gal2) (Tschopp et al. 1986); then it undergoes a series of changes through Leloir pathway to convert to D-glucose, in which high energy is utilized (Timson 2007). That is why the metabolism of galactose imposes more energy consumption to yeast and leads to lower fermentation yields. A fermentation efficiency of 0.18–0.29 g ethanol/g substrate has been reported for galactose (Bro et al. 2005, Meinita et al. 2012b), in comparison with the efficiency of 0.48g ethanol/g substrate for glucose. On the other hand, galactose metabolism is slower than glucose metabolism. Some research has been conducted to enhance microorganisms to adapt with galactose metabolism to obtain higher fermentation efficiency (Ostergaard et al. 2000, Bro et al. 2005, Hong et al. 2011, Lee et al. 2011). Macroalgal carbohydrates are sulfated in various degrees, and this specification is another bottleneck in fermentation of macroalgal sugars, because sulfate bonds can inhibit the metabolism. It is suggested that removing the sulphate bonds and acclimation of yeast or bacteria may improve the fermentation of such sugars (Cho and Kim 2014, Kim et al. 2014). Alginate is another important polysaccharide that mainly is found in brown seaweeds (20–40%), and mannans that can be present in lower amounts in brown seaweeds are facing

difficulty, and very low yield of fermentation efficiencies are recorded; however, ongoing studies on recombinant yeast or bacteria may facilitate the use of these polysaccharides in bioethanol production (Takeda et al. 2011). High salinity in marine biomass, specifically calcified seaweeds, is another issue that causes unpleasant effects on fermentation mainly with increasing osmosity in seaweed hydrolysate, as well as inducing sedimentation of added dissolved minerals and nutrient, based on personal experiences. Intensive washing of the seaweed biomass with fresh water which might remove salinity and isolating marine fermentative yeast which are adapted to high salinity might tackle this issue.

OPPORTUNITIES OF THE MACROALGAL BIOFUEL Presently, with available technologies it is obvious that the cultivation of algal biomass is unlikely to be economically viable, especially if the only outcome product is energy (Bruhn et al. 2011, Pittman et al. 2011) since the raw seaweed cost is not cheap; moreover, seaweeds’ products are among the most expensive feedstock in the agrimarket, i.e., agar wholesale price reported 17 USD/kg in 2015 (Table 4.4), and the wholesale price for some seaweeds species may rise to 3.5 USD/kg DW. Thus, it can be asserted that, as long as fossil fuels exist, cultivation of seaweeds for bioethanol as main product is not economically feasible; however, the seaweed industry’s (i.e., agar, carrageenan, alginate or even protein)

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Handbook of Algal Technologies and Phytochemicals

TABLE 4.4 Average Sales Volume and Prices for Main Macroalgal Products 2009 Product Name Agar Alginates Carrageenans

2015

Volume (tons)

Price (USD/kg)

Volume (tons)

9,600 26,500 50,000

18 12 10.5

14,500 24,644 57,500

Price (USD/kg) 17 14 NA

Source: Porse and Rudolph (2017); NA, not available.

by-products are cheap, environmentally safe and secure feedstocks for bioethanol plants. During an interesting study, using the brown seaweed Sargassum angustifolium, harvested from Persian Gulf, not only alginate as a valuable phycocolloids and bioethanol from solid residues were achieved, but also the residual nitrogen rich liquor consumed in replacement of “Yeast Extract”, a general nutrient that is in use to provide nitrogen in the fermentation process (Ardalan et al. 2018). In addition, taking into account mentioned biofuel-scale (25 million km2) cultivation of seaweeds, magnificent advantages such as supplying 2.5 × 109 tons of protein for human and animal feed can be achieved (Bjerregaard et al. 2016), which is nearly eight times higher than global protein demands by 2050 (Henchion et al. 2017). Moreover, being rich in nitrogen, phosphorus, potassium and trace elements, the organic residues of seaweed processing can be utilized as agricultural fertilizers; additionally, by the composting process, storage and distribution of seaweed waste will be possible. In accordance with that historical strategy, we strongly believe that if infrastructures are provided for mass-cultivation of seaweeds in mentioned biofuel-scale magnitude, bioethanol from seaweeds can be economically feasible.

CONCLUSION Finite fossil fuel resources and increasing global population and consequently extra energy demands in coming decades and environmental concerns left us no choice but exploring sustainable energy resources. In theory, one type of renewable energy is not expected to solve the energy crisis in near future, and also biomass never was the sole option, due to its hardship, but in respect to global warming and environmental concerns, achieving more CO2 sequestering approaches and establishing of environmentally friendly resources is a must. It is assumed that marine macroalgae is one of the promising candidates. Nonetheless, in respect to the available technology in seaweed processing, inadequate worldwide

attention towards macroalgal biofuel, limitations for developing mass-cultivation of seaweeds and infrastructural deficiencies it is believed that additional organized research is demanded in a few areas. Firstly, mass-cultivation techniques and harvesting of seaweeds needs to be improved, since the aquaculture encounters its specific limitations and deadlocks. Secondly, while dealing with massive biomass the post-harvest technology must be robust. A more efficient biofuel extraction technique is another area that needs to be optimized, and finally the environmental, social and economic convergence is highly required.

ACKNOWLEDGMENTS This work did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. Dr. ARR acknowledges Vignan’s Foundation for Science, Technology and Research University for providing the facility for this work.

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5

Biodiesel Production from Microalgal Biomass Challenges and Perspectives Srijoni Banerjee and Debabrata Das

CONTENTS Introduction���������������������������������������������������������������������������������������������������������������������������������������������������������������51 Microalgae for Biodiesel Production������������������������������������������������������������������������������������������������������������������������53 Microalgae Cultivation����������������������������������������������������������������������������������������������������������������������������������������������54 Open Cultivation System (Raceway Ponds)�������������������������������������������������������������������������������������������������������������54 Closed Cultivation Systems��������������������������������������������������������������������������������������������������������������������������������������54 Biosynthesis of Lipid/Triacylglycerol in Microalgae�����������������������������������������������������������������������������������������������55 Metabolic Engineering for the Enhancement of Lipid Production���������������������������������������������������������������������������55 Microalgal Harvesting����������������������������������������������������������������������������������������������������������������������������������������������55 Microalgal Lipid Extraction��������������������������������������������������������������������������������������������������������������������������������������56 Microalgal Lipid to Biodiesel�����������������������������������������������������������������������������������������������������������������������������������57 Scale-up Strategies for Biodiesel Production from Microalgae�������������������������������������������������������������������������������59 Challenges and Future Research Perspectives����������������������������������������������������������������������������������������������������������59 Conclusion����������������������������������������������������������������������������������������������������������������������������������������������������������������59 Acknowledgment������������������������������������������������������������������������������������������������������������������������������������������������������60 References�����������������������������������������������������������������������������������������������������������������������������������������������������������������60

BOX 5.1  SALIENT FEATURES The detrimental rise in the world’s energy demand, rapid exhaustion of fossil fuel reserves and environmental damages have necessitated the shift towards sustainable and renewable energy sources. Microalgal biodiesel has garnered considerable interest as a propitious energy alternative. Algae are the most efficient photosynthetic organisms on earth which makes them a prospective feedstock for different purposes. Microalgal biomass is considered as a third-generation feedstock for biofuel production. In the present era, several approaches are being adapted for the maximization of biodiesel production from microalgae. Increasing the CO2 fixation rate of microalgal strain and channeling the metabolic flux towards lipid production using a metabolic engineering approach is one of the major challenging arenas. Different scale-up strategies of microalgal biodiesel production using a suitably designed photobioreactor coupled with innovative

downstream processing strategies have provided needed impetus to the algal biodiesel technology. Extensive scientific and industrial research has been carried so far for suitable photobioreactor design for microalgal cultivation and different harvesting techniques. Recent advances on microalgal biodiesel production, harvesting and lipid extraction to biodiesel conversion procedures, integrating metabolic engineering approaches for the maximization of lipid content in microalgae, different scale-up strategies and challenges to achieve the commercializing of microalgal biodiesel production are discussed in detail in the present chapter.

INTRODUCTION In the present era, increasing consumption of fossil fuels as conventional energy has caused serious concerns about energy security and environmental degradation. The so-called conventional energy sources are

51

52

unsustainable and are the cause of global warming. To address these problems, it is essential to produce energy from sustainable and renewable energy sources. The associated issues are intimately connected with economic development and prosperity, quality of life, global stability and require from all stakeholders tough decisions and long term strategies. Biomass-based biofuel production is gaining importance day by day (Khan et al. 2009). However, the first- and second-generation biofuel feedstock mainly derived from edible food crops has culminated in a fuel vs food dilemma. In this scenario, microalgal biomass provides an ecofriendly approach to third-generation biofuel production (Mata et al. 2010). Microalgal species provide multiple advantages such as higher photosynthetic efficiency than higher plants; they require lesser area and minimal amount of nutrient for cultivation and can also grow in sea water, fresh water and wastewater (Cheng et al. 2006; Chisti 2007). Many algal species have high lipid content (Metzger and Largeau 2005) and thus could be ideal for oleo-fuel generation (Figure 5.1). In recent time, several approaches are being adapted for the maximization of biodiesel production from microalgae. Increasing the carbon fixation rate of microalgal strain and channeling the metabolic flux towards lipid production using metabolic engineering approach,

FIGURE 5.1  Different generation of biofuel feedstock.

Handbook of Algal Technologies and Phytochemicals

scale-up of microalgal biodiesel production using suitably designed photobioreactor coupled to innovative downstream processing strategies have provided needed impetus to the algal biodiesel technology. Extensive scientific and industrial efforts have been made for the development of cost effective and high efficiency cultivation systems. Microalgal biomass harvesting is one of the major steps in the scale-up process of microalgal biodiesel production. Improvement of the algal harvesting techniques is another major area to be considered for the microalgal biodiesel production. Algal biodiesel consists of monoalkyl esters that are derived from microalgal lipids by different techniques namely transesterification (using acid or alkali or nanoparticles as catalysts), hydrothermal liquefaction, pyrolysis, etc. (Roy and Das 2016). The transesterification process also needs to be addressed for biodiesel production process commercialization. The present chapter describes the recent advances on biodiesel production from microalgae adopting diverse methods of cultivation, harvesting and algal lipid extraction to biodiesel conversion techniques. Integration of metabolic engineering approaches to enhance the biodiesel production by microalgae, scale-up strategies and challenges to achieve commercial feasibility of micro algal biodiesel production is also reviewed.

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Biodiesel Production from Microalgal Biomass

MICROALGAE FOR BIODIESEL PRODUCTION Microalgae are mainly unicellular organisms which exist individually or in chains or groups. Microalgae can sequester CO2 from the atmosphere and produce 30 times more oil per unit area of land as compared to terrestrial oilseed (Mata et al. 2010). Some of the lipid rich microalgae, like Neochloris, Nannochloropsis, Chlorella, Duneliella and Porphyridium, are promising candidates for biodiesel production (Table 5.1). Usually, under stressed conditions, oleaginous microalgal strains accumulate lipid up to 70% w/w of dry cell weight (DCW) (Table 5.1) (Banerjee et al. 2002). The highest lipid content of 75% w/w has been observed in

Botryococcus brauni; however, it has low productivity due to slow growth (Banerjee et al. 2002). Depending upon the species, the growth characteristics, the productivity of biomass and lipid would vary. The composition of fatty acid is also an important feature of biodiesel production as it determines thermal capacity. Different species are found to have a different fatty acid composition; for example, Ankistrodesmus sp. contains mostly C16:4 and C18:4, Isochrysis sp. contains C18:4 and C22:6, while Nannochloris sp. contains C16:3 and C20:5. The composition of fatty acids in microalgae is dependent upon its growth and cultivation conditions. In nutrientlimiting conditions, C18:1 is accumulated in all species. However, an exception is B. braunii, which accumulates

TABLE 5.1 Potential Microalgal Strains for Biodiesel Production Habitat Fresh Water

Marine Water

Microalgal Species

Lipid Content (%, w/w DCW)

Lipid Productivity (mg L−1 d−1)

Botryococcus sp. Chaetoceros muelleri Chaetoceros calcitrans Chlorella emersonii Chlorella protothecoides Chlorella sorokiniana Chlorella vulgaris Chlorella pyrenoidosa Chlorella sp. Chlorococcum sp. Cylindrotheca sp. Ellipsoidion sp. Haematococcus pluvialis Neochloris oleoabundans Scenedesmus obliquus Scenedesmus quadricauda Scenedesmus sp. Schizochytrium sp. Dunaliella salina Dunaliella tertiolecta Dunaliella sp. Isochrysis galbana Isochrysis sp. Nannochloris sp. Nannochloropsis oculata Nannochloropsis sp. Nitzschia sp. Pavlova salina Pavlova lutheri Phaeodactylum tricornutum Porphyridium cruentum Spirulina platensis Tetraselmis sp. F&M-M34

25.0–75.0 33.6 14.6–16.4/39.8 25.0–63.0 14.6–57.8 19.0–22.0 5.0–58.0 2 18.0–57.0 19.3 16–37 27.4 25 29.0–65.0 11.0–55.0 1.9–18.4 19.6–21.1 50–77 6.0–25.0 16.7–71.0 17.5–67.0 7.0–40.0 7.1–33 20.0–56.0 22.7–29.7 12.0–53.0 30.9 30.9 35.5 18.0–57.0 9.5 4.0–16.6 14–18

– 21.8 17.6 10.3–50.0 1214 44.7 11.2–40.0 – 18.7 53.7 47.3 – 90.0–134.0 – 35.1 40.8–53.9 – 116 – 33.5 – 37.8 60.9–76.5 84.0–142.0 60.9–76.5 30.9 49.4 40.2 44.8 34 – 43

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Handbook of Algal Technologies and Phytochemicals

C20:5 in such conditions. Apart from growth and cultivation conditions, the assimilation of CO2 also influences the fatty acid profile in algal cells, e.g. Scenedesmus sp. and B. braunii show higher biomass productivity when grown under 10% (v/v CO2-air) (Yoo et al. 2010). Based on their adaptability, microalgae can be (a) autotrophic, (b) mixotrophic and (c) heterotrophic. Algae used for biodiesel production should be selected to grow preferably on photoautotrophic condition to reduce the cultivation cost of microalgae and to sequester CO2 from the atmosphere. Some of the major factors which are responsible for microalgae cultivation are growth rate, cultivation techniques, substrate utilization, ease of biomass harvesting, lipid content of microalgal cells and the quality and robustness of the process. Biodiesel can be produced from microalgae by a biorefinery approach (Ravishankar et al. 2012). After lipid extraction from the microalgal biomass, the residual biomass can be used for ethanol and methane production and also can be burned for energy cogeneration. Other valuable products like natural dyes, pigments and antioxidants can be extracted from microalgae. Algal biodiesel can perform as well as conventional diesel by reducing the emission of particulate matter, SOX, NOX and hydrocarbons in the atmosphere. Algal biodiesel can be used in automobile engines without any engine modification as it has similar properties to commercial diesel fuels and ASTM (American Society for Testing and Materials) standards (Table 5.2) (Ghosh et al. 2017).

MICROALGAE CULTIVATION Microalgal cultivation method can be mainly by two types: either open cultivation systems or closed cultivation systems (Chen et al. 2011). Often the microalgal TABLE 5.2 Properties of Biodiesel Fuel Properties

ASTM

Diesel

Biodiesel

Kinematic viscosity at 40°C (mm−2 s−1) Density (kg m−3) Calorific value (MJ kg−1) Acid value (mg KOH g−1) Iodine (g I2 100 g−1) Flash Point (°C) Pour point (°C) Cetane no

1.9–6

4.1

4.5

– –

825 45

880 40

< 0.8

0.4

0.6

– 130 –15 >= 47

79.4 115 –25 49

80 120 –18 53

cultivation for the biofuel production is coupled to utilization of wastewater and use of industrial flue gases as a source of CO2 to produce biomass in an economical manner while at the same time addressing environmental issues.

OPEN CULTIVATION SYSTEM (RACEWAY PONDS) Open cultivation systems are used in many algal industries for large scale production of microalgal biomass for food, feed, nutraceuticals and value-added products (Ranga Rao et al. 2012; Ravishankar et al. 2012). Open cultivation systems are easier to construct and operate as compared to closed cultivation systems. The most commonly used open cultivation systems are shallow big ponds, circular ponds and raceway ponds. In these ponds, the CO2, nutrients and water are circulated continuously using paddlewheels. The algae are exposed to sunlight for these systems. Due to the simple structure, these open ponds have very low production and operational costs. However, these open cultivation systems have some drawbacks like limited sunlight penetration to the cells, evaporative losses, atmospheric CO2 diffusion and requirement of large areas of land. Furthermore, due to the limited control in its operation, contamination with other fast-growing heterotrophs is inevitable. In addition, the inefficient stirring mechanisms decrease the overall mass transfer rates leading to poor biomass productivity. These limitations have thus restricted the commercial production of algae in open systems since they may not be suitable for several forms (Kumar et al. 2015).

CLOSED CULTIVATION SYSTEMS Closed cultivation systems in the form of photobioreactors (PBRs) are more efficient than the open cultivation systems as they can be operated in highly controlled condition. PBRs provide a controlled environment for the growth of algae to enhance its biomass productivity (Olaizola 2003). In PBRs, the different culture conditions such as CO2 supply, water supply, temperature, light intensity, culture density, pH, mixing regime, etc., can be maintained with respect to the type of algal species used. For large scale production of microalgae, the photobioreactors should have several characteristics like: (a) low doubling time, (b) high volumetric productivity, (c) ease of operation and scalability, (d) effective mixing and gas liquid mass transfer and (e) energy efficiency in terms of biomass productivity per energy input

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Biodiesel Production from Microalgal Biomass

(Zijffers et al. 2008). The most important parameter that affects PBR design is the light penetration, which means high surface-to-volume ratio (Posten 2009). Light penetration efficiency of a photobioreactor maximizes the biomass and product formation. There are several types of photobioreactors like: bubble column PBR, airlift PBR (Barbosa et al. 2003), flat panel PBR (Zhang et al. 2001), horizontal tubular PBR, helical PBR (Morita et al. 2002) and stirred tank PBR.

BIOSYNTHESIS OF LIPID/ TRIACYLGLYCEROL IN MICROALGAE Lipid biosynthesis in microalgae contains three independently regulated steps: (a) fatty acid synthesis in plastid, (b) glycerolipid assembly in the endoplasmic reticulum and (c) packing of lipid bodies. Photosynthetic microalgae sequester CO2 and fix it into sugars in presence of photon energy. By Calvin cycle, 3-phosphoglycerate (3-PGA) is formed which is followed by formation of pyruvate in glycolytic pathway. Pyruvate releases CO2, and forms acetyl-CoA (acetyl coenzyme) in presence of pyruvate dehydrogenase (PDH). The synthesis of fatty acid is catalyzed through multifunctional enzyme complex such as ACCase (acetyl-CoA carboxylase) which produces malonyl-CoA from acetyl-CoA and bicarbonate (Greenwell et al. 2010). Malonyl-CoA group is transferred to malonylacetyl carrier protein (ACP) catalyzed by an acyl carrier protein malonyl transferase. Malonyl-ACP with the help of fatty acid synthase (FAS) and after a series of carbon chain lengthening and desaturation reactions eventually forms free fatty acid chains, mainly C16 and C18 fatty acids. The elongation reaction terminates by either of the following: the removal of acyl group from ACP by the action of acyl-ACP thioesterases (FATs) or direct transfer of the ACP to glycerol-3-phosphate (G3P) backbone by acyltransferase in Kennedy pathway. FATs hydrolyze the thioester bond of the acylACP and release free fatty acids (FFAs) to the cytosol by acyl-CoA synthetase (Chen et al. 2012; Maity et al. 2014). Kennedy pathway involves stepwise addition of FFAs, adding to each hydroxyl group of glycerol beginning with G3P. Transfer of first FFA chain to position one of G3P is catalyzed by glycerol-3-phosphate acyltransferase (GPAT) to form lyso-phosphatidic acid (LPA) and then after successive dephosphorylation step produces phosphatidic acid (PA), diacylglycerol (DAG) and finally triacylglycerol (TAG) (Hu et al. 2008; Chen et al. 2012) (Figure 5.2). These two distinct pathways assembled and connected in the cytosol to form the TAG-lipid body.

METABOLIC ENGINEERING FOR THE ENHANCEMENT OF LIPID PRODUCTION Metabolic engineering deals with regulating or turning the metabolic pathway inside the cell to trigger the target metabolite production. Different strategies like flux balance analysis, engineering different enzymes towards lipid biogenesis, carbon partitioning, overexpression of gene, transcription factor engineering and proteomics approaches (Banerjee et al. 2016) can be adopted for triggering the target metabolite. Genetic manipulation in microalgae is challenging as editing tools are species specific and cannot be used interchangeably because of codon usage, defensive strategies, uptake of nucleic acid and porosity of cell (Banerjee et al. 2016). Microalgae metabolic engineering figured the origin of fourth-generation biodiesel production with an aim of transgenic microalgae development (Lu et al. 2011). Until today lipid metabolism with a detailed description of biosynthetic pathways that modify the chain length or saturation/unsaturation of fatty acids has not been meticulously examined for microalgae. However, scientists reported that many genes responsible for the lipid metabolism in higher plants have also found to be homologous with the microalgal genome sequences (Radakovits et al. 2010). Research attempts were made several times to up-regulate the ACCase enzyme in lipid metabolism pathway to induce or maximize lipid production but the success story is still awaited. In the model organism Chlamydomonas reinhardtii the endogenous gene source of the fatty acidacyl carrier protein thioesterases enzyme was modified to produce shorter chain length fatty acids (Blatti et al. 2012). CRISPRi (clustered regularly interspaced short palindromic repeats interference) was used for the first time to regulate expression of exogenously supplied rfp gene as a proof-of-concept and endogenous PEPC1 gene as a proof-of-function in Chlamydomonas reinhardtii to enhance the lipid production (Kao and Ng 2017). 13C metabolic flux analysis of Chlamydomonas reinhardtii revealed that Acetyl-coA which was synthesized in plastid during lipid metabolism is directly incorporated in fatty acid synthesis (Boyle et al. 2017). Proteomics study of Neochloris oleoabundans under nitrogen limited condition revealed that acyl carrier protein and protein biotin carboxylase is up-regulated under nitrogen deprived conditions (Morales-Sánchez et al. 2016).

MICROALGAL HARVESTING A major challenge in microalgal biodiesel production is the harvesting of biomass and downstream procession which accounts for nearly 80% of the total process cost.

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Handbook of Algal Technologies and Phytochemicals

FIGURE 5.2  Lipid/triacylglycerol (TAG) biosynthesis pathway in microalgae.

Microalgal cells are very small in size. A small amount of biomass has to be separated from the large volume of culture media (Amaro et al. 2011). Many harvesting technologies like centrifugation, filtration, flocculation and floatation, alone and/or in combination have been used (Ben-Amotz and Avron 1990; Brennan and Owende 2010). In recent years, a wide range of flocculation approaches have been explored (Vandamme et al. 2013; Lal et al. 2018). These approaches mainly cover traditional flocculation methods using chemical flocculating agents or bioflocculants or safe nanoparticles. Chemical flocculants like ferric chloride and alum are used for microalgal harvesting but have some drawbacks. This causes high concentration of metal in the microalgal biomass. Flocculants based on natural biopolymers like chitosan are a safer alternative for microalgal harvesting (Vandamme et al. 2010). Microalgal cell surfaces are negatively charged, hence the positive charged biopolymers like chitosan are often considered. Autoflocculation can be achieved by increasing the pH of culture above 9 or decreasing it below 5 (Vandamme et al. 2012). During CO2 sequestration by microalgae, decrease in pH of the medium causes the spontaneous aggregation of the cells which lead to flocculation. Bioflocculation is a sustainable and cost-effective technique for microalgae

harvesting. Nanoparticle engineering, provides very intriguing potential solutions in microalgal harvesting process. Improvement of cell separation efficiency and processing time from culture broth, the multiple reuse of magnetic nanoparticle flocculants and integrated harvesting/cell-disruption technique in reactor vessel make the use of magnetic nanoparticle as flocculant a plausible alternative for cost-effective microalgal harvesting and biodiesel production (Akia et al. 2014; Alves et al. 2014).

MICROALGAL LIPID EXTRACTION Lipids are a diverse group of biological substances made up primarily of polar (free fatty acids, phospholipids and sphingolipids) and non-polar compounds (triglycerides, diglycerides, monoglycerides and sterols). They bind covalently to carbohydrates and proteins to form glycolipids and lipoproteins, respectively. The possibility of lipids binding to other molecules and the ability of different solvent mixtures to solubilize lipid classes has led to the concept of total lipid extraction. Several methods are developed for total lipid extraction depending on the combination of solvents (Bligh and Dyer 1959; Booij and Van den Berg 1994; Smedes and Askland 1999). Because of large variations in algal cell shape, size,

57

Biodiesel Production from Microalgal Biomass

cell wall structure and characteristics of algal lipids, various lipid extraction methods may work differently on different algal species (Shen et al. 2009). Converti et al. (2009) demonstrated that the most effective lipid extraction method in Nannochloropsis oculata was the combination of ultrasound with the Folch method. Lee et al. (2010) tested various methods, including autoclaving, bead-beating, microwave, sonication and a 10 w/v % NaCl solution, to identify the most effective cell disruption method in microalgae like Botryococcus sp., Chlorellavulgaris and Scenedesmus sp. The total lipids from these microalgae were extracted by following Bligh and Dyer method (1959). The choice of the Bligh and Dyer method was justified on the basis that chloroform and methanol quantitatively extracted all lipid classes and produced the highest lipid yields. Some recent studies investigated different direct transesterification processes from wet algal biomass. The microalgal cell disruption is a key factor for intracellular lipid extraction from the wet biomass. Autoclaving, microwave irradiation, ultrasonication, bead milling, high pressure homogenization and enzymatic treatments were incorporated on wet microalgal biomass for lipid extraction. These pre-treatment techniques for lipid extraction from wet microalgal biomass help in reducing the overall process cost of biodiesel production (Selena Dickinson et al. 2017). An outline of the process from algal biomass production to biodiesel is presented in Figure 5.3.

FIGURE 5.3  Overall microalgal lipid extraction process.

MICROALGAL LIPID TO BIODIESEL Biodiesel is produced by transesterification process, where triglycerides are transformed into fatty acid methyl ester (FAME) in presence of a monohydroxy alcohol, such as methanol and a catalyst (alkali, acid, enzyme, nanoparticle) with glycerol as a byproduct (Hoydonckx et al. 2004). The transesterification reaction is represented by the general equation as given in Figure 5.4, where R1, R2 and R3 are long hydrocarbon chains. The fatty acids obtained from microalgal lipid after transesterification are mainly composed of carbon chain length of C12–C22 (5.3) (Ghosh et al. 2017) (Table 5.3). The process of transesterification is affected by various factors like the molar ratio of alcohol to oil, type and the amount of catalyst, reaction time and temperature and purity of reactants. However, transesterification is an equilibrium reaction in which an excess of alcohol is required to drive the transesterification reaction for completion. The presence of a sufficient amount of methanol during the transesterification reaction is essential to break the glycerine–fatty acid linkages (Al-Widyan and Al-Shyoukh 2002). Being polar and shortest chain alcohol, methanol can quickly react with triglycerides. The catalyst is usually used to improve the reaction rate and yield. The transesterification reaction can be catalyzed by alkalis, acids, enzymes and nanoparticles. The KOH and NaOH are commonly used as nbase catalyst for biodiesel preparation. However, during the separation of the final products from glycerol, KOH has been found

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Handbook of Algal Technologies and Phytochemicals

FIGURE 5.4  Transesterification reaction.

TABLE 5.3 Common Fatty Acids Present in Microalgae and Their Chemical Structure Common Name

Systemic Name

Chemical Structure

Common Acronym

Lauric acid Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Archidic acid Behenic acid Erucic acid Lignoceric acid

Dodecanoic acid Tetradecanoic acid Hexadecanoic acid Octadecanoic acid Octadecenoic acid Octadecadienoic acid Octadecatrienoic acid Eicosanoic acid Decosanoic acid Docosenoic acid Tetracosanoic acid

CH3(CH2)10COOH CH3(CH2)12COOH CH3(CH2)14COOH CH3(CH2)16COOH CH3(CH2)7CH=CH(CH2)7COOH CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH CH3(CH2)18COOH CH3(CH2)20COOH CH3(CH2)7CH=CH(CH2)11COOH CH3(CH2)22COOH

C 12:0 C 14:0 C 16:0 C 18:0 C 18:1 C 18:2 C 18:3 C 20:0 C 22:0 C 22:1 C 24:0

to be more convenient (Guan et al. 2009). However, one limitation to the alkali-catalyzed process is its sensitivity to the purity of reactants. The alkali-catalyzed system is very sensitive to both water and FFA. The presence of water may cause saponification under alkaline condition. Acids used for transesterification include sulfuric, phosphoric, hydrochloric and organic sulfonic acids. It was reported that ester conversion reached 98% at a molar ratio of 30:1 (methanol:oil) with 3% sulfuric acid as a catalyst at 60°C. As acid-catalyzed transesterification is a relatively slow process, many researchers have combined both acidic and alkaline catalysts in a twostep reaction in which the acid treatment converts the FFA into esters while the alkaline catalyst is performing the transesterification. This process has been developed by Canacki and Garpen (2001) using yellow and brown grease having FFA content of more than 10%. Nanoparticles play a significant role as catalysts for the production of biodiesel, owing to their higher catalytic activity, large specific surface area, high resistance to saponification reaction and good rigidity. A higher level of biodiesel yield was obtained using nanoparticles as catalyst. Efficient and recyclable heterogeneous catalysts like metal oxide catalysts (e.g., ZnO), metal supported by metal oxide catalysts (e.g., Au–ZnO), alloy (e.g., Cu–Co) and metal oxide supported by metal oxide

(e.g., KF–CaO–Fe3O4) can be used for the transesterification process. Enzymatic catalysis with the use of lipases is also possible (Azean and Yilmaz 2012), however, its costs are very high (Thanh et al. 2012). Hydrothermal liquefaction technique for algal biodiesel production is gaining importance in recent times. With hydrothermal liquefaction (HTL), whole wet microalgae biomass can be converted into biodiesel, and during the refining process liquid bio-crude can be also produced (Douglas et al. 2015). During these processes, water-based microalga slurries react in a high temperature and high pressure (HTHP) reactor at a temperature of 200–350°C and sufficient pressure (~20 MPa) in water as the liquid phase. This converts whole hydrocarbon compounds present in the algal biomass in the form of bio-crude. This bio-crude is further distilled using a true boiling point distillation unit to achieve various fractional biofuel products. During the HTL process, harvested algal biomass doesn’t require drying which can save energy (Gollakota et al. 2018). Moreover, HTL converts all sorts of hydrocarbon compounds present in the feedstock into bio-crude which further increases its yield. Furthermore, the transesterification process results in single biofuel product as biodiesel, whereas bio-crude delivers various fractions of biofuels ranging from green gasoline, green aviation fuel and green

59

Biodiesel Production from Microalgal Biomass

diesel. Recently, Elliott (2011) has conducted a study on HTL of algal biomass and achieved more than 80% conversion. In another study by Jazrawi et al. (2013), Chlorella and Spirulina biomass were converted into bio-crude using HTL process and 47% of bio-crude conversion was achieved. A higher bio-crude yield of up to 68% may be obtained which depends on the biochemical composition of feedstock. There are very limited reports on microalgae HTL process to predict accurate conversion percentage of feed into biodiesel.

SCALE-UP STRATEGIES FOR BIODIESEL PRODUCTION FROM MICROALGAE Scale-up is a process to enlarge the production quantities with similar or higher productivity. Scale-up of microalgal biodiesel production is a crucial step due to the difficulty in assessing the process parameter affecting the scale-up. There is a gap between theoretical biological potential of microalgae and the biomass productivity achieved in scale-up studies mainly in outdoor cultivation. Parameters like mixing, gas holdup, temperature and pH control, biofouling, proper light illumination and contamination should be addressed for successful scale-up of microalgal biodiesel production. Mixing is an important parameter for higher biomass production. Proper mixing is required to avoid poor mass transfer, biomass settling, anaerobic dead zone minimization and proper distribution of nutrients. All the parameters responsible for scaling-up have to be optimized in lab scale before going for scaleup studies. Recent advancements in computational fluid dynamics (CFD) can be effectively used to model PBRs for microalgae cultivation, thus reducing the burden of sole experimentation. Hydrodynamic studies, which form an integral part in designing of PBRs, can be adequately analyzed with the aid of CFD techniques (Grace and Taghipour 2004; Hutmacher and Singh 2008). There are various challenges in the design and optimization of PBRs and open cultivation system that adversely affect the scaleup studies which can be solved by CFD analysis.

CHALLENGES AND FUTURE RESEARCH PERSPECTIVES The main challenge of microalgal biodiesel production is large scale biomass cultivation and biomass generation. Biomass production maximization requires screening of a suitable strain, adequate CO2 and nutrient supplement, proper light distribution for photosynthesis and optimal temperature for enhancing the enzyme kinetics. Large scale microalgal biomass production with the optimization of physico-chemical parameters has to be

evaluated. Apart from the physico-chemical parameters, hydrodynamic parameters like mixing time, shear stress and mass transfer in large-scale photobioreactor are dramatically different with that in the lab-scale reactor. Mathematical modeling can be used to understand the complex phenomena inside the photobioreactor, which can be a great help to overcome the limitations related to design and scale-up of photobioreactors. Flow hydrodynamics in PBRs has significant effect on the microalgae growth. CFD simulations can provide the details of fluid hydrodynamics. CFD can be used to study operating and geometry factors in PBRs that influence the flow dynamics, such as the inlet gas flow rate, mixing, mass transfer and reactor geometry. The use of artificial intelligence (AI), particularly artificial neural network model (ANN model), and statistical and evolutionary learning-based techniques are some innovative approaches in manipulating and optimizing productivity and costs in algal biofuel production. Dewatering and microalgal biomass harvesting are other bottlenecks for commercialization of microalgal biodiesel production. Harvesting, dewatering and extraction of oil from microalgae involves the highest cost in terms of downstream processing of algal biofuels. Lipid extraction from wet microalgal biomass and direct transesterification processes can be adopted to reduce the cost of microalgal biodiesel production. Genetic engineering like recombinant DNA technology towards genetic modification or alteration of the existing lipid/triglycerol production pathway or metabolic flux analysis can be also adapted to enhance the lipid/triglycerol production from microalgae (Corteggiani Carpinelli et al. 2014; Jia et al. 2015; Mansfeldt et al. 2016).

CONCLUSION Biodiesel production from microalgal biomass has promising potential for commercialization. Many companies and researchers have developed techniques for microalgal biodiesel production. Large-scale production under realistic approaches has to be focused for microalgal biomass production maximization. Life cycle assessment study has to be analyzed to find out the environmental impacts of the process and sustainability of the process. Furthermore, the microalgal cultivation can be combined with wastewater treatment and CO2 sequestration from thermal power plants; these are important milestones to be reached for a cleaner, carbon neutral sustainable future. The biorefinery concept encompasses the vision where the spent biomass after lipid extraction could be used for biohydrogen, bioethanol and biogas production. Glycerol is the major byproduct during lipid extraction from microalgae and can be also reused for microalgal

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cultivation improvement. This biorefinery approach increases the overall conversion efficiency of the microalgal biodiesel production. Thus, the technology of algal biodiesel production is fast evolving to overcome the bio-process hurdles in order to realize the challenging dream of being a profitable industry.

ACKNOWLEDGMENT The authors acknowledge Ministry of New and Renewable Energy and Ministry of Human Resource Development, Govt. of India, for their financial support and Indian Institute of Technology, Kharagpur, for the laboratory facilities.

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Mansfeldt, C. B., Richter, L. V., Ahner, B. A., Cochlan,W. P., Richardson, R. E. Use of de novo transcriptome libraries to characterize a novel oleaginous marine Chlorella species during the accumulation of triacylglycerols. PLOS ONE 2016. 11(2): 0147527. Mata, T. M., Martins, A. A., Caetano, N. S. Microalgae for biodiesel production and other applications: A review. Renew Sust Energ Rev 2010. 14: 217–232. Metzger, P., Largeau, C. Botryococcus braunii: A rich source for hydrocarbons and related ether lipids. Appl Microbiol Biotechnol 2005. 66: 486–496. Morales-Sánchez, D., Kyndt, J., Ogden, K., Martinez, A. Toward an understanding of lipid and starch accumulation in microalgae: A proteomic study of Neochloris oleoabundans cultivated under N-limited heterotrophic conditions. Algal Res 2016. 20: 22–34. Morita, M., Watanabe, Y., Saiki, H. Photosynthetic productivity of conical helical tubular photobioreactor incorporating Chlorella sorokiniana under field conditions. Biotechnol Bioeng 2002. 77(2): 155–162. Olaizola, M. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomol Eng 2003. 20(4): 459–466. Posten, C. Design principles of photo‐bioreactors for cultivation of microalgae. Eng Life Sci 2009. 9(3): 165–177. Radakovits, R., Jinkerson, R. E., Darzins, A., Posewitz, M. C. Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 2010. 9(4): 486–501. Rao, A. R., Ravishankar, G. A., Sarada, R. Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production. Bioresour Technol 2012. 123: 528–533. Ravishankar, G. A., Sarada, R., Vidyashankar, S., Kumudha, A. 2012 Cultivation of micro-algae for lipid and hydrocarbon and utilization of spent biomass for livestock feed for bio-active constituents. In: Biofuel Co-Products as Livestock Feed, ed. Makkar, P.S., 486. FAO (UN). Roy, S., Das, D. 2016 Liquid fuels production from algal biomass. In: Algal Biorefinery: An Integrated Approach, ed. Das, D. 277–318. Springer International Publishing AG, Switzerland. Shen, Y., Pei, Z., Yuan, W., Mao, E. Effect of nitrogen and extraction method on algae lipid yield. Int J Agric Biol Eng 2009. 2: 51–57. Smedes, F., Askland, T. K. Revisiting the development of the Bligh and Dyer total lipid determination method. Mar Pollut Bull 1999. 38: 193–201. Thanh, L. T., Kenji, O., Luu, V., Yasuaki, M. Catalytic technologies for biodiesel fuel production andutilization of glycerol: A review. Catalysts 2012. 2: 191–222. Vandamme, D., Foubert, I., Fraeye, I., Meesschaert, B., Muylaert, K. Flocculation of Chlorella vulgaris induced by high pH: Role of magnesium and calcium and practical implications. Bioresour Technol 2012. 105: 114–119. Vandamme, D., Foubert, I., Meesschaert, B., Muylaert, K. Flocculation of microalgae using cationic starch. J Appl Phycol 2010. 22: 525–530.

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Carbon Dioxide Sequestration by Microalgae G.V. Swarnalatha, A. Shekh, P.V. Sijil, C.K. Madhubalaji, V.S. Chauhan, and R. Sarada

CONTENTS List of Abbreviations�������������������������������������������������������������������������������������������������������������������������������������������������64 Microalgae and Their Importance�����������������������������������������������������������������������������������������������������������������������������64 Microalgae Cultivation Systems�������������������������������������������������������������������������������������������������������������������������������64 Factors Affecting CO2 Utilization by Microalgae�����������������������������������������������������������������������������������������������������64 Light���������������������������������������������������������������������������������������������������������������������������������������������������������������������64 pH�������������������������������������������������������������������������������������������������������������������������������������������������������������������������65 Temperature����������������������������������������������������������������������������������������������������������������������������������������������������������65 Mixing������������������������������������������������������������������������������������������������������������������������������������������������������������������65 Carbon Concentration Mechanisms (CCM) in Microalgae��������������������������������������������������������������������������������������66 Need for CCM in Microalgae�������������������������������������������������������������������������������������������������������������������������������66 Types of Carbonic Anhydrases�������������������������������������������������������������������������������������������������������������������������66 Functions of Carbonic Anhydrase in Microalgae���������������������������������������������������������������������������������������������67 Ribulose-1, 5-bisphosphate Carboxylase Oxygenase (RuBisCO)�����������������������������������������������������������������������67 Location of RuBisCO in Microalgae���������������������������������������������������������������������������������������������������������������67 Accumulation of HCO3–�����������������������������������������������������������������������������������������������������������������������������������68 Effect of CO2 Supplementation on Carbon Concentrating Mechanism (CCM)�������������������������������������������������������68 Effect of CO2 Supplementation on the Calvin Cycle������������������������������������������������������������������������������������������������68 Effect of CO2 Supplementation on Carbohydrate Metabolism���������������������������������������������������������������������������������68 Glycolysis and Gluconeogenesis��������������������������������������������������������������������������������������������������������������������������68 TCA Cycle and Oxidative Phosphorylation���������������������������������������������������������������������������������������������������������69 Effect of CO2 Supplementation on Protein Metabolism���������������������������������������������������������������������������������������69 Effect of CO2 Supplementation on Lipid Metabolism�����������������������������������������������������������������������������������������70 Industrial Application������������������������������������������������������������������������������������������������������������������������������������������������70 Future Needs�������������������������������������������������������������������������������������������������������������������������������������������������������������71 References����������������������������������������������������������������������������������������������������������������������������������������������������������������� 71 BOX 6.1  SALIENT FEATURES Continuously increasing CO2 remains one of the major environmental concerns to be addressed. Various physical, chemical, and biological means of CO2 sequestration are being tried and tested. Using microalgae for CO2 sequestration has significant advantages since elevated CO2 supports its high biomass productivity. Microalgae are considered as the best biomass producers on earth due to their high growth and productivities per unit area, no dependence on agricultural land and freshwater, and CO2 assimilation potential. Microalgae, due to their carbon concentrating mechanism (CCM), are able to survive under high CO2, utilize it, and convert it into chemical energy.

The enzyme carbonic anhydrase and RuBisCO are the major components of the CCM. Moreover, CO2 sequestration and the biomass production by microalgae is the function of cultivation parameters like temperature, light intensity, pH, and mixing. Various metabolic pathways like Calvin cycle, glycolysis, TCA cycle, and protein and lipid synthesis in microalgae gets influenced through CO2 supply. Further, microalgal biomass can be utilized for the production of various high value metabolites, food and feed grade products, and fuels. CCM and its components, cultivation parameters, effect of CO2 supply on various metabolic pathways, and industrial applications of microalgae are discussed in this chapter. 63

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LIST OF ABBREVIATIONS % Percent α Alpha β Beta γ Gamma µ Micro µg Microgram µM Micromolar Mg Milligram Kg Kilogram M Molar N Normality L Liter nm Nanometer cm Centimeter mm Millimeter s Second(s) min Minute(s) h Hours − HCO3 Bicarbonate Mol Mole Atm Atmosphere Srel Ratio of rates of carboxylase to oxygenase reactions cat Corresponding substrate-saturated rates of catalysis PGA Phospho glyceric acid Cys Cysteine ADP Adenosine di-phosphate GAPDH Glyceraldehyde-3-phosphate dehydrogenase PK Pyruvate kinase GTP Guanosine triphosphate

MICROALGAE AND THEIR IMPORTANCE The term microalgae represents a large and diversified group of simple, typically autotrophic organisms. In general, microalga exists in unicellular to multi-cellular forms from different phylogenetic groups, representing many taxonomic divisions. They are mainly classified into 11 classes by taxonomic concepts and standards based on their photosynthetic pigments, cell wall composition, and reserve food material. Microalgae play a key role in carbon fixation process, which accounts for about 50% of the global carbon fixation and also have an enormous biotechnological potential (Field et al. 1998). Microalgae are versatile in nature in maintaining their structure by using the energy metabolisms like respiration and nitrogen fixation (Demeyer et al. 1982; Grossman et al. 1994). They also represent an exclusive biological resource for a wide range of pigments, such

Handbook of Algal Technologies and Phytochemicals

as chlorophylls (essential for photosynthesis), carotenoids (red, orange, or yellow lipid-soluble pigments), and accessory pigments like phycobilins-phycocyanin (blue pigment) and phycoerythrin (red pigment) (Fay 1983). The spectral light absorption is different from the other photosynthetic organisms, since high photosynthetic activity rates are measured both in the spectral region from 665 to 680 nm (for CO2, chlorophyll a) and 620 to 560 nm (for phycocyanin and phycoerythrin). This shows a very high photosynthetic activity as light is absorbed by both the chlorophyll and phycobiliproteins (Campbell et al. 1998). Additionally, they have a highly developed intra cytoplasmic system for photosynthesis as the preferred metabolic pathway. The microalgae use free CO2 and other forms of inorganic carbon source like bicarbonate ions for photosynthesis and transport it across the plasma membrane. This transported inorganic carbon serves as a reservoir for photosynthesis. The enzyme carbonic anhydrase converts bicarbonate to CO2 (Badger and Price 2003) and makes it available for RuBisCo to fix it into the carbohydrates.

MICROALGAE CULTIVATION SYSTEMS Various types of cultivation systems with different configurations are being used for microalgae culturing; viz. simple open circular ponds, open raceway ponds, and complex enclosed systems called closed photobioreactors, etc. Recent reports suggests that the higher surface area, short light paths, external light provisions, and small dark zones are the factors that influence CO2 capture in photobioreactor during algal growth. The latest research has been conceded to microalgal culture in photobioreactors with the higher surface area, short light paths, external light provisions, and small dark zones. Both open raceway ponds (cheapest) and different closed photobioreactors (flat panel, airlift, bubble column, stirred tank reactors) are considered for CO2 sequestration.

FACTORS AFFECTING CO2 UTILIZATION BY MICROALGAE Light The photosynthetic growth of the microalgae and their CO2 fixation ability is influenced by the light intensity. Light intensity is a limiting factor which influences photosynthesis process, CO2 fixation, biomass production, and overall metabolic activity and growth of microalgae. High light intensities also cause photo inhibition for surface layer of algae. The daily growth rate of microalgae with respect to biomass is increased by avoiding photo inhibition (Chisti 2007). Increased removal of CO2 by

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Carbon Dioxide Sequestration by Microalgae

C. vulgaris and D. tertiolecta was noted with increase in light intensity (10, 20, and 50 Wm−3 under 4% CO2 supplementation (Hulatt and Thomas 2011)). Also, 4.2-fold increased biomass production was noted for cyanobacterium Synechococcus sp. with increasing light intensity from 156 to 1250 lx (2.5–20 µE m−2 s−1). In general, increasing light intensity generally increases microalgal CO2 fixation rates. Oxygen evolution and CO2 capture will have a positive correlation with increasing light intensity till reaching the light saturation point (Fan et al. 2007). Chlorella sp. was investigated with sunlight, xenon lamps, and fluorescent lamps, and it has variable effects on CO2 fixation rates. Researchers concluded that the highest CO2 fixation rate (0.865 g L−1 d−1), biomass productivity (0.437 g L−1 d−1), and biomass concentration (0.842 g L−1) were achieved with white fluorescent lamps (Hirata et al. 1996). The self-shading effect of microalgal cells also affects the light distribution in the culture. Self-shading, therefore, leads to decreased productivities. However, in the case of airlift photobioreactor systems, this limitation can be overcome by sparging air from the bottom which increases the light delivery to the cells due to Brownian motion. pH

In general, pH plays one of the key roles in microalga growth. The pH and CO2 are interrelated parameters for the algal growth. The optimum range is 8.5–9.0. Microalgae easily capture CO2 from atmosphere and produce more biomass when grown in alkaline conditions. The increase of pH results in formation of bicarbonate from CO2 in water which is used by microalgae widely. The pH of the culture medium decreases with the dissolution of CO2 and SOX from the flue gas. Enhanced CO2 fixation by microalgae is limited by CO2 mass transfer which in turn is a function of CO2 concentration and flow rate (Pires et al. 2012). Photosynthetic efficiency and carbon concentrating mechanisms defines the microalgal affinity for the carbon species (CO2 or HCO3−). Supply of CO2 majorly affecting the pH of the medium influences the cell growth. Even though the different microalgal species have varied pH tolerances, lower pH-tolerating species can grow at higher CO2 concentrations (Moheimani 2013).

Temperature Temperature is one of the vital factors in microalgal growth, particularly in the maintenance of cell morphology and physiology. The metabolic activities of the culture may increase or decrease as a function of temperature.

Economic cultivation of microalgae for biomass production is combined with CO2 capture. Enhanced biomass production is associated with availability of dissolved inorganic carbon in the nutrient medium. However, CO2 solubility is influenced by culture/atmospheric temperature. An ideal temperature range for microalgal biomass production is reported to be 25–35°C. The optimal growth of microalgae is determined by the intracellular enzyme activity and reaction rates which in turn are dependent on temperature. Various microalgal species have their optimal growth temperature in the range of 15–26°C (Kumar et al. 2010). Since the temperature of the flue gas from power plants is around 120°C, thermotolerant microalgae can be employed to sequester the CO2 from flue gas. The thermotolerant microalgae has the ability to tolerate and grow up to the temperature of 55°C with more than 40% CO2 (Wang et al. 2008). Some species, viz. Cyanidiium caldarim, Galdieria partita, and Cyanidioschyzon melorae, have been identified as thermotolerant and can grow at 50°C (Kurano et al. 1995). Thermotolerant Synechococcus (a unicellular cyanobacterium) is known to grow at the temperature range of 48–55°C (Eberly and Ely 2012). According to Henry’s law, at 50°C Henry’s constant is 1.817 × 10 −2 mol/atm, whereas with lowering temperatures the constant increases to 2.965 × 10 −2 mol/atm (Eberly and Ely 2012), leading to higher CO2 availability and fixation by microalgae (Eberly and Ely 2012). Increase in temperature decreases the solubility, and it will be balanced with the increased metabolic rates.

Mixing Mixing is of great importance for microalgae cultivation. It mainly improves the mass transfer limitations and also maintains the efficient distribution of various gases like CO2, air, oxygen, etc. Mixing prevents sedimentation of algae, imparts homogenous mixing of nutrients, and makes sure that all cells are equally exposed to light which helps to enhance the overall biomass productivity. The microalgal strain, culture system (open raceway ponds and closed photobioreactor), scale of the cultivation, and the cultivation environment (indoor/outdoor) determine the type of mixing to be employed. Therefore, it is vital to apply efficient mixing so that maximum microalgal biomass productivities can be achieved. Mixing by direct bubbling of air in tubular PBRs is also an option; in pneumatically agitated PBRs mixing in bubble column PBRs can be random and erratic when gas is sparged from the bottom of the PBR (Ugwu and Aoyagi 2012). Raceway mixing in open ponds is achieved via a defined circulation of the culture

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through continuously operating paddle wheels (Ugwu and Aoyagi 2012).

CARBON CONCENTRATION MECHANISMS (CCM) IN MICROALGAE Need for CCM in Microalgae The microalgal photosynthesis is responsible for ∼50% of the world’s primary productivity (Behrenfeld et al. 2001). The carbon flux in algae involves the species using C3 pathway where dissolved inorganic carbon is fixed directly by RuBisCO (Equation 6.1). The low affinity of RuBisCO for CO2 is aggravated by its oxygenase role (Equation 6.2).



Ribulose-1,5-bisphosphate + CO2 + H 2 O ® 2 ´ glycerate-3-P Ribulose-1,5-bisphosphate + O2 ® glycerate-3-P + glycolate-2-P

(6.1)

(6.2)

The phosphoglycolate is dephosphorylated to glycolate by the enzyme phosphoglycolate phosphatase; otherwise it inhibits the carboxylase activity of RuBisCO. This glycolate via photorespiration enters into further metabolism (Beardall and Raven 2004; J. A. Raven 2000), or it is lost by excretion from algal cells. In some algae, dissolved inorganic carbon assimilation from the environment occurs by alternative pathways. RuBisCO is known to have both competitive carboxylase and oxygenase functions. The two competitive reactions of RuBisCO in autotrophic cells depend on the concentrations of O2 and CO2 at active site of the RuBisCO. The relative rates of carboxylase and oxygenase reactions are calculated by selectivity factor (Equation 6.3).

Srel =

K 0.5 ( O2 ) × K cat (CO2 )

K 0.5 (CO2 ) × K cat (O2 )

. (6.3)

Srel = Ratio of rates of carboxylase to oxygenase reactions Kcat (CO2) and Kcat (O2) = Corresponding substratesaturated rates of catalysis K0.5 (CO2) = Half saturation constant for the carboxylase K0.5 (O2) = Half saturation constant for oxygenase The type of RuBisCO present varies in different autotrophs. Form 1B, Form 1D, and Form II RuBisCO with varying Srel values are distributed amongst various

autotrophs. Form 1B is predominant in higher plants, β-cyanobacteria, and green algae with Srel values from 35 to 90. Form 1D is predominantly observed in α/βproteobacteria, cryptophytes, rhodophytes, heterokonts, and haptophytes, which have much higher Srel values. On the other hand, Form II RuBisCO is found to be present in proteobacteria (Srel: 9–15) and dinophytes (Srel: ∼30) (Badger et al. 1998, 2002; Raven 1997). A low K0.5 (CO2) and a high Srel correlate with a low Kcat (CO2), and vice versa (Raven and Beardall 2003). There are inherent inefficiencies in physiology of CO2 assimilation like oxygen inhibiting CO2 fixation, low affinities for external CO2, and high CO2 compensation points. Most of the algae and aquatic plants and all cyanobacteria possess mechanisms that conquer the limitations of RuBisCO (Beardall and Raven 2004) to CO2 limiting conditions. These are all referred to as CCMs even though they function with diverse mechanisms. The occurrence of a CCM is examined in several thousand species of cyanobacteria and eukaryotic algae (Giordano et al. 2005). The CCMs in algae and cyanobacteria involve active transport of HCO3− and/or CO2 across one or more membranes that separate medium from RuBisCO. The membrane across which active transport occurs has a low permeability to the dissolved inorganic carbon transported to the side of the membrane nearby to RuBisCO (Raven and Beardall 2003). In eukaryotic microalgae, plasma membrane or the inner plastid envelope membrane or both could be involved in the active transport of dissolved inorganic carbon (Moroney and Chen 1998; Raven and Beardall 2003). A range of CAs in eukaryotic algal cells is responsible for maintaining equilibration between CO2 and HCO3− in the various cell compartments (J. V. Moroney et al. 2001; Mitra et al. 2004). In cyanobacteria, plasmalemma or thylakoid membranes are involved in either CO2 or HCO3− transport as a part of CCM (Hewett-Emmett and Tashian 1996) and in higher plants. Carbonic anhydrase plays a major role in decarboxylation reactions including photosynthesis and respiration (Smith and Ferry 2000). It is involved in transportation of inorganic carbon (Ci) to actively photosynthesizing cells or away from actively respiring cells (Henry et al. 1996). Even though there are several forms of CAs detected in cyanobacteria, many cyanobacterial genomes were known to lack identifiable CA genes (Badger and Price 2003). Types of Carbonic Anhydrases There are five classes of CA, i.e., α, β, γ, δ, and ε (Shekh et al. 2013; So and Espie 2005). The α-class was found in animals, plants, and eubacteria. They are protein monomers with antiparallel β-strands with active site (Liljas et al. 1972; So and Espie 2005). Active site majorly contains

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hydrophobic amino acid side chains with Zn2+ ion coordinated by three histidine residues (Liljas et al. 1972). β-CAs were found in chloroplasts of plants and sub-cellular compartments of many organisms. The amino acids present in β-CAs are different than in α-CAs. β-CAs are active only when they oligomerize and the catalytic core of a β-CA can be dimer, tetramer, or octamer (Mitsuhashi et al. 2000). The hydrophobic site resulted from dimerization serves as active site for CO2 binding. The central metal Zn2+ is surrounded by a combination of His, Cys, and Glu residues (Mitsuhashi et al. 2000). The α-helical structures are present in β-CAs. The γ-CA was first isolated from methanogenic archaeon Methano sarcinathermophila (Shekh 2012). Cam (for carbonic anhydrase of M. thermophile), a type of γ-CA from M. thermophile, is active when it is trimerized (Kisker et al. 1996). Each monomer resembles a triangle when it is cross-sectioned. The αβ-helix was found in γ-CA where β-helix was a left-handed structure containing seven complete turns with an α-helix at the end (Kisker et al. 1996). The active sites are present at the interface of two β-helices. The interface is stabilized by hydrogen bonds, salt bridges, and hydrophobic interactions. Among γ-CAs, cam is the only one which shows CA activity. Several cam homologs of plants and bacteria lack CA activity. These include cytochrome C maturation operon (CcmM), a γ class CA from the cyanobacteria Synechocystis PCC6803, and Synechococcus PCC7942. The δ-class (TWCA1) is found in marine diatom Thalassiosira weissflogii (Roberts et al. 1997). Chlamydomonas reinhardtii has five classes of CAs, out of which three α-CAs and two β-CAs are identified (Moroney et al. 2001). Functions of Carbonic Anhydrase in Microalgae Direct uptake of HCO3– and facilitated diffusion by CA are the transportation modes in plasma membrane. The HCO3– is directly taken by the cells in Scenedesmus obliquus, and they are able to photosynthesize at pH 10 and above (Thielmann et al. 1990). The preferred Ci is CO2 in Chlorella saccharophila, but it also takes up HCO3– (Williams and Colman 1995). Periplasmic CA encoding genes are identified in D. salina and

C. reinhardtii (Fujiwara et al. 1990). Under low CO2 conditions, the periplasmic CA1 proteins are induced in C. reinhardtii LIP-36, and a transport protein in chloroplast envelope is induced under low CO2 in C. reinhardtii. It also acts as exchanger (e.g., ATP for ADP transporters). It helps in accumulating HCO3– in the chloroplast. Based on the location there are two types of CAs, i.e., external and internal. The function of external CA, which is located outside the cell membrane, is to transfer CO2 from surrounding water into cell. The internal CA is located in chloroplasts and transports CO2 within the cell. Internal CA is soluble, whereas external CA is surface active, part of it soluble in water and part of it soluble in the lipid bilayer of the membrane. Internal CA is used in CO2 fixation where it provides CO2 to RuBisCO. RuBisCO has a high N:C ratio so it catalyzes backward reaction and results in waste photorespiration (Raven and Johnston 1991). CA maintains a high CO2:O2 ratio in the chloroplast near RuBisCO. External CA is mainly involved in conversion of bicarbonate to CO2. As bicarbonate is charged and cannot cross the lipid membrane, so it has to be converted to CO2. In a typical diatom, a diffusion reaction model was developed (Riebesell et al. 1993). It proves that CO2 supply is a rate limiting phenomenon for photosynthesis. In other species, external CA converts bicarbonate to CO2 which can be further used for photosynthesis. Active transport of bicarbonate is also observed in some other species (Tortell et al. 1997).

Ribulose-1, 5-bisphosphate Carboxylase Oxygenase (RuBisCO) Location of RuBisCO in Microalgae RuBisCO is present as a soluble protein in the chloroplast stroma of higher plants. But in microalgae, it is present in the pyrenoid of the chloroplast. In cyanobacteria it is present in carboxysomes that are surrounded by a protein shell and are rich in electron dense particles. The primary location of RuBisCO is carboxysomes in cyanobacteria (McKay et al. 1993) (Table 6.1).

TABLE 6.1 Location of RuBisCO in Organisms with Different Types of Photosynthesis Photosynthesis Type C3 Photosynthesis (higher plants) C4 Photosynthesis (higher plants) Eukaryotic microalgae Cyanobacteria

CO2 Concentrating Ability No Yes Yes Yes

RuBisCO Location Chloroplast stroma of most cells in leaf Chloroplast stroma of bundle-sheath cells Pyrenoid of the chloroplast Carboxysomes

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Accumulation of HCO3 – The microalgae which are growing under elevated levels of CO2 are inefficient in Ci acquisition (Matsuda et al. 1998). Usually, algae are very efficient in acquiring Ci from the low CO2 environment. This indicates that the transport mechanism available in microalgae is inducible in nature. However, the amount of RuBisCO is the same when there is fluctuation of CO2 from high to low conditions. Within the cell, the HCO3– concentrated by the cyanobacteria is 100-fold more in presence of light (Miller et al. 1990), whereas eukaryotic algae accumulate only 20-fold.

EFFECT OF CO2 SUPPLEMENTATION ON CARBON CONCENTRATING MECHANISM (CCM) The studies on the effect of CO2 on the metabolic pathways are still in their infancy. Hence, the understandings of the detailed mechanisms of CO2 sequestration and its regulatory network might help to improve the CO2 utilization along with enhanced production of value-added products including lipids and carotenoids, etc. (Zhu et al. 2017). RuBisCO is one of the major enzymes responsible for fixation of atmospheric CO2 to organic carbon in microalgae. This enzyme shows less affinity towards CO2, and O2 is a competitive substrate for CO2 which may lead to photorespiration. Therefore, when O2 is increased and CO2 is decreased in the atmosphere, it affects the carboxylase activity of RuBisCO due to decreased ratio of CO2:O2. The source of CO2 required for the photosynthesis for the aquatic organisms is from surrounding water in which the CO2 diffuses 10,000 times slower than it does in the air. In order to enhance the CO2 concentration in the vicinity of RuBisCO for improved photosynthesis, these aquatic organisms have an inorganic CCM inside the cell (Hwangbo et al. 2018) which enhances the uptake of CO2 (Swarnalatha et al. 2015). The CO2 tolerance and biofixation rates of various microalgal strains are given in Table 6.2. The importance of CCM in microalgae is to elevate the CO2 concentration near the active site of RuBisCO at lower CO2 concentration. However, the higher CO2 concentration of this mechanism is less active. According to Winck et al. (2016), elevated CO2 during microalgae cultivation triggers carbon fixation by the alternative synthesis of compounds. These compounds may inhibit photosynthesis or enhance energy losses through photorespiration. In contrast, at low CO2 concentrations, CCM related genes are overexpressed to enhance carbon uptake (Winck et al. 2016). Carbonic anhydrases

Handbook of Algal Technologies and Phytochemicals

and inorganic carbon transporters are two major factors of CCM (Baba and Shiraiwa 2012). It is known that the carbonic anhydrase catalyzes the reversible inter-conversion of CO2 to carbonic acid. This helps to increase CO2 concentration near the site of RuBisCO, compensating the low affinity of RuBisCO for CO2 followed by an increased carbon uptake. The carbon transporters located in the plasma membranes and chloroplast are suggested as main proteins in the carbon uptake process (Swarnalatha et al. 2015; Winck et al. 2016). The results showed that the gene encoding the carbonic anhydrase enzymes (CAH1, CAH4, CAH5) and a formate/nitrite transporter that increases HCO3– transport in the stroma (LCIA or low-CO2 inducible protein A) are higher at low CO2 concentration (0.04%) (Fang et al. 2012).

EFFECT OF CO2 SUPPLEMENTATION ON THE CALVIN CYCLE The supplementation of CO2 and its assimilation in microalgal cells are dependent on the photosynthetic CO2 fixation, known as the Calvin cycle. It has been observed that the CO2 supplementation up-regulated the genes encoding the major enzymes in the Calvin cycle. Phosphoglycerate kinase (PGK) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) are up-regulated. It catalyzes the phosphorylation and reduction of 3-carbon intermediates, respectively, in the presence of ATP and NADPH to generate glyceraldehyde-3-phosphate. This up-regulation of the Calvin cycle by CO2 supplementation shows higher CO2 fixation by microalgae resulting in increased biomass production. Even so, it is reported that the higher CO2 concentration inhibited the photosynthetic efficiency of microalgae. According to Winck et al. (2016), at 10% CO2 supplementation sucrose was reduced, and the xylose was accumulated, which is a clear indication of inhibition of photosynthesis. These results also suggest that photorespiration or an alternative pathway with similar substrates and products may be modulated in cells at a high CO2 concentration. It has been observed that the gene encoding the ferredoxin was up-regulated by CO2 supplementation which is suggested to enhance the Calvin cycle and carbohydrate synthesis (Peng et al. 2016; Zhu et al. 2017).

EFFECT OF CO2 SUPPLEMENTATION ON CARBOHYDRATE METABOLISM Glycolysis and Gluconeogenesis CO2 supplementation is known to significantly affect the carbohydrate metabolism. The study carried out by

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Carbon Dioxide Sequestration by Microalgae

TABLE 6.2 The CO2 Tolerance and Bio-fixation Rate of Microalgal Strains Species Chlorella sp. Mutant Chlorella pyrenoidosa Chlorella sp. Chlorella vulgaris Chlorella kessleri Scenedesmus sp. Scenedesmus obliquus Scenedesmus obliquus Scenedesmus dimorphus Svenedesmus obtusus Desmodesmus sp. Kirchneriella cornuta Acutodesmus sp. H. pluvialis mutant Haematococcus pluvialis Nostoc flagelliform Anabaena sp. Tetraselmis suecica Nannochloropsis gaditana

CO2 Concentration (%)

CO2 Bio-fixation Rate (g/l/d)

15 10 5–15 4 18 20 10 15 15 20 30 30 30 15 16–34 20 10 5–15 8

1.54 0.260 0.097 0.71 0.163 0.42 0.288 0.770 0.174 0.092 0.207 0.198 0.186 2.57 0.143 0.17 1.01 0.111 1.77

Peng (2016) in Coccomyxa subellipsoidea C-169 showed that supplementation of CO2 at 2% up-regulated most of the genes related to glycolysis and suppressed most of the genes related to gluconeogenesis. The genes coding the enzymes phosphofructokinase (PFK) and pyruvate kinase (PK), which are the key regulatory steps in glycolysis, were found to be significantly up-regulated by CO2 supplementation. At the same time, genes encoding enzymes for the unique reactions in gluconeogenesis, PEP carboxykinase (PEPCK) (catalyzes the conversion of oxaloacetate into PEP), and fructose-1, 6-bisphosphatase (FBP) (catalyzes the conversion of fructose-1, 6-biphosphate into fructose- 6-phosphate) were significantly repressed. This finding indicates that the CO2 supplementation indirectly helps in synthesis of building blocks and energy molecules required for enhanced growth and lipid synthesis (Peng et al. 2016; Zhu et al. 2017).

TCA Cycle and Oxidative Phosphorylation The CO2 supplementation up-regulated the gene encoding the components of pyruvate dehydrogenase complex, which is a multienzyme complex catalyzing the conversion of pyruvate into acetyl Co-A for TCA cycle. Similarly, it has been observed that the genes encoding nearly all the enzymes of TCA cycle were up-regulated

References (Cheng et al. 2013) (Tang et al. 2011) (Kassim and Meng 2017) (Adamczyk, Lasek, and Skawińska 2016) (Wang et al. 2008) (Westerhoff et al. 2010) (Tang et al. 2011) (Bagchi and Mallick 2016) (Vidyashankar et al. 2013) (Sarat Chandra et al. 2016) (Swarnalatha et al. 2015) (Swarnalatha et al. 2015) (Swarnalatha et al. 2015) (Li et al. 2017) (Huntley and Redalje 2007) (Lv et al. 2014) (Chiang, Lee, and Chen 2011) (Kassim and Meng 2017) (Adamczyk, Lasek, and Skawińska 2016)

by the CO2 supplementation which includes citrate synthase, isocitrate dehydrogenase, aconitase, oxoglutarate, dehydrogenase, succinyl-CoA synthetase, and fumarase. This implies that the CO2 supplementation improves the TCA cycle providing more NADH, ATP, and GTP. In addition, the genes encoded for the enzymes for anapleurotic reactions including PEP carboxylase and pyruvate carboxylase (catalyzing the carboxylation of PEP and pyruvate, respectively) were up-regulated on CO2 supplementation providing more oxaloacetate to replenish the TCA cycle. Correspondingly, the genes involved in electron transport and oxidative phosphorylation were significantly up-regulated on CO2 supplementation. This includes several subunits of the Complex I (NADH dehydrogenase), III (cytochrome bc1 complex), IV (cytochrome c oxidase), and ATP synthase leading improved electron flow to the O2 and increased ATP production. This indicated that CO2 supplementation led to increased metabolic energy to sustain the increased growth of microalgae (Peng et al. 2016; Zhu et al. 2017).

Effect of CO2 Supplementation on Protein Metabolism The C:N ratio plays an important role in microalgal growth. CO2 supplementation to microalgal cultures

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was noted to up-regulate the genes involved in nitrogen acquisition and assimilation (Peng et al. 2016). The genes encoding nitrate/nitrite transporter and ammonium transporters were observed to be up-regulated by CO2 supplementation to microalgae. In addition, the genes encoding enzymes involved in the assimilation of extracellular nitrogen into ammonium including nitrate reductase and nitrite reductase were up-regulated by CO2 supplementation (Peng et al. 2016).

Acetyl Co-A Carboxylase catalyzes the first step in the fatty acid biosynthesis which generates malonyl Co-A from acetyl Co-A. This malonyl Co-A gets further transferred to an acyl carrier protein which undergoes a series of reactions catalyzed by fatty acid synthase complex (FAS) towards fatty acid biosynthesis. Peng et al. (2016) observed that the genes coding ACC and FAS I were up-regulated by CO2 supplementation indicating its role in lipid accumulation upon CO2 supplementation.

Effect of CO2 Supplementation on Lipid Metabolism

INDUSTRIAL APPLICATION

The reports indicate that the CO2 supplementation enhances the lipid content in various microalgal strains. According to Peng et al. (2016), the enhancement of lipid accumulation resulted in the significant down-regulation of the genes encoding enzymes for fatty acid degradation and the up-regulation of fatty acid synthesis.

Microalgae are well known to produce non methane hydrocarbons (ethane, ethylene, propane, propylene, butane, pentane, hexane, and ethylene) (Schobert and Elstner 1980; Shaw et al. 2003), organo-halogens (chloroform, trichloroethylene, bromo methane, chloromethane, and iodo methane), and aldehydes (propanal, hexanal, n-heptanal, formaldehyde, acetaldehyde, furfural, and

TABLE 6.3 Potential Uses of Microalgal Biomass Application

Examples

References

Human food

Source of single-cell protein and use in the supplementation of products such as pastas, soups, and beverages Frequent use of some species in the feeding of fish and shellfish Source of chlorophyll a, phycocyanin, β-carotene, γ- linolenic acid, eicosapentaenoic acid, and stable isotope biochemical Use of the biomass as a source of nitrogen and phosphorous in tillable land Production of CH4 in fermenters by the digestion of biomass Production of biodiesel from the lipid fraction of the cells Production of synthesis gas from the biomass Production source of carbonates and bicarbonates Polymers source of exocellular sugars and proteins Production of hydrocarbons, aldehydes, and organohalogens

(Rodriguez-Garcia and Guil-Guerrero 2008) (Olvera-Novoa et al. 1998) (Spolaore et al. 2006)

Animal feed High-value molecules Fertilizers Natural gas production Biodiesel production Syngas production Inorganic salts Renewable production Volatile organic compounds

(Chae, Hwang, and Shin 2006) (Yen and Brune 2007) (Miao and Wu 2006) (Amin 2009) (Lee, Apel, and Walton 2004) (Ishida et al. 1997) (Muñoz et al. 2004)

TABLE 6.4 Important Algal Species for Production of High Value Metabolites of Biological Significance Algal Sp.

High Value Metabolites

Reference

Haematococcus pluvialis Dunaliella sp. Spirulina

Astaxanthin

(Del Campo et al. 2007; Ambati et al. 2014; Ranga Rao et al. 2018) (Del Campo et al. 2007) (Becker 2007; Becker and Venkataraman 1982)

Botryococcus Porphyridium Chlorella Scenedesmus

β-carotene, glycerol, protein High protein, essential amino acids, vitamin B complex and γ- linolenic acid, β-carotene, phycocyanin, chlorophyll Hydrocarbon Phycoerythrin Lutein, protein, minerals Protein, essential amino acids

(Dayananda et al. 2005) (Dufossé et al. 2005; Kathiresan et al. 2007) (Becker and Venkataraman 1982) (Becker and Venkataraman 1982)

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valeraldehyde) (Nuccio et al. 1995; Scarratt and Moore 1996; Schobert and Elstner 1980). These compounds are synthesized and released extracellularly by microalgae. In addition, the microalgae are also known to produce extracellular proteins and carbohydrates. All these compounds are used in pharmaceutical and food industries (Su et al. 2003). The production of metabolites and biomolecules such as high-value pigments, proteins, carbohydrates, and biopolymers has commercial potential (Table 6.3 and Table 6.4). Microalgae are therefore considered as the biorefinery system being able to produce all these valueadded compounds. Since microalgae are known to capture CO2, it helps in CO2 abatement and simultaneously provides effective solutions to the energy crisis. The CO2 sequestration rates are low and are not viable for processes that aim only to obtain carbon credits and value-added products. Closed photobioreactors have higher rates of biotransformation of CO2 into bio products and the greatest potential for commercial application (Eriksen 2008). However, there are still many hurdles to overcome before these processes can become fully scalable.

FUTURE NEEDS Microalgal CO2 sequestration provides an opportunity to reduce environmental impacts of CO2, use of fossil fuels for energy, and to produce various food and feed grade high value metabolites at a cheaper cost. However, efficient CO2 assimilation by microalgae remains one of the research areas to focus upon by microalgal biotechnologists. the following are some of the future directions to further improve the microalgal CO2 sequestration. 1. Screening of robust microalgal strains for high CO2 tolerance, high biomass production, and compatibility to proliferate in a geographicalregion-specific climate from which the local climatic conditions can be considered. 2. Mixed microalgal and algal bacterial consortium can be tested in open raceway pond as well as in closed photobioreactor to check its potential for CO2 sequestration. 3. A hybrid system comprising carbonic anhydrase (known for CO2 dissolution) and microalgae can be studied to enhance the CO2 solubility in cultivation medium to further improve the CO2 sequestration and biomass production potential of microalgae. 4. To further improve the economics of the system, microalgae cultivation using CO2 can be integrated with the wastewater treatment.

5. Improved microalgal strain development through genetic engineering for high CO2 tolerance remains an area to be studied in detail. 6. Effective and efficient CO2 delivery systems need to be devised for open ponds and closed photobioreactors to avoid CO2 losses.

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7

Modulation of Lipid Biosynthesis by Stress in Diatoms Bing Huang, Virginie Mimouni, Annick Morant-Manceau, Justine Marchand, Lionel Ulmann, and Benoît Schoefs

CONTENTS Introduction���������������������������������������������������������������������������������������������������������������������������������������������������������������77 Stress Modifies the Lipid Composition and Membrane Remodeling�����������������������������������������������������������������������78 Nutrients ��������������������������������������������������������������������������������������������������������������������������������������������������������������78 Nitrogen������������������������������������������������������������������������������������������������������������������������������������������������������������78 Phosphorus�������������������������������������������������������������������������������������������������������������������������������������������������������79 Light Intensity������������������������������������������������������������������������������������������������������������������������������������������������������79 Temperature����������������������������������������������������������������������������������������������������������������������������������������������������������80 Salinity������������������������������������������������������������������������������������������������������������������������������������������������������������������80 Arsenic������������������������������������������������������������������������������������������������������������������������������������������������������������������80 Regulation Mechanisms��������������������������������������������������������������������������������������������������������������������������������������������80 Bioengineering of Enzymes Involved in Pathways Providing Acetyl-CoA for Fatty Acid Biosynthesis������������80 Overproducing Enzymes Involved in Fatty-Acid Biosynthesis����������������������������������������������������������������������������81 Overproducing Enzymes Involved in TAG Formation�����������������������������������������������������������������������������������������82 Silencing or Deleting Genes Controlling Competing Pathways��������������������������������������������������������������������������83 Increasing the Stability of the Lipid Droplets������������������������������������������������������������������������������������������������������83 Conclusions and Perspectives�����������������������������������������������������������������������������������������������������������������������������������84 References�����������������������������������������������������������������������������������������������������������������������������������������������������������������85

BOX 7.1  SALIENT FEATURES Diatoms are photosynthetic microalgae and are among the most abundant, diverse and efficient aquatic primary producers. Because lipids are compounds with a high density of energy, diatoms reorient the carbon metabolism towards lipid production under stress. Beside its interest for basic sciences, the understanding of carbon reorientation is crucial for microalgal biotechnology. This review starts with a short overview of the lipid biochemistry in diatoms taking into account the particular cellular context of these organisms. Then, the effects of stress on the production of triacylglycerol accumulation and membrane remodeling are reviewed. Regulatory branch points influencing the supply of intermediate molecules and metabolic regulators controlling the fate of carbon into the different metabolic pathways will be highlighted. Altogether, the knowledge about the

modulation of lipid biosynthesis in diatoms opens a new avenue for the biotechnological use of stress to produce algae enriched in specific lipids or lipids themselves. The progress in this field is described in the last part of the chapter.

INTRODUCTION Recently, diatoms received a great deal of attention because under stress, the carbon metabolism is reoriented toward the production of lipids and especially triacylglycerols (TAG) (Heydarizadeh et al., 2013; Heydarizadeh et al., 2017). In the most favorable case, i.e. when a carbon source is available, the lipid cellular quota can account for up to 60% DW (Sayanova et al., 2017; Heydarizadeh et al., 2017). A unique feature of TAGs accumulated by diatoms is their enrichment in medium-chain fatty acids and very long chain-polyunsaturated fatty acids (VLC-PUFAs). VLC-PUFAs are essential ω3 fatty acids 77

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with applications in pharmaceutical and nutraceutical industries (Mimouni et al., 2012). A hierarchical cluster analysis of PUFA profiles of 144 Heterokonts, and including 19 different PUFA species, was carried out using a large published dataset of fatty acid profiles for microalgae (Zulu et al., 2018). This study suggests that the inter- and intra-specific diversity of reported PUFA profiles is related to the existence or the absence of certain FA-modifying enzymes, or just different substrate specificities of enzymes significantly shape FA profiles. This genetic diversity makes diatoms an attractive model to study lipid metabolism and to identify a large set of biotechnologically relevant enzymes (Lang et al., 2011). It is out of the scope of this chapter to propose a comprehensive review of lipid biosynthesis, and the interested readers are advised to read recent reviews on this topic (Zulu et al., 2018; Mimouni et al., 2018). Instead, the most important steps for the understanding of stress actions are presented below. In diatoms, both the prokaryotic pathway, in the chloroplast envelopes, and the eukaryotic pathway, in the endoplasmic reticulum, coexist, and they look homologous to their counterpart in land plants (Tanaka et al., 2015). The FA biosynthesis starts with the acetyl-CoA, a compound produced in several sub compartments, i.e. mitochondria, peroxisome and plastid (Heydarizadeh et al., 2017). Whether these pools can be connected is still under debate as all transporters have not yet been found (Marchand et al., 2018). In the plastid, acetyl-CoA is synthesized either from free acetate (Lin and Oliver, 2008) or through the pyruvate decarboxylation, a reaction catalyzed by pyruvate dehydrogenase complex (PDC). Regardless of the route used, it is converted to malonyl-CoA through the catalytic action of ATP-dependent acetyl-CoA carboxylase (ACCase). In diatoms, plastidial and cytosolic forms of the ACCase were identified (Tanaka et al., 2015). The next steps of the prokaryotic pathway consist of a sequential condensation of two-carbon units, which is catalyzed by the enzymes of the FA synthase (FAS) complex. Two major classes of FAS, i.e. FASI and FASII, are known. FASII complex being active in the mitochondria and plastids, it is the major supplier of FAs in plastids (Ryall, Harper and Keeling, 2003). FA synthesis requires four enzymatic reactions consisting of the addition of two carbon units from malonyl-ACP to the growing acyl chain (Li-Beisson et al., 2010). Synthesized acyl-ACP could be hydrolyzed in the inner envelope of the plastid resulting in the formation of free FAs (FFAs), that, in turn, can be exported to ER for the synthesis of glycerolipids (Chapman and Ohlrogge, 2012) through the “eukaryotic pathway.” The length of FAs found in diatom lipids varies mainly from 14 to 24 carbon atoms (Yang et al., 2017). The individual

Handbook of Algal Technologies and Phytochemicals

proportion of each FAs differs between taxa and can be modulated by changes in culture conditions. Besides EPA and DHA, diatoms are also able to synthesize other VLCPUFAs composed of 24 to 28 carbon atoms, although usually present as traces (Stonik and Stonik, 2015). TAGs synthesis takes place in the ER (Moriyama et al., 2018). Acyl-CoA dependent (Kennedy pathway) and independent pathways have been described. In the former pathway, G-3-P is involved in TAG biosynthesis as previously described (Liu and Benning, 2013). The last reaction is catalyzed by acyl-CoA:DAG acyltransferase (DGAT), which is coded by several isogenes. In the latter pathway, phosphatidylcholine (PC) is used as an acyl donor for the formation of TAG from DAG (Xu et al., 2016). The TAG composition in FA differs between diatom taxa suggesting different regulation pathways of DGAT. The acyl-CoAs used in this pathway can be obtained either from de novo synthesized 16:0-, 16:1- and 20:5-CoAs FAs in plastids (Popko et al., 2016) or from the remodeling of phospholipids by the enzymes of the Lands' cycle, which supplies the acyl-CoA pool with mainly 18:2 and 18:3-CoAs FAs. Synthesized TAGs are assembled into lipid droplets (LDs) at specialized ER sites before being released into the cytoplasm (Maeda et al., 2017).

STRESS MODIFIES THE LIPID COMPOSITION AND MEMBRANE REMODELING Nutrients Nitrogen Nitrogen is often considered as the most critical nutrient triggering the accumulation of neutral lipids (Sharma, Schuhmann and Schenk, 2012). While under nutrientreplete conditions, the light environment strongly controls the quantum efficiency of carbon-related biomass production (ΦC) and the metabolic costs of carbon production; under nitrogen-limited conditions, the light environment has less or even no influence on ΦC and the metabolic costs (Jakob et al., 2007). The excess of absorbed energy is dissipated as heat and in alternative electron-consuming reactions, while the carbon flux shifts from proteins to carbohydrates and lipids (Wagner et al., 2017). Under nitrogen limitation the degradation of intracellular compounds may offer the carbon skeletons for FAs de novo synthesis (Ge et al., 2014). Genes related to nitrogen assimilation are up-regulated, whereas genes related to lipid biosynthesis are mostly down-regulated, suggesting that TAG accumulation is a consequence of remodeling of intermediate metabolism rather than induction of lipid biosynthesis genes. Moreover, nitrogen depletion in Phaeodactylum tricornutum leads to

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the remodeling of membranes together with polar lipids recycling towards TAG formation (Huang et al., 2019). In P. tricornutum, non-phosphorous polar lipids, betaine lipids (BLs), seem to be the major source for TAG synthesis (Popko et al., 2016). In the Pt4 strain, the proportions of polyunsaturated EPA and DHA decrease due to the de novo synthesis of C16 FAs in neutral lipids while EPA is transferred from glycolipids (GLs) to phospholipids (Popko et al., 2016). Compared with replete conditions, nitrogen-starvation triggered in the Pt1 strain an increase of the amount of EPA in TAG and a decrease in MGDG, while EPA’s amount remained unchanged in phospholipids (Abida et al., 2015). Phosphorus Phosphorus is an essential element for all organisms because it is involved in the composition of essential molecules such as phospholipids that play an important role in phosphate storage. In low-phosphorus conditions, diatoms reduce the cell phosphorus demand by 10–30% thanks to the replacement of phospholipids with BLs (Abida et al., 2015) such as diacylglycerylcarboxyhydroxymethylcholine (DGCC), diacylgyceroltrimethylhomoserine (DGTS) and diacylglycerylhydroxymeth yltrimethyl-β-alanine (DGTA) (Cañavate et al., 2016). Counterbalancing between phospholipids and BLs in response to phosphorus availability has been demonstrated in P. tricornutum (Cañavate et al., 2016). Under phosphorus depletion, membrane phospholipid degradation was supported by the upregulation of phospholipases, while the expression of a putative DGTA synthase was increased by 30–80-fold (Cruz de Carvalho et al., 2016). The phospholipid biosynthesis pathway may not be completely inactive as a shift of lipid classes could occur via phospholipid-recycling mechanisms (Huang et al., 2019). In Chaetoceros gracilis, which contains phospholipids and DGCC, phosphorus deprivation decreases the phospholipid content without increasing BL (Cañavate, Armada and Hachero-Cruzado, 2017), revealing that the dynamics between phospholipids and BL are taxon-dependent. Under low phosphorus conditions, Thalassiosira pseudonana substitutes PC with the DGCC, and phosphatidylglycerol (PG) with sulfoquinovosyl diacylglycerol (SQDG) (Van Mooy et al., 2009). The increase of several diglycosylceramide lipids as much as ten-fold may represent another substitution possibility (Hunter et al., 2018). Under phosphorus stress, the TAG content increases (Cruz de Carvalho et al., 2016). However, genes related to lipid biosynthesis are mostly not differentially regulated or show diverging responses under phosphorus depletion (Huang et al., 2019). Valenzuela et al. (2012) suggested a “push”

scenario under nitrogen- or phosphorus-limited conditions, in which neutral lipid accumulation could be a passive pathway that depended on the relative growth rate and on the total cellular carbon content. Contrary to nitrogen depletion, under phosphorus limitation TAG accumulation in P. tricornutum does not rely on the degradation of intracellular compounds but on the efficiency of carbon fixation (Huang et al., 2019).

Light Intensity Light intensity impacts lipid biosynthesis and FA composition. Whereas low light (LL) generally induces the synthesis of polar lipids mainly associated with chloroplast membranes, high light intensity (HL) decreases the total polar lipid content with a simultaneous increase in the amount of neutral lipids, especially TAG (Sharma, Schuhmann and Schenk, 2012), suggesting the involvement of plastid membrane degradation (Goodson et al., 2011). In P. tricornutum, the typical constitutive lipids of thylakoid membranes are the galactolipids MGDG, DGDG and SQDG, and the phospholipids PG (Wilhelm et al., 2014). When P. tricornutum is exposed to LL, even if MGDG remains the most abundant galactolipid of thylakoid membranes, SQDG concentration increased. In contrast, under HL, the SQDG became higher than MGDG concentration (Lepetit et al., 2012). Numerous studies revealed that MGDG has a photoprotective activity under saturating light intensities (Wilhelm et al., 2014). Because SQDG has a lower solubilization capacity for diadinoxanthin than MGDG (Goss et al., 2009), SQDG can be considered as an antagonist to MGDG. In T. pseudonana grown under HL, a large increase of the level of saturated FA, especially 14:0 and 16:0, was observed while the amounts of monounsaturated (notably 18:1ω9) and polyunsaturated FAs (mainly 20:5ω3 and 22:6ω3) decreased by 70–100% (Dong et al., 2016). This altered FA composition may raise the capability to resist to a HL stress, for instance by alleviating ROS formation at PSII. P. tricornutum appeared to be an exception because the highest TAG yield was reached under LL (60 μmol photon m−2 s−1) (Remmers et al., 2017). A four-day dark stress enhanced TAG productivity by up to 50% compared to the light in P. tricornutum. The amounts of 16:0, 16:1 and EPA were increased, whereas no changes in the FA composition were reported at various light intensities (Remmers et al., 2017). Because nitrogen limitation and HL have antagonist effects on diatom lipid productivity, Huerte-Ortega et al. (2018) investigated the combined effect of these two factors on the induction of lipid accumulation in P. tricornutum and demonstrated that the combination is a good strategy

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to enhance lipid biosynthesis without compromising the growth rate, which can be of great interest for the production of biofuels.

Temperature Low temperatures have been used to enhance lipid biosynthesis and modify the composition of VLCPUFAs (Yang et al., 2013). Colder temperature generally increases the unsaturation of FAs and may therefore increase the relative amounts of EPA or DHA (Ravet, Brett and Arhonditsis, 2010). In the industrially cultured diatom Odontella aurita, growth and lipid composition were regulated according to culture temperature but also according to growth phase. Indeed, EPA and DHA levels were increased at low temperatures (8°C) by comparison to those observed at 16 and 24°C, with a decrease in saturated FAs (Pasquet et al., 2014). Otherwise, it has been shown that cultivation of P. tricornutum at 30°C resulted in the exchange of EPA in galactolipids by shorter C18 FAs (Feijão et al., 2018), suggesting that this mechanism participates in the adaptation to high temperature.

Salinity

Handbook of Algal Technologies and Phytochemicals

is accomplished by the phosphate transport system, aquaglyceroporins and hexoses permeases (Wang et al., 2015). At the cellular level, arsenate competes with phosphate in phosphorylation and oxidative phosphorylation reactions (Fujiwara et al., 2000), generates an oxidative stress and participates in division inhibition (Levy et al., 2009). Arsenite interferes with protein synthesis leading to membrane degradation and cell death (Wang et al., 2015). Arsenate competing with phosphate in microalgal metabolism has been tested in the diatom P. tricornutum and Thalassiosira pseudonana. Arsenic was found in several cell fractions, including the lipid one (Duncan et al., 2013). The dominant arsenic species contained in the lipid-soluble fraction were sulfate-, glycerol- and phosphate arsenoribosides with 50 to 80% of the total arsenic of this fraction. Arsenoribosides are considered as the final As species in microalgae (Edmonds and Francesconi, 2003). Because arsenoriboside-containing phospholipid has been identified (García-Salgado et al., 2012), it has been proposed that this species could be the product of arsenolipid hydrolysis present in the lipidsoluble fraction (Duncan et al., 2013). Few studies have been dedicated to the toxicological effect of arsenic on diatoms. In rice, arsenite and silica share the same transport system (Seyfferth and Fendorf, 2012). Silica being a major nutrient in diatoms, it has been hypothesized that silica and As could also be co-transported in diatoms (Ding et al., 2017). In natural waters, mixed arsenic species such as arsenite or arsenate but also organoarsenicals such as monomethylarsenate or dimethylarsenate co-exist (Hellweger et al., 2003), allowing a place for combined or interactive effects between As forms. In the diatom Nitzschia palea, a higher toxicity of arsenite and dimethylarsenate than when individually used has been reported.

Permeability and fluidity of membranes can be altered by osmotic stress. In the marine diatom Nitzschia laevis, Chen, Jiang and Chen (2008) have shown that an increase of the NaCl concentration from 10 to 20 g/L induces a decrease of neutral lipids content and an increase in polar lipids, in particular PL and GL. The degree of FA unsaturation of neutral and polar lipids also increased sharply. However, a decrease in unsaturation of FA incorporated into both neutral and polar lipids was induced when microalgae were submitted to NaCl concentration above 20 g/L, suggesting that high salt concentrations decrease membrane permeability and fluidity as an adaptation to prevent cell damages.

REGULATION MECHANISMS

Arsenic

Bioengineering of Enzymes Involved in Pathways Providing Acetyl-CoA for Fatty Acid Biosynthesis

Arsenic is a metalloid released by human activities and can be found in fresh and marine water (Wang et al., 2015). In microalgae, arsenic compounds are found in cytosolic and membrane fractions, as inorganic arsenic species such as arsenite and arsenate or as different forms such as arsenobetaine, arsenocholine, arsenosugars and in lipid-soluble fractions as arsenolipids (Duncan et al., 2013). These molecular species are less toxic than inorganic ones (Hsieh and Jiang, 2012) except for arsenolipids (Meyer et al., 2014). The accumulation of organoarsenical compounds in microalgae

In microalgae, there is only fragmented information about carbon flux toward the generation of acetyl-CoA for FA synthesis and in particular about the regulation of the different acetyl-CoA-generating routes (Shtaida, Khozin-Goldberg and Boussiba, 2015). The study of Ma et al. (2014) has shed some new light on the regulatory mechanisms of PDC. PDC catalyzes the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA. The activity of the mitochondrial PDC, unlike its plastid counterpart, is repressed by pyruvate dehydrogenase kinase (PDK). The silencing of the mitochondrial

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PDK-encoding gene (PtPDK) resulted in an increase of up to 82% of the neutral lipid content, revealing an important contribution of the mitochondrial acetyl-CoA pool to the lipid metabolism in diatoms (Ma et al., 2014). The malic enzyme (ME) has been demonstrated to be a major regulator of lipid synthesis. ME is indeed a key enzyme of the pyruvate metabolism since it catalyzes the irreversible oxidative decarboxylation of malate to pyruvate, yielding NADPH and CO2. The production of pyruvate and NADPH is vital for FA biosynthesis. The overexpression of P. tricornutum mitochondrial ME1 gene (PtME1) significantly enhanced (2.5-fold) the total lipid content while keeping the same growth rate as the wild type (WT). The neutral lipid content was further increased by 31% under nitrogen-deprivation, still 66% higher than that of WT with also larger oil bodies (Xue et al., 2015). The increase was mostly due to the accumulation of neutral lipids holding SFAs and PUFAs (Zhu et al., 2018). Additionally, increased ME1 activity was accompanied by elevated NADPH content in all transformants, indicating that ME1 activity plays an important role in the NADPH levels required for lipid synthesis and FA desaturation in P. tricornutum (Zhu et al., 2018). Glycerol is an important raw material for TAG synthesis. Glycerol-3-phosphate dehydrogenase (GPDH) is part of an important pathway in which the reduction of dihydroxyacetone phosphate (DHAP) from the glycolytic pathway is catalyzed into glycerol-3-phosphate (G3P) in a reversible manner. G3P is an essential precursor for DAG and TAG biosynthesis by the Kennedy pathway and may act as a bridge for carbon transfer between carbohydrate and lipid metabolism (Zulu et al., 2018). When glycerolipid synthesis is highly induced (nitrogenor/and phosphorus-starvation), the utilization of G3P by the Kennedy pathway for TAG production might even be predominant on glycerol production. GPDH activity may therefore be important for lipid biosynthesis (Driver et al., 2017). In P. tricornutum, overexpressing the gene encoding the GPDH resulted in a 6.8-fold increase of the glycerol concentration per transformant cell compared with the WT. There was also a 60% increase in neutral lipid content despite a 20% decrease in cell concentration. FA profiling was also modified with the significant increase in the levels of C16- and C18-MUFA (Yao et al., 2014). Carbon skeletons from enhanced branched-chain amino acid (BCAA) degradation feed into the TCA cycle and can thus contribute significantly to TAG biosynthesis (Pan et al., 2017). Indeed, Ge et al. (2014) highlighted that many proteins involved in BCAA catabolic processes were significantly altered during TAG accumulation in P. tricornutum. The silencing of the most

significantly upregulated gene, of the BCAA catabolic pathway, encoding the beta-subunit of methylcrotonylCoA carboxylase (MCC2), resulted in decreased TAG accumulation. MCC2 inhibition also gave rise to an incomplete utilization of extracellular nitrogen, thus lowering biomass at the stationary growth phase (Ge et al., 2014). The overexpression of the gene encoding the 3-hydroxyisobutyryl-CoA hydrolase (HIBCH), which is involved in the valine/isoleucine degradation process, resulted in lower isoleucine contents. This accelerated degradation enhances carbon skeletons to the TCA cycle, giving rise to a higher TAG accumulation (Pan et al., 2017). The silencing of the gene encoding the propionyl-CoA carboxylase (PCC) redirected propionylCoA to acetyl-CoA via a modified β-oxidation pathway, resulting in a higher TAG cellular quota. Altogether these results highlight the interconnections between the leucine/valine and isoleucine degradation pathways and their influence on the lipid metabolism.

Overproducing Enzymes Involved in Fatty-Acid Biosynthesis Optimizing the flux of carbon into TAG by overproducing enzymes involved in fatty acid synthesis is called the “push strategy.” The first committed reaction of FA production is the formation of malonyl-CoA from acetylCoA by ACCase. Dunahay et al. (1996) were the first to overexpress an ACCase in Cyclotella cryptica, but no significant enhancement in lipid production in transgenic cells was observed despite a significant increase in ACCase activity. This suggests that the building-up of precursors to the ACCases may play a more significant role in TAG synthesis than the actual ACCase levels (Sayanova et al., 2017) or that a secondary rate-limiting step emerged when ACC was overexpressed (Courchesne et al., 2009). Thioesterases have also been key targets for lipid bioengineering because they determine the FA chain length. The overexpression of the gene encoding the plastidic thioesterase in P. tricornutum (PtTE) enhanced by two-fold the lipid levels but without modifying the FA composition (Gong et al., 2011). The overexpression of the thioesterase from the land plant Umbellularia californica in P. tricornutum impacted the FA chain length with an increase of 6.2 and 15% in 12:0 and 14:0 respectively. Seventy-five to 90% of these mid-chain FAs were found in TAGs (Radakovits, Eduafo and Posewitz, 2011). Other examples of modifying the FA composition in P. tricornutum involve the targeting of desaturases and elongases that are involved in the synthesis

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of long chain-polyunsaturated fatty acids (LC-PUFAs) (Dolch and Marechal, 2015). In P. tricornutum, the overexpression of the endogenous Δ5 desaturase, catalyzing the formation of 20:5 from 20:4, conducted to a 65% increase of neutral lipid levels as well as a modification of the FA composition because monounsaturated FA (MUFA) and PUFA levels were increased by 75 and 64%, respectively (Peng et al., 2014). The overexpression of a heterologous Δ5 desaturase from the green picoalga Ostreococcus tauri into P. tricornutum resulted in DHA levels enhanced by eight-fold. DHA was also shown to accumulate in TAG, which exhibited a novel FA composition (Hamilton et al., 2014). Since P. tricornutum naturally produces very low amounts of DHA, this result suggests that this elongation step limits DHA production (Zulu et al., 2018). Homologous overexpression in T. pseudonana of three elongases led to an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs DHA) depended on the type of elongase that was overexpressed (Cook and Hildebrand, 2016).

Overproducing Enzymes Involved in TAG Formation As in most eukaryotes, diatoms produce TAGs mainly via the acyl-CoA dependent Kennedy pathway (Mühlroth et al., 2013). This pathway begins with the acylation of G3P by acyl-CoA:glycerol-3-phosphate acyl transferase (GPAT), resulting in the formation of lysophosphatidic acid (LPA), which may be further acylated to produce phosphatidic acid (PA) by the acyl-CoA dependent acylCoA:LPA acyltransferase (LPAAT) enzyme (also called acyl-glycerol-3-phosphate acyltransferase [AGPAT]) (Balamurugan et al., 2017). PA may be dephosphorylated by phosphatidic acid phosphatase (PAP) to produce DAG, which in turn, by incorporating the third acyl-CoA thanks to DGAT, will form TAG (Zulu et al., 2018). Different bioengineering studies were performed on diatoms to understand the regulation power of these enzymes on the synthesis of lipids. The strategy to overproduce enzymes involved in TAG assembly is called the “pull strategy.” The overexpression of a GPAT in transgenic P. tricornutum promoted the formation of oil bodies and stimulated the neutral lipid content by two-fold with an overall increased productivity (Niu et al., 2016). A significantly higher proportion of unsaturated FAs was also found in transformants compared to the WT. The study of Balamurugan et al. (2017) showed that the overexpression of AGPAT1 (LPAAT) in P. tricornutum

Handbook of Algal Technologies and Phytochemicals

significantly increased the expression of other key genes such as DGAT2 and GPAT and consequently TAG content while decreasing total carbohydrates and soluble proteins. Moreover, the FA composition in the TAGs of transformants was altered as EPA and DHA levels were increased. DGAT enzymes play an important role in regulating TAG accumulation since they have been identified as the rate-limiting enzyme for oil accumulation in numerous oleaginous organisms (Lung and Weselake, 2006). Different types of DGAT can exist depending on the organism but two major isoforms, encoded by DGAT1 and DGAT2 genes, have been identified as responsible for the bulk of TAG synthesis in most organisms (Guihéneuf et al., 2011). In diatoms, only two DGAT genes were reported (Balamurugan et al., 2017; Dinamarca et al., 2017). Guihéneuf et al. (2011) characterized a DGAT1 gene in P. tricornutum. Its heterologous expression in neutral lipid-deficient yeast restored DGAT1 activity, resulting in lipid body formation in yeast cells. The recombinant yeast appeared to display a preference for incorporating saturated C16 and C18 fatty acids into TAG. Niu et al. (Niu et al., 2013) characterized a DGAT2 from P. tricornutum, and homologous overexpression triggered an increase in the neutral lipid content by 35% compared to the wild type with also a substantial increase (of 76%) in the proportion of PUFAs such as EPA. Moreover, the growth rate of transgenic microalgae remained similar. A similar experiment with another DGAT2 gene (DGAT2D) from P. tricornutum revealed a two-fold higher total lipid content and an incorporation of carbon into lipids more efficiently in transformants than in the WT while growing only 15% slower (Dinamarca et al., 2017). Flux analysis revealed that the increase in lipids was mainly achieved through pyruvate, suggesting that the conversion of pyruvate to acetyl-CoA would provide substrates for the TCA cycle, increasing simultaneously the production of precursors for ACCase toward FA biosynthesis (Dinamarca et al., 2017). Gong et al. (2013) identified a distinct DGAT2 gene in P. tricornutum (PtDGAT2B), the expression of which in TAG-deficient yeast strain completely restored TAG synthesis and lipid body formation while the proportion of unsaturated C16 and C18 fatty acids in yeast TAG was increased. Under nitrogen-replete conditions, PtDGAT2B was strongly upregulated before the onset of TAG accumulation, suggesting that this gene may be a contributor to TAG synthesis in nitrogen-replete cells. In T. pseudonana, similar findings were reported. Xu et al. (2013) functionally characterized a TpDGAT2 in yeast and showed that the TpDGAT2 activity resulted in an altered FA profile, i.e. an increase of C18:1 and

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the incorporation of EPA and DHA into TAG. The overexpression of this gene in T. pseudonana resulted in a significant increase of TAG accumulation and a shift in fatty acid composition without a negative effect on growth or biomass accumulation (Manandhar-Shrestha and Hildebrand, 2015).

Silencing or Deleting Genes Controlling Competing Pathways Lipid catabolism is an important part of the metabolic processing of lipids because it provides energy to the cells. It is intimately involved in both lipid homeostasis and accumulation (Trentacoste et al., 2013). From the biochemical point of view, lipid catabolism involves the release of FFAs by lipases and the subsequent breakdown of these FAs through β-oxidation. Trentacoste et al. (2013) demonstrated in T. pseudonana that the knockdown of a multifunctional lipase/phospholipase/ acyltransferase increased lipid yields without affecting growth under continuous growth and nutrient limitation conditions. FA composition analyses, lipid classes and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis (Trentacoste et al., 2013). In P. tricornutum, Barka et al. (2016) identified a TAG lipase (Tgl1) mediating the first initial step of TAG breakdown from storage lipids. Knockdown mutant strains for this Tgl1 showed a reduction up to 20% of the tgl1-mRNA-level compared to that of the WT, accompanied by a strong increase of TAG in the lipid extracts. In spite of the TAG accumulation, the polar lipid species pattern appeared to be unchanged, confirming the TAG-lipase function of Tgl1 (Barka et al., 2016). Still in P. tricornutum, Li, Pan and Hu (2018) identified another TAG-lipase (OmTGL), localized in the third outermost plastid membrane (Om), and highlighted that the knockdown of OmTGL considerably enhanced neutral lipid content (up to 68–70%). Moreover, FA profiles showed that mostly C20:5 FA as well as glycolipids and phospholipids were degraded under nitrogen stress in the mutants. This study shows that this lipase is a good target for regulating not only TAG accumulation but also LC-PUFA flux to TAGs. It is well documented that TAG accumulation is a consequence of re-allocation of carbon from intermediates of the TCA cycle but also from the degradation of chrysolaminarin (Zhu et al., 2016). Diatoms can produce both lipids and chrysolaminarin as energy reserves, with lipid-to-chrysolaminarin ratios that depend on growth conditions. In the most common situation, chrysolaminarin is the primary energy compound, but how its

production switches to FAs or TAG under specific conditions is not completely understood. Daboussi et al. (2014) were the first to report that a block of the chrysolaminarin pathway by disruption of the UDP-glucose pyrophosphorylase gene (UGPase) using nucleases (meganucleases and TALEN) in P. tricornutum resulted in a 45-fold increase in TAG accumulation. Zhu et al. (Zhu et al., 2016), by knocking down the same gene in P. tricornutum (69% of the UGPase activity was inactivated), highlighted a significant decrease in chrysolaminarin content and an increase in lipid synthesis. In T. pseudonana, Hildebrand, Manandhar-Shrestha and Abbriano (2017) demonstrated that knockdown transformants were accumulating less chrysolaminarin and increased their TAG level, suggesting that UGPase plays an important role in carbon allocation. Their data also suggest that the effect of chrysolaminarin levels on TAG accumulation is triggered by growth cessation and is transient. Silencing nitrate assimilation can also be a strategy to direct the carbon flux toward lipids since they require little nitrogen (Levitan et al., 2015b). In diatoms, the rate‐limiting reaction in the assimilation of nitrate is the reduction of the molecule to nitrite, a reaction catalyzed by the nitrate reductase. The silencing of the nitrate reductase in P. tricornutum revealed an accumulation of over 40% more fatty acids with a 50% lower expression and activity of the enzyme in the transformants compared to the wild type. In contrast to nitrogen‐stressed WT cells, which grow at about 20% of the rate of nitrogen‐replete cells, growth of the transformants was only reduced by about 30% (Levitan et al., 2015b).

Increasing the Stability of the Lipid Droplets Lipid droplets (LD) have a universal architecture across all kingdoms and consist of a phospholipid monolayer and a hydrophobic core in which neutral lipids (mostly TAGs) are stored. LD proteins (LDP) likely function to protect TAG from cytosolic lipases. Increasing the stability of lipid droplets is called the “protect strategy” and has already been adapted on P. tricornutum. Wang et al. (2017) identified a potential lipid droplet protein PtLDP1 predominant in nitrogen-deprived P. tricornutum cells and confirmed its localization into lipid droplets. They showed that overexpressing and silencing the gene coding PtLDP1 led to a significant change in lipid droplet size, neutral lipid or TAG accumulation and also transcriptional levels of key lipogenic genes. These results suggest an important role for PtLDP1 in LD biogenesis and regulation of lipid synthesis. Still in P. tricornutum, Yoneda et al. (2016) identified a major LD protein (StLDP), different from PtLDP1 and containing a

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central hydrophobic domain also found in oleosin of A. thaliana and LDSP of Nannochloropsis. They reported that StLDP expression levels were consistent with the changes of sizes of LDs under N depletion, suggesting a potential role for StLDP in the maintenance, distribution and degradation of TAG in LDs (Yoneda et al., 2016). In Arabidopsis, knockout of the oleosin gene resulted in increased sizes of LDs because the oleosin deficiency significantly inhibited the fusion of LDs (Shimada et al., 2008). The overexpression of StLDP in P. tricornutum led to a significant increase in neutral lipid content between the wild type and the mutants under nitrogen starvation, probably because of the higher protection of TAG from LDP and the interruption of hydrolyzes by lipases (Yoneda et al., 2018). They also observed that the proportion of cells forming three or four lipid droplets increased in mutants compared to WT during the initial nitrogen-deficient period, suggesting that StLDP facilitates the sequestration of TAG.

CONCLUSIONS AND PERSPECTIVES In flowering plants, it has been shown that multigene strategies are much more successful at enhancing lipid levels in comparison to all the single gene strategies described previously (Vanhercke et al., 2014). Some research groups have adapted these multigene strategies as a way of enhancing lipid production in microalgae. For instance, the heterologous co-expression of a Δ6 desaturase and Δ5 elongase from Ostreococcus tauri in P. tricornutum resulted in much higher DHA levels than when the Δ6 desaturase was expressed on its own (Hamilton et al., 2014). Also, Hamilton et al. (2016) generated transgenic strains in which the ∆5 elongase from Ostreococcus tauri was co-expressed with a glucose transporter from the moss Physcomitrella patens in P. tricornutum. The double mutant has the capacity to grow in the dark in liquid medium supplemented with glucose and accumulates substantial levels of omega-3 LC-PUFAs (DHA), highlighting the potential of commercial production under mixo- and hetero-trophic conditions (Hamilton et al., 2016). This multigene strategy still needs further development in diatoms. The use of regulatory factors such as transcription factors (TFs) to control the abundance or activity of multiple enzymes has also provoked growing interest (Capell and Christou, 2004). They are classified into more than 50 families according to their conserved structure and their DNA binding domains (Thiriet-Rupert et al., 2016). TFs modulate transcriptional timing and levels by influencing positively or negatively the efficiency of the transcriptional machinery (Grotewold, 2008). TFs can be classified into

Handbook of Algal Technologies and Phytochemicals

a pyramidal hierarchy, where the high-level TFs influence the low-level ones (Grotewold, 2008). A high-level TF has important impacts on other TFs and in consequence regulates a broad range of genes (Courchesne et al., 2009). Frequently, a combination of TFs may regulate a single metabolic pathway (Santos and Stephanopoulos, 2008). The TFs approach consists in overexpressing a particular TF or multiple TFs or even modifying TF binding domains within target gene promoters in order to affect a large number of genes involved in lipid-related metabolic pathways, resulting in an integrated up- or down-regulation of these pathways simultaneously (Courchesne et al., 2009). This emerging metabolic engineering approach has been demonstrated to be able to improve the production of lipids in plants (Vanhercke et al., 2013). In microalgae, this approach represents an attractive alternative that is likely to bring out the breakthroughs in producing metabolically engineered strains for cost-effective TAG production (Courchesne et al., 2009). Recent studies have begun to use omic approaches to identify differentially regulated TFs in microalgal species, with a focus on lipid metabolism. For example, in microalgae, TFs have been identified as regulators of TAG biosynthesis by acting on components of FA and glycerolipid synthesis, and lipid degradation/remobilization. One such TF is PSR1 from the green alga Chlamydomonas reinhardtii, which is a key component in controlling nutrient starvation-induced TAG biosynthesis (Bajhaiya et al., 2016). Overexpression of PSR1 increases TAG accumulation without inhibiting growth (Ngan et al., 2015), and PSR1 overexpression can also increase starch biosynthesis (Bajhaiya et al., 2016). PSR1 regulates carbon storage metabolism by controlling specific lipid and starch metabolism genes and therefore seems to function as a global regulator of microalgal carbon storage (Bajhaiya, Ziehe Moreira and Pittman, 2017). The overexpression of a bZIP TF and a bHLH TF in Nannochloropsis salina resulted in enhanced growth with concomitant increase in lipid contents (Kang et al., 2015; Kwon et al., 2018). TAG can also be increased in microalgae by heterologous expression of a plant TF (GmDOF4) known to regulate soybean lipid content. For example, in the green alga Chlorella ellipsoidea, the TF GmDOF4, known to regulate soybean lipid content, was also found to modulate FA synthesis and alters the FA composition of TAG of transformant cells (Bajhaiya, Ziehe Moreira and Pittman, 2017). Also, the heterologous overexpression of the Arabidopsis thaliana AtWRI1 in Nannochloropsis salina led to neutral and total lipid contents greater (around 45%) in transformants than in the WT under both normal and stress conditions. They also identified TAGL, DAGK, PPDK, LPL, LPGAT1 and PDH as AtWRI1‐regulated genes (Kang et al., 2017).

Modulation of Lipid Biosynthesis by Stress in Diatoms

Controlling cell quiescence was also identified as a good way to increase nutrient starvation-induced TAG biosynthesis in Chlamydomonas. CHT7 is a Chlamydomonas TF that acts as a regulatory switch between quiescence (during starvation) and proliferation (during nutrient repletion) such that mutating CHT7 promotes starvationinduced TAG accumulation without limiting biomass (Tsai et al., 2014). Numerous TFs have been identified in the genome of diatoms. For instance, a total of 204 and 247 TFs were identified in the genomes of P. tricornutum and T. pseudonana, respectively (Thiriet-Rupert et al., 2016). Transcriptomic analyses indicated that 20 TFs were differentially expressed between nitrogen-replete WT and nitrate reductase knocked down transformants (NR21) of P. tricornutum, suggesting that these TFs play a significant role either in sensing and signal transduction of nitrogen stress or in lipid accumulation (Levitan et al., 2015a). To date, the bioengineering of TFs directly linked to lipid metabolism has not yet been performed on diatoms. By overexpressing the transcription factor bZIP14, Matthijs et al. (2017) showed that it is a regulator of the TCA cycle that plays a central role in carbon reallocation during nitrogen starvation in P. tricornutum. They however did not link it to an increase in lipid content. Studies targeting TFs for the engineering of oil yield in diatoms will definitely increase in the next few years.

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Sayanova, O., Mimouni, V., Ulmann, L., Morant-Manceau, A., Pasquet, V., Schoefs, B. and Napier, J.A. (2017) Modulation of lipid biosynthesis by stress in diatoms. Philosophical Transactions of the Royal Society Series B: Biological Sciences 372(1728), 1728, #20160407. Seyfferth, A.L. and Fendorf, S. (2012) Silicate mineral impacts on the uptake and storage of arsenic and plant nutrients in rice (Oryza sativa L.). Environmental Science and Technology 46(24), 13176–13183. Sharma, K.K., Schuhmann, H. and Schenk, P.M. (2012) High lipid induction in microalgae for biodiesel production. Energies 5(5), 1532–1553. Shimada, T.L., Shimada, T., Takahashi, H., Fukao, Y. and Hara-Nishimura, I. (2008) A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana. The Plant Journal 55(5), 798–809. Shtaida, N., Khozin-Goldberg, I. and Boussiba, S. (2015) The role of pyruvate hub enzymes in supplying carbon precursors for fatty acid synthesis in photosynthetic microalgae. Photosynthesis Research 125(3), 407–422. Stonik, V. and Stonik, I. (2015) Low-molecular-weight metabolites from diatoms: Structures, biological roles and biosynthesis. Marine Drugs 13(6), 3672–3709. Tanaka, T., Maeda, Y., Veluchamy, A., Tanaka, M., Abida, H., Maréchal, E., Bowler, C., Muto, M., Sunaga, Y., Tanaka, M., Yoshino, T., Taniguchi, T., Fukuda, Y., Nemoto, M., Matsumoto, M., Wong, P.S., Aburatani, S. and Fujibuchi, W. (2015) Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome. The Plant Cell 27(1), 162–176. Thiriet-Rupert, S., Carrier, G., Chénais, B., Trottier, C., Bougaran, G., Cadoret, J.P., Schoefs, B. and Saint-Jean, B. (2016) Transcription factors in microalgae: Genomewide prediction and comparative analysis. BMC Genomics 17(1), 282. Trentacoste, E.M., Shrestha, R.P., Smith, S.R., Glé, C., Hartmann, A.C., Hildebrand, M. and Gerwick, W.H. (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proceedings of the National Academy of Sciences of the United States of America 110(49), 19748–19753. Tsai, C.H., Warakanont, J., Takeuchi, T., Sears, B.B., Moellering, E.R. and Benning, C. (2014) The protein Compromised Hydrolysis of triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas. Proceedings of the National Academy of Sciences of the United States of America 111(44), 15833–15838. Valenzuela, J., Mazurie, A., Carlson, R.P., Gerlach, R., Cooksey, K.E., Peyton, B.M. and Fields, M.W. (2012) Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum. Biotechnology for Biofuels 5(1), 40. Van Mooy, B.A.S., Fredricks, H.F., Pedler, B.E., Dyhrman, S.T., Karl, D.M., Koblížek, M., Lomas, M.W., Mincer, T.J., Moore, L.R., Moutin, T., Rappé, M.S. and Webb,

Modulation of Lipid Biosynthesis by Stress in Diatoms

E.A. (2009) Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity. Nature 458(7234), 69–72. Vanhercke, T., El Tahchy, A., Liu, Q., Zhou, X.R., Shrestha, P., Divi, U.K., Ral, J.P., Mansour, M.P., Nichols, P.D., James, C.N., Horn, P.J., Chapman, K.D., Beaudoin, F., Ruiz-López, N., Larkin, P.J., de Feyter, R.C., Singh, S.P. and Petrie, J.R. (2014) Metabolic engineering of biomass for high energy density: Oilseed-like triacylglycerol yields from plant leaves. Plant Biotechnology Journal 12(2), 231–239. Vanhercke, T., El Tahchy, A., Shrestha, P., Zhou, X.R., Singh, S.P. and Petrie, J.R. (2013) Synergistic effect of WRI1 and DGAT1 coexpression on triacylglycerol biosynthesis in plants. FEBS Letters 587(4), 364–369. Wagner, H., Jakob, T., Fanesi, A. and Wilhelm, C. (2017) Towards an understanding of the molecular regulation of carbon allocation in diatoms: The interaction of energy and carbon allocation. Philosophical Transactions of the Royal Society Series B-Biological Sciences 372(1728), #20160410. Wang, X., Hao, T.-B., Balamurugan, S., Yang, W.-D., Liu, J.S., Dong, H.-P. and Li, H.-Y. (2017) A lipid droplet-associated protein involved in lipid droplet biogenesis and triacylglycerol accumulation in the oleaginous microalga Phaeodactylum tricornutum. Algal Research 26, 215–224. Wang, Y., Wang, S., Xu, P., Liu, C., Liu, M., Wang, Y., Wang, C., Zhang, C. and Ge, Y. (2015) Review of arsenic speciation, toxicity and metabolism in microalgae. Reviews in Environmental Science and Bio/Technology 14(3), 427–451. Wilhelm, C., Jungandreas, A., Jakob, T. and Goss, R. (2014) Light acclimation in diatoms: From phenomenology to mechanisms. Marine Genomics 16(5–15), 5–15. Xu, C., Andre, C., Fan, J. and Shanklin, J. (2016) Cellular organization of triacylglycerol biosynthesis in microalgae. In: Lipids in Plant and Algae Development. Y. Nakamura and Y. Li-Beisson (eds.) Springer International Publishing, Cham: pp. 207–221. Xu, J., Kazachkov, M., Jia, Y., Zheng, Z. and Zou, J. (2013) Expression of a type 2 diacylglycerol acyltransferase from Thalassiosira pseudonana in yeast leads to

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incorporation of docosahexaenoic acid β-oxidation intermediates into triacylglycerol. The FEBS Journal 280(23), 6162–6172. Xue, J., Niu, Y.F., Huang, T., Yang, W.D., Liu, J.S. and Li, H.Y. (2015) Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation. Metabolic Engineering 27, 1–9. Yang, Y.H., Du, L., Hosokawa, M., Miyashita, K., Kokubun, Y., Arai, H. and Taroda, H. (2017) Fatty acid and lipid class composition of the microalga Phaeodactylum tricornutum. Journal of Oleo Science 66(4), 363–368. Yang, Z.K., Niu, Y.F., Ma, Y.H., Xue, J., Zhang, M.H., Yang, W.D., Liu, J.S., Lu, S.H., Guan, Y. and Li, H.Y. (2013) Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation. Biotechnology for Biofuels 6(1), 67. Yao, Y., Lu, Y., Peng, K.-T., Huang, T., Niu, Y.-F., Xie, W.H., Yang, W.-D., Liu, J.-S. and Li, H.-Y. (2014) Glycerol and neutral lipid production in the oleaginous marine diatom Phaeodactylum tricornutum promoted by overexpression of glycerol-3-phosphate dehydrogenase. Biotechnology for Biofuels 7(1), 110. Yoneda, K., Yoshida, M., Suzuki, I. and Watanabe, M.M. (2016) Identification of a major lipid droplet protein in a marine diatom Phaeodactylum tricornutum. Plant and Cell Physiology 57(2), 397–406. Yoneda, K., Yoshida, M., Suzuki, I. and Watanabe, M.M. (2018) Homologous expression of lipid droplet proteinenhanced neutral lipid accumulation in the marine diatom Phaeodactylum tricornutum. Journal of Applied Phycology 30(5), 2793–2802. Zhu, B.H., Shi, H.P., Yang, G.P., Lv, N.N., Yang, M. and Pan, K.H. (2016) Silencing UDP-glucose pyrophosphorylase gene in Phaeodactylum tricornutum affects carbon allocation. New Biotechnology 33(1), 237–244. Zhu, B.H., Zhang, R.H., Lv, N.N., Yang, G.P., Wang, Y.S. and Pan, K.H. (2018) The role of malic enzyme on promoting total lipid and fatty acid production in Phaeodactylum tricornutum. Frontiers in Plant Science 9, 826. Zulu, N.N., Zienkiewicz, K., Vollheyde, K. and Feussner, I. (2018) Current trends to comprehend lipid metabolism in diatoms. Progress in Lipid Research 70, 1–16.

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Microalgal Biomass, Lipids, and Fatty Acids Production through Open or Closed Cultivation Systems Challenges and Future Perspectives Ambati Ranga Rao and Gokare A. Ravishankar

CONTENTS Introduction���������������������������������������������������������������������������������������������������������������������������������������������������������������91 Algal Species That Produce High Growth and Productivities����������������������������������������������������������������������������������92 Selection of Algal Species for High Biomass Production Capability������������������������������������������������������������������92 Culture Parameters and Nutrients for Algal Growth �������������������������������������������������������������������������������������������92 Lipid Production and Its Productivities��������������������������������������������������������������������������������������������������������������������93 Innovations in Downstream Processing��������������������������������������������������������������������������������������������������������������������93 Industrial Feasibility�������������������������������������������������������������������������������������������������������������������������������������������������95 Conclusion and Future Perspectives�������������������������������������������������������������������������������������������������������������������������95 Acknowledgment������������������������������������������������������������������������������������������������������������������������������������������������������96 Bibliography��������������������������������������������������������������������������������������������������������������������������������������������������������������96

BOX 8.1  SALIENT FEATURES Globally, people are facing various problems such as climatic changes, global warming effects, and also oil supply crises. Biofuels are in demand in order to reduce gaseous emissions such as nitrogen, carbon dioxide, and sulfur oxides. Biofuels are produced from different crops such as soybean, sunflower, and palm. However, the use of algae can be a suitable alternative biomass feedstock for biofuel production because of its accumulation of high amounts of oils which could be extracted, processed, and refined into transportation fuels using available technology. Algae have a fast growth rate, permit the use of non-arable land and non-potable water, use far less water, and do not compete with food crops; their production is not seasonal, and they can be harvested on a daily basis. Further, algal species accumulate high amounts of lipids and fatty acids under various culture parameters. Select the best algal species for biofuel production depends on growth, biomass yield, and lipids and fatty acids production. This book chapter provides information on biomass

and lipid and fatty acid production of various algal species such as Chlorophyta, Bacillariophyta, Cryptophyta, Cynophyta, Dinoflagellata, Haptop hyta, Eustigatophyceae, Ochrophyta, Rhodophyta, and Labyrinthulomycota for biofuel applications. Further culture parameters and nutrients for algal growth; cultivation of algae in photoautotrophic, heterotrophic, and mixotrophic for biomass and lipid production; downstream processing; and industrial feasibility of algae are provided.

INTRODUCTION Global warming and the depletion of fossil fuels are most important issues confronting the world. Therefore, the production of ecofriendly and renewable biofuel from plants and animals has been attempted as an alternative source. The biodiesel is advantageous to mitigate carbon dioxide and as a substitute for petroleum (Chisti 2007; Vasudevan and Briggs 2008). Algae have several advantages when compared to other plants or crops, viz., a high growth rate, biomass yield, and oil content coupled to ease of biomass production with harnessing

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of solar energy (Lee 2001). Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms that can grow in varied culture conditions. The microalgae are nearly ten times more efficient in biological fixation of atmospheric CO2 than higher plants (Cheng et al. 2006). The important groups of microalgae are diatoms, green algae, blue green algae, and golden algae. These are potential feed stock for the production of high value products. Under favorable culture conditions, the algae can synthesize lipids up to 60% on the dry wt basis (Lv et al. 2010). Fatty acid produced by algae includes medium chain, long chain, very long chain, and fatty acid derivatives. Under stress conditions, algae accumulate lipids in the form of triglycerides. Triglycerides are deposited in lipid bodies located in the cytoplasm of the algal cells. The conclusive advantage of microalgae for biodiesel production vis-à-vis other available feed stock has been discussed by Li et al. (2008a) and Mata et al. (2010). Algal oils contain a high degree of polyunsaturated fatty acids when compared to vegetable oils, which makes them susceptible to oxidation in storage and therefore limits utilization (Ahmed et al. 2011). Biodiesel produced through algal biomass can be used directly in conventional diesel engines. Physical and chemical properties of algal biodiesel are similar to petroleum and diesel. It has several advantages over petroleum diesel, as it is derived from biomass in a renewable, biodegradable, and quasi-carbon neutral manner. It is also nontoxic and contains reduced levels of particulates, carbon monoxide, hydrocarbons, and SOx. Algal biodiesel is more suitable for use in the aviation industry where low freezing points and high energy densities are key (Chisti 2008). Another major advantage of algal biodiesel is the reduced CO2 emissions, up to 78% compared to emissions from petroleum diesel (Fulke et al. 2010). In view of this background, throughout the world, efforts are continuing to improve productivity of biomass, lipid, and fatty acid for increasing the value of feedstock for biodiesel production. The present book chapter covers cultivation of algal species under various culture conditions for biomass, lipid, and fatty acid production for commercial applications to meet global energy needs.

ALGAL SPECIES THAT PRODUCE HIGH GROWTH AND PRODUCTIVITIES Selection of Algal Species for High Biomass Production Capability Selection of algae is based on screening of isolates for high growth rate, lipid biosynthesis, environmental tolerances, and high value-added products. They may be

Handbook of Algal Technologies and Phytochemicals

metabolically engineered to have the desired attributes of high productivity of lipid under the cultivation conditions. A set of culture conditions as close to those expected in the large-scale culture facility needs to be simulated for enhanced productivity. Selecting the high carbon dioxide tolerant algal strains, before adopting them for commercial cultivation, improves the practical feasibility of the process from an economic point of view as a necessary prerequisite. Among the microalgal species, the maximum biomass production was 2.5 and 3.3 (g/L) reported in Phaeodactylum tricormutum and Chaetoceros gracilis (Araujo et al. 2011). The higher biomass productivity (g/L/d) was 2.02 and 3.7 in Chaetoceros gracilis and Nitzschia laevis (Araujo et al. 2011; Chen et al. 2008). Among the micro algal species of the Chlorophyta family, the maximum biomass was 42.6 g/L achieved in Chlorella zofingiensis under fed batch fermenter cultivation (Liu et al. 2010). The biomass content 10.96–17.85 (g/L) was achieved in Botyrococcus braunii and 12–14 g/L in Chlorella protothecoides culture under photobioreactor and flask mode cultivation (Cheng et al. 2009; Li et al. 2007; Orpez et al. 2009). The higher biomass productivity (g/L/d or g/m2/d) was 12.2 and 41 in Chlorella sorokiniana and Chlorella vulgaris under outdoor production system (Cuasresma et al. 2009). In micro algal species of the Cryptophyta family, the biomass productivity was 13.4 g/m2/d, which was achieved in Rhodomonas sp. (Huerlimann et al. 2010). Among the micro algal species of the Cynophyta family, the maximum biomass was 5.2–7.7 g/L, and productivity was 0.39–0.76 g/L/d in Synechocystis and Anabaena sp. (Lopez et al. 2009; Sheng et al. 2011). Among the microalgal species of the Eustigmatophyceae family, the maximum biomass content was 2.5 (g/L), whereas productivity was 0.42 (g/L/d) and 44.8 g/m2/d in Nanochlorophsis sp., as reported by different authors (Gouveia and Oliveira, 2009; Doan et al. 2011; Patil et al. 2007).

Culture Parameters and Nutrients for Algal Growth Algae require nutrients, light, pH, aeration and mixing, temperature, salinity, nitrogen, carbon dioxide, vitamins, and trace elements for their growth (Chisti 2007). Temperature, light, and nutrient supply have a profound influence on the composition of microalgal biomass. Optimal combination of nutrients, light, and temperature results in higher biomass yield and lipid accumulation in algae. Light being a primary source for algal photosynthesis, it plays an important role in the biomass productivity

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which depends on the depth of culture and the density of the algal culture. The blue or the red light spectrum is preferred as these are the most active portions of the light spectrum for photosynthesis. Many algal species are able to grow under different light and dark conditions. High light intensities favor production of lipids. About 45% of the total light spectrum consists of photosynthetically active radiation and thus can be utilized by algal flora to capture CO2 (Gao et al. 2007). Most of the alga forms can grow at temperatures varying between 16–27°C. The temperature of the culture medium has a profound influence on fatty acid production in algae. Increase in saturated fatty acids has been observed with increasing temperature and vice versa for unsaturated fatty acids for lower temperatures (Renaud et al. 2002). Most of the algal species grow in a pH range 7–10, but optimum range is 8.2–8.7. Algae are incredibly tolerant to changes in salinity. Variations in salinity affect microalgae in different ways such as osmosis stress, ion stress, and changes of the cellular ion ratios due to the selective ion permeability of the membrane. Salinity of 20–24 g/L has been found to be optimal, which can improve the production of lipids and fatty acid composition in algae (FAO 1996). For high-density algal culture, the addition of carbon dioxide allows increases in pH, reaching limiting values up to pH 9 during algal growth. Agitation of culture is very important for algal growth. It is necessary to overcome sedimentation of algae and to ensure that all cells are exposed to equal light intensity and nutrients. Nitrogen, phosphorus, ammonia, vitamins, and trace elements are used for algal growth. Microalgal nutrient requirements are met from usage of the fresh water or wastewater. Algae grown in different ratios of carbon, phosphorus, and nitrogen resulted in various growth rates, lipid contents, and cell sizes. Algae treated with wastewater result in the removal of nitrogen and phosphorus from effluents (Shi et al. 2007). Both nitrogen and phosphorus deficiency are known to enhance the accumulation of lipids and fatty acids in algae (Kumar Saha et al. 2003). A general trend towards accumulation of lipids in response to nitrogen deficiency has been observed in numerous species of algae (Merzlyak et al. 2007). It is important to find out an optimal combination of nutrients that gives higher growth rates, biomass yield, and lipid productivities.

LIPID PRODUCTION AND ITS PRODUCTIVITIES Lipids contain different classes such as total lipids (TL), neutral lipids (NL), phosphoplipids (PL), triglyceridies

(TAG) and glycolipids (GL), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglyerol (SQDG), and diacylglycerlytrimethylhomoserine (DGTS). Lipid classes NL, PL, and GL were reported in Nitzschilaevis of Bacillarophyta family (Chen et al. 2008). TL, NL, PL, TAG, and GL were reported in Chlorella sps, Choricystis minor, Dunalliella tertilecta, Nechlorisole abundans, and Scendesmus of the Chlorophyta family (Liu et al. 2010, 2011; Ota et al. 2009, 2011; Pruvost et al. 2011; Takagi et al. 2006). NL, GL, TL, TAG, MGDG, DGDG, SQDG, DGTS, and PL were reported in Pavlova lutheri, Cylindrotheca closterium, and Monodus substeraneus of the Haptophyta family (Guedes et al. 2010; KhozinGoldberg and Cohen 2006; Pruvost et al. 2011) and NL, GL, and PL in Schizochytrium mangrovei of the Labyrinthulomycota family (Fan et al. 2007). Many microalgae strains have high lipid content, around 25–75% dry weight basis; it is possible to increase the concentration by optimizing the growth and culture parameters such as the control of nitrogen level, light intensity, temperature, salinity, carbon dioxide concentration, and harvesting procedure (Chiu et al. 2009; Dayananda et al. 2005; Ranga Rao et al. 2007a, 2007b; Widjaja et al. 2009; Wu and Shi 2007). The open pond cultivation system is most appropriate for mass microalgae production due to low costs (Dayananda et al. 2011; Ranga Rao et al. 2012). Nitrogen is the main factor to improve the lipid accumulation in micro algal culture and also changes in fatty acid composition (Widjaja et al. 2009). Wu and Hsieh (2008) reported that the influence of salinity, nitrogen, and light intensity on lipid productivity enhanced lipid content up to 76%. Table 8.1 represents the lipid content and its productivity in microalgal species.

INNOVATIONS IN DOWNSTREAM PROCESSING Various benefits and significant progress have been made in the development of the algal biofuels. Some technological drawbacks remain when using algal feedstock in the downstream processing for conversion of algal biomass into biofuels, which subsequently diminish the economics of fuel production (Roberts et al. 2013). Major practical limitations for the algal biofuel industry have remained in the extraction of biofuels from wet algal biomass. Moreover, most of the reported literature has focused on upstream processes, i.e., algal cultivation technologies (Zhou et al. 2013). Biodiesel production from microalgae involves four steps, namely, cultivation, harvesting, lipid extraction, and transesterification.

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TABLE 8.1 Biomass and Lipid Producing Microalgal Species under Photoautotrophic, Heterotrophic, and Mixotrophic Cultivation Algal Species

Biomass (g/L)

Lipid (%)

Achnanthes sp. Chaetoceroscalcitrans (CS 178) Botryococcus sp. (TRG) Botryococcusbraunii (LB-572) Botryococcusbraunii (SAG-30.81, CFTRI-Bb-1; CFTRI-Bb-2) Chlorella protothecoides (UTEX 25) Chlorella protothecoides (UTEX25) Chlorella vulgaris (ATC, Brazil) Scenedesmus obliquus (SAG 276-3a) Synechocystis (PCC 6803) Heterosigma sp.

0.09 – – 1.45 1.5–1.9

Phototrophic 44.5 39.8 25.8, 35.9 24–28 25–33

6.4–10.8 6.4–10.8 – 2 1.73 0.07

48.1–63.8 48.1–63.8 16.6–52.5 53 59.5 39.9

Lipid Productivity (mg/L/d or g/m2/d

References

– 17.6 46.9 – –

Doan et al. (2011) Rodolfi et al. (2009) Yeesang and Cheirsilp (2011) Ranga Rao et al. (2007a) Ranga Rao et al. (2007b)

343.2–629.3 343.2–629.3 597.6–3727.5 131 –

De la Hoz Siegler et al. (2011) De la Hoz Siegler et al. (2011) Araujo et al. (2011) Mandal and Mallick (2011) Sheng et al. (2011) Doan et al. (2011)

Chlorella protothecoides Chlorella protothecoides (UTEX 25) Chlorella protothecoides (UTEX) Chlorella protothecoides Chlorella vulgaris (UTEX-259) Chlorella zofingiensis (ATCC 30412) Chlorella protothecoides Chlorella protothecoides (UTEX) Schizochytriummangrovei (isolated)

Heterotrophic cultivation – 43–46 6.4–10.8 48.1–63.8 6 49 15.5 46.1 0.25–1.60 21–38 10.1 52 3.74–3.92 54.7–55.3 17 46 12.2 68

1700–1600 343.2–629.3 586.8 – – 374.4 – 1600 1659

Cheng (2008) De la Hoz Siegler et al. (2011) Gao et al.(2010) Li et al. (2007) Liang et al. (2009) Liu et al. (2011) Xu et al. (2006) Cheng et al. (2009) Fan et al. (2007)

Chlorella protothecoides (249) Chlorella protothecoides Chlorella vulgaris Chlamydomonas reinhardtii (D1 mutant)

Mixotrophic cultivation 0.97–4.41 18.2–52.38 – 40 – 20–44.5 – 52

12.0–25.0 – – 11.6

The downstream processing constituting the last three steps (Figure 8.1) contributes to approximately 60% of the total biodiesel production cost (Kim et al. 2017; Roberts et al. 2013; Roux et al. 2017). In general, suspended algae cultivation involves substantial challenges of biomass harvesting or dewatering that can account for nearly 20–30% of the total cost. However, this may vary based on the type of harvesting technology used, the nature or types of microalgae, and the density of the microalgal culture (Christenson and Sims 2012). Biomass concentrating methods play an important role to increase the solid concentration of microalgae in suspension and volume reduction, which may contribute a considerable amount in the downstream processing. Typically, concentrating processes involve the use of flocculants, centrifugation, gravity sedimentation, or

Heredia-Arroyo et al. (2010) Ruiz et al. (2009) Scarsella et al. (2009) Torri et al. (2011)

flotation, or processes such as auto-flocculation, electroflocculation, or microbial flocculation (Lee et al. 2013; Salim et al. 2011). Flocculation has lower energy requirements than centrifugation and increases the settling rate by aggregating suspended particles to increase the biomass concentration (Lee et al. 2013). The pretreatment of the harvested microalgal biomass is essential for the effective extraction of lipids to augment biofuel yields (Seo et al. 2015). This pretreatment, besides being quite an energy-intensive step, involves a large amount of chemicals, such as acids, bases, organic solvents, and/or physical treatments including cell disruption, autoclaving, sonication, etc., incorporated in either stepwise or simultaneous mode (Seo et al. 2015). At present, solvent extraction methods are most commonly used for lipid extraction as they provide

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Microalgal Biomass, Lipids and Fatty Acids Production 

FIGURE 8.1  Scheme for biodiesel production using microalgae.

the highest lipid recovery. Use of mechanical methods is not a wise option because of poor recovery and the possible degradation of lipids. Solvent-free methods appear promising at the laboratory scale at present, and more research has to be carried out for minimal use of solvents for large-scale commercialization. A more promising way for effective and efficient lipid extraction could be to use combinative methods such as enzymatic and mechanical/solvent extraction methods. Solventfree methods such as enzymatic degradation when combined with other methods will reduce solvent usage/ energy consumption and also increase recovery efficiency. The extracted microalgal lipid is converted into the final product as fatty acid methyl esters (FAME) or biodiesel. The transesterification of microbial lipids to produce biodiesel is conventionally catalyzed by catalysts, such as acid, alkali, solid catalysts, or enzymes. This final stage also accounts for the major cost to the overall biodiesel production process (Tan et al. 2012). The feasibility of algae-based biofuel and biochemical production is largely dependent on technologies that have the potential to be integrated into the existing upstream and downstream steps so as to develop a holistic process for biofuel production (Kim et al. 2017; Roberts et al. 2013). Otherwise, downstream processing steps are performed independently and demand different chemicals and/or equipment, which account for the high processing cost (Seo et al. 2015). The integration of various biomass components as well as the involvement of technologies in the form of the biorefinery model presents an immense opportunity to advance the field of microalgae-based biofuel production. Moreover, it has been reported that the production of more than one product improves the economic efficiency by 33%, compared to one strain and one product specific processes (Laurens et al. 2015).

INDUSTRIAL FEASIBILITY The industrial production of the algal biofuels is attempted worldwide with an aim to find alternative source of bioenergy at affordable costs. The challenges

for industrial production are many. The algal biofuel production is always linked to the available alternate options which are mutually competitive thus influencing the industrial feasibility. However, the ease of adoption of algae-based biodiesel to the existing fossil fuel-dependent machines would make it attractive. The innovations in algal production technologies need to keep pace with the immediate needs for alternate fuels. The growth of algal biomass is still largely inadequate and so also the production of lipids. The technology now uses the destructive method of algal biomass utilization which would limit the process. High production of the lipids coupled to its extracellular release would be advantageous for recovery in continuous cultivation as well as utilization of biomass. Synthetic biology approach is being adopted to increase the productivity of algal biomass to produce lipids. This step of the process linked to the innovative cultivation methodology integrated with efficient downstream processing is the buzzword for the industrial feasibility. There are many industries who are attempting the aforementioned approaches (Table 8.2) with the ultimate objective of delivering the affordable algal biofuels. However, the innovation has been continuous and needs to be supported by a multiprong approach from the strain development to sustainable production in a competitive manner.

CONCLUSION AND FUTURE PERSPECTIVES It is beyond doubt that algae hold tremendous promise for biofuel production harnessing solar energy. Further efforts on algal fuels should concentrate on reducing the costs in large scale production systems. Several innovations are needed to improve the process parameters. Final word has not been said to write off the current approaches, however continuous innovations will pose perpetual challenges. The face of the technology is expected to constantly change with the evolution of the innovative steps. The global efforts should continue to reach the goal of sustainable solutions via algal biofuels for the energy security of the world.

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TABLE 8.2 Some Examples of Companies Engaged in Algal Biofuels Company

Project

Product

Technology

Market

Algenol Biofuels Sonoran Desert (Mexico)

$850 million committed to build algae farm that sells ethanol fuel for $3 per gallon A demonstration facility that could produce up to 3,000 gallons of algal biofuels per acre per year Demo: a 300-acre integrated algal biorefinery

$850 million committed to build algae farm that would sell ethanol fuel for $3 per gallon Lipid for biodiesel

The company claims its process lets it make around 6,000 gallons per acre per year Specialized photobioreactors; extraction through benzene or ether coupled to sonication

Projected production costs will be around 85 cents per gallon

“Green crude”, of composition as crude oil, and compatible with existing refineries Plans $60 to $80 per barrel of biofuel

Biomass production with biorefinery approach

Solix Biofuels Coyote Gulch, Colorado

Sapphire Energy

Solazyme, South San Francisco

Seambiotic, Ashkelon, Israel

Exxon Mobile, USA

Along with Sustainable Oils (camelina-based biofuel) and Honeywell subsidiary UOP (biodiesel) It formed a partnership with NASA to optimize the growth rates of its microalgae

Collaboration with Synthetic Genomics, Inc

Its 1,000-square-meter facility produces roughly 23,000 grams of algae per day—three tons of algal biomass would yield around 100 to 200 gallons of biofuel Bio-gasoline

ACKNOWLEDGMENT The first author would like to thank Vignan’s Foundation for Science, Technology and Research University for their support of this work.

BIBLIOGRAPHY Ahmad, A.L., Yasin, N.H.M., Derek, C.J.C., and Lim, J.K. Microalgae as a sustainable energy source for biodiesel production: A review. Renew Sus Energy Rev 2011. 15: 584–593. Ambati, R.R., Gogisetty, D., Aswathnarayana Gokare, R., Ravi, S., Bikkina, P.N., Su, Y., and Lei, B. Botryococcus as an alternative source of carotenoids and its possible applications–an overview. Crit Rev Biotechnol 2018. 38: 541–558. Ambati, R.R., Phang, S.M., Ravi, S., and Aswathanarayana, R.G. Astaxanthin: Sources, extraction, stability, biological activities and its commercial applications-a review. Mar Drugs 2014. 12: 128–152.

Designer algal cultures metabolically engineered strains grown in large fermenters

Status not known

Plans to make 100 million gallons by 2018 and 1 billion gallons per year by 2025 Solazyme plans to supply fuel to the Air Force and the U.S. Navy as demonstration

Open ponds using flue gases like carbon dioxide and nitrogen from a nearby coal plant as feedstocks

No information

Synthetic biology approach to achieve high productivities of biomass and lipids

To supplement supplies of conventional gasoline for aviation fuels

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Cheng, Y., Zhou, W., Gao, C., Lan, K., Gao, Y., and Wu, Q. Biodiesel production from Jerusalem artichoke (Helianthus tuberosus L.) tuber by heterotrophic microalgae Chlorella protothecoides. J Chem Technol Biotechnol 2009. 84: 777–781. Chisti, Y. Biodiesel from microalgae. Biotechnol Adv 2007. 25: 294–306. Chisti, Y. Response to Reijnders: Do biofuels from microalgae beat biofuels from terrestrial plants? Trends Biotechnol 2008. 26: 351–352. Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., and Lin, C.S. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour Technol 2009. 100: 833–838. Christenson, L.B., and Sims, R.C. Rotating algal biofilm reactor and spool harvester for wastewater treatment with biofuels by‐products. Biotechnol Bioeng 2012. 109: 1674–1684. Cuaresma, M., Janssen, M., Vílchez, C., and Wijffels, R.H. Productivity of Chlorella sorokiniana in a short lightpath (SLP) panel photobioreactor under high irradiance. Biotechnol Bioeng 2009. 104: 352–359. Dayananda, C., Kumudha, A., Sarada, R., and Ravishankar, G.A. Isolation, characterization and outdoor cultivation of green microalgae Botryococcus sp. Sci Res Essays 2011. 5: 2497–2505. Dayananda, C., Sarada, R., Bhattacharya, S., and Ravishankar, G.A. Effect of media and culture conditions on growth and hydrocarbon production by Botryococcus braunii. Process Biochem 2005. 40: 3125–3131. De la Hoz Siegler, H., Ben-Zvi, A., Burrell, R.E., and McCaffrey, W.C. The dynamics of heterotrophic algal cultures. Bioresour Technol 2011. 102: 5764–5774. Doan, T.T.Y., Sivaloganathan, B., and Obbard, J.P. Screening of marine micro algae for biodiesel feed stock. Biomass Bioenerg 2011. 35: 2534–2544. Eriksen, N.T. Production of phycocyanin a pigment with applications in biology, biotechnology, foods and medicine. Appl Microbiol Biotechnol 2008. 80: 1–14. Fan, K.W., Jiang, Y., Faan, Y.W., and Chen, F. Lipid characterization of mangrove thraustochytrid – Schizochytrium mangrovei. J Agric Food Chem 2007. 55: 2906–2910. FAO Corporate Document Repository. 1996. Manual on the Production and Use of Live Food for Aquaculture. Feng, D., Yang, Z., Chen, Z., Yu, X., and Zhang, W. Acetic acid supplement on total lipid production of marine microalga Isochrysis zhanjian gensis. J Biotechnol 2008. 136: S594–S595. Fulke, A.B., Mudliar, S.N., Yadav, R., Shekh, A., Srinivasan, N., Ramanan, R., Krishnamurthi, K., Devi, S.S., and Chakrabarti, T. Bio-mitigation of CO2, calcite formation and simultaneous biodiesel precursors production using Chlorella sp. Bioresour Technol 2010. 101(21): 8473–8476. Gao, C., Zhai, Y., Ding, Y., and Wu, Q. Application of sweet sorghum for biodiesel production by heterotrophic microalga Chlorella protothecoides. Appl Energ 2010. 87: 756–761.

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Gao, K.S., Wu, Y.P., Li, G., Wu, H.Y., Villafañe, V.E., and Helbling, E.W. Solar UV radiation drives CO2 fixation in marine phytoplankton: A double edged sword. Plant Physiol 2007. 144: 54–59. González López, C.V., Acién Fernández, F.G., Fernández Sevilla, J.M., Sánchez Fernández, J.F., Cerón García, M.C., and Molina Grima, E. Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in CO2 removal processes. Bioresour Technol 2009. 100: 5904–5910. Gouveia, L., and Oliveira, A.C. Microalgae as a raw material for biofuels production. J Ind Microbiol Biotechnol 2009. 36: 269–274. Guedes, A.C., Meireles, L.A., Amaro, H.M., and Malcata, F.X. Changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity. J Am Oil Chem Soc 2010. 87: 791–801. Heredia-Arroyo, T.H., Wei, W., and Hu, B. Oil accumulation via heterotrophic/mixotrophic Chlorella protothecoides. Appl Biochem Biotechnol 2010. 162: 1978–1995. Hsieh, C.H., and Wu, W.T. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour Technol 2009. 100: 3921–3926. Huerlimann, R., de Nys, R.D., and Heimann, K. Growth, lipid content, productivity, and fatty acid composition of tropical microalgae for scale up production. Biotechnol Bioeng 2010. 107: 245–257. Khozin-Goldberg, I., Bigogno, C., Shrestha, P., and Cohen, Z. Nitrogen starvation induces the accumulation of arachidonic acid in the freshwater green alga Parietochloris incisa (Trebuxiophyceae). J Phycol 2002. 38: 991–994. Khozin-Goldberg, I., and Cohen, Z. The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemical. 2006. 67: 696–701. Kim, J., Yoo, G., Lee, H., Lim, J., Kim, K., Kim, C.W., Park, M.S., and Yang, J.W. Methods of downstream processing for the production of biodiesel from microalgae. Biotechnol Adv 2013. 31: 862–876. Kumar Saha, S.K., Uma, L., and Subramanian, G. Nitrogen stress induced changes in the marine cyanobacterium Oscillatoria willei BDU130511. FEMS Microbiol Ecol 2003. 45: 263–272. Laurens, L.M.L., Nagle, N., Davis, R., Sweeney, N., Van Wychen, S., Lowell, A., and Pienkos, P.T. Acid-catalyzed algal biomass pretreatment for integrated lipid and carbohydrate-based biofuels production. Green Chem 2015. 17: 1145–1158. Lee, A.K., Lewis, D.M., and Ashman, P.J. Harvesting of marine microalgae by electroflocculation: The energetics, plant design, and economics. Appl Energy 2013. 108: 45–53. Lee, Y.K. Microalgal mass culture systems and methods: Their limitation and potential. J Appl Phycol 2001. 13: 307–315. Li, Q., Du, W., and Liu, D. Perspectives of microbial oils for biodiesel production. Appl Microbiol Biotechnol 2008. 80: 749–756.

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Li, X., Xu, H., and Wu, Q. Large-scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors. Biotechnol Bioeng 2007. 98: 764–771. Liang, Y., Sarkany, N., and Cui, Y. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol Lett 2009. 31: 1043–1049. Liu, J., Huang, J., Fan, K.W., Jiang, Y., Zhong, Y., Sun, Z., and Chen, F. Production potential of Chlorella zofingienesis as a feedstock for biodiesel. Bioresour Technol 2010. 101: 8658–8663. Liu, J., Huang, J., Sun, Z., Zhong, Y., Jiang, Y., and Chen, F. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis assessment of algal oils for biodiesel production. Bioresour Technol 2011. 102: 106–110. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., and Chen, H.L. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour Technol 2010. 101: 6797–6804. Mandal, S., and Mallick, N. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl Microbiol Biotechnol 2009. 84: 281–291. Mandal, S., and Mallick, N. Waste Utilization and biodiesel production by the green microalga Scenedesmus obliquus. Appl Environ Microbiol 2011. 77: 374–377. Mata, T.M., Martins, A.A., and Caetano, N.S. Microalgae for biodiesel production and other applications: A review. Renew Sustain Energy Rev 2010. 14: 217–232. Merzlyak, M.N., Chivkunova, O.B., Gorelova, O.A., Reshetnikova, I.V., Solovchenko, A.E., KhozinGoldberg, I., and Cohen, Z. Effect of nitrogen starvation on optical properties, pigments, and arachidonic acid content of the unicellular green alga Parietochloris incisa (Trebouxiophyceae, Chlorophyta). J Phycol 2007. 43: 833–843. Ota, M., Kato, Y., Watanabe, H., Watanabe, M., Sato, Y., Smith, R.L., and Inomata, H. Fatty acid production from a highly CO2 tolerant alga, Chlorocuccum littorale, in the presence of inorganic carbon and nitrate. Bioresour Technol 2009. 100: 5237–5242. Ota, M., Kato, Y., Watanabe, M., Sato, Y., Smith, R.L., Rosello-Sastre, R., Posten, C., and Inomata, H. Effects of nitrate and oxygen on photoautotrophic lipid production from Chlorococcum littorale. Bioresour Technol 2011. 102: 3286–3292. Patil, V., Kallquvist, T., Olsen, E., Vogt, G., Gislerod H.R. Fatty acid composition of 12 microalgae for possible use in aqua culture feed. Aquacult Int 2007. 15: 1–9. Pruvost, J., Van Vooren, G., Le Gouic, B., Couzinet-Mossion, A., and Legrand, J. Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application. Bioresour Technol 2011. 102: 150–158. Ranga Rao, A., Deepika, G., Ravishankar, G.A., Sarada, R., Narasimharao, B.P., Lei, B., and Su, Y. Industrial potential of carotenoid pigments from microalgae: Current trends and future prospects. Crit Rev Food Sci Nutr 2018: 1–22.

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Ranga Rao, A., Ravishankar, G.A., and Sarada, R. Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production. Bioresour Technol 2012. 23: 528–533. Ranga Rao, A., Sarada, R., Ravishankar, G.A., and Phang, S.M. Industrial production of microalgal cell-mass and bioactive constituents from green microalga-Botryococcusbraunii. In: J. Liu, Z. Sun, and Henri Gerken (eds), Recent Advances in Microalgal Biotechnology, OMICS Group, CA, USA, 2016. pp: 103–126; ISBN: 978-63278-066-9. Ranga Rao, A., Dayananda, C., Sarada, R., Shamala, T.R., and Ravishankar, G.A. Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Bioresour Technol 2007a. 98: 560–564. Ranga Rao, A., Sarada, R., and Ravishankar, G.A. Influence of CO2 on growth and hydrocarbon production in Botryococcus braunii. J Microbiol Biotechnol 2007b. 17: 414–419. Renaud, S.M., Thinh, L.V., Lambrinidis, G., and Parry, D.L. Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures. Aquaculture 2002. 211: 195–214. Richmond, A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology, Blackwell Science, Oxford, UK, 2004: 178–214. Roberts, G.W., Fortier, M.P., Sturm, B.S.M., and StaggWilliams, S.M. Promising pathway for algal biofuels through wastewater cultivation and hydrothermal conversion. Energy Fuels 2013. 27: 857–867. Roux, J.M., Lamotte, H., and Achard, J.L. An overview of microalgae lipid extraction in a biorefinery framework. Energy Procedia 2017. 112: 680–688. Salim, S., Bosma, R., Vermuë, M.H., and Wijffels, R.H. Harvesting of microalgae by bio-flocculation. J Appl Phycol 2011. 23: 849–855. Scarsella, M., Parisi, M.P., DUrso, A., De Filippis, P., Opoka, J., and Bravi, M. Achievements and perspectives in hetero and mixotrophic culturing of microalgae. In: Icheap-9: 9th International Conference on Chemical and Process Engineering, Pts 1-3, (Ed.) Pierucci, S., 2009. 17: 1065–1070. Seo, Y.H., Sung, M., Kim, B., Oh, Y.K., Kim, D.Y., and Han, J.I. Ferric chloride based downstream process for microalgae based biodiesel production. Bioresour Technol 2015. 181: 143–147. Sheng, J., Vannela, R., and Rittmann, B.E. Evaluation of methods to extract and quantify lipids from Synechocystis PCC 6803. Bioresour Technol 2011. 102: 1697–1703. Shi, J., Podola, B., and Melkonian, M. Removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: An experimental study. J Appl Phycol 2007. 19(5): 417–423. Takagi, M., Karseno, and Yoshida, T. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J Biosci Bioeng 2006. 101: 223–226.

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Torri, C., Samorì, C., Adamiano, A., Fabbri, D., Faraloni, C., and Torzillo, G. Preliminary investigation on the production of fuels and bio-char from Chlamydomonas reinhardtii biomass residue after bio-hydrogen production. Bioresour Technol 2011. 102(8): 8707–8713. Tran, D.T., Yeh, K.L., Chen, C.L., and Chang, J.S. Enzymatic transesterification of microalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase. Bioresour Technol 2012. 108: 119–127. Vasudevan, P.T., and Briggs, M. Biodiesel production current state of the art and challenges. J Ind Microbiol Biotechnol 2008. 35(5): 421. Widjaja, A., Chien, C.C., and Ju, Y.H. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J Taiwan Inst Chemeng 2009. 40: 13–20. Wu, W.T., and Hsieh, C.H. Cultivation of microalgae for optimal oil production. J Biotechnol 136: S521–S521.

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Wu, Z., and Shi, X. Optimization for high-density cultivation of heterotrophic Chlorella based on a hybrid neural network model. Lett Appl Microbiol 2007. 44(1): 13–18. Xu, H., Miao, X., and Wu, Q. High quality biodiesel production from a microalgae Chlorella protothecoides by heterotrophic growth in fermenters. J Biotechnol 2006. 126: 499–507. Yeesang, C., and Cheirsilp, B. Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour Technol 2011. 102: 3034–3040. Zhou, Y., Schideman, L., Yu, G., and Zhang, Y. A synergistic combination of algal wastewater treatment and hydrothermal biofuel production maximized by nutrient and carbon recycling. Energy Environ Sci 2013. 6: 3765–3779.

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Microalgae for Sustainable Fuel Technology Coupling Photobioreactor and Bioelectrochemical System for Microalgae Cultivation and Hydrogen Generation Surajbhan Sevda, Dipak A. Jadhav, S.P. Jeevan Kumar, and T.R. Sreekrishnan

CONTENTS Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������101 Microalgae Description�������������������������������������������������������������������������������������������������������������������������������������������102 Algae Used as Feedstock����������������������������������������������������������������������������������������������������������������������������������������103 Utilization of Harvested Algae Biomass in Bioelectrochemical System����������������������������������������������������������������103 Coupling Photobioreactor and Bioelectrochemical System�����������������������������������������������������������������������������������104 Conclusion��������������������������������������������������������������������������������������������������������������������������������������������������������������105 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 105

INTRODUCTION To limit the carbon footprint, the future energy resources should be carbon neutral and renewable. Such sources include biomass as a sustainable resource for energy production. Algae can be considered as one of such potential feedstocks for bio-based economy (Gajda et al., 2015). During conventional wastewater treatment processes, a large quantity of carbon dioxide (CO2) is emitted into the atmosphere during oxidation of organic matter, and it causes a harmful effect on environment. If this amount of CO2 can be captured by algae for photosynthesis, huge algal biomass can be produced for production of value-added products (Gude et al., 2013). Such microalgal biomass can be further utilized for the production of methane, hydrogen, ethanol, butanol and electricity during anaerobic processes. Considering the energy value, the highest energy yields (kJ/g of dry wt. microalgal biomass) reported in the literature were 14.8 as ethanol, 14.4 as methane, 6.6 as butanol, 1.2 as hydrogen and electricity of 0.98 W/m2 (Lakaniemi et al., 2013). Microalgal biomass is considered as a potential substrate for anaerobic energy conversion processes, especially for methanogenic digestion, fermentation and for electrogenic bacteria (Lakaniemi et al., 2013). Additionally, algal biomass with/without pre-treatment serves as feedstock for bio-electrochemical systems (BES) for production

of energy. The energy can be recovered in the form of direct electricity in microbial fuel cell (MFC) or in the form of hydrogen in microbial electrolysis cell (MEC) during treatment of organic matter. Also, CO2 released during anodic oxidation captured for photosynthesis with release of oxygen as terminal electron acceptor for cathodic reduction in microbial carbon capture cell (MCC) or with microbial solar cell (MSC). The algae grown in the photobioreactor (or in cathodic chamber) can serve as potential candidates for CO2 capture, biomass recovery, feedstock for anodic reactions, methanogen suppressors and by-product recovery in various aspects of BES (Jadhav et al., 2017; Rajesh et al., 2015), as discussed in Table 9.1. The potential applications of algae in bioelectrochemically mediated oxidation reactions in MFC and MEC for electricity/hydrogen production is a novel and sustainable approach from an environmental point of view. The production yield depends upon algal characteristics, type of species selected, biomass conditions, operating conditions and design constraints. To improve the performance, BES can be coupled with a photobioreactor (PBR). In double chamber MFC, algal biomass served as a biocatalyst as well as substrate and produced protons during anodic oxidation (Figure 9.1). Further, these protons are utilized by heterotrophic bacteria to

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TABLE 9.1 Utilization of Algae in MFC for Different Applications Application

MFC Details

Findings

References

Algae as substrate for electricity generation Oxygen production for cathodic reactions Methanogen inhibitor

Spirulina platensis algal biofilm on anode Scenedesmus obliquus algal biocathode; H-type MFC Clayware MFC with Chaetoceros pretreated inoculum

Lin et al. (2013)

CO2 sequestration

Chlorella vulgaris biocathode; glucose as an anodic substrate C. vulgaris algae

Current generation proportional to chlorophyll content 15.7 mg/L of oxygen used as terminal electron acceptor for MFC Long chain saturated fatty acid from algae inhibits methane formation CO2 generated during anaerobic process fixed at algal biocathode Ethanol = 2.13 g/L/hr; biodiesel = 12 m3/year

Ethanol and biodiesel production

Kakarla and Min (2014) Rajesh et al. (2015)

Wang et al. (2010) Kerls (2012)

FIGURE 9.1  Schematic representation of CO2 capture, biomass production and cathodic reactions involved in microbial carbon capture cell.

produce hydrogen gas or water in the cathodic chamber (Saratale et al., 2017).

MICROALGAE DESCRIPTION Changing climatic conditions coupled with detrimental environmental consequences and population explosion mean that renewable and carbon neutral energy sources are essential. This includes biomass-based products such as crops, plants, algae and organic wastes. Among these biomass products, algae have gained wide acceptability from many researchers as a potential source for biofuel, biochemical and bioelectricity production (Chisti, 2007). Microalgae are predominantly autotrophic in nature, which means they convert solar power for synthesis of both biomass and high value products (Deirue et al., 2012).

Although microalgae have been widely explored for biodiesel and biorefinery products, still cost has impeded translation of the technologies into commercial viability. Recent studies on microalgae-based MFC have shown them to have huge potential and to be promising substrates for MFC synthesis (Venkata Mohan et al., 2008; Rosenbaum et al., 2010). Microbial fuel cells are aimed to generate energy by converting the chemical energy to electrical energy through biological pathway. The biological substrates include sewage sludge, municipal wastewater and agricultural wastes. Several studies have been conducted on the coupling of microalgae and MFC (Raschitor et al., 2015; Yu et al., 2015), that lead to microalgae-MFC (mMFC) development (Cao et al., 2009; Wieczorek et al., 2015). In mMFC, photosynthetic microorganisms

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readily convert the solar energy into electrical energy (electricity) via metabolic reactions (Bombelli et al., 2011). Moreover, application of mMFCs not only generate bioelectricity but also sequestrate CO2 from air and get rid of nitrogen from air and contaminants from waters (Figure 9.2) (Li et al., 2012; Wang et al., 2010).

ALGAE USED AS FEEDSTOCK In general, the conventional microbial fuel cell produces CO2 at the anodic chamber because of oxidized products formed from organic feeds. In contrast, at the cathode either ferricyanide or O2 is placed to accept the excess protons derived from the anodic chamber and electrons from the external circuit. The half-cell reactions at the anode and cathode is illustrated below. At anode,

Organics ® CO2 + H + ( to cathode ) + e - (external circuit)

(9.1)

whereas at cathode, O2 + H + (from anodic chamber )



Organics + O2 ® CO2 + H 2 O + external power

(9.3)

All the above reactions are similar to MFC, but the carbon dioxide formed in Equation (9.3), can be assimilated into biomass by photosynthetic microalgae with the aid of incident light and chlorophyll as shown below.

CO2 + H 2 O + light ® biomass + O2 (9.4)

The oxygen released from photosynthetic reaction of Chlorella vulgaris at cathode surface serve as terminal electron acceptor for cathodic reduction reaction (Powell et al., 2009; Wang et al., 2010). Wu et al. (2013) had reported a strong correlation between dissolved oxygen (DO) and cell voltage in mMFC. Similarly, biocathode pertaining to algae has been tested in mMFC (Liu et al., 2013) and a study reported that a photo-biocathode has been applied for CO2 reduction in a completely anoxic mMFC (Cao et al., 2009). These studies substantiate that the microalgae remaining either in attached or suspended form can be used for mMFC applications.

(9.2)

UTILIZATION OF HARVESTED ALGAE BIOMASS IN BIOELECTROCHEMICAL SYSTEM

Half-cell reactions of both these half-cells lead to a process of converting chemical energy to electrical energy as given below.

Microalgae received special potential for converting light and inorganic carbons (e.g., CO2 and HCO3−) into high-value organic matter during photosynthesis, which



+ e − (external circuit) → H 2 O

FIGURE 9.2  Microalgae-based microbial fuel cells. Dotted lines depict putative integration of anodic and cathodic chambers with carbon flows. (Adapted from Duu-Jong Lee et al., 2015.)

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is nature’s most primitive way of converting carbon into biomass. Using BES, the harvested biomass can be utilized for hydrogen production or energy recovery. Only specific groups of algae (e.g. C. reinhardtii) and cyanobacteria have potential to harvest solar energy resource to drive molecular H2 production for BES applications (Mao and Verwoerd, 2013). The concept of electrocatalytic photoMFC for coupling the photosynthetic hydrogen production with in situ hydrogen oxidation through an electrocatalytic conversion was tested in 1964 (Berk and Canfield, 1964). The performance of microbial carbon capture cell (MCC) is governed by selection of algal species, operating conditions, anodic parameters, cathodic parameters, design constraints, etc. (Gautam, 2016). The performance of MFC with different operating conditions is shown in Table 9.2. Considering the type of algal species selected at biocathode of MCC, higher coulombic efficiency (15.23 ± 1.30%) and biomass production (66.4 ± 4.7 mg/(L*day)) in MCC indicated the superiority of Chlorella over Anabaena algae for carbon capture and oxygen production to facilitate the cathodic reduction (Jadhav et al., 2017). Concentration of algae also affects the power production. Maximum power density has increased from 4.1 to 5.6 W/m3 as optical density (OD) of cathodic algae suspension increased from 0.21 to 0.85 (658 nm) (Wang et al., 2010). Duration and intensity of light affects the growth of microalgae during photosynthesis. High light intensity

promotes higher photosynthetic activity and production of oxygen (Gouveia et al., 2014), which will be available for the cathodic reactions, resulting in an enhancement of voltage output. Algal MFC has shown potential for electricity generation at light intensity of 96 µE/(m2s) by producing a maximum power of 62.7 mW/m2 (Gouveia et al., 2014). Grobbelaar et al. (1996) showed that the rate of photosynthesis increases exponentially with frequencies of light/dark cycle; also, longer dark periods in relation to light period further improve the rate of photosynthesis, but not vice versa. Higher concentration of CO2 is also toxic for algae, but some species showed tolerance to high levels of CO2 and moderate levels of SOx and NOx (up to 150 ppm) (Matsumoto et al., 1997) which showed potential for use of flue gas from industry as carbon source for algae. Thus, photosynthetic algae in the cathodic chamber perform multiple functions including oxygen supply, nutrient removal and biomass production (Xiao and He, 2014).

COUPLING PHOTOBIOREACTOR AND BIOELECTROCHEMICAL SYSTEM Previously, several researchers working on photo-algal bioreactor (PBR) developed integrated photobio-electrochemical systems by incorporating a BES within an algae-based bioreactor or coupling MFC with PBR. Such a system has been found to be useful in the generation of electricity, energy recovery and algal biomass

TABLE 9.2 Performance of Algal MFC Under Different Operating Conditions Wastewater Treatment

Algal Biocathode

Anode Chamber Detail

Electrical Output

Scenedismus obliquus

250 ml, 0.5 mg/cm2 Pt-coated carbon paper; wastewater and acetate (2 g/l)

ND

Kakarla, and Min (2014)

Chlorella vulgaris

100 ml, 0.1 mg/cm2 Pt-catalystactivated sludge inoculum,sodium acetate (1 g/L) 750 ml, anaerobic mixed consortia, 3g/L glucose 50 ml, 0.5 mg/cm2 of platinum, 20 mM of acetate Activated sludge, fruit processing industry effluent – 220 ml; glucose, preacclimated bacteria; 0.1 mg/cm2 Pt/C catalyst

153 mW/m2; Reduction current = 9.3 mA 187 mW/m2 in light and 21 in dark

ND

Liu et al. (2015)

57 mW/m2 (spring) vs. 1.1 mW/m2 (summer) 62.7 mW/m2; OV: 280 mV 14.40 mW/m2; 116.96 mA/m2 12.947 mW/ m2 5.2 W/m3

ND ND

Venkata Mohan et al. (2014) Gouveia et al. (2014)

80% COD removal ND ND

González del Campo et al. (2013) Lan et al. (2013) Wang et al. (2010)

Mixed microalgal culture Chlorella vulgaris Chlorella vulgaris Chlamydomonas reinhardtii Chlorella vulgaris ND: Not defined

Reference

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Microalgae for Sustainable Fuel Technology

FIGURE 9.3  Flow chart representing applicability of integrated MFC–photobioreactor system.

production with enhancement in efficiency (Figure 9.3). In this integrated type, MFC is connected to a PBR, in which CO2 is pumped directly from the MFC to the PBR or can be integrated in one unit. Conventional fermentative hydrogen production processes have limitations of low substrate conversion efficiency and lower hydrogen production due to accumulation of by-products as the end products. These limitations can be overcome by coupling such processes with MFC for effective hydrogen production, electricity generation and efficient wastewater treatment. Li et al. (2013) reported a 15-fold increment in hydrogen production yield and improvement in wastewater treatment efficiency with a PBR-MFC integrated system as compared to a PBR system alone due to the removal of inhibitory by-products and H+- ions from the PBR effluent by the MFCs. The acid rich effluent of acidogenic sequential batch biofilm reactor (AcSBBR) producing H2 by fermenting vegetable waste can be effectively used as substrate for MFC achieving COD removal of 84.6% and power of 0.11 W/m2 (Mohankrishna et al., 2010). For electricity generation application, Strik et al. (2008) developed photosynthetic algal MFC (PAMFC) by coupling photobioreactor with flat plate MFC which is capable to produce maximum current density of 539 mA/m2 (power density = 110 mW/m2). When MFC is combined with PBR, the integrated system was capable to remove total phosphorus and ammonium by 99.3% and 99.0%, respectively. PMFC with Spartina anglica sp. is capable of producing the highest long-term current and power density (50 mW/m2) over 33 days operating duration (Timmers et al., 2010). The microbial solar cells (MSCs) or plant MFC captured CO2 from atmosphere for fixation and generated organic matter can be utilized as a substrate for anodic oxidation. According to application, MSCs are utilized for phototrophic biofilms,

plant MFC, green roof MFC or in combination with photobioreactors (Strik et al., 2011).

CONCLUSION The recent progress in the BES-microalgae system shows the sustainable recovery of biomass enhancing higher wastewater treatment along with bioelectricity generation. The photobioreactor and BES combination provides a better solution for clean energy generation with wastewater treatment. The usages of microalgae in the cathodic chamber decrease the overall cost as this can replace the use of expensive catalyst in the cathodic chambers. The combined system is capable of production of biohydrogen and biofuel along with treatment of wastewater with bioelectricity generation. This combined system represents a low cost and environmentally friendly biological wastewater treatment with bioelectricity generation compared to the conventional wastewater treatment processes.

REFERENCES Berk, R. S., & Canfield, J. H. (1964) Bioelectrochemical energy conversion. Applied Microbiology, 12, 10–12. Bombelli, P., Bradley, R. W., Scott, A. M., Philips, A. J., McCormick, A. J., Cruz, S. M., Anderson, A., Yunus, K., Bendall, D. S., Cameron, P. J., & Davies, J. M., 2011. Quantitative analysis of the factors limiting solar power transduction by Synechocystis sp. PCC 6803 in biological photovoltaic devices. Energy Environ. Sci. 4, 4690–4698. Cao, X. X., Huang, X., Liang, P., Boon, N., Fan, M. Z., Zhang, L., & Zhang, X. Y. (2009) A completely anoxic microbial fuel cell using a photo-biocathode for cathodic carbon dioxide reduction. Energy and Environmental Science, 2(5), 498–501.

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Chisti, Y. (2007) Biodiesel from microalgae. Biotechnology Advances, 25(3), 294–306. Deirue, F., Seiter, P. A., Sahut, C., Cournac, L., Roubltier, G., & Froment, A. K. (2012) An economic, sustainability, and energenic model of biodiesel production from microalgae. Bioresource Technology, 112, 191–200. Gajda, I., Greenman, J., Melhuish, C., & Ieropoulos, I. (2015) Self-sustainable electricity production from algae grown in a microbial fuel cell system. Biomass and Bioenergy, 82, 87–93. Gautam, P. K. (2016) Study of effect of different operating parameters on performance of low cost clayware microbial carbon capture cell. M.Tech. Dissertation, IIT Kharagpur, West Bengal, India. González del Campo, A. G., Cañizares, P., Rodrigo, M. A., Fernández, F. J., & Lobato, J. (2013) Microbial fuel cell with an algae assisted cathode: A preliminary assessment. Journal of Power Sources, 242, 638–645. Gouveia, L., Neves, C., Sebastião, D., Nobre, B. P., & Matos, C. T. (2014) Effect of light on the production of bioelectricity and added-value microalgae biomass in a Photosynthetic Alga Microbial Fuel Cell. Bioresource Technology, 154, 171–177. Grobbelaar, J. U., Nedbal, L., & Tichý, V. (1996) Influence of high frequency light/dark fluctuations on photosynthetic characteristics of microalgae photo acclimated to different light intensities and implications for mass algal cultivation. Journal of Applied Phycology, 8(4–5), 335–343. Gude, V. G. G., Kokabian, B., & Gadhamshetty, V. (2013) Beneficial bioelectrochemical systems for energy, water, and biomass production. Journal of Microbial and Biochemical Technology, 6(2), S6-005. Jadhav, D. A., Jain, S. C., & Ghangrekar, M. M. (2017) Simultaneous wastewater treatment, algal biomass production and electricity generation in clayware microbial carbon capture cells. Applied Biochemistry and Biotechnology, 183(3), 1076–1092. Kakarla, R. M., & Min, B. (2014) Photoautotrophic microalgae Scenedesmus obliquus attached on a cathode as oxygen producers for microbial fuel cell (MFC) operation. Int. J. Hydrogen Energ. 39, 10275–10283. Kerls, M. (2012). Simultaneous electricity, bioethanol, and algal biodiesel production using microbial fuel cell. M.S. dissertation, Texas Tech University. Lakaniemi, A. M., Tuovinen, O. H., & Puhakka, J. A. (2013). Anaerobic conversion of microalgal biomass to sustainable energy carriers–A review. Bioresource Technology, 135, 222–231. Lan, J. C. W., Raman, K., Huang, C. M., & Chang, C. M. (2013) The impact of monochromatic blue and red LED light upon performance of photo microbial fuel cells (PMFCs) using Chlamydomonas reinhardtii transformation F5 as biocatalyst. Biochemical Engineering Journal, 78, 39–43. Lee, D. J., Chang, J. S., & Lai, J. Y. (2015) Microalgae–microbial fuel cell: A mini review. Bioresource Technology, 198, 891–895.

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Li, J., Liu, L. G., Zhang, R. D., Luo, Y., Zhang, C. P., & Li, M. C. Power generation from glucose and nitrobenzene degradation using the microbial fuel cell, Environ. Sci. 31 (2010) 2811–2817. Li, J., Zou, W., Xu, Z., Ye, D., Zhu, X., & Liao, Q. (2013) Improved hydrogen production of the downstream bioreactor by coupling single chamber microbial fuel cells between series-connected photosynthetic biohydrogen reactors. International Journal of Hydrogen Energy, 38(35), 15613–15619. Liu, S., Song, H., Li, X., & Yang, F. (2013). Power generation enhancement by utilizing plant photosynthate in microbial fuel cell coupled constructed wetland system. International Journal of Photoenergy, 2013. Liu, T., Rao, L., Yuan, Y., & Zhuang, L. (2015) Bioelectricity generation in a microbial fuel cell with a self-sustainable photocathode. The Scientific World Journal, 2015, 1–8. Mao, L., & Verwoerd, W. S. (2013) Selection of organisms for systems biology study of microbial electricity generation: A review. International Journal of Energy and Environmental Engineering, 4(1), 17. Matsumoto, H., Hamasaki, A., Sioji, N., & Ikuta, Y. (1997) Influence of CO2, SO2 and NO in flue gas on microalgae productivity. Journal of Chemical Engineering of Japan, 30(4), 620–624. Mohanakrishna, G., Venkata Mohan, S. V., & Sarma, P. N. (2010) Utilizing acid-rich effluents of fermentative hydrogen production process as substrate for harnessing bioelectricity: An integrative approach. International Journal of Hydrogen Energy, 35(8), 3440–3449. Powell, E. E., Mapiour, M. L., Evitts, R. W., Hill, G. A. (2009) Growth kinetics of Chlorella vulgaris and its use as a cathodic half cell. Bioresour. Technol. 100, 269–274. Rajesh, P. P., Jadhav, D. A., & Ghangrekar, M. M. (2015) Improving performance of microbial fuel cell while controlling methanogenesis by Chaetoceros pretreatment of anodic inoculum. Bioresource Technology, 180, 66–71. Raschitor, A., Soreanu, G., Fernandez-Marchante, C. M., Lobato, J., Cañizares, P., Cretescu, I., & Rodrigo, M. A. (2015) Bioelectro-Claus processes using MFC technology: Influence of co-substrate. Bioresource Technology, 189, 94–98. Rosenbaum, M., He, Z., & Angenent, L. T. (2010) Light energy to bioelectricity: Photosynthetic microbial fuel cells. Current Opinion in Biotechnology, 21(3), 259–264. Saratale, R. G., Kuppam, C., Mudhoo, A., Saratale, G. D., Periyasamy, S., Zhen, G., Koók, L., Bakonyi, P., Nemestóthy, N., & Kumar, G. (2017) Bioelectrochemical systems using microalgae–A concise research update. Chemosphere, 177, 35–43. Strik, D. P., Terlouw, H., Hamelers, H. V., & Buisman, C. J. (2008) Renewable sustainable biocatalyzed electricity production in a photosynthetic algal microbial fuel cell (PAMFC). Applied Microbiology and Biotechnology, 81(4), 659–668.

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Strik, D. P., Timmers, R. A., Helder, M., Steinbusch, K. J., Hamelers, H. V., & Buisman, C. J. (2011) Microbial solar cells: Applying photosynthetic and electrochemically active organisms. Trends in Biotechnology, 29(1), 41–49. Timmers, R. A., Strik, D. P., Hamelers, H. V., & Buisman, C. J. (2010) Long-term performance of a plant microbial fuel cell with Spartina anglica. Applied Microbiology and Biotechnology, 86(3), 973–981. Venkata Mohan, S. V., Mohanakrishna, G., Reddy, B. P., Saravanan, R., & Sarma, P. N. (2008) Bioelectricity generation from chemical wastewater treatment in mediatorless (anode) microbial fuel cell (MFC) using selectively enriched hydrogen producing mixed culture under acidophilic microenvironment. Biochemical Engineering Journal, 39(1), 121–130. Venkata Mohan, S. V., Srikanth, S., Chiranjeevi, P., Arora, S., & Chandra, R. (2014) Algal biocathode for in situ terminal electron acceptor (TEA) production: Synergetic association of bacteria–microalgae metabolism for the functioning of biofuel cell. Bioresource Technology, 166, 566–574.

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Wang, X., Feng, Y., Liu, J., Lee, H., Li, C., Li, N., & Ren, N. (2010) Sequestration of CO2 discharged from anode by algal cathode in microbial carbon capture cells (MCCs). Biosensors and Bioelectronics, 25(12), 2639–2643. Wieczorek, N., Ali Kucuker, M., & Kuchta, K. (2015) Microalgae-bacteria flocs (MaB-Flocs) as a substrate for fermentation biogas production. Bioresource Technology, 194, 130–136. Wu, X. Y., Song, T. S., Zhu, X. J., Wei, P., Zhou, C. C. (2013) Construction and operation of microbial fuel cell with Chlorella vulgaris biocathode for electricity generation. Appl. Biochem. Biotechnol. 171(8), 2082–2092. Xiao, L., & He, Z. (2014) Applications and perspectives of phototrophic microorganisms for electricity generation from organic compounds in microbial fuel cells. Renewable and Sustainable Energy Reviews, 37, 550–559. Yu, N., Dieu, L. T. J., Harvey, S., & Lee, D. Y. (2015) Optimization of process configuration and strain selection for microalgae-based biodiesel production. Bioresource Technology, 193, 25–34.

Section III Other Products of Economic Value

10

Seaweed as Source of Plant Growth Promoters and Bio-Fertilizers An Overview Sananda Mondal and Debasish Panda

CONTENTS Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������112 Chemical Composition of Seaweeds����������������������������������������������������������������������������������������������������������������������112 Seaweed Extracts as Source of Bio-fertilizer����������������������������������������������������������������������������������������������������������112 Phytohormones in Liquid Seaweed Extracts ���������������������������������������������������������������������������������������������������������113 Auxins�����������������������������������������������������������������������������������������������������������������������������������������������������������������113 Cytokinins�����������������������������������������������������������������������������������������������������������������������������������������������������������113 Gibberellins��������������������������������������������������������������������������������������������������������������������������������������������������������114 Abscisic Acid������������������������������������������������������������������������������������������������������������������������������������������������������115 Ethylene��������������������������������������������������������������������������������������������������������������������������������������������������������������115 Betaines��������������������������������������������������������������������������������������������������������������������������������������������������������������115 Polyamines���������������������������������������������������������������������������������������������������������������������������������������������������������116 Effect of Seaweed Extracts on Germination, Growth, Physiology and Yield���������������������������������������������������������116 Effect of Seaweed Extracts on Seed Germination����������������������������������������������������������������������������������������������117 Root Development and Mineral Absorption�������������������������������������������������������������������������������������������������������117 Effect on Shoot Growth and Photosynthesis �����������������������������������������������������������������������������������������������������118 Effect on Crop Yield�������������������������������������������������������������������������������������������������������������������������������������������118 Role in Vegetative Propagation ��������������������������������������������������������������������������������������������������������������������������118 Role in Abiotic Stresses Tolerance in Crop Plants ��������������������������������������������������������������������������������������������118 Conclusion �������������������������������������������������������������������������������������������������������������������������������������������������������������119 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 119

BOX 10.1  SALIENT FEATURES The seaweeds are a natural source of bio-fertilizer, plant growth regulators and bio-stimulants. The huge biomass of these marine macro-algae has the potential to become a renewable and economic source of input for sustainable agriculture. The extracts of marine macro-algae are extensively used as liquid fertilizers in crop production. The seaweed extracts are also rich in growth-promoting hormones like auxins, gibberellins, cytokinins, abscisic acid, ethylene, polyamines and quaternary ammonium molecules like betaine. The seaweed extracts are rich in micronutrients, vitamins, amino acids and antibiotics. The seaweed extracts can induce tolerance of crops to several abiotic stresses

like drought, heat and cold. This may be attributed to the presence of the stress hormone abscisic acid and osmoprotectants like proline and glycine betaine in the algal extracts. The extracts of marine algae like Ascophyllum nodosum, Macrocycstis pyrifera, Ecklonia maxima, Durvillea Antarctica, Durvillea protatorum, Porphyra perforate, Fucus vesiculosus, Caulerpa paspaloides, Sargassum heterophyllum, Chara globularis, Sargassum muticum, Enteromorpha prolifera, Cyanidium caldarium, Laminaria digitata, Ulva lactuca, Nereocystis spp., Kappaphycus spp. and Gracilaria spp. have been used as liquid bio-fertilizers and biostimulants. The seaweed extracts as source of plant nutrients and plant growth regulators are discussed in this chapter.

111

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Handbook of Algal Technologies and Phytochemicals

INTRODUCTION

CHEMICAL COMPOSITION OF SEAWEEDS

Seaweeds include the macro-algae found in the intertidal zones of marine ecosystems around the world (Mohanty et al., 2013). These macro-algae which include several species of green, brown and red algae are also abundant in several locations along the coastline of India. Seaweeds are found growing luxuriantly along the south-east coast of Tamilnadu from Mandapam to Kanyakumari, Gujarat coast, Lakshadweep and Andamanand Nicobar Islands. These marine algae are also been reported from coastal areas in the vicinity of Bombay, Ratnagiri, Goa, Karwar, Varkala, Kovalam, Visakhapatnam, Vizhinjam and in some lakes such as Chilka and Pulicat (Rath and Adhikary, 2005; Kaliaperumal et al., 1987). Seaweeds are reported to have plant-growth promoting activity and have been utilized around the world as organic manures and fertilizers in agriculture and horticulture (Craigie, 2011). The extracts of marine macro-algae, viz., brown, red and green algae, are known to have positive effect on growth and yield of crops. The brown algae are the most commonly used seaweeds in agriculture. Seaweed extracts contain different phytohormones like auxins, gibberellins, cytokinins, abscisic acid, ethylene, betaine and polyamines and other growth promoters along with trace elements, vitamins, amino acids, antibiotics and micronutrients which enhance the yield and yield attributes of crops, when applied exogenously (Panda et al., 2012). Seaweeds and seaweed extracts which are important components of organic farming are a promising avenue for yield maximization through their biostimulatory role on crop plants. A relatively small proportion of the total number of seaweed species are of significant importance in agriculture as mulches and manure (Craigie, 2011; Khan et al., 2009). In general, seaweed extracts, even at low concentrations, are capable of inducing an array of physiological plant responses, such as promotion of plant growth, improvement of flowering and yield and also enhanced quality of products, improved nutritional content of edible product as well as shelf life. The application of different types of seaweed extract has been reported to enhance tolerance of plants to a wide range of abiotic stresses such as salinity, drought and temperature extremes. However, the macronutrients such as N, P and K found in seaweeds are known to be insufficient to elicit physiological responses at the typical concentrations that the seaweed extracts were applied in the field (Blunden, 1991; Khan et al., 2009). Seaweeds and their extracts are integral to sustainable farming because of their multifarious utility in various fields of agriculture including nutrient and crop management, growth promotion and plant protection, etc.

The chemical constituents of seaweed extract include complex polysaccharide, fatty acids, vitamins, phytohormones and mineral nutrients, etc. Seaweeds contain a diverse range of organic compounds which includes several common amino acids inter alia aspartic acid, glutamic acid and alanine in commercially important species. Alginic acid, laminarin and mannitol represent nearly half of the total carbohydrate content of commercial seaweed preparations (Panda et al., 2012). Seaweeds also contain a wide range of vitamins which might be utilized by crops. Vitamins C, B1 (thiamine), B2 (riboflavin), B12, D3, E, K, niacin, pantothenic, folic and folinic acids occur in algae. Although vitamin A is not present, the presence of its precursor carotene and another possible precursor, fucoxanthin, has been found (Stephenson, 1968). Apart from the above organic and inorganic constituents, there is evidence of presence of substances of a more stimulatory and antibiotic nature. The organic constituents of seaweed extract include plant hormones which elicit strong physiological responses in low doses (Crouch and van Staden, 1993). Seaweeds are rich in mineral elements such as K, Mg, Ca, Na, Fe, Cu, Zn and Mn. The liquid extracts of many seaweed extracts are also rich in antioxidants such as phenolic substances and flavonoids. Seaweed extracts also contain osmoprotectants such as proline and glycine betaine which imparts abiotic stress tolerance in plants.

SEAWEED EXTRACTS AS SOURCE OF BIO-FERTILIZER Several species of marine algae are being utilized for preparation of seaweed liquid fertilizers (SLF). The nutrient content of the SLF varies from species to species. The SLFs are a great source of natural bio-fertilizers rich in macro- as well as micronutrients. The seaweeds, particularly kelp and high nitrogen rich green seaweed (Ulva ohnoi), can be either applied to the soil as mulch or can be added to the compost (Cole et al., 2016). Some of the commonly used algae species for preparation of SLF are Ascophyllum nodosum, Macrocycstis pyrifera, Ecklonia maxima, Durvillea Antarctica, Durvillea protatorum, Porphyra perforate, Fucus vesiculosus, Caulerpa paspaloides, Sargassum heterophyllum, Chara globularis, Sargassum muticum, Enteromorpha prolifera, Cyanidium caldarium, Laminaria digitata, Ulva lactuca, Nereocystis spp., Kappaphycus spp. and Gracilaria spp. Extensive studies on the chemical composition of various extracts made from a diversity of seaweeds revealed that the nutrient content (typically

Seaweed as Source of Plant Growth Promoters and Bio-Fertilizers 

macronutrients including N, P and K) of the extracts was insufficient to elicit physiological responses at the typical concentrations that the seaweed extracts were applied in the field (Blunden, 1991; Khan et al., 2009). Some of the commonly found minerals elements in SLF are K, Mg, Ca and Na. The seaweed extracts also contain trace elements such as Fe, Cu, Zn and Mn (Chojnacka et al., 2012; Sivasankari et al., 2006). Seaweed liquid fertilizer can be applied to the crops as foliar spray or applied to the soil or the seeds. SLF applied as soil amendment improves soil health by enhancing the nutrient status of the soil and favoring microbial growth in the rhizosphere. Different species of seaweeds as sources of biofertilizers and organic manures and the roles of different mineral elements found SLF are presented in Tables 10.1 and 10.2.

PHYTOHORMONES IN LIQUID SEAWEED EXTRACTS Various phytohormones and plant growth regulators are found in different seaweed concentrates and marine TABLE 10.1 Seaweed Extracts as Source of Seaweed Liquid Fertilizers Sl. No.

Species

Algal Type

Application

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Sargassum wightii Sargassum muticum Sargassum crassifolium Sargassum polyphyllum Sargassum polycystum Sargassum johnstonii Sargassum ilicifolium Sargassum heterophyllum Ulva lactuca Padina pavonia Caulerpa racemosa Caulerpa scalpelliformis Gracilaria edulis Gracilaria corticata Gracilaria verrucosa Gelidella aerosa Kappaphycus alvarazii Chaetomorpha linum Turbenaria conoides Spyridia hypnoides Dictyota dichotoma Nereocystis sp. Cyanidium caldarium Chara globularis

Brown algae Brown algae Brown algae Brown algae Brown algae Brown algae Brown algae Brown algae Green algae Brown algae Green algae Green algae Red algae Red algae Red algae Red algae Red algae Green algae Brown algae Red algae Brown algae Brown algae Red algae Green algae

SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF SLF

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macro-algal extracts, viz., auxins, gibberellins, cytokinins, abscisic acid, ethylene, betaine and polyamines, and simulate plant growth when applied exogenously (Panda et al., 2012). Different species of seaweeds as sources of bio-stimulants and the roles of liquid seaweed extracts in promotion of crop growth are presented in Tables 10.3 and 10.4.

Auxins Auxins or auxin-like compounds are known to occur endogenously in many marine algae. The presence of indole-3-acetic acid (IAA) has been recorded in a number of marine algae such as Nereocystis spp., Ecklonia maxima, Macrocystis pyrifera, Ascophyllum nodosum, Porphyra perforata, Fucus vesiculosus, Caulerpa paspaloides and Sargassum heterophyllum etc. Auxin-like substances like phenyl-3-aceticacid (PAA) and hydroxyl phenyl acetic acid (OH-PAA) have been reported from Undaria pinnatifida extracts (Abe et al., 1974) and 3-hydroxyacetyl-indole in Prionitis lanceolate extracts (Bernart and Gerwick, 1990). IAA is also found in hydrolyzed and liquified A. nodosum commercial preparation. The seaweed concentrate of the brown alga, Ecklonia maxima, exhibits a remarkable root-promoting ability when applied to cuttings, which is attributed to endogenous indoles like indole-3-carboxylic acid (ICA); N,N-dimethyltryptamine; indole-3-aldehyde (IAId) and in addition, iso-indole, 1, 3-dione (N-hydroxyethylphthalimide) in the extracts (Crouch and van Staden, 1991). In higher plants, IAA occurs as an inactive conjugate with carboxyl groups, glycans, amino acids and peptides, which are converted to free active IAA upon hydrolysis (Bartel, 1997), whereas in marine algae, it occurs as conjugates of indole and amino acids (Stirk et al., 2004).

Cytokinins Seaweed extract is one of the sources of phytohormone cytokinin. Cytokinins have been detected in fresh seaweeds (Hussein and Boney, 1969) as well as seaweed extracts (Brain et al., 1973). Various available forms of cytokinins are present in seaweed extracts include transzeatin, trans-zeatin riboside and dihydro derivatives of these two forms (Stirk and van Staden, 1997). Seawater taken from the Fucus-Ascophyllum zone is found to contain cytokinin in the form of zeatin (6-3-methyl2-butenylamino purine). Different forms of cytokinins like iso-pentenyl adenosine, zeatin, zeatin riboside, dihydrozeatin, iso-pentenyladenine, 2-hydroxy-6-methylaminopurine and 2-hydroxy-1-methylzeatin have been isolated from Sargassum muticum, Porphyra

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TABLE 10.2 Physiological Roles of Mineral Elements Found in Seaweed Liquid Fertilizers Sl. No.

Mineral Element

1

K

2

Mg



3

Ca



4

Fe

5

Cu



6

Zn



7

Mn

8

Na



Important Physiological Roles

References

1. It plays important role in maintaining water balance and stomatal movement. 2. It acts as an activator for many enzymes involved in carbohydrate metabolism. 3. It imparts disease tolerance to the crops. 1. It is an important constituent of the chlorophyll molecule and essential for photosynthesis and carbohydrate metabolism. 2. It plays an important role in assembling of ribosomal units on mRNA to form polysomes during protein synthesis. 3. It is an activator of the enzymes involved in phosphate transfer reactions and involved in the synthesis of nucleic acid. 1. It is an important constituent of middle lamella and plays an important role in formation of the cell wall. 2. It acts as a secondary messenger in metabolic regulations (extra-cellular cell signaling). 3. It is involved in chromatin or mitotic spindle organization and is essential for mitosis. 4. It is an activator of many enzymes. 1. It is an important constituent of porphyrin structures. 2. Essential for synthesis of chlorophyll. 3. It is an important constituent of ferredoxin which plays an important role in biological nitrogen fixation and light reaction of photosynthesis. 1. It is a component of several enzymes such as phenolase, lactase and ascorbic acid oxidase. 2. Copper plays important role in photosynthesis. Chloroplast contains a Cu-containing protein called plastocyanin which is important in light reaction of photosynthesis. 1. It is involved in biosynthesis of auxin Indole-3-acetic acid (IAA). 2. It acts as an activator of the enzymes such as carbonic anhydrase, alcohol dehydrogenase and hexose kinase or triosphosphate dehydrogenase. 3. It plays an important role in protein synthesis. 1. It is essential for respiration. 2. It functions as an activator of many enzymes. 3. It plays important role along with Cl in photolysis of water during photosynthesis. 4. It is essential for nitrogen metabolism and nitrate reduction. 5. It acts as an activator of the enzyme nitrate reductase and hydroxylamine reductase. 1. It acts as a functional element in plants. 2. It is required for maximal biomass growth of many plants. 3. It has the ability to replace K in a number of ways. 4. It acts as an osmaticum for cell enlargement and an accompanying cation for longdistance transport.

Jain (2008)

perforata and Chara globularis of seaweeds. Mostly crop responses to different seaweed extracts are thought to be primarily due to the response of plant hormone cytokinin. Seaweed extracts with cytokinin activity are capable of producing physiological change, even when applied at low concentrations used under field conditions. Several cytokinins like trans-zeatin, trans-zeatin riboside and their dihydroderivatives; iso-pentenyladenine; iso-pentenyl adenosine and several cytokinin glucosides have been identified and quantified in several seaweed extracts (Panda et al., 2012).

Jain (2008)

Jain (2008)

Jain (2008)

Jain (2008)

Jain (2008)

Jain (2008)

Subbarao et al. (2003)

Gibberellins In seaweeds the existence of gibberellin-like substances is well known nowadays. The presence of gibberellic acid in Enteromorpha prolifera and Ecklonia radiata has been reported by Jennings (1968). At least two compounds have been recorded that behave like the gibberellins GA3 and GA7, although these may be vitamins A1 and A4 (Stephenson, 1968). A terpenoid, α-tocopherol, a major component of the E group of vitamins present in seaweeds, may mimic gibberellin activity (Gopala, 1984;

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Seaweed as Source of Plant Growth Promoters and Bio-Fertilizers 

TABLE 10.3 Extracts of Some Seaweeds with Growth Stimulant Role in Agriculture and Horticulture Sl. No.

Species

Algal Type

Application

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Ascophyllum nodosum Macrocycstis pyrifera Ecklonia maxima Durvillea antarctica Durvillea protatorum Porphyra perforata Fucus vesiculosus Caulerpa paspaloides Sargassum heterophyllum Nereocystis spp. Chara globularis Sargassum muticum Enteromorpha prolifera Cyanidium caldarium Laminaria digitata Ulva lactuca

Brown algae Red algae Brown algae Brown algae Brown algae Red algae Brown algae Green algae Brown algae Red algae Brown algae Brown algae Green algae Red algae Brown algae Green algae

Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant bio-stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Plant growth stimulant Growth retardant

Jensen, 1969). Gibberellin-like compounds are also readily found in a diversity of seaweeds. It is thought that these compounds may break down during the manufacturing process. Gibberellin activity has been found in some freshly made-up seaweed preparations (Panda et al., 2012).

Abscisic Acid The presence of Abscisic acid (ABA) has been confirmed in Laminaria digitata, Ascophyllum nodosum (Hussian and Boney, 1973) and Ulva lactuca (Hartmann and Kester, 1983). A higher level of ABA is found in some commercial extracts of A. nodosum. ABA is also present in the green algae Enteromorpha compressa (Niemann and Dorfiiing, 1980). The water-soluble growth inhibitors extracted from Laminaria digitata and A. nodosum resulted in marked inhibition of lettuce hypocotyl growth (Hussain and Boney, 1973).

Ethylene Literature suggests that still now very few studies on ethylene have been conducted, but the precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) was found in the seaweed concentrate prepared from the brown kelp E. maxima. The level of the ethylene-releasing compound was estimated as 9.29 nmol ml−1 (Nelson

and Van Staden, 1985). The presence of ethylene in seaweed concentrate, however, is yet to be demonstrated.

Betaines Betaines are the compounds found in the extracts of seaweeds which behave like cytokinins. Betaines have been isolated from many of the species of brown algae used for the production of seaweed extracts. Ascophyllum nodosum extracts contain c-aminobutyric acid betaine, d-aminovaleric acid betaine and laminine whilst Laminaria species have a range of betaines including glycine betaine (Blunden et al., 1986). In plants, betaines serve as a compatible solute that alleviates osmotic stress induced by salinity and drought stress; however, other roles have also been suggested (Blunden and Gordon, 1986), such as enhancing leaf chlorophyll content of plants following their treatment with seaweed extracts (Blunden et al., 1997). This increase in chlorophyll content may be due to a decrease in chlorophyll degradation (Whapham et al., 1993). Yield enhancement effects due to improved chlorophyll content in leaves of various crop plants have been attributed to the betaines present in the seaweed (Blunden et al., 1997; Genard et al., 1991). It has been indicated that betaine may work as a nitrogen source when provided in low concentration and may serve as an osmolyte at higher concentrations (Naidu et al., 1987).

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TABLE 10.4 Physiological Role of Seaweed Extracts Similar to Plant Growth Regulators Sl. No.

Plant Growth Regulators in Seaweed Extract

Implied Physiological Roles Similar to Plant Growth Regulators

1

Auxin as IAA and conjugates of indole and amino acids

2

Cytokinins as trans-zeatin, trans-zeatin riboside, and dihydro derivatives of these two forms

3

Gibberellins

4 5

Ethylene Abscisic acid

6

Betaines

7

Polyamines

Extract of Ecklonia maxima exhibited remarkable rootpromoting activity on mung bean. Increased rooting in marigold (Tagetus patula) by treatment with Ecklonia maxima extracts (10% SWC Kelpak) for about 18 h. Seaweed extracts improve water and nutrient uptake by roots with improved water and nutrient efficiency, thereby causing enhanced general plant growth and vigor. Photosynthates partitioning and nutrient mobilization in treated plants. Seaweed extracts contain substantial amounts of cytokinins which are known to mitigate abiotic stress-induced free radicals • by anti-oxidative enzyme activities which scavenges several reactive oxygen species (ROS) • direct scavenging by preventing ROS formation by inhibiting xanthine oxidation Yield increases in seaweed-treated plants are thought to be associated with cytokinin present in the extracts Using the lettuce hypocotyl bioassay, the presence of Gibberellins in the extracts of Ascophyllum nodosum has been detected. A. nodosum extract induces amylase activity independent of GA3 and might act in concert with GA-dependent amylase production leading to enhanced germination and seedling vigor in barley. Not known. The water-soluble growth inhibitors extracted from Laminaria digitata and A. nodosum resulted in marked inhibition of lettuce hypocotyl growth. Increase in chlorophyll content in treated plants by reducing chlorophyll degradation. Not known.

Polyamines The polyamines are a group of compounds that act as plant growth regulators but are not classified as plant hormones. These are a class of compounds which have several amino groups replacing hydrogen usually in alkyl chain, e.g. putrescine, spermidine and spermine. Polyamines are known to have a significant effect on the stability of various conformational states of RNA and DNA and are often associated with important phases in the cell division cycle. They also impart membrane stability to different cellular membranes. Several polyamines have been determined in the unicellular thermoacidophilic red alga, Cyanidium caldarium (Hamana

References Crouch et al. (1992), Crouch and van Staden (1991) Crouch et al. (1990) Crouch and van Staden (1991)

Stirk and van Staden (1997) McKersie and Leshem (1994) Fike et al. (2001), Ayad (1998), Fike et al. (2001) Featonby-Smith and van Staden (1984)

Williams et al. (1976) Rayorath et al. (2008b)

– Hussain and Boney (1973), Tietz et al. (1989), Kingman and Moore (1982) Whapham et al. (1993) –

et al., 1990). As polyamines affect a wide range of physiological growth processes, the occurrence of these compounds in seaweed products could influence plant growth. At present they have not been recorded in commercial seaweed products.

EFFECT OF SEAWEED EXTRACTS ON GERMINATION, GROWTH, PHYSIOLOGY AND YIELD Seaweed extracts affect various aspects of germination, growth, physiology and development including overall health of the plants. The effect of seaweed extracts on

Seaweed as Source of Plant Growth Promoters and Bio-Fertilizers 

crop plants can be discussed on the aspects like germination; root development and mineral absorption; shoot growth and photosynthesis; crop yield and vegetative propagation (Table 10.4) (Figure 10.1).

Effect of Seaweed Extracts on Seed Germination Pre-sowing seed treatment with seaweed extracts improves germination and seedling growth in many crops. Alkaline extract of Ascophyllum esculentum was found to improve germination of tomato seeds (Ali et al., 2016). Liquid seaweed extracts of Ulva lactuca and Padina gymnospora (2%) enhanced germination in tomato seeds by lowering mean germination time and increasing germination energy, germination index and seedling vigor (Hernandez-Herrera et al., 2014). The seaweed liquid extracts (SLE) of the brown algae Sargassum vulgare, Colpomenia sinuosa and Padina pavonica at lower concentration was found to enhance germination rate of fenugreek (Trigonella foenum-graecum L.). The extracts of Sargassum vulgare (5% SLE)

FIGURE 10.1  Effect of seaweed liquid fertilizer (SLF).

117

recorded significantly higher germination rate of the seeds (El-Sheekh et al., 2016)

Root Development and Mineral Absorption Seaweed extracts are known to promote root growth and development (Metting et al., 1990; Jeannin et al., 1991). The root growth stimulatory effect is more pronounced when extracts were applied at an early growth stage in maize, and the response was similar to that of auxin, an important root growth-promoting hormone (Jeannin et al., 1991). Seaweed concentrate (SWC) application is found to reduce transplant shock in seedlings of marigold, cabbage and tomato by increasing root size and vigor. SWC treatment enhanced both root:shoot ratios and biomass accumulation in tomato seedlings and wheat by stimulating root growth, indicating that the components in the seaweed had a considerable effect on root development. The root growth-promoting activity is observed when the seaweed extracts are applied either to the roots or as a foliar spray (Finnie and van Staden,

118

1985). Bio-stimulants in general are capable of affecting root development by both improving lateral root formation and increasing total volume of the root system (Vernieri et al., 2005). An improved root system could be influenced by endogenous auxins as well as other compounds in the extracts. Seaweed extracts improve nutrient uptake by roots, resulting in root systems with improved water and nutrient efficiency, thereby causing enhanced general plant growth and vigor.

Effect on Shoot Growth and Photosynthesis Seaweeds and seaweed products enhance plant chlorophyll content (Blunden et al., 1997). Application of a low concentration of Ascophyllum nodosum extract to soil or on foliage of tomatoes produced leaves with higher chlorophyll content than those of untreated controls. This increase in chlorophyll content is due to reduction in chlorophyll degradation, which might be caused in part by betaines in the seaweed extract. Glycine betaine delays the loss of photosynthetic activity by inhibiting chlorophyll degradation during storage conditions in isolated chloroplasts. The extracts of Ascophyllum nodosum have been shown to affect the root growth of Arabidopsis at very low concentrations (0.1 g l−1), whereas plant height and number of leaves were affected at concentrations of 1 g l−1 (Rayorath et al., 2008a). Application of seaweed extracts improves plant mineral uptake by the roots and in the leaves.

Effect on Crop Yield Seaweed extracts probably encourage flowering by initiating robust plant growth (Abetz and Young, 1983). Yield increases in seaweed extract-treated plants may be attributed to the hormonal substances present in the extracts, especially cytokinins (Featonby-Smith and van Staden, 1984). Cytokinin in seaweed concentrate helps to shift distribution of photosynthate from vegetative parts (roots, stem and young leaves) to the developing fruit and promotes fruit development (Panda et al., 2012). Seaweed extract induces early flowering and increases fruit yield when sprayed on tomato plants during the vegetative stage, producing large sized fruits with superior quality (Crouch and van Staden, 1992). Seaweed concentrates trigger early flowering and fruit set in a number of crop plants. Seaweed extract of Ecklonia maxima is found to increase the number of flowers and seeds flower−1 head in marigold when applied immediately after transplanting (Aldworth and van Staden, 1987). Application of Ascophyllum nodosum extract has been shown to increase the yield of cauliflower, lettuce

Handbook of Algal Technologies and Phytochemicals

and maize (Abetz and Young, 1983; Jeannin et al., 1991). Foliar application of Ecklonia maxima extracts is known to enhance yield in bean, wheat, barley and peppers (Arthur et al., 2003; Beckett and van Staden, 1989; Featonby-Smith and van Staden, 1987; Nelson and van Staden, 1984). Ascophyllum nodosum extract has also been shown to have positive effects on the yield of Thompson seedless grape (Norrie and Keathley, 2006).

Role in Vegetative Propagation Seaweed products are used in conventional vegetative propagation (Atzmon and van Staden, 1994; Crouch and van Staden, 1991; Kowalski et al., 1999) in many crop species. Increased rooting has been observed in marigold (Tagetus patula) treated with 10% the seaweed concentrate Kelpak (Ecklonia maxima) for about 18 h (Crouch and van Staden, 1991). Similarly, Kelpak, when applied at a 1:100 dilution, is found to increase the number of rooted cuttings and improve the vigor of the roots in difficult-to-root cuttings of Pinus pinea (Atzmon and van Staden, 1994). Foliar application of commercial liquid seaweed extract from Ascophyllum nodosum, supplemented with BA and IBA, enhances the number of propagules (crown divisions) plant−1 in the ornamental herbaceous perennial Hemerocallis sp. (Leclerc et al., 2006).

Role in Abiotic Stresses Tolerance in Crop Plants Different abiotic stresses experienced by the crop plants such as drought, salinity and high temperature can reduce the yield of major crops. Seaweed extracts from Ascophyllum nodosum have been shown to contain betaines, including gamma-amino butyric acid betaine, 6-aminovaleric acid betaine and glycinebetaine which have antioxidant activity. Plants sprayed with seaweed extracts also exhibit enhanced salt and freezing tolerance (Mancuso et al., 2006). Commercial formulations of Ascophyllum extracts are known to improve freezing tolerance in grapes. Seaweed extracts which contain substantial amounts of cytokinins are known to mitigate stress-induced free radicals by direct scavenging and by preventing ROS formation by inhibiting xanthine oxidation (Fike et al., 2001). The heat tolerance in seaweed extract-induced plants might be attributed largely to the cytokinin components in the seaweed extracts (Zhang and Ervin, 2008). It has also been reported that commercial seaweed extracts like Kelpak mediate stress tolerance by enhancing K+ uptake in plants. The extracts of seaweeds like Ascophyllum nodosum and Ecklonia maxima are known to alleviate

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Seaweed as Source of Plant Growth Promoters and Bio-Fertilizers 

TABLE 10.5 Physiological Effects Elicited by Seaweed Extracts and Possible Mechanism(s) of Bioactivity Mode of Application Seed treatment Seedling dip Foliar spray

Incorporation of marine bio-products Soil drenching Addition of extracts to hydroponics

Effects

Possible Mechanisms

Aerial Application Growth responses • Improved shoot and root growth • Higher flowering and fruit set • Better yield Biotic stress resistance • Resistance to fungal, bacterial and viral pathogens • Resistance to insect-pests Abiotic stress resistance Salt and drought resistance Freezing and chilling resistance Enhanced photosynthesis Enhanced nutritional quality Soil Application Suppression of soil borne diseases and nematodes Abiotic stress resistance Improved modulation Promote plant growth promoting rhizobacteria Water and low temperature stress resistance

Modulation of phytohormones Increased photosynthetic efficiency and carbon assimilation Delayed senescence Anti-microbial Anti-feedant and insect repellant Up-regulation of disease resistance genes, e.g. PR genes Reduced transpiration Enhanced stomatal conductance Up-regulation of subset of stress resistance metabolome Altered metabolism Up-regulation of biosynthetic enzyme Anti-microbial Enhanced growth of friendly microbes Anti-infective Altered metabolism Modulation of root exudates Differential expression of signal molecules and bio-synthetic enzymes Altered root architecture Efficient water and nutrient uptake

Source: Khan et al. (2009).

several abiotic stresses in crop plants when applied exogenously (Panda et al., 2012) (Table 10.5).

CONCLUSION The seaweeds are not only a source of bio-fertilizers but also bio-stimulatory substances including phytohormones. Though the macronutrients present in SLFs are insufficient to elicit growth in crop plants, the micronutrients present in seaweed extracts favor growth and yield in many crops. The huge biomass of this marine resource is needed to be exploited more for its utilization in agriculture. The exhaustive use of seaweeds in agriculture can further augment crop productivity, which can be achieved through further research into their biochemical nature and mechanism of action. SLFs are also known to protect plants against a number of biotic and abiotic stresses and can offer an effective solution for

crop production under adverse conditions. Therefore, research into developing sustainable methods to alleviate these stresses should be a priority. The use of seaweed extracts in agriculture not only reduces the application of harmful agrochemicals but also helps in protecting the environment. This huge mass of renewable resources can be exploited to achieve food security through sustainable agriculture with minimal use of inorganic fertilizers.

REFERENCES Abe, H., Uchiyama, M. and Sato, R. Isolation of phenylacetic acid and its p-hydroxy derivative as auxin-like substances from Undaria pinnatifida. Agric. Biol. Chem. 1974. 38: 897–898. Abetz, P. and Young, C.L. The effect of seaweed extract sprays derived from Ascophyllum nodosum on lettuce and cauliflower crops. Bot. Mar. 1983. 26: 487–492.

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Aldworth, S.J. and van Staden, J. The effect of seaweed concentrate on seedling transplants. S. Afr. J. Bot. 1987. 53: 187–189. Arthur, G.D., Stirk, W.A., van Staden, J. and Scott, P. Effect of a seaweed concentrate on the growth and yield of three varieties of Capsicum annuum. S. Afr. J. Bot. 2003. 69: 207–211. Ayad, J.Y. The effect of seaweed extracts (Ascophyllum nodosum) extract on antioxidant activities and drought tolerance of tall fescue (Festuca arundinacea Schreb.). Ph.D. Thesis, Texas Tech University, Lubbock. 1998. Bartel, B. Auxin biosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997. 48: 51–66. Beckett, R.P. and van Staden, J. The effect of seaweed concentrate on the growth and yield of potassium stressed wheat. Plant Soil 1989. 116: 29–36. Bernart, M. and Gerwick, W.H. 3-(hydroxyacetyl) indole, a plant growth regulator from the Oregon red alga Prionitislanceolata. Phytochemistry 1990. 29: 3697–3698. Blunden, G. Agricultural uses of seaweeds and seaweed extracts. In: Guiry, M.D. et al. (Eds.), Seaweed Resources in Europe: Uses and Potential. 1991: 65–81. Blunden, G. Jenkins, T. and Liu, Y. Enhanced leaf chlorophyll levels in plants treated with seaweed extract. J. Appl. Phycol. 1997. 8: 535–543. Blunden, G., Cripps, A.L.G., Mason, S.M., T.G. and Turner, C.H. The characterization and quantitative estimation of betaines in commercial seaweed extracts. Bot. Mar. 1986. 29: 155–160. Blunden, G. and Gordon, S.M. Betaines and their sulphono analogues in marine algae. In: Round, F.E., Chapman, D.J. (Eds.), Progress in Phycological Research, Vol. 4. Biopress Ltd., Bristol. 1986: 39–80. Brain, K.R., Chalopin, M.C., Turner, T.D., Blunden, G. and Wildgoose, P.B. Cytokinin activity of commercial aqueous seaweed extract. Plant Sci. Lett. 1973. 1: 241–245. CGIAR. Farming Systems Research at International Agricultural Research Centre Roam Secretariat. Agriculture Department, FAO, Rome. 1978. Chojnacka, K., Saeid, A. Witkowska, Z. and Tuhy, L. Biologically active compounds in seaweed extracts-the prospects for the application. Open Conf. Proc. J. 2012. 3(1): 20–28. Cole, A.J., Roberts, D.A., Garside, A.L., De Nys, R. and Paul, N.A. Seaweed compost for agricultural crop production. J. Appl. Phycol. 2016. 28: 629–642. Craigie, J.S. Seaweed extract stimuli in plant science and agriculture. J. Appl. Phycol. 2011. 23: 371–393. Crouch, I.J., Beckett, R.P. and van Staden, J. Effect of seaweed concentrate on the growth and mineral nutrition of nutrient stressed lettuce. J. Appl. Phycol. 1990. 2: 269–272. Crouch, I.J., Smith, M.T., van Staden, J., Lewis, M.J. and Hoad, G.V. Identification of auxins in a commercial seaweed concentrates. J. Plant Physiol. 1992. 139: 590–594. Crouch, I.J. and van Staden, J. Evidence for rooting factors in a seaweed concentrate prepared from Ecklonia maxima. J. Plant Physiol. 1991. 137: 319–322.

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Crouch, I.J. and van Staden, J. Effects of seaweed concentrate on the establishment and yield of greenhouse tomato plants. J. Appl. Phycol. 1992. 4: 291–296. Crouch, I.J. and van Staden, J. Evidence for the presence of plant growth regulators in commercial seaweed products. Plant Growth Regul. 1993. 13: 21–29. El-Sheekh, Ismail, M.M. and Hamouda, M.M. Influence of some brown seaweed extracts on germination and cytological responses of Trigonella foenum-Graecum L. Biotechnol. Ind. J. 2016. 12(9): 104. Featonby-Smith, B.C. and van Staden, J. The effect of seaweed concentrate and fertilizer on growth and the endogenous cytokinin content of Phaseolus vulgaris. S. Afr. J. Bot. 1984. 3: 375–379. Featonby-Smith, B.C. and van Staden, J. Effects of seaweed concentrate on grain yield in barley. S. Afr. J. Bot. 1987. 53: 125–128. Fike, J.H., Allen, V.G., Schmidt, R.E., Zhang, X., Fontenot, J.P., Bagley, C.P., Ivy, R.L., Evans, R.R., Coelho, R.W. and Wester, D.B. Tasco-Forage: I. Influence of a seaweed extract on antioxidant activity in tall fescue and in ruminants. J. Anim. Sci. 2001. 79: 1011–1021. Finnie, J.F. and van Staden, J. Effect of seaweed concentrate and applied hormones on in vitro cultured tomato roots. J. Plant Physiol. 1985. 120: 215–222. Genard, H., Le Saos, J., Billard, J.P., Tremolieres, A. and Boucaud, J. Effect of salinity on lipid composition, glycine betaine content and photosynthetic activity in chloroplasts of Suaedamaritima. Plant Physiol. Biochem. 1991. 29: 421–427. Gopala, R.P. Gibberellin-like behaviour of α-tocopherol in green gram Vigna radiata. Geobios. 1984. 11: 21–25. Hamana, K., Matsuzaki, S., Nitsu, M., Samejima, K. and Nagashima, H. Polyamines of unicellular thermoacidophillic red alga, Cyanidium caldarium. Phytochemistry 1990. 29: 377–380. Hartmann, H.T. and Kester, D.E. 1983. Plant Propagation: Principles and Practices (4th Ed.). Prentice Hall, Englewood Cliffs, NJ. 1983: 234–297. Hussain, A. and Boney, A.D. Isolation of kinetin-like substances from Laminaria digitata. Nature 1969. 223: 504–505. Hussain, A. and Boney, A.D. Hydrophilic growth inhibitors from Laminaria and Ascophyllum. New Phytol. 1973. 72: 403–410. Jain, V.K. Fundamentals of Plant Physiology. S. Chand & Company Ltd., New Delhi. 2008: 626. Jeannin, I., Lescure, J.C. and Morot-Gaudry, J.F. The effects of aqueous seaweed sprays on the growth of maize. Bot. Mar. 1991. 34: 469–473. Jennings, R.C. Gibberellins as endogenous growth regulators in green and brown algae. Planta 1968. 80: 34–42. Jensen, A. Tocopherol content of seaweed and seaweed meal I: Analytical methods and distribution of tocopherols in benthic algae. J. Sci. Food Agric. 1969. 20: 449–453. Kaliaperumal, N., Chennubhotla, V.S.K. and Kalimuthu, S. Seaweed resource of India. Seaweed Res. Utiln. 1987. 41: 51–99.

Seaweed as Source of Plant Growth Promoters and Bio-Fertilizers 

Khan, W., Rayirath, U.P., Subramanian, S., Jithesh, M.N., Rayorath, P., Mark Hodges, D.M., Critchley, A.T., Craigie, J.S., Norrie, J. and Prithiviraj, B. Seaweed extracts as biostimulants of plant growth and development. J. Plant Growth Regul. 2009. 28: 386–399. Kingman, A.R. and Moore, J. Isolation, purification and quantification of several growth regulating substances in Ascophyllum nodosum (Phaeophyta). Bot. Mar. 1982. 25: 149–153. Kowalski, B., Jäger, A.K. and Van Staden, J. The effect of a seaweed concentrate on the in vitro growth and acclimatization of potato plantlets. Potato Res. 1999. 42: 131–139. Leclerc, M., Caldwell, C.D., Lada, R.R. and Norrie, J. Effect of plant growth regulators on propagule formation in Hemerocallis spp. and Hosta spp. Hortic. Sci. 2006. 41: 651–653. Mancuso, S., Azzarello, E., Mugnai, S. and Briand, X. Marine bioactive substances (IPA extract) improve ion fluxes and water stress tolerance in potted Vitis vinifera plants. Adv. Hortic. Sci. 2006. 20: 156–161. Metting, B., Zimmerman, W.J., Crouch, I.J. and van Staden, J. Agronomic uses of seaweed and microalgae. In: Akatsuka, I. (Ed.), Introduction to Applied Phycology. SPB Academic Publishing, Hague, the Netherlands. 1990: 269–627. Mohanty, D. Adhikary, S.P. and Chattopadhyay, G.N. Seaweed liquid fertilizer (slf) and its role in agriculture productivity. Ecoscan 2013. III: 1–10. Naidu, B.P., Jones, G.P., Paleg, L.G. and Poljakoff-Mayber, A. Proline analogues in Melaleuca species: Response of Melaleuca lanceolata and M. uncinata to water stress and salinity. Aust. J. Plant Physiol. 1987. 14: 669–677. Nelson, W.R. and van Staden, J. The effect of seaweed concentrate on wheat culms. J. Plant Physiol. 1984. 115: 433–437. Nelson, W.R. and van Staden, J. 1-aminocyclopropane-l-carboxylic acid in seaweed concentrate. Bot. Mar. 1985. 28: 415–417. Niemann, D.I. and Dorfing, K. Growth inhibitors and growth promotors in Enteromorpha compressa (Chlorophyta). J. Appl. Phycol. 1980. 16: 383. Norrie, J. and Keathley, J.P. Benefits of Ascophyllum nodosum marine-plant extract applications to ‘Thompson seedless’ grape production. Acta Hortic. 2006. 727: 243–248. Panda, D., Pramanik, K. and Nayak, B.R. Use of seaweed extracts as plant growth regulators for sustainable agriculture. Int. J. Bioresour. Stress Manag. 2012. 3(3): 404–411.

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Rath, J. and Adhikary, S.P. Algal Flora of Chilika Lake. Daya Publishing House, New Delhi. 2005: 206. Rayorath, P., Jithesh, M.N., Farid, A., Khan, W., Palanisamy, R., Hankins, S.D., Critchley, A.T. and Prithiviraj, B. Rapid bioassays to evaluate the plant growth promoting activity of Ascophyllum nodosum (L.) Le Jol. using a model plant, Arabidopsis thaliana (L.) Heynh. J. Appl. Phycol. 2008a. 20: 423–429. Rayorath, P., Khan, W., Palanisamy, R., MacKinnon, S.L., Stefanova, R., Hankins, S.D., Critchley, A.T. and Prithiviraj, B. Extracts of the brown seaweed Ascophyllum nodosum induce gibberellic acid (GA3)independent amylase activity in barley. J. Plant Growth Regul. 2008b. 27(4): 370–379. Sivasankari, S., Venkatesalu, V., Anantharaj, M. and Chandrasekaran, M. Effect of seaweed extracts on the growth and biochemical constituents of Vigna sinensis. Bioresour. Technol. 2006. 97(14): 1745–1751. Stephenson, W.A. Seaweed in Agriculture and Horticulture. Faber and Faber, London. 1968. Stirk, W.A. and van Staden, J. Isolation and identification of cytokinins in a new commercial seaweed product made from Fucus serratus L. J. Appl. Phycol. 1997. 9: 327–330. Stirk, W.A., Arthur, G.D., Lourens, A.F., Novák, O., Strnad, M. and Staden, Jv. Changes in cytokinin and auxin concentrations in seaweed concentrateswhen stored at an elevated temperature. J. Appl. Phycol. 2004. 16: 31–39. Subbarao, G.V., Ito, O., Berry, W.L. and Wheeler, R.M. Sodium-A functional plant nutrient. Crit. Rev. Plant Sci. 2003. 22(5): 391–416. Tietz, A., Ruttkowski, U., Kohler, R. and Kasprik, W. Further investigations on the occurrence and the effects of abscisic acid in algae. Biochem. Physiol. Pflanz. 1989. 184: 259–266. Vernieri, P., Borghesi, E. Ferrante, A. and Magnani, G. Application of biostimulants in floating system for improving rocket quality. J. Food Agric. Environ. 2005. 3: 86–88. Whapham, C.A., Blunden, G., Jenkins, T. and Hankins, S.D. Significance of betaines in the increased chlorophyll content of plants treated with seaweed extract. J. Appl. Phycol. 1993. 5: 231–234. Williams, D.C., Brain, K.R., Blunden, G. Wildgoose, P.B. and Jewers, K. Plant growth regulatory substances in commercial seaweed extracts. Proceedings of International Seaweed Symposium 1976. 8: 59–63. Zhang, X. and Ervin, E.H. Impact of seaweed extract-based cytokinins and zeatin riboside on creeping bentgrass heat tolerance. Crop Sci. 2008. 48: 364–370.

11 A Nutraceutical Supplement in Aquaculture Algae

Helena M. Amaro, I. Sousa-Pinto, and A. Catarina Guedes CONTENTS Abbreviations����������������������������������������������������������������������������������������������������������������������������������������������������������123 Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������123 General Attributes of Algal Species in Aquaculture�����������������������������������������������������������������������������������������������124 Use of Algae as a Bioactive Supplement for Aquaculture Fish Feed���������������������������������������������������������������������124 Use of Algae as a Bioactive Supplement for Aquaculture Bivalves Feed���������������������������������������������������������������126 Use of Algae as a Bioactive Supplement for Aquaculture Crustaceans Feed���������������������������������������������������������127 Conclusion��������������������������������������������������������������������������������������������������������������������������������������������������������������128 Acknowledgments���������������������������������������������������������������������������������������������������������������������������������������������������128 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 129

ABBREVIATIONS AA Arachidonic Acid DHA Docosahexaenoic Acid FA Fatty Acids LC-PUFA Long-Chain Polyunsaturated Fatty Acids PUFA Polyunsaturated Fatty Acids ROS Reactive Oxygen Species SCD Single Cell Detritus Se Selenium SOD Superoxide Dismutase

INTRODUCTION Aquaculture is one of the fastest growing food industries in the world. Over the last 30 years, the fish production in aquaculture had a worldwide annual growth rate of c. 8.8%, being predicted that by 2022 that it will reach 35%, providing an additional 22 million tons of fish. But, by 2030, it is predicted that an additional 40 million tons of aquatic food will be required for human consumption (Maisashvili et al., 2015). Intensive aquaculture involves high stocking densities, which in consequence may result in low water quality and other stressors. These intense production conditions can make aquaculture animals more susceptible to diseases, leading to an increase in death rate resulting in disastrous consequences in terms of costs of production. To avoid possible bacterial diseases, usually, diets are supplemented with antibiotics, but unfortunately, antibiotics have been misused resulting in

rampant antibiotic resistance posing human health and environmental issues. For these reasons, several bioactive compounds from natural origins, like organic acids and algae extracts immunostimulants, are being sought as strategies to control pathogens, not only to reduce the use of antibiotics but also to increase disease resistance of aquatic organisms, limiting their negative effects and allowing aquaculture to reach its full potential (Mendoza Rodriguez et al., 2017). Nutritional composition of algae is described as containing mainly proteins, carbohydrates, lipids and trace nutrients, including vitamins, and trace elements, thus entailing compounds with all characteristics to be a great natural supplement in animal feed (Yaakob et al., 2014). Moreover, diets based on microalgae are known to have played an important role in aquaculture and have been recognized as a valuable natural source of amino acids, containing significant levels of high-quality protein and LC-PUFA (long-chain polyunsaturated fatty acids), such as docosahexaenoic acid and eicosapentaenoic acid, which are essential fatty acids (FA) for maintaining marine fish health. Moreover, FA longer than ten carbon atoms have been reported to induce bacterial protoplast lysis, useful antimicrobial activity for removing potential fish pathogens (Guedes et al., 2011; Holdt and Kraan, 2011; Meena et al., 2013), besides the vitamins, essential amino acids, carotenoids and other substances with high biological and physiological activities (Becker, 2007; Xu and Yang, 2007; Stara et al., 2014). On the other hand, macroalgae have been recognized as a valuable natural source of biologically active amino acids, 123

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proteins and peptides, containing significant levels of high-quality protein. This protein raw material may be a potential source of bioactive peptides with potential health-promoting and disease-preventing properties. Both micro- and macroalgae have been considered a rich source of different types of carotenoids, highly bioactive compounds, such as antioxidant compounds and so prevent oxidative stress in aquaculture organisms (Pan et al., 2011; Kouba et al., 2014; Stara et al., 2014). Carotenoids and β-glucan have been proven to prevent problems linked to oxidation and to have immune stimulant properties, respectively (Meena et al., 2013). Moreover, macroalgae present a higher content in polysaccharides, important bioactive compounds (Holdt and Kraan, 2011). Currently, microalgae are used in aquaculture as live feeds for all growth stages of several organisms such as bivalves (oysters, scallops, clams and mussels), juvenile stages of abalone, crustaceans, fish and for zooplankton used in other aquaculture food chains (Brown, 2002). The most frequently used microalgae in aquaculture are Chlorella, Tetraselmis, Scenedesmus, Pavlova, Phaeodactylum, Chaetoceros, Nannochloropsis, Skeletonema and Thalassiosira (Sirakov et al., 2015). However, a combination of different algal species provides better balanced nutrition and improves fish growth better than a diet composed of only one algal species (Spolaore et al., 2006). Particularly for larval feed, microalgae from the genera Chaetoceros, Thalassiosira, Tetraselmis, Isochrysis and Nannochloropsis can be included. These organisms are fed directly or indirectly – providing the microalgae through artemia, rotifers and daphnia, which are, in turn, fed to the target larval organisms. With regard to use of macroalgae, the most used species applied as feed in aquaculture are Porphyra, Ulva, Gracilaria, Laminaria and Chondrus, used to partially replace diets composition in percentages from 5 to 10%, mostly the lipid and protein counterpart (Valente et al., 2006; Soler-Vila et al., 2009; Thanigaivel et al., 2015a; Carboni et al., 2016; Peixoto et al., 2016). Also, these macroalgae contain in their composition bioactive compounds able to perform immunomodulatory effects, such as laminaran (β-1,3 glucan) and -M-alginate, or antioxidant effects, such as pigments like fucoxanthin, astaxanthin and tocopherol (Vetvicka et al., 2013). In a general way, the use of algae (micro- and macroalgae) as a nutraceutical in aquaculture has been applied due its positive effect on weight gain, increased triglyceride and protein deposition in muscle, improved resistance to disease, increasing fish digestibility, physiological activity, starvation tolerance and carcass quality (Fleurence et al., 2012).

Handbook of Algal Technologies and Phytochemicals

GENERAL ATTRIBUTES OF ALGAL SPECIES IN AQUACULTURE Algal species can vary significantly in nutritional value, as a function of the prevailing culture conditions, especially microalgae. Only a reduced number of species have been used, primarily for historical reasons and ease of cultivation – rather than supported by scientific evidence of any superior performance as nutritional or therapeutical supplements. Hence, formulations more carefully selected of micro- and macroalgal origin may offer the opportunity for development of improved nutritional packages aimed at larval animals. Algae and their extracts should, in general, possess a number of key attributes to be useful for aquacultured species: they should be of an appropriate size and shape for ingestion and ready digestion; they should be stable to fluctuations in temperature, light and nutrient profile, as often occur in hatchery systems; and they should exhibit appropriate nutritional qualities, including absence of toxins – and in the case of microalgae, besides all this, they should exhibit fast growth rates and be amenable to mass culture too (Guedes and Malcata, 2012).

USE OF ALGAE AS A BIOACTIVE SUPPLEMENT FOR AQUACULTURE FISH FEED Apparently, the world consumption of fish per capita has doubled since the 1960s to 2013 and continues to increase over the years. Therefore, it is crucial to find sustainable forms of fish production in aquaculture, with special attention to the improvement of their health (FAO, 2016). As referred to before, usage of disinfectants that are used in aquaculture, may cause oxidative stress in the animals due the generation of reactive oxygen species (ROS) endangering the production of fish in aquaculture systems. The use of natural antioxidant compounds can aid to avoid these oxidative processes; indeed, carotenoids produced by algae and known for their antioxidant capacity can be a solution. For example, increase of oxidative stress resultant upon chemical treatment can be managed by inducing resistance by supplementation of microalgal biomass to the fish diet, with a content of carotenoids around 214 mg per kilogram of feed (Stara et al., 2014). Furthermore, the replacement of fishmeal by algae (micro- and macroalgae) has proven to be effective in terms of growth and nutrition, because they are a rich source of high-quality protein, vitamins and micronutrients (trace elements), besides bioactive compounds such

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as carotenoids, that can be applied to aquaculture feeds (Subhadra, 2011), as summarized in Table 11.1. Concerning the partial replacement of diets by microalgae, a study of Olvera-Novoa et al. (2008) proved the usage of Spirulina sp. as a supplement to 40% of the fishmeal protein in tilapia diets without any adverse effects. Similar results were obtained by Badwy et al. (Badwy et al., 2008) who partially replaced fishmeal with two different dried microalgae (Chlorella spp. and Scenedesmus spp.) in Nile tilapia (Oreochromis niloticus) diets and observed that formulation up to 50% of algae led to better growth, weight and protein efficiencies ratio (Yaakob et al., 2014). Another example is related with selenium (Se), which is a trace element essential for all living organisms as it is a constituent of selenoproteins (Sweetman et al., 2010). In aquaculture, Se is used as a supplement to feed, improving its nutritional profile to satisfy requirements of aquaculture fish (Penglase et al., 2011). For example, the promotion of dietary Se helps fish to tolerate stress (Rider et al., 2009) and moderates toxicity of heavy metals such as mercury and cadmium (Raymond and Ralston, 2009). A study of Kouba et al. (2014) compared the benefits of including a Se-enriched Chlorella microalgae or sodium selenite in fishmeal of common barbel (Barbus barbus L.) as diet supplements. This assay

proved that Se from Chlorella is more readily accumulated and biologically active while being less toxic than sodium selenite. Also, inclusion of seaweeds in fish diets proved to be a good nutraceutical supplement. Indeed, in the last years, several seaweed species have been investigated as a dietary ingredient for a wide range of fish species, but it was observed that the percentage of inclusion varies with both species of seaweed and fish. For example, it was observed that a low-level incorporation of Ulva on fishmeal improved growth, nutrient absorption, physiological activity, disease resistance, carcass quality and reduced stress response in fish (Mustafa et al., 1995; Wassef et al., 2005; Valente et al., 2006). In Nile tilapia, inclusion of 5% of Ulva on fishmeal improved growth performance, feed efficiency, nutrient utilization and body composition (Ergün et al., 2009). Supplementation of Red tilapia (Oreochromis sp.) was more efficient in influencing growth performance by inclusion of up to 15% of Ulva sp. in the feed (El-Tawil, 2010). Also, positive effects were observed in modulation of digestive enzyme of European seabass (Dicentrarchus labrax) when Ulva spp. was used as supplementation (Peixoto et al., 2016). Also, Porphyra sp. demonstrated potential to enhance fish growth when incorporated at 10% as supplement in

TABLE 11.1 Nutraceutical Evidences of Use of Algae as a Supplement in Aquaculture Fish Production Algal Compound

Alga Source

Fish

Action

References

1% Alginic acid (Ergosan) 1% Alginic acid (Ergosan) Extracts of carrageenan and alginic acid Carrageenan extract

L.digitata

Snakehead Channastriata Sea bass

Immune responses enhancement Activation of innate immune responses Modulates immunoestimulation High total immunoglobulin was higher Increases accumulation and reduces stress Increases respiratory burst and immune system stimulation Antibacterial and prophylactic actions against Pseudomonas spp. Antibacterial action against Aeromonas salmonicida Modulation of digestive enzymes

Miles et al. (2001)

Selenium

L.digitata and Ascofillum nodosum Alfa Aesar, from algalorigin Alfa Aesar, Ward Hill, MA, USA Se-enriched Chlorella

Barbusbarbus

Aqueous extracts and β-glucans

Ulva rígida and Chondrus crispus

Scophthalmus maximus and Salmosalar

Aqueous and ethanol extracts

Padina gymnospora Sargassum cinereum

Oreochromis mossambicus

Aqueous and ethanol extracts Whole seaweed

Gracilaria folifera

Oreochromis mossambicus Dicentrarchus labrax

Ulva spp.

Sciaenopsocellatus Sciaenop socellatus

Bagni et al. (2005) Mendoza Rodriguez et al. (2017) Mendoza Rodriguez et al. (2017) Kouba et al. (2014) Dalmo and Seijelid (1995); Castro et al. (2004) Thanigaivel et al. (2015a)

Thanigaivel et al. (2015b) Peixoto et al. (2016)

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Rainbow trout (Oncorhynchus mykiss) (Soler-Vila et al., 2009). Gracilaria spp. exhibited potential of application as supplement (Peixoto et al., 2016) at 2.5% in European seabass diet, resulting in enhanced antioxidant capacity. Most of studies examining the immunostimulatory ability of algae have been carried out in vitro with algal extracts (Díaz-Rosales et al., 2005; Díaz-Rosales et al., 2007). Studies on oral administration are scarce and usually include only algal extracts or isolated compounds instead of whole algae (Valente et al., 2006; Díaz-Rosales et al., 2008) For instance, algal polysaccharides such as carrageenan, a linearly sulfated polysaccharide derived from red seaweed, and alginic acid, an anionic polysaccharide obtained from brown seaweed, are a group of compounds which have been shown to improve immune stimulation in some fish, such as common carp – Cyprinus carpio (Fujiki and Yano, 1997). Alginic acid also has been reported to increase immune responses of snakehead Channa striata (Miles et al., 2001), sea bass (Bagni et al., 2005) and kelp grouper Epinephelus burneus (Harikrishnan et al., 2011) and Sciaenops ocellatus (Mendoza Rodriguez et al., 2017). Alginic acid can be found in the intercellular mucilage and cell walls of species such as Undaria pinnatifida and Macrocystis pyrifera (Chapman and Chapman, 1980). In vitro studies have stated that Ulva rigida and Chondrus crispus extracts and β-glucans increased respiratory burst and immune system stimulation in turbot (Scophthalmus maximus) and Atlantic salmon (S. salar) phagocytes, through rapid release of ROS and signaling proteins (Dalmo and Seijelid, 1995; Castro et al., 2004). Also, in addition to immune competency, a correlation has been reported concerning phenolic content and antioxidant capacity of seaweeds (Devi et al., 2011), owing to ROS scavenging activity or lipid peroxidation inhibition (Heo et al., 2005). Similarly, in orange-spotted grouper (Epinephelus coicoides), feeding of sodium alginate from Macrocystis pyritera and carrageenan from C. crispus presented an increase in: respiratory burst, superoxide dismutase (SOD) and phagocytic activities when exposed to Vibrio alginolyticus (Cheng et al., 2007). In Nile tilapia (Oreochromis mossambicus) it was observed that administration of seaweeds extracts from Gracilaria folifera, Padina gymnospora and Sargassum cinereum may be effective as therapeutic and prophylactic treatments against Pseudomonas spp. infection (Thanigaivel et al., 2015a, 2015b). All these studies prove the beneficial effects of the use of immune-stimulant functional ingredients, from

Handbook of Algal Technologies and Phytochemicals

natural sources such as algae, as meals or as extracts and isolated compounds, as a powerful tool to improve aquaculture systems (Peixoto et al., 2016).

USE OF ALGAE AS A BIOACTIVE SUPPLEMENT FOR AQUACULTURE BIVALVES FEED The aquaculture of mollusks, particularly bivalves such as oysters, mussels, clams and scallops, has an important commercial role, and its exploitation is increasing on a global scale. Although total mollusk production only accounts for 9.7% of total volume food fish production, their economic value represented 22.4% or equivalent to €26.2 billion. In the aquaculture sector, some countries, like Japan and Korea, rely deeply on their mollusk production, achieving 50% of their total food fish production (Mau and Jha, 2018). Bivalves do not require supplementary feeding beyond the natural algae content (Helm and Bourne, 2004). In the recent years, bivalves have witnessed a higher demand of production, leading to a production massification, and together with that a need to improve culture operations efficiency is in demand. It is well known that bivalves feed essentially exclusively on marine microalgae throughout all their life cycle and that biochemical composition of the diet influences their physiology (Anjos et al., 2017; Yang et al., 2017). Presently the quality issues are getting due attention, in this context the water quality is a critical factor that affects directly the bivalves’ production. For example, the increasing of toxins in water, leads to high rates of diseases and death, hampering the production (Helm and Bourne, 2004; FAO, 2016). Thus, in aquaculture production, the quality of feed has a preponderant role, improving bivalves’ health, preventing diseases and stress and supporting high productivity (Helm and Bourne, 2004; Anjos et al., 2017). A bivalve autonomous aquaculture production entails three main stages: 1) production of seeds in hatcheries, 2) juveniles growth, and 3) broodstock. Stage 1 is undeniably related with the quality of the food provided (Helm and Bourne, 2004; Anjos et al., 2017), and the effect of food on broodstock conditioning is species-specific both in quality and in quantity (Utting and Millican, 1998). The larvae of most bivalve species have similar food preferences; suitable algal species include C. calcitrans, T. pseudonana, I. galbana and T. suecica (for larvae > 120 µm in length) (FAO, 1996). Although microalgae are the primary food source for bivalves, they can also feed on other small particles such as bacteria, detritus and even zooplankton (Arapov et al., 2010).

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In nature, detritus input in bivalve diet is important during the periods when abundance of phytoplankton is too low to satisfy bivalve energy needs (Arapov et al., 2010). Evidence of scallop C. farreri showed that macroalgae Ulva pertusa, during its bloom, contributed from 8.7 to 11.0% to the carbon budget of intertidal oyster and mussel (Xu and Yang, 2007). Although in aquaculture bivalves; diet is predominantly a microalgae mixture, composed of one or more diatoms and one flagellate (Anjos et al., 2017), seaweed can be also an effective diet source (Table 11.2). Indeed, Porphyra haitanensis were found to be a successful substitution diet in nursery production of Crassostrea belcheri (Carboni et al., 2016; Tanyaros and Chuseingjaw, 2016), suggesting that macroalgae can be an important food source for this animal class. Also, Ulva species were identified as an important food source of intertidal bivalves such as oyster (Xu and Yang, 2007), due to their high content in carbohydrates and proteins. In vitro studies reported a positive correlation between phenolic content and antioxidant capacity of macroalgae, using strategies such as ROS scavenging activity or lipid peroxidation inhibition (Heo et al., 2005; Devi et al., 2011). Particularly U. rigida has received particular attention as a novel source of natural bioactive compounds, such as polyphenols, polysaccharides terpenoids, fatty acids and vitamins (Yildiz et al., 2012; Mezghani et al., 2013), some of them responsible, among others, for antioxidant and immuno-stimulating features. The production of single cell detritus (SCD) from several genera of macroalgae, such as Laminaria, Macrocystis and Sargasun, can be obtained either by enzymatic or bacterial action or both. The SCD incorporation has been tested in the last two decades, and these particles were shown to be easily ingested, especially by larvae of the clam Ruditapes philippinarum (Uchida and Murata, 2002). SCD from Laminaria saccharina derived by enzymatic and bacterial digestion had profound influence as hatchery diets for Ruditapes decussatus resulting in increases by 54 to 68% live weight and

length, respectively, when compared to a phytoplanktonbased diet (Camacho et al., 2004). Even the highest quality live microalgae will rarely have an optimal nutrient composition (Becker, 2013), and so currently multiple cultures of different microalgal species are grown, which increases production costs and commercial risk in the case of contamination. For example, the diatom Thalassiosira pseudonana is widely cultivated to feed variety of mollusks, including the Pacific oyster Crassostrea gigas and rock scallops (Yaakob et al., 2014). In this regard, it is possible to use their natural source of feed to improve bivalves’ health by manipulating the diet’s composition, either by providing the whole algae or incorporating bioactive extracts of micro- and macroalgae in diets (Yaakob et al., 2014). Microencapsulation is also being adopted to enhance efficiency of the diets. This technology can offer an efficient way to deliver feed or supplementary nutraceutical diets in order to improve bivalve nutrition, growth and production, while reducing the mortality, costs of production and financial risks. A study of Willer and Aldridge (2017) aimed to demonstrate that a new form of BioBullets, containing lipid-walled microparticles of Schizochytrium microalga, could be successfully captured and ingested by a commercially farmed bivalve. Results showed that microparticles were successfully ingested and broken down by the gut. This technology seems to be very promising to improve diets by including a nutraceutical diet for aquaculture bivalves.

USE OF ALGAE AS A BIOACTIVE SUPPLEMENT FOR AQUACULTURE CRUSTACEANS FEED Aquaculture of crustaceans accounted in 2014 for 6.9 million tons of crustaceans (corresponding to ca. €31.6 billion), being mostly located in South America and Asia. Among the 62 crustacean species produced in aquaculture, most are species of shrimp (Metapenaeus

TABLE 11.2 Nutraceutical Evidence of Use of Algae as a Supplement in Aquaculture Bivalves’ Production Bivalve

Algae

Action

Reference

Pinctada fucatamartensii

Chlorella sp. Spirulina platensis Thalassiosira pseudonana Chaetoceros calcitrans

Enhancement of antioxidant capacity and immune response Antioxidant protection Improvement of immune functions

Yang et al. (2017) Yaakob et al. (2014) Delaporte et al. (2003)

Laminaria saccharina

Improvement of weight and length

Camacho et al. (2004)

Crassostrea gigas Crassostrea gigas Ruditapes philippinarum Ruditapes decussatus

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and Penaeus) and crawfish (Pacifastacus) (FAO, 2016). Particularly penaeid shrimp culture has developed quickly over the last three decades to become an important economic activity in many Asian countries (Varadharajan and Pushparajan, 2013). Although it is quite variable, in nature the feed of shrimps in inshore was described to include phytoplankton, zooplankton, nematodes, polychaetes, detritus, crustacean, amphipods, isopods, mud, miscellaneous (Varadharajan and Pushparajan, 2013). In aquaculture, a typical shrimp diet is wheat flour (35%), soybean meal (20%) and fishmeal (25%) (Hunter, 1996); and these ingredients provide the protein, amino acids and energy in the diet. However, the compositions of diets must include a high percentage of protein, and the main protein supplements are sourced from animal, yeast or plant proteins. Excellent major protein sources for shrimp diets include squid, soybean meal, shrimp meal, fishmeal, krill and scallop waste, however microalgae were also found to be a valid alternative (FAO; Venero et al., 2008). Indeed, the inclusion of 10% of Spirulina platensis in the diet of juvenile Pacific white shrimp Litopenaeus vannamei (Boone) showed enhancement of final weight with stronger pigmentation (Hanel et al., 2007). In aquaculture the most serious disease-causing organisms at larval stage are Zoothamnium, Fungi (Lagenidium) and bacteria (Vibrio) (Kungvankij, 1984). The inclusion of algal extracts with recognized antimicrobial power may be a viable solution to reduce these problems. Furthermore, algae are added in other crustaceans’ larval stages, such as during the non-feeding nauplius stage so that algae are available immediately upon molting into the protozoa stage. Algal species most often used are Tetraselmis chui, Chaetoceros gracilis and Skeletonema costatum (FAO, 1996). Moreover, the effects of supplementation of other species of microalgae were tested, such as Hypnea cervicornis and Cryptonemia crenulata, rich in protein, resulting in significant increase in shrimp growth rates (Harun et al., 2010). Concerning the important role of polyunsaturated fatty acids (PUFAs) in shrimp nutrition, it is known that docosahexaenoic acid (DHA) is required for the production of ecdysone for molting, growth and egg production. The mechanism involves the biological membranes rich in di-22:6 (n-3) phosphoglycerides which is relatively constant in the face of changing environmental variables like temperature, pressure, and salinity (Hemaiswarya et al., 2011). Besides, DHA and arachidonic acid (AA) have a relevant role in improvement of immune parameters, such as total hemocyte count, phenoloxidase activity, superoxide dismutase activity and bactericidal activity (Nonwachai et al., 2010).

Handbook of Algal Technologies and Phytochemicals

In this context, several studies have been done to study the effects of the addition of microalgae on shrimp diets. For example, it has been reported that the addition of 0.5% Spirulina meal in a complete diet for shrimp (L. vannamei juveniles), with 14% of Peruvian fishmeal, proved to be a nutritionally efficient feeding attractant, due the high levels of DHA and AA of Spirulina (Silva-Neto et al., 2012). In parallel, through short-term feeding studies, two marine algal products (MAP) from Nanofrustulum spp. (Bacillariophyceae) and Tetraselmis spp. (Chlorophyceae) named MAP3 (rich in neutral lipids) and MAP8 (with all lipids) were found suitable as substitutes for protein source in the feeds of Whiteleg shrimp (Kiron et al., 2012).

CONCLUSION Aquaculture is considered an integral component of the bioeconomy in Europe; and the diversification in aquacultured species is essential for development of a competitive industry. The ‘blue’ biomass derived from algae, with efficient use of sunlight and simple growth requirements, is globally recognized as potential feedstock and a source of valuable chemical constituents with applications in a variety of bio-products to animal feed, opening new markets for these products and diminishing pressure on land resources. Microalgae have been recognized as a valuable natural source of amino acids and long-chain polyunsaturated fatty acids (PUFAs), essential for fish health. Also, macroalgae has been recognized as a valuable natural source of biologically active amino acids, proteins and peptides, containing significant levels of high-quality protein and high levels of polysaccharides, well known bioactive compounds. Both micro- and macroalgae are a rich source of pigments (carotenoids, chlorophylls, phycobilins), also highly bioactive. However, the benefits of using micro- and macroalgae together as feed supplement, taking advantage of their different biochemical compositions, including nutritional elements and potential bioactivities, for inclusion as functional feeds in aquaculture diet, has not been a very explored alternative. These properties, together with the increasing consumer awareness of the advantages of using natural ingredients in food products, has triggered an increased demand both by consumers and the industry, opening promising new markets for algal-based products.

ACKNOWLEDGMENTS This work was funded by project ZEBRALGRE ( P T D C /C V T-W E L /52 0 7/ 2 014 - P O C I - 01- 0145 FEDER-016797), funded by FEDER funds through

Algae

COMPETE2020—POCI (Programa Operacional Competitividade e Internacionalização) and by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P.; and by the project GENIALG - GENetic diversity exploitation for Innovative macro-ALGalbiorefinery, funded by H2020 (EC Grant agreement no: 727892).

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Techno-Economic Analysis of Multiple Scenarios for the Production of Microalgal Chemicals and Polymers Giannis Penloglou and Costas Kiparissides

CONTENTS Abbreviations���������������������������������������������������������������������������������������������������������������������������������������������������������� 134 Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������134 Methodology�����������������������������������������������������������������������������������������������������������������������������������������������������������135 Investigated Scenarios����������������������������������������������������������������������������������������������������������������������������������������135 Photo-Bioreactors Design�����������������������������������������������������������������������������������������������������������������������������������135 Design Considerations����������������������������������������������������������������������������������������������������������������������������������������135 Description of Scenarios�����������������������������������������������������������������������������������������������������������������������������������������136 Scenarios-S1�������������������������������������������������������������������������������������������������������������������������������������������������������136 Scenarios-S2�������������������������������������������������������������������������������������������������������������������������������������������������������136 Scenarios-S3 and Scenario-S4����������������������������������������������������������������������������������������������������������������������������136 Techno-Economic Analysis������������������������������������������������������������������������������������������������������������������������������������137 Comparison of Scenarios������������������������������������������������������������������������������������������������������������������������������������137 Effect of Plant Capacity��������������������������������������������������������������������������������������������������������������������������������������138 Techno-Economic Analysis of Scenario-S1a�����������������������������������������������������������������������������������������������������139 Sensitivity Analysis of Scenario-S1a������������������������������������������������������������������������������������������������������������������140 Conclusion��������������������������������������������������������������������������������������������������������������������������������������������������������������141 Acknowledgment����������������������������������������������������������������������������������������������������������������������������������������������������142 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 142

BOX 12.1  SALIENT FEATURES The utilization of microalgal biomass for the production of environmentally friendly chemicals and polymers in industrial-scale plants is presently inhibited by several technical and economic barriers. Thus, the applied (thermo)chemical, enzymatic, microbial, etc., technologies for the conversion of microalgal biomass to valuable products should be designed and analyzed to optimize the efficiency of the related process steps and minimize the relevant production cost. In this chapter, the economic potential of two Botryococcus braunii strains to produce high added value metabolites, as well as their derivatives, are thoroughly discussed. The conceptual design and optimization of several flowsheets led to the identification of eight scenarios for the production of

exopolysaccharides, hydrocarbons, 1,4-pentanediol, poly(ethylene 2,5-furandioate), adipic acid and ethylene/propylene. The model-based simulation and sensitivity analysis of the eight scenarios disclosed the importance of both biomass cultivation and metabolites separation steps. These process steps are the clear ‘hotspots’ for both capital and operation expenditures. Overall, the scenarios that consider exopolysaccharides and hydrocarbons as the final products are more promising than the ones that employ their further conversion to chemicals and polymers. Taking into account that the optimization of all the involved process steps is a prerequisite and that the targeted products should be of higher than commodities value, the developed microalgal conversion pathways present a strong potential to operate in the near future under economic sustainability.

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ABBREVIATIONS AFC Total annual fixed costs CAPEX Capital expenditures DC Total direct costs DPC Total direct production costs EPS Extracellular polysaccharides FDCA 2,5-furandicarboxylic acid HCs Hydrocarbons HMF Hydroxymethylfurfural I Total fixed capital investment IC Total indirect costs Fixed capital investment IF Working capital IW MF Methylfuran OC Total other costs OPEX Operating expenditures PBR Photo-bioreactor PenDO 1,4-pentanediol PEF Poly(ethylene 2,5-furandioate) TPC Total production costs TPCP Specific production cost TRL Technology readiness level TGC Total general costs

INTRODUCTION Nowadays, microalgal biomass is in the center of research and industrial interest as a renewable source for the production of environmentally friendly fuels, chemicals and polymers (Rizwan et al. 2018). Beyond that microalgae constitute one of the solutions to the dependence on fossil resources, they present numerous advantages: high peracre productivity; non-food feedstock; use of otherwise non-productive/non-arable land; utilization of non-fresh water; production of multiple products; recycling of CO2 and other waste streams (Delrue et al. 2013; Enamala et al. 2018). Presently, several pathways exist for the utilization of microalgal biomass, however, the level of maturity of the relevant plants (focusing mainly on biofuels) is still in demo-level, expecting to extend in the flagship-level in the forthcoming years (Hoffman et al. 2017; Richardson et al. 2012; Sudhakar and Premalatha 2012). Selected microalgal components can be converted into different products, through (thermo)chemical, enzymatic or microbial processes (Ruiz et al. 2016). The nature of the employed value chain is determined by the economic potential of the relevant technology and may vary according to the raw materials costs (Marzocchella et al. 2010). If coupled with optimized technologies, such as an efficient ‘milking’ process integrated with a highyield photo-bioreactor (PBR) through recycling steps,

Handbook of Algal Technologies and Phytochemicals

the demands of these plants can be reduced (Chaudry et al. 2018). In this context, the development of a microalgaebased plant needs to be analyzed from a site location and a resource availability perspective (Davis et al. 2011). Critical requirements, such as land, climate, water resources, CO2 and other nutrients supply, must be aligned in terms of their geolocation, characteristics, availability and affordability (Delrue et al. 2012; Iancu et al. 2012). To ensure their technical, environmental and economic sustainability, the siting and resource factors must be matched to the characteristics of the microalgal species and the cultivation systems (Norsker et al. 2011). Given the multiple technology options and their interdependencies, an integrated techno-economic analysis approach is crucial in guiding research efforts towards a viable and sustainable microalgal industry (Sun et al. 2011). Systems modeling and analysis at different Technology Readiness Levels (TRLs), ranging from individual unit operations to an integrated plant, can provide guidance to successfully select appropriate technologies (Banerjee and Ramaswamy 2017). The large potential of microalgae as feedstock resulted in a growing interest, with a number of innovation activities competing in bringing the technology to the market (Peng et al. 2018). Although intensive work has been done, cost information is scarce, economic data are scattered and, generally, there is great uncertainty over the capital and operating costs (Amer et al. 2011). Accordingly, the future potential of microalgae has become the focus of lively debate. The uncertainties of the economics originate from large differences in the technical and economic assumptions and the inconsistency in the likely future improvements in annual microalgae productivity and product yields (West and Posarac 2008). Thus, the differences among the literature reports, regarding the production cost and product prices, are almost one order of magnitude (Riberio and Silva 2013). In this framework, a mathematical model for the conceptual design of a microalgae-based plant for the production of ‘extracellular’ polysaccharides (EPS) and hydrocarbons (HCs) and their conversion to chemical and polymers is developed in this study. The model is built up accounting for multiple alternative technologies for the process steps, thus enabling the comparison of economics under different value chains. The variants of the flowsheets reflect the options offered by making use of different strains, upstream technologies, downstream processing and conversion processes. Beyond the mass/energy balances of each process step and the techno-economic evaluation of the different value

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chains, a sensitivity analysis is performed to determine the parameters that present the largest effect on the production cost. Finally, the best-case scenarios are identified and the major economic performance parameters are calculated to provide input for the final assessment of the value chains. It should be noticed that the developments were based on experimental data and information generated within the EU-funded project SPLASH.*

METHODOLOGY Investigated Scenarios Following the success stories of SPLASH, eight scenarios (value chains) are considered: Scenario-S1a: a plant for the production of high volume–low value EPS: capacity 20,000 tons/ year. Scenario-S1b: a plant for the production of low volume–high value EPS: capacity 200 tons/year. Scenario-S2a: a plant for the production of high volume–low value HCs: capacity 10,000 tons/ year. Scenario-S2b: a plant for the production of low volume–high value HCs: capacity 500 tons/year. Scenario-S3a: an EPS-derivatives plant for the production of 1,4-pentanediol (PenDO) and its polymerization to polyesters/polyamides: capacity 2,000 tons/year. Scenario-S3b: an EPS-derivatives plant for the production of 2,5-furandicarboxylic acid (FDCA) and its polymerization to poly(ethylene 2,5-furandioate) (PEF): capacity 10,000 tons/year. Scenario-S3c: an EPS-derivatives plant for the production of adipic acid and its polymerization to polyesters/polyamides: capacity 10,000 tons/year. Scenario-S4: a HCs-refinery plant for the production of bioethylene/biopropylene: capacity 100,000 tons/year.

Photo-Bioreactors Design In compliance with the cultivation facilities of SPLASH, only closed PBR units were considered. From the available strains, Botryococcus braunii CCALA 778 was cultivated for the production of EPS and Botryococcus braunii Showa was cultivated for the production of HCs. * h t t p: //e u - s p l a s h . e u / h t t p s: //c o r d i s . e u r o p a . e u / p r o j e c t / rcn/104994_en.html

For both strains, two steps of biomass cultivation were assumed: a preculture and a main PBR. The latter is the main cultivation step, where the biomass growth and the accumulation of bioproducts is performed (Penloglou et al. 2016). Both PBRs were made from Plexiglas tubes and installed outside in a sunny area. Gaseous CO2 was continuously supplied as the carbon source. Degassing units were installed between tube-sections to exhaust the produced O2 and the excessive CO2. Temperature was always controlled at < 30°C, using an in-situ cooling sprinkler system spraying water on the PBRs. The cultivation medium was simulated to contain only the nitrogen source (KNO3). All the other nutrients were considered in excess, though their consumption was taken into account in the cost analysis. The biomass molecular formula was calculated based on the molecular composition of proteins (C4.43H7O1.44N1.16), lipids (C40H74O5), carbohydrates (C6H12O6), EPS (C6H10O5) and HCs (C36H62) (Karapatsia et al. 2016). The preculture PBR operated in eight-day batch cycles; at the end of each cycle its content was transferred to the main PBR. The main PBR operated in batch mode for five days until the desired level of biomass concentration (working density) was attained. Then, it switched to a semi-continuous mode for 45 days, with a medium renewal rate equal to the current cells’ specific growth rate. During this period, the preculture PBR provided fresh seed culture to the main PBR to replenish the biomass ‘bleeding’ stream.

Design Considerations The model was used to simulate the PBRs, the biomass dewatering/harvesting, the EPS/HCs ‘milking’ and the production/recovery of chemicals and polymers, based on available experimental, empirical and literature data. Moreover, the unique characteristics of the selected strains were taken into account. Firstly, the ability of B. braunii cells to float due to the accumulation of EPS/ HCs (‘attached’ to the cell membranes) enables their efficient ‘milking’ and the recycle of the product-free biomass to the PBRs (Blifernez-Klassen et al. 2018). Secondly, a B. braunii culture is in general prone to contamination, thus it should be ‘reinforced’ with new fresh axenic cell population on a regular basis (García-Cubero et al. 2018); this requires a constantly operating preculture PBR (Turu et al. 2016). The facility is assumed to operate for 350 days/year due to favorable climatic conditions (in Greece); the downtime is required for maintenance, new cultures and contingencies. For the calculation of the capital expenditures (CAPEX), the major and auxiliary equipment costs were calculated based on the simulation results. The costs

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of equipment, consumables and materials were obtained directly from suppliers when possible, otherwise, from standard engineering estimates or literature, updated to the base year. In addition, the equipment is depreciated over ten years and Lang factors are used to estimate the CAPEX breakdown (Beal et al. 2015; Ruiz et al. 2016). For the calculation of the operating expenditures (OPEX), the costs of raw materials, inoculum, nutrients, CO2, utilities, major and auxiliary equipment operation and wastewater treatment were based on the simulation results. Industrial prices for raw materials and utilities supply were also considered. The demanded area for the plant was leased at a cost estimated for the years of operation. The extra land required for buildings, offices, laboratories or roads was considered as 20% of the calculated PBRs area. Finally, the labor cost calculation was based on the Wessel equation.

A, the cultivation area; and Area B, the HCs ‘milking’. The downstream processing includes a pre-concentration step, which operates to partially dewater the PBR outflow stream. The HCs ‘milking’ is performed via wet extraction, using cyclohexane as the solvent. The HCsfree biomass and the recovered water are recycled to the main PBR. The extracted HCs are condensed in a vacuum distillation process, and the used solvent is recycled to the extraction step.

Scenarios-S3 and Scenario-S4 In the three alternatives of Scenario-S3, a third area is added to the EPS platform flowsheet: Area C, the conversion of EPS to chemicals and polymers.

DESCRIPTION OF SCENARIOS Scenarios-S1 The two alternatives of Scenario-S1 consider EPS as the marketable product of the plant (Figure 12.1). Two major areas can be identified: Area A, the microalgae cultivation in two PBRs (preculture and main); and Area B, the EPS recovery/‘milking’ and drying. The flowsheet is identical for S1a and S1b, as their difference is only on the plant scale. The EPS ‘milking’ is performed by a two-step filtration process: micro- and ultra-filtration. The EPS-free biomass and the separated water are recycled to the main PBR. Finally, the end product is prepared after drying the recovered EPS in a spray dryer.

Scenarios-S2 Similarly, Scenarios-S2a and -S2b represent a single process operating in different scales (Figure 12.2): Area

In S3a, PenDO is produced through the conversion of fucose/rhamnose, produced from the hydrolysis of EPS, in a three-step process (Huang et al. 2017): dehydration of sugars to 5-metylfurfural (5-MF); decarbonylation of 5-MF to 2-methylfuran (2-MF); and conversion of 2-MF to PenDO (Han 2016). Upon the separation of PenDO in an azeotropic distillation step, it is polymerized to polyesters/polyamides. In S3b, the production of FDCA and polymerization to PEF is performed via the conversion of EPS-derived glucose in a two-step process (Eerhart et al. 2014): dehydration of glucose to 5-hydroxymethylfurfural (HMF); and catalytic oxidation of HMF to FDCA, which is separated in the following crystallization step (Eerhart et al. 2015). The final process step is the polymerization of FDCA to PEF. In S3c, an enzymatic process step is developed to produce adipic acid from glucose in a threestep process: enzymatic oxidation of glucose to

CO2/Air Supply

Biomass Bleeding

Biomass Cultivation Mixer

Preculture Photo-bioreactor

Main Photo-bioreactor for EPS Production Biomass Recycle Water Recycle

Separation

Microalgal Biomass

Milking Process

EPS Spray Drying

Ultrafitration

FIGURE 12.1  Flowsheet for the production of EPS in Scenarios-S1a and -S1b.

Microfiltration

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Design and Economics of Microalgal Products CO2/Air Supply

Biomass Bleeding

Biomass Cultivation Mixer

Preculture Photo-bioreactor Cyclohexane

Water Recycle

Solvent Recovery Mixer

Main Photo-bioreactor for EHCs Production

Biomass Recycle

Microalgal Biomass

Milking Process

Separation Pre-concentration Filtration HCs Vacuum Distillation

Extraction

FIGURE 12.2  Flowsheet for the production of HCs in Scenarios-S2a and -S2b.

6-oxogalactose; catalytic oxidation to galactaric acid; and catalytic hydro-deoxygenation to adipic acid (Bueno et al. 2015). The recovered, via distillation, adipic acid is then polymerized to polyesters/polyamides (Gunukula and Anex 2017). In S4, the production of ethylene/propylene is based on the HCs platform. The recovered HCs are subjected to steam cracking for their conversion, initially to green naphtha and further to ethylene/ propylene (Brown et al. 2012). Upon a cooling and compression step the produced alkenes are recovered through a cryogenic separation process (Zacharopoulou and Lemonidou 2018).

TECHNO-ECONOMIC ANALYSIS The eight scenarios were simulated with the aid of Aspen Plus 8.0. In what follows, their techno-economic analysis is presented to identify the most promising scenario(s) for further investigation.

Comparison of Scenarios Firstly, the scenarios were simulated according to the selected values of the key-process parameters (Table 12.1), which were experimentally discovered within SPLASH. Moreover, the selection of the plant capacities for the scenarios was based on a market analysis, performed also in SPLASH. The biomass capacities were simulated to meet the production demands of each scenario. Note that these values depend on the specifications of the employed processes: when the EPS and HCs are considered as the final products, the ratio of biomass to product capacity is approximately three to four

tons of biomass/ton of product. On the other hand, when they are converted to chemicals and polymers, the ratio increases to 3.5–5. The calculated CAPEX and OPEX values of the scenarios are also reported in Table 12.1. It is obvious that these values are strongly affected by the biomass and product capacities. The evaluation of the scenarios was initially performed by the comparison of the production cost values with the product prices/values. Specifically for S1a, S2a and S2b, the product values completely cover the production costs, thus presenting a promising economic potential. For S1b, the ratio of cost per value can be increased if the ‘bleeding’ biomass is considered as an additional revenue. In the remaining four scenarios, the production cost is larger than the product values, thus additional process optimization is needed. Finally, the calculated products prices for process viability are also reported in Table 12.1. Beyond S1a, S2a and S2b, where the product price for viability is smaller than the product value from the market analysis, in S1b, S3a, S3b, S3c and S4 the product price should be significantly increased; thus, an additional risk for market penetration is raised. In addition, the ratio of products values to the production costs for all scenarios is also depicted in Table 12.1. It is expected that the scenarios which demonstrate a value > 1 will have the potential to operate under economic viability. Notice that the direct utilization of EPS and HCs as final products is economically more promising, compared to the cases where these primary products are further converted to chemicals and polymers. Moreover, the operating window for economic viability, with respect to the selected process parameters, is extremely narrow. Thus, process optimization is a prerequisite for sustainability. In general, the ratio of the

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TABLE 12.1 Overview of the Selected Key-process Parameters, the Capital Expenditures, the Production Cost and the Product Price/Value for the Eight Scenarios Scenario

S1a

S1b

S2a

S2b

S3a

S3b

S3c

S4

Plant Capacity (ktons/year) Biomass Capacity (ktons/year) EPS/HCs Content (% w/w) Milking Efficiency (% w/w) Conversion/Recovery (% w/w) CAPEX (Μ€) OPEX (M€/year) Production Cost (€/kg) Product Value (€/kg) Product Value/Production Cost Product Price for Viability (€/kg)

20

0.2

10

0.5

2

10

10

100

66.3

0.66

40.2

2.01

10.3

51.7

37.8

386.4

40

40

35

35

40

40

40

35

40

40

35

35

40

40

40

35

95

95

95

95

50

50

75

95

94.88

12.67

50.22

13.11

37.92

90.21

74.13

220.3

66.13

3.76

37.40

4.89

19.99

72.35

62.24

317.8

3.31

18.81

3.74

9.78

9.99

7.23

6.22

3.18

6

15

4

20

5

3

2.5

1.5

1.81 5.34

0.79 37.5

1.07 4.85

2.04 17.25

0.50 15.2

0.41 9.43

0.40 8.08

0.47 3.46

product price/value to the production cost should be > 0.4–0.5 so that a plant can be economically viable, upon process optimization. By focusing on each scenario separately, the following conditions for economic viability can be identified: Scenario-S1a: one of the most promising scenarios; the production of bulky EPS can be viable. Scenario-S1b: promising scenario, though a larger capacity and/or product price is needed. Scenario-S2a: marginally viable scenario; to avoid any risk, the process should be further optimized. Scenario-S2b: one of the best scenarios overall due to the large value of specialty HCs. Scenarios-S3a, -S3b, -S3c and -S4: viability is far from possible, even after process optimization; the product prices and/or the plant capacities should be significantly increased, thus introducing market penetration risks. Based on the above, one scenario for each production platform can be considered as promising: Scenario-S1a and Scenario-S2b. Thus, the production of bulky EPS

in relatively large scale and the production of specialty HCs in small scale can be both economically sustainable. Due to this observation, the two scenarios will be further evaluated. In Table 12.2, the comparison of the eight scenarios is presented also on a flowsheet area basis. From the percentage distribution of CAPEX and OPEX to the involved areas in each scenario, it can be concluded that Area A has the major contribution on both cost categories. The biomass cultivation in the PBRs is the economic ‘hotspot’ for all scenarios. Even in Scenarios-S3 and -S4, the contribution of Area A is larger than 50% for the CAPEX and 40% for the OPEX. The second most important area is Area B; on the other hand, the contribution of Area C is minor.

Effect of Plant Capacity In what follows, the selected Scenarios-S1a and -S2b are compared with respect to the impact of the plant capacity on their economic performance. To do so, the production costs were calculated in a large capacity range (100–20,000 tons/year). As can be seen in Figure 12.3, the production costs of EPS and HCs are similar. Taking

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Design and Economics of Microalgal Products

TABLE 12.2 Distribution of CAPEX and OPEX to the Flowsheet Areas for the Eight Scenarios Area↓

Contribution to the Total Cost (%)

Scenario→

S1a

S1b

S2a

Area A Area B Area C

55.59 44.41 –

61.21 38.79 –

Area A Area B Area C

75.38 24.62 –

75.47 24.53 –

86.33 13.67 –

S2b

S3a

S3b

S3c

S4

CAPEX 74.44 81.58 25.56 18.42 – –

54.52 32.29 13.19

49.85 34.22 15.93

52.28 34.99 12.73

75.66 15.20 9.14

OPEX 83.59 16.41 –

45.36 29.63 25.01

47.46 31.02 21.52

39.57 39.16 21.27

77.42 12.01 10.57

30 EPS HCs

Production Cost (€/kg)

25

20

15

10

5

0

0

2500

5000

7500

10000

12500

15000

17500

20000

Plant Capacity (tons/year)

FIGURE 12.3  Effect of plant capacity on the production cost of EPS and HCs.

into account that the two scenarios differ only in Area B, while Area A is identical, it is obvious that the PBRs present a larger contribution to the production cost than the ‘milking’ stage. In general, the production cost of both platforms strongly depends on the plant capacity. To minimize this impact, the selected capacities should be > 5,000 tons/ year. From the selected scenarios, only S1a satisfies this condition; the capacity of S2b lies in a range, where a small change of this value will have a major impact on the production cost. Moreover, if the capacity is significantly increased, an additional risk is raised regarding the marketable value of the products and the introduction of large product amounts to the market. As a result,

Scenario-S1a is selected for further investigation, as the most promising case.

Techno-Economic Analysis of Scenario-S1a Initially, the major and auxiliary equipment costs were calculated for Scenario-S1a. In the total value (€16,115,191), the contribution of major equipment (PBRs with cooling and gas supply systems, micro- and ultrafiltration and spray dryer) is 66.21%, while the remaining 33.79% comes from the cost of auxiliary equipment (pumps, heat exchangers, storage units, etc.). Notice that the cost of PBRs is > 50%, without including the cooling system cost. Thus, in order to reduce the total

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equipment cost and, in extent, the total capital investment, the optimization of the biomass cultivation/EPS accumulation step should be performed by enhancing the microalgae growth rate and/or by maximizing the ability of the selected species to selectively accumulate EPS. From the remaining costs, the costs of ultra-filtration and micro-filtration are the second (24.58%) and third (11.02%) largest; the cost of the spray dryer is of minor importance. The remaining direct, indirect and other capital costs, as well as the working capital cost, are reported in Table 12.3. The total value of CAPEX is €94,884,525, and the complete breakdown is: direct costs 62.50%, indirect costs 12.57%, other costs 11.89% and working capital 13.04%. Regarding the OPEX, the total costs of the raw materials and utilities were identified by the simulation of the total plant and the individual operation of the major process steps. The total value for these categories is €40,601,637/year (63.68% for raw materials and 36.32% for utilities). The contributions of CO2 and nutrients (47.78% and 37.65%, respectively) are the largest among the remaining materials. Thus, it is safe to conclude that the feasibility potential of the plant will improve, provided that alternative low-cost sources for carbon, nitrogen and other nutrients will be used. For example, the CO2 contained in industrial flue-gases and the nutrients (nitrogen TABLE 12.3 CAPEX Breakdown for Scenario-S1a Cost Major Equipment Auxiliary Equipment Installation Instrumentation/Control Piping Insulations Electrical Buildings/Services Land Improvements Service Facilities

Estimation

Simulated (100%) Simulated 47% 36% 68% 8% 11% 18% 10% 70% Total Direct Costs (DC) Engineering/Supervision 33% Construction Expenses 41% Total Indirect Costs (IC) Legal Fees 4% Contractors’ Fees 22% Contingencies 33% Total Other Costs (OC) DC + IC+OC Fixed Capital Investment (IF) Working Capital (IW) 15%·IF Total Fixed Capital Investment (I)

Value (€) 10,670,393 5,444,798 7,574,140 5,801,469 10,958,330 1,289,215 1,772,671 2,900,682 1,611,490 11,280,429 59,302,828 5,317,917 6,607,109 11,925,025 644,596 3,545,278 7,090,556 11,280,429 82,508,282 12,376,242 94,884,525

and phosphorous) from fertilizers could be exploited. Notice also that the contribution of process water is minimal, due to the already designed recycle streams. The breakdown of the utilities costs revealed that the major contributions are due to the electricity (56.57%) and steam (42.60%) demands. On the other hand, the operating cost for cooling water is minimal, due to the in-situ designed cooling system for the PBRs; the relevant capital cost was already included in the CAPEX calculations. In addition, the power and heat demands could be reduced if a power/heat co-generation unit (e.g., anaerobic digestion) was integrated to the process. The other direct costs, namely the land lease, the auxiliary equipment operation cost, the labor cost, the consumables and the wastewater treatment, were either simulated or calculated based upon the plant simulation. The remaining direct, annual fixed and general costs were also calculated, according to Table 12.4. Their respective contributions to the total OPEX are 74.95%, 21.58% and 3.47%.

Sensitivity Analysis of Scenario-S1a The developed simulation tool was also employed for the sensitivity analysis of the production cost of ScenarioS1a, against a number of key-process variables. As can be seen in Figure 12.4, the biomass growth rate and the EPS content/yield presented the major impact on the production cost. These two parameters are directly associated with the biomass cultivation in the PBRs. From the remaining parameters, the ones that correspond to the downstream processing (‘milking’ and drying efficiencies) were proven also important. Finally, from the raw materials and utilities, only CO2 presented a relatively modest effect on the production cost. In a second sensitivity analysis level, the Taguchi method of fractional factorial statistical design was employed, with the aid of Minitab 17.0 software. The selection of the parameters was based on the first level sensitivity analysis (Figure 12.4): maximum specific growth rate, EPS accumulation yield, ‘milking’ efficiency and drying efficiency. In particular, the selected levels of these parameters and the production cost (as the response variable) values for the nine simulation runs can be seen in Table 12.5. The production cost significantly varies with respect to the selected parameters values. According to the ANOVA analysis, the individual effects of the four selected parameters on the production cost were calculated (Figure 12.5). In addition, the optimal values of the parameters that minimize the EPS production cost correspond to the maximum level for each one. The relative

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Design and Economics of Microalgal Products

TABLE 12.4 OPEX Breakdown for Scenario-S1a Cost

Estimation

1.Raw Materials Simulated 2.Land Lease Calculated 3.Auxiliary Equipment Simulated 4.Labor Calculated 5.Supervision 15%·A4 6.Utilities Simulated 7.Consumables Calculated 8.Wastewater Treatment Calculated 9.Maintenance 5%·IF 10.Operating Supplies 15%·A9 11.Laboratory 10%·A4 12.Overheads 1%·CW Total Direct Production Costs (DPC) 1.Total Taxes 2%·IF 2.Insurances 1%·IF 3.Depreciation 10%·IF 4.Contingencies 70%·(A4 + A5 + A9) Total Annual Fixed Costs (AFC) 1.Administration 20%·A4 2.Marketing 3%·CW 3.Interests 8%·B3 Total General Costs (TGC) Total Production DPC + AFC + TGC Costs (TPC) Specific Production Cost (TPCP)

Value (€/year) 25,857,338 339,338 388,643 813,192 121,979 14,744,299 966,894 1,014,408 4,125,414 618,812 81,319 490,717 49,562,462 1,650,166 825,083 8,250,828 3,542,409 14,268,486 162,638 1,472,152 660,066 2,294,857 66,125,805

ranking of the parameters and their percentage contributions to the total variance of the production cost is: EPS yield, 58.49%; ‘milking’ efficiency, 34.72%; drying efficiency, 3.60%; specific growth rate, 3.19%. The cumulative contribution of the EPS yield and ‘milking’ efficiency is 93.21%. It is evident that focus should be given primarily to the optimization of these process parameters, which are related with the upstream cultivation of microalgae and the downstream separation of EPS.

CONCLUSION The conceptual process design of two microalgal platforms (EPS- and HCs-based) led to the identification of eight scenarios for the production of primary and secondary chemicals and polymers. The techno-economic analysis of these scenarios provided a useful insight on the bioprocesses and their potential economic viability. In general, the consideration of EPS and HCs as the final products is economically more promising, due to the simpler plants, with respect to the demanded process areas and equipment, and the larger value of the products. Generally, the ratio of product value to production cost should be > 0.4–0.5 so that a plant can be economically viable, upon process optimization. For all the scenarios, the area with the major contribution on both CAPEX and OPEX is the one of the PBR units, followed by the recovery/‘milking’ area. The conversion to chemicals and polymers area presents a significantly smaller effect to the economics. As a result, the clear ‘hot-spot’ of the integrated process is the PBR units.

€3.31 /kg

Utilities Cost

20% Increase 20% Decrease

Inoculum Cost Nutrients Cost CO2 Cost EPS Yield Drying Efficiency Growth Rate Milking Efficiency EPS Capacity –20 –15 –10 –5

0

5

10

15

20

25

30

35

40

45

50

Percentage Change of the Production Cost (%)

FIGURE 12.4  First level sensitivity analysis of the key-process parameters impact on the production cost for Scenario-S1a.

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TABLE 12.5 Simulation Runs for the Second Level Sensitivity Analysis of the Key-Process Effect on the Production Cost for Scenario-S1a

Run #

Specific Growth Rate (day−1)

EPS Yield (%w/w)

Milking Efficiency (%w/w)

Parameters Drying Efficiency (%w/w)

Production Cost (€/kg)

0.3 0.3 0.3 0.35 0.35 0.35 0.4 0.4 0.4

30 40 50 30 40 50 30 40 50

30 40 50 40 50 30 50 30 40

90 95 100 100 90 95 95 100 90

5.32 3.35 2.45 4.01 3.01 3.27 3.57 3.78 2.83

1 2 3 4 5 6 7 8 9

7

Production Cost (€/kg)

6 5

Level

Specific Growth Rate 0.30 day–1 0.35 day–1 0.40 day–1

EPS Yield 30% 40% 50%

Milking Efficiency 30% 40% 50%

Drying Efficiency 90% 95% 100%

4 3

parameter. In any case, it is important to consider complementary products, such as the ‘bleeding’ biomass, as the means to increase the revenue of the plant. It can be concluded that the developed cultivation and ‘milking’ technologies have the potential to operate under economic sustainability, provided that all the involved processes will be optimized and the targeted products are of higher than commodities value.

2

ACKNOWLEDGMENT

1

This work was financially supported by the European Commission under the project: ‘Sustainable Polymers from Algae Sugars and Hydrocarbons-SPLASH’ (Project ID: 311956, Funded under the 7th Framework Programme-KBBE.2012.3.4-02). The authors acknowledge the contribution of the SPLASH partners regarding the provided information and experimental data on strains, upstream and downstream processes, etc.

0

Specific Growth Rate

EPS Yield

Milking Efficiency

Drying Efficiency

FIGURE 12.5  Main effect plots for the second level sensitivity analysis of the selected key-process parameters impact on the production cost for Scenario-S1a.

Both the CAPEX and OPEX of this process step are the largest among the other steps. Thus, the PBRs should be the primary target for process optimization, as their efficient design and operation is a prerequisite for economic viability. From the selected key-process parameters, the biomass specific growth rate and the EPS/HCs yield (associated with the cultivation area) are the ones with the major effect on the production cost, followed by the ‘milking’ and drying efficiencies (associated with the downstream processing). The impact of raw materials and utilities on the production cost is modest, with the CO2 and the utilities being the most important ones. In principal, a microalgae-based plant should operate in a > 5,000 tons/year capacity to be less dependent on this

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13

Kappaphycus Farming for SocioEconomic Development of Coastal People in India P.V. Subba Rao and C. Periyasamy

CONTENTS Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������145 Origin of Kappaphycus Cultivation������������������������������������������������������������������������������������������������������������������������146 World Scenario��������������������������������������������������������������������������������������������������������������������������������������������������������146 Indian Scenario�������������������������������������������������������������������������������������������������������������������������������������������������������147 Environmental Conditions Essential for Good Growth������������������������������������������������������������������������������������������147 Methods of Farming������������������������������������������������������������������������������������������������������������������������������������������������148 Floating Bamboo Raft Method (FBR Method)�������������������������������������������������������������������������������������������������������148 Monoline Method����������������������������������������������������������������������������������������������������������������������������������������������������148 Off-Bottom Monoline Method (OMM)��������������������������������������������������������������������������������������������������������������148 Off-Bottom Monoline Net Fenced Method (OMNF Method)���������������������������������������������������������������������������149 Off-Bottom Monoline Tubular Method (OMT Method)����������������������������������������������������������������������������������������149 Off-Bottom Monoline Net Bag Method (OMNB Method)������������������������������������������������������������������������������������149 Economics of Kappaphycus Cultivation�����������������������������������������������������������������������������������������������������������������149 Conclusion��������������������������������������������������������������������������������������������������������������������������������������������������������������150 Acknowledgment����������������������������������������������������������������������������������������������������������������������������������������������������151 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 151

BOX 13.1  SALIENT FEATURES The cultivation technology of Kappaphycus alvarezii in India was developed in the coastal waters of Gulf of Mannar near Mandapam, Tamil Nadu, from 1995 to 2000 by Marine Algal Research Station, Mandapam Camp of Central Salt and Marine Chemicals Research Institute (CSIR), Bhavnagar, and the technology was transferred to PepsiCo India Holdings Private Limited in 2001. Dr. P. V. Subba Rao (father of Kappaphycus cultivation in India), the then scientist in charge at CSMCRIMarine Algal Research Station was instrumental in introducing this seaweed leading to its commercialization. Aquaculture Foundation of India, Chennai, played a key role for commercialization of this cultivation technology by introducing selfhelp groups. The net annual income per hectare ranged from Rs 8, 50, 000/- (US$12,142.86) to Rs 14,00,000/- (US$20,000.00) depending on the method adopted. Farming of this seaweed would

ensure an alternative viable source of livelihood for small scale fisher folk to eradicate their poverty thereby improving their socio-economic standards and also pave the way to establish carrageenanbased industries.

INTRODUCTION Seaweeds have been used for food, feed and fodder from time immemorial, and they also form a source of phytochemicals like agar, algin and carrageenan. Agar is obtained from species of Gelidium, Gelidiella and Pterocladia, algin from species of Macrocystis, Laminaria and Sargassum and carrageenan from species of Chondrus, Gigartina, Sarcotheca, Eucheuma and Kappaphycus (Levring et al. 1969; Chapman and Chapman 1980; Mc Hugh 2003). Of 9,900 seaweed species enumerated in the world (Khan et al. 2009), 221 species are being used either for food or for extraction of phytochemicals (Zemke-White and Ohno 1999). 145

146

At  present only ten species or genera, viz., Laminaria japonica and Undaria pinnatifida brown algae, Porphyra, Eucheuma, Kappaphycus and Gracilaria – red algae – Monostroma and Enteromorpha – green algae – and two micro algae – Dunaliella salina and Spirulina platensis, are intensively cultivated to meet the industrial demand as their natural resources are insufficient (Wikfors and Ohno 2001). Among the phytochemicals, carrageenan especially kappa carrageenan has occupied a pivotal role for its use in more than 250 applications in pharmaceutical and food industries since it forms a stronger gel than the iota carrageenan. It is a cell wall polysaccharide (a family of linear sulphated polysaccharides) used as a gelling, viscosity increasing, texture modifying and cell immobilizing agent in various foods and pharmaceutical, industrial and biotechnological applications (Bixler 1996; Ask and Azanza 2002; Bixler and Porse 2011; Villanueva et al. 2011). It is obtained from Kappaphycus alvarezii (Doty 1973; Bixler and Porse 2011).

ORIGIN OF KAPPAPHYCUS CULTIVATION As natural resources of this seaweed were found to be meager to meet the industrial demand, its cultivation was first introduced in Sulu seawaters of the Philippines in 1960 by Doty (1973), adopting off-bottom monoline method. The first Kappaphycus farm was established in 1969 jointly by Marine Colloids Inc. and Professor Maxwell Doty, University of Hawaii, in the province of Tawi-Tawi, in the south of the Philippines (Parker 1974; Doty and Alvarez 1975; Trono et al. 2000; Ask and Azanza 2002). Doty and Alvarez (1975) developed some improved techniques, and the same were followed by others in Philippines waters for many years (Trono et al. 2000; Ask and Azanza 2002). Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, the fast-growing strain was discovered from among the cultivated populations in 1985 by Doty (1985), and it has been further domesticated. Commercial cultivation of this seaweed was developed jointly by Marine Colloid Corporation (purchased by FMC Corporation in 1997, and now FMC Bio Polymers has been bought by Dupont in early 2017) and Maxwell Doty, University of Hawaii, in Tawii Tawii province of the Philippines (Subba Rao et al. 2008; Hurtado et al. 2013).

WORLD SCENARIO Kappaphycus alvarezii cultivation along with that of Kappaphycus striatum has been introduced in more than 20 tropical countries worldwide (Kumar et al.

Handbook of Algal Technologies and Phytochemicals

2016). Its cultivation was commercially accomplished in the following countries: China (Qian et al. 1996), Indonesia (Adnan and Porse 1987; Luxton 1993; Neish 2013), Madagascar (Mollion and Braud 1993), Malaysia (Neish 2003), Philippines (Doty 1973; Parker 1974; Doty and Alvarez 1975) and Tanzania (Lirasan and Twide 1993; Msuya et al. 2014). The cultivation was either not successful or only on a very small level in some countries of Central and South America (de Góes and Reis 2012; Hayashi et al. 2014), Southeast Asia and the South Pacific islands (Neish 2003). Its production by cultivation alone in the world has been reported to be 183,000 tons dry (Bixler and Porse 2011). The economical commercial farming needed a Daily Growth Rate of 3.5% and more for the 45 days growth period usually followed worldwide (Adnan and Porse 1987; Luxton et al. 1987; Ask and Azanza 2002; Subba Rao et al. 2008) In 2012 world aquaculture production including food fishes, aquatic plants mostly seaweeds and nonfood products (Pearls, Shells, etc) was 90.43 million tons wet of which seaweeds constituted 23.78 million tons wet from 33 countries and territories worldwide contributing 26.297% of total aquaculture production while capture was only 1.1 million tons wet (4.626% of the total aquatic plants production) (FAO 2014). As per an FAO (2013) report rapid expansion of Kappaphycus and Eucheuma cultivation in different countries from 2000 had resulted the production increase from 944,000 tons wet in 2000 (48% of the total red seaweed production) to 5,623,000 tons wet in 2010 (63% of total red seaweed production) with the increase in farm gate value from US$72 million to US$1.4 billion. Their production level also surpassed that of Japanese kelp (Laminaria japonica) production in 2010 (FAO 2014). In 2014 Eucheuma and Kappaphycus production accounted for 10,992 thousand tons wet (FAO 2016). In 2000 major carrageenan seaweed producing countries (production in thousand tons wet) included the Philippines – 679 (71.9%), Indonesia – 197 (20.9%), United Republic of Tanzania – 51 (5.4%), Kiribati – 11 (1.2%) and Fiji – 5 (0.6%), whereas in 2010, they included Indonesia – 3,399 (60.5%), the Philippines – 1,795 (31.9%), Malaysia – 208 (3.7%), United Republic of Tanzania – 132 (2.3%), China – 64 (1.1%) and other countries – 1 (0.5%) (FAO 2013). Carrageenan seaweed production in India was on a much smaller scale accounting for 21 tons dry in 2001 to 714 tons dry in 2009 (Krishnan and Narayanakumar 2010, 2013). Both floating raft and off-bottom monoline methods of cultivation were used in Indonesia (Neish 2013), the Philippines (Hurtado 2013), Mexico (Robledo et al. 2013), United Republic

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of Tanzania (Msuya 2013) and India (Kumar et al. 2016; Periyasamy and Subba Rao 2018) and only the off-bottom monoline method was used in other countries, viz., Malaysia (FAO 2013), China (Li et al. 1990), Kirbati, (Neish 2003; FAO 2013) and Fiji (Luxton et al. 1987; FAO 2013). The crop yields (tons dry ha−1 yr−1) of K. alvarezii were found to vary in different countries: 6 to 40 in Indonesia (Neish 2013), 27 to 54 in the Philippines (Hurtado 2013) and 31 to 57 in Mexico (Robledo et al. 2013). Further, this alga gave a crop yield (tons dry ha−1 yr−1) of 25 for net bag method, 40 for raft method and 45 for open culture method in eight harvests in the Mandapam region of southeast coast of India whereas at Okha, Northwest coast of India, it yielded 22 in five harvests for the raft method (Subba Rao and Mandri 2006). The production fluctuation of carrageenan seaweeds is constrained by a number of environmental factors such as seasonality, disease (“ice ice”), inclement weather (cyclones, hurricanes and typhoons), rising surface water temperature and grazing by fishes and other organisms.

INDIAN SCENARIO In India, Kappaphycus alvarezii was introduced in September 1995 at Thonithurai, near Pamban Bridge (Mandapam), in Gulf of Mannar waters of Bay of Bengal, southeast coast of India by Dr. P. V. Subba Rao (father of Kappaphycus cultivation in India), the then scientist in charge at Marine Algal Research Station (MARS), Mandapam Camp, Tamil Nadu, a unit of Central Salt and Marine Chemicals Research Institute (CSMCRI) Bhavnagar, Gujarat (a national laboratory of Council of Scientific and Industrial Research – CSIR, Ministry of Science and Technology, Government of India, New Delhi). Indian Kappaphycus owed its origin to the Philippines although it was brought from Japan in 1984 for research and cultivation, after following the necessary quarantine and introduction procedures (Mairh et al. 1996). Its introduction and domestication in Indian waters along with the feasibility of its successful cultivation on the Okha Mandal coast at Mithapur, Okha and Beyt Dwaraka, on the northwest coast of India was described by Subba Rao et al. (2008). After repeated domestication and experimentation at Mandapam, PepsiCo India Holdings Private Limited, Gurgaon, took up this cultivation technology. Further, this cultivation has got a boost with the sanction of a project by the Department of Biotechnology (DBT), New Delhi (2006 to 2009) to Aquaculture Foundation of India (AFI), Chennai, which introduced a five-member selfhelp groups (SHGs) to rehabilitate the tsunami affected

fisher folk in Tamil Nadu. In July 2008, the PepsiCo India Holdings Private Limited, Gurgaon, transferred this project to Aquagri Processing Private Limited, New Delhi. Currently, several companies like Linn Plantae Private Limited, Madurai, Snap alginate and natural products, Vellore, and Sea 6 Private Limited, Tuticorin, are involved in Kappaphycus cultivation by purchasing this seaweed through buyback arrangement with SHGs. Subsequently, this cultivation was carried out at different locations on the Indian coast at Vellar Estuary, Tamil Nadu (Thirumaran and Anantharaman 2009), Vizhinjam, Kerala (Bindu 2011) and Saurashtra coast (Gunalan et al. 2011; Kotiya et al. 2011; Kumar et al. 2016), different places in Ramanathapuram district, Tamil Nadu (Periyasamy et al. 2014a,b, 2016a), different places in Vizianagaram and Visakhapatanam districts, Andhra Pradesh (Periyasamy et al. 2016b; Periyasamy and Subba Rao 2017). However, commercial farming has been going on only in Tamil Nadu along the coastal waters of four districts, viz., Ramanathapuram, Pudukkottai, Tuticorin and parts of Tanjore and generated self-employment for hundreds of thousands of fisher folk (Periyasamy et al. 2014a, 2015; Periyasamy and Subba Rao 2017), and the growth period evaluated for economical commercial farming required would be 45 days considering sustainable growth and carrageenan content (Periyasamy et al. 2019). A comprehensive review on the origin and development of farming of this seaweed in Indian waters was furnished by Krishnan and Narayanakumar (2010), Periyasamy et al. (2016a) and Mantri et al. (2017).

ENVIRONMENTAL CONDITIONS ESSENTIAL FOR GOOD GROWTH This seaweed grows profusely in the sea where the bottom is sandy or rocky, salinity in the range of 28 to 33‰, sea water temperature 30 ± 3°C, water depth around 1.5 meters, nutrients: phosphate range from 0.37 to 3.22 μmol L−1 and nitrate from 0.39 to 1.22 μmol L−1, moderate light intensity and wave action (Neish 2003; Neish 2008; Subba Rao et al. 2008; Periyasamy et al. 2017). A seed plant of 150 g in 45 days grows to > 600 g in calm waters like Palk Bay waters of Bay of Bengal where the raft method and off-bottom monoline method are ideal (Periyasamy et al. 2016a) and grows to > 8 times in open sea waters with fairly high wave action like Okha coastal waters in the Arabian Sea where the monoline net bag method is ideal (Subba Rao et al. 2008). However Indian coastal waters have ideal climatic conditions for growing this seaweed.

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METHODS OF FARMING Kappaphycus farming was carried out in coastal waters of Tamil Nadu, Andhra Pradesh and Gujarat using different cultivation methods specific to the habitat, and they include floating bamboo raft method (FBR method), (Figure 13.1); off-bottom monoline method (OM method), (Figure 13.2); off-bottom monoline net fenced method (OMNF method) (Figure 13.3); off-bottom monoline tubular method (OMT method) (Figure 13.4); and off-bottom monoline net bag method (OMNB method) (Figure 13.5).

FLOATING BAMBOO RAFT METHOD (FBR METHOD) The Floating bamboo raft method of Trono and Ohno (1989) and Subba Rao et al. (2008) was adopted with some modifications. This method was standardized after conducting several field trials with respect to raft size, rope size, knot, seed density, space between seedlings, space between ropes, etc. Finally 3 × 3 m sized bamboo

Handbook of Algal Technologies and Phytochemicals

(3–4” diameter) rafts were used (Figure 13.1). Each raft had 20 seeded 3 mm polypropylene (pp) ropes (20 × 3 m = 60 m) aligned parallel to each other at 15 cm intervals, and each rope contained 20 knots. Seedlings of 150 g each were tied at 15 cm space intervals to the rope at the knots with the help of high-density polyethylene (HDPE) strings with “Made loop” (Ask et al. 2003). Thus, each raft had 400 seedlings constituting 60 kg (400 × 150 g) seed material. The planted rafts were placed in the cultivation ground with the help of anchors on both sides. This method has been widely used by the growers in coastal waters of Tamil Nadu for commercial farming (Periyasamy et al. 2016a).

MONOLINE METHOD Off-Bottom Monoline Method (OMM) The off-bottom monoline method adopted by Doty (1973) and subsequently modified by others (Luxton et al. 1987; Li et al. 1990) was widely used worldwide for Kappaphycus cultivation. This method involved a single

FIGURE 13.1  Floating bamboo raft method (FBR method).

FIGURE 13.3  Off-bottom monoline net fenced method (OMNF method).

FIGURE 13.2  Off-bottom monoline method (OM method).

FIGURE 13.4  Off-bottom monoline tubular method (OMT method).

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entire seeded monoline was covered with tubular net (0.5 m × 0.5 m size – HDEP, 20 × 20 mm opening and 1.5 mm thickness) of size 60 m × 0.75 m or two bits of size 30 m × 0.75 m. The net was tied properly at both ends with 2 mm PP (Polypropylene) rope. The planted lines were placed in a plot area of 60 m × 45 m at intervals of 1.0 m. This method is widely used in the coastal waters of Andhra Pradesh and North Tamil Nadu (Periyasamy et al. 2016b; Periyasamy and Subba Rao 2017).

FIGURE 13.5 Off-bottom monoline net bag method (OMNB method).

lengthy rope of 100 m or less depending on availability of cultivation ground. In India, the length of rope used was 60 m or two bits of 30 m each for easy handling and also with respect to availability of cultivation ground as described by Periyasamy et al. (2016b). Monoline rope of 60 m had 400 knots (HDEP braider – Made Loop) at intervals of 15 cm (Figure 13.2). At each knot, 150 g seed material was inserted, and thus one monoline contained 60 kg of seed material (150 gm × 400 knots). The planted lines were placed in a plot area of 60 m × 45 m at intervals of 1.0 m. This method was used in the coastal waters of Pudukottai where grazing was not seen.

Off-Bottom Monoline Net Fenced Method (OMNF Method) The off-bottom monoline net fenced (PEN culture) method of Trono (1994) with some modifications was adopted as described by Periyasamy and Subba Rao (2018). The procedure of plantation was the same as narrated in the off-bottom monoline method. However, in this method the whole plot (60 m × 45 m) was fenced with HDEP fishing net (20 mm mesh size and 1.5 mm thickness) (Figure 13.3). This method is widely used in the coastal waters of South Tamil Nadu particularly in the Ramanathapuram, Pudukkottai and Tanjore districts where grazing was noticed (Periyasamy et al. 2016a).

OFF-BOTTOM MONOLINE TUBULAR METHOD (OMT METHOD) The off-bottom monoline tubular method of Hayashi et al. (2010) was followed. In general, the monoline rope length was 60 m with 120 knots (HDEP braider – Made Loop) at an interval of 50 cm (Figure 13.4). At each knot 500 g seed material was inserted, and thus one monoline contained 60 kg seed material (500 gm × 120 knots). The

OFF-BOTTOM MONOLINE NET BAG METHOD (OMNB METHOD) The off-bottom monoline net bag method as described by Subba Rao (2004) and Kumar et al. (2016) was adopted. The length of rope and mode of plantation were similar to that of the off-bottom monoline tubular method (Figure 13.5). However, each seedling was covered with a net bag (0.5 m × 0.5 m size – HDEP, 20 × 20 mm opening and 1.5 mm thickness). The seed material used was 60 kg per rope (120 knots of 500 g each). Net bags were tied properly with the monoline rope using 2 mm PP rope. The planted lines were placed in a plot area of 60 m × 45 m at an interval of 1 m. This method was widely used in the coastal waters of South Tamil Nadu, Andhra Pradesh and parts of Gujarat (Periyasamy et al. 2016b; Kumar et al. 2016; Periyasamy and Subba Rao 2017). In all the above methods 45 seeded rafts or monolines (tubular/net bag tied) were floated in five rows of nine each (rafts in each row were fastened to each other) and placed horizontal to the shore in the sea in such a way that they were not exposed even at low tides and were aligned parallel to each other. Periodical maintenance of the rafts or monolines was done manually by removing the unwanted weeds to ensure good growth. The harvesting was done after 45 days of growth, and reseeding was done on day of harvest and placed in the cultivation ground. Every day one raft or monoline was harvested and the harvested raft or monoline was replanted. The harvested seaweed was dried on a coir mat in open sunlight for two to three days. The dried seaweed was cleaned and packed as per the buyer’s advice (Subba Rao et al. 2008; Periyasamy et al. 2014b).

ECONOMICS OF KAPPAPHYCUS CULTIVATION The successful model of Kappaphycus cultivation consisted of five-member self help group (SHG). The costs of one raft, one monoline, one monoline net fenced, one monoline tubular and one monoline net bag methods

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were Rs. 1500/-, Rs 700/-, Rs. 1000, Rs. 1250 and Rs. 2000, respectively. Each SHG required 225 rafts or monolines (45 rafts or monolines × five members) for their regular operation. Investment for one SHG was Rs. 337500/-, Rs 157500/-, Rs. 225000/-, Rs. 281250 and Rs. 450000/- for the FBR, OMM, OMNF, OMT and OMNB methods respectively. At the rate of handling five rafts or monolines/day, 225 rafts or monolines would be handled in a staggered manner in 45 days by each SHG, i.e. to harvest one raft or line/person/day. On the 46th day the fully grown five rafts or monolines would be harvested, replanted with tender plants from harvest and placed in the cultivation ground by SHG. Balance material (after replantation) would be dried, cleaned and packed. This operation would be continued daily for their regular income. The net annual income per hectare was arrived at Rs 14, 00, 000/-, (US$20,000.00) Rs 9, 60, 000/(US$13,714.29), Rs 9, 00, 000/- (US$12,857.14), Rs 8, 50, 000/- (US$12,142.86) and Rs 8, 50, 000/- (US$12,142.86) for the FBR, OMM, OMNF, OMT and OMNB methods, respectively, for each of the SHG (Table 13.1). The highest income per hectare in FBR method was due to 400 rafts equal to 400 monolines whereas other methods contained only 200 monolines. The income might

increase if the growers maintain the farm well coupled with per kg cost increase by the buyer. This income might slightly vary from place to place depending upon the climatic conditions and people’s attitudes.

CONCLUSION Indian peninsula (08°04’–37°06’N and 68°07’–97°25’E) has a coast line of 7,516 km excluding its island territories (1,256 islands) with 2 million km2 Exclusive Economic Zone (EEZ) (Subba Rao 2000). A thorough investigation of the coastal waters of Tamil Nadu in 2014 by Aquaculture Foundation of India (AFI) Madurai revealed many congenial locations along the coastal waters of districts of Thiruvallur, Kancheepuram, Villupuram, Cuddalore and Nagapattinam on Palk bay side and Kanniyakumari on Gulf of Mannar side for Kappaphycus cultivation by a study undertaken through an International Fund for Agricultural Development (IFAD) funded for Post Tsunami Sustainable Livelihood Programme (PTSLP), Chennai, Tamil Nadu (Periyasamy et al. 2017). Similarly, a number of places in the coastal waters of the Visakhapatnam and Vizianagaram districts of Andhra Pradesh are found to be encouraging for this seaweed cultivation based on pilot scale

TABLE 13.1 Income Estimate for One Hectare Using Different Methods of Cultivation Sl. No 1

2 3

4

5 6 7

8

Details

FBR

OMM

OMNF

OMT

OMNB

Cost of one raft (including all infrastructure cost) No. of rafts to be floated in one hectare Total cost (Rs.) (Sl. No. 1 × Sl. No. 2) (1US $ = Rs. 70.00) Annual output expected from one raft or line (kg DW raft−1 line−1 year−1) Total annual output expected (kg DW raft−1 line−1 year−1) Cost of dry weed per kg (Rs.)

1,500

700

1,000

1,250

2,000

400

200

200

200

200

6,00,000 (US $ 8571.43)

1,40,000 (US $ 2000)

2,00,000 (US $ 2857.14)

2,50,000 (US $ 3571.43)

4,00,000 (US $ 5714.29)

100

110

110

110

125

40,000

22,000

22,000

22,000

25,000

50 (US $ 0.71) 20,00,000

50 (US $ 0.71) 11,00,000

50 (US $ 0.71) 11,00,000

50.00 (US $ 0.71) 11,00,000

50.00 (US $ 0.71) 12,50,000

14,00,000 (US $ 20,000)

9,60,000 (US $ 13,714.29)

9,00,000 (US $ 12,857.14)

8,50,000 (US $ 12,142.86)

8,50,000 (US $ 12,142.86)

Annual income expected from one hectare – Sl. No. 5 × 6 (Rs. year−1) Net income from one hectare – Sl. No. 7 – Sl. No. 3 (US $1 = Rs. 70.00)

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experiments conducted by AFI, Madurai, in 2015 through funding by the National Fisheries Development Board (NFDB), Hyderabad (Periyasamy et al. 2016b; Periyasamy and Subba Rao 2017). Moreover, two-thirds of Indian coastal waters are conducive for Kappaphycus cultivation, and as such the cultivation could be done in 200,000 ha or 0.01% of the 2 million km2 Exclusive Economic Zone (EEZ) (Krishnamurthy 2005). Total rural employment has been growing at the slow rate of 0.58% per year with the rural population growing at 1.7% per year (Singh 2005). The large-scale cultivation of this seaweed in this context could provide a face lift to rural employment by providing urban facilities in rural areas. It could play a catalytic role in rejuvenating the coastal rural economy. As a whole Kappaphycus cultivation is not complicated but very simple and easy. Capital investment for this cultivation is less than any other aquaculture practice. It does not involve any inputs that are harmful to the environment, and on the other hand it is beneficial to the marine organisms thereby increasing their diversity, and this is an eco-friendly operation with sustainable income to the coastal poor (Periyasamy et al. 2016a). Though Kappaphycus farming appears to be labor intensive it fetches lucrative earning as the market for its products is diversified. Farming of this seaweed may be preferred as an alternative viable source of livelihood for smallscale fisher folk (Smith and Smith 1980; Smith 1987; Periyasamy et al. 2015). Further, this farming could also reduce the fishing pressure and helps in carbon sequestration to combat global warming. Before implementing commercial farming, the growers (farmers) have to be well educated on cultivation protocol involving farm maintenance, harvesting, drying, quality maintenance, packing and selling as suggested by Periyasamy et al. (2016c). If this protocol is followed scrupulously the SHGs/growers will get good revenue through this farming which would solve the problem of unemployment in rural India and also pave the way for starting Kappaphycus-based industries. The successful model of cultivation in Tamil Nadu could be promoted to all other coastal states of India. It has been estimated that India has the potential to produce one million tons of dried seaweed, Kappaphycus alvarezii (Krishnan and Narayanakumar 2013). The Indian government should promote Kappaphycus cultivation as a mission mode program to eradicate poverty in the coastal villages and initiate indigenous production of carrageenan (refined/ semi-refined) for internal use thereby saving the foreign exchange hitherto being incurred for importing the same and also for export of this chemical to earn foreign exchange.

ACKNOWLEDGMENT The authors would like to thank the funding agencies such as DBT, New Delhi; NFDB, Hyderabad; IFAD – PTSLP, Chennai, for their financial support to execute the cultivation from time to time. All the stakeholders of Kappaphycus farming are also acknowledged for their valuable suggestions to expand the farming.

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Luxton, D. M. 1993. Aspects of the farming and processing of Kappaphycus and Eucheuma in Indonesia. Hydrobiologia 260–261: 365–371. Luxton, I. M., Robertson, M., Kindley, M. J. 1987. Farming of Eucheuma in the South Pacific islands of Fiji. Hydrobiologia 151–152: 359–362. Mairh, O. P., Zodape, S. T., Tewari, A., Rajaguru, M. R. 1996. Culture of marine red alga Kappaphycus striatum (Schmitz) Doty on the Saurashtra region, West Coast of India. Indian J Mar Sci 24: 24–31. Mantri, V. A., Eswaran, K., Shanmugam, M., Ganesan, M., Veeragurunathan, V., Thiruppathi, S., Reddy, C. R. K., Seth, A. 2017. An appraisal on commercial farming of Kappaphycus alvarezii in India: Success in diversification of livelihood and prospects. J Appl Phycol 29(1): 335–357. Mc Hugh, D. J. 2003. A guide to the seaweed Industry. FAO Fish Tech Pap 441: 105. Mollion, J., Braud, J. P. 1993. A Eucheuma (Solieriaceae, Rhodophyta) cultivation test on the south-west coast of Madagascar. Hydrobiologia 260–261: 373–378. Msuya, F. E. 2013. Social and economic dimensions of carrageenan seaweed farming in the United Republic of Tanzania. In: Valderrama, D., Cai, J., Hishamunda, N., Ridler, N. (Eds.), Social and Economic Dimensions of Carrageenan Seaweed Farming. Fisheries and Aquaculture Technical Paper No. 580. FAO, Rome. pp 61–89. Msuya, F. E., Buriyo, A., Omar, I., Pascal, B., Narrain, K., Ravina, J. J. M., Mrabu, E., Wakibia, J. G. 2014. Cultivation and utilization of red seaweeds in the Western Indian Ocean (WIO) Region. J Appl Phycol 26: 699–705. Neish, I. 2003. ABC of Eucheuma Seaplant Production. Monograph 1-0703. Suriya Link. pp 1–80. Neish, I. 2008. Good agronomy practices for Kappaphycus and Eucheuma: Including an over view of basic biology. SEAPlant.net. Monograph HB2F 1008 V3 GAP. pp 1–72. Neish, I. C. 2013. Social and economic dimensions of carrageenan seaweed farming: In Indonesia. In: Valderrama, D., Cai, J., Hishamunda, N., Ridler, N. (Eds.), Social and Economic Dimensions of Carrageenan Seaweed Farming. Fisheries and Aquaculture Technical Paper No. 580, FAO, Rome. pp 61–89. Parker, H. S. 1974. The culture of the red algal genus Eucheuma in the Philippines. Aquaculture 3: 425–439. Periyasamy, C., Anantharaman, P., Balasubramanian, T. 2014a. Social upliftment of coastal fisher women through seaweed (Kappaphycus alvarezii (Doty) Doty) farming in Tamil Nadu, India. J Appl Phycol 26: 775–781. Periyasamy, C., Anantharaman, P., Balasubramanian, T., Subba Rao, P. V. 2014b. Seasonal variation in growth and carrageenan yield in cultivated Kappaphycus alvarezii (Doty) Doty on the coastal waters of Ramanathapuram, Tamil Nadu, India. J Appl Phycol 26: 803–810.

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Periyasamy, C., Anantharaman, P., Subba Rao, P. V. 2015. Experimental farming of Kappaphycus alvarezii (Doty) Doty with income estimates in different sites of Mandapam region, Palk Bay, Southeast coast of India. J Appl Phycol 27: 935–944. Periyasamy, C., Subba Rao, P. V. 2017. Growth rate and carrageenan yield of cultivated Kappaphycus alvarezii (Doty) Doty in the coastal waters of Bay of Bengal at Chepala Timmapuram, Andhra Pradesh, East Coast of India. J Appl Phycol 29(4): 1977–1987. Periyasamy, C., Subba Rao, P. V. 2018. Cultivation of Kappaphycus alvarezii (Doty) Doty for optimum growth and carrageenan yield using two different methods in the coastal waters of Palk bay, Tamil Nadu, Southeast coast of India. Aquaculture (Communicated). Periyasamy, C., Subba Rao, P. V., Anantharaman, P. 2016a. Kappaphycus cultivation – A boon for the livelihood of coastal fisherfolk in India. Seaweed Res and Utiln 38(1): 75–83. Periyasamy, C., Subba Rao, P. V., Anantharaman, P. 2016c. Protocols for successful commercial farming of Kappaphycus alvarezii, a potential carrageenophyte in Indian waters. Seaweed Res and Utiln 38(2): 89–97. Periyasamy, C., Subba Rao, P. V., Anantharaman, P. 2019. Harvest optimization to assess sustainable growth and carrageenan yield of cultivated Kappaphycus alvarezii (Doty) Doty in Indian waters. J Appl Phycol 31(1): 587–597. Periyasamy, C., Subba Rao, P. V., Selvavinayagam, K. T., Anantharaman, P. 2017. Feasibility of seaweed cultivation at new locations in the coastal waters of Tamil Nadu. Seaweed Res Utiln39(1): 28–38. Periyasamy, C., Subba Rao, P. V., Selvavinayagam, K. T., Srinivasa Rao, A. 2016b. Pilot scale field cultivation of Kappaphycus alvarezii (Doty) Doty at two different locations in Bay of Bengal waters of Andhra Pradesh, East Coast of India. Seaweed Res and Utiln 38(1): 85–94. Qian, P. Y., Wu, C. Y., Wu, M., Xie, Y. K. 1996. Integrated cultivation of the red alga Kappaphycus alvarezii and the pearl oyster Pinctadamartensi. Aquaculture 147: 21–35. Robledo, D., Gasca-Leyva, E., Fraga, J. 2013. Social and economic dimensions of carrageenan seaweed farming in Mexico. In: Valderrama, D., Cai, J., Hishamunda, N., Ridler, N. (Eds.), Social and Economic Dimensions of Carrageenan Seaweed Farming. Fisheries and Aquaculture Technical Paper No. 580. FAO, Rome. pp 61–89. Singh, G. 2005. Trigger for rural development. Employment News. pp 30–32.

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Smith, I. R. 1987. The economics of small scale seaweed production in the South China Sea region. FAO Fisheries Circular No. 806: 26. Smith, I. R., Pestano Smith, R. 1980. Seaweed farming and alternative income for small-scale fisherman: A case study. Proc Indo Pac Fishers Comm 19: 715–729. Subba Rao, D. 2000. The Bay of Bengal. In: Sheppard, C. (Ed.), Seas at Millennium. Elsevier Science, London. pp 1–14. Subba Rao, P. V. 2004. A report on studies on field cultivation and harvesting of seaweeds – Porphyra, Enteromorpha, Eucheuma and their use in processed foods. DBT report. CSMCRI, Bhavnagar and CFTRI, Mysore. pp 67. Subba Rao, P. V., Mandri, V. A. 2006. Indian seaweed resources and sustainable utilization: Scenario at the dawn of a new century. Curr Sci 91(2): 164–174. Subba Rao, P. V., Suresh Kumar, K., Ganesan, K., Thakur, M. C. 2008. Feasibility of cultivation of Kappaphycus alvarezii (Doty) Doty at different localities on the Northwest coast of India. Aquacult Res 39: 1107–1114. Thirumaran, G., Anantharaman, P. 2009. Daily growth rate of field farming seaweed Kappaphycus alvarezii (Doty) Doty ex P. Silva in velar Estuary. World J Fish Mar Sci 1(3): 144–153. Trono, G. C. Jr. 1994. Eucheuma and Kappaphycus: Taxonomy and Cultivation. Ohno, M., Critchley, A. T. (Eds.), Seaweed Cultivation and Marine Ranching, First Edition. JICA, Yokosuka. Japan. pp 75–88. Trono, G. C., Ohno, M. 1989. Seasonality in the biomass production of the Eucheuma strains in Northern Bohol, Philippines. In: Umezaki, I. (Ed.), Scientific Survey of Marine Algae and Their Resources in Philippine Islands. Monbusho International Scientific Research Program, Japan. pp 71–80. Trono, J. G. C., Lluisma, A. O., Montano, M. N. E. 2000. Primer on Farming and Strain Selection of Kappaphycus and Eucheuma in the Philippines. Marine Science Institute United Nations Developmental Programme and Philippine Council for Aquatic and Marine Research and Development, Quezon City, Philippines. Villanueva, R. D., Romero, J. B., Montaño, M. N. E., de la Peña, P. O. 2011. Harvest optimization of four Kappaphycus species from the Philippines. Biomass Bioenerg 35: 1311–1316. Wikfors, G. H., Ohno, M. 2001. Impact of algal research in aquaculture. J Appl Phycol 11: 968–974. Zemke-White, L. W., Ohno, M. 1999. World seaweed utilization: An end of century summary. J Appl Phycol 11: 369–376.

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Diversity and Utilization of Marine Cyanobacteria N. Thajuddin and G. Subramanian

CONTENTS Abbreviations����������������������������������������������������������������������������������������������������������������������������������������������������������155 Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������155 Morphology�������������������������������������������������������������������������������������������������������������������������������������������������������������156 Marine Cyanobacteria���������������������������������������������������������������������������������������������������������������������������������������������156 Possible Applications in Biotechnology�����������������������������������������������������������������������������������������������������������������161 Conclusion and Recommendations�������������������������������������������������������������������������������������������������������������������������166 Acknowledgment����������������������������������������������������������������������������������������������������������������������������������������������������167 Bibliography������������������������������������������������������������������������������������������������������������������������������������������������������������ 167

BOX 14.1  SALIENT FEATURES The basic and fundamental requirement for initiating marine cyanobacterial biotechnology is to first enumerate the available cyanobacterial diversity and understand their ecobiological properties. In addition to their widespread geographic distribution, cyanobacteria have probably played a major role throughout the biological history of the earth, cyanobacteria being a prokaryotic organism having potentials of both bacteria and algae, and they can be easily manipulated like bacteria which shows promise in the field of cyanobacterial biotechnology. This review is intended to focus on the biodiversity of cyanobacteria in marine environments of India and future research priorities and opportunities in the commercial utilization in varied areas such as mariculture, food, feed, fertilizer, medicine, industry and combating pollution.

ABBREVIATIONS ESBL GLA PHA EPS

Extended-Spectrum β-Lactamase Gamma Linolenic Acid Poly Hydroxy Alkalonates Exopolysaccharide production

INTRODUCTION Cyanobacteria are one of the largest sub-groups of Gram-negative photosynthetic (especially oxygenic)

autotrophic prokaryotes (Adams, 2000; Whitton and Potts, 2000). Members of this group possess chlorophyll a and phycobiliproteins such as phycocyanin and phycoerythrin, which are responsible for the blue-green pigmentation often evident in this group. As a result of their pigmentation and oxygenic photosynthesis, cyanobacteria were traditionally referred to as blue-green algae. Several names have been used for these organisms, viz., myxophyceae, cyanophyceae, cyanophyta, cyanoprokaryotes and cyanochloronta (Thajuddin and Subramanian, 2005; Anand et al., 2019). They were originally considered as algae because of their microscopic morphology, pigmentation and oxygen evolving photosynthesis in which two photosystems (PS II and PS I) are connected in series. Their distribution is rivaled only by true bacteria. They can grow well in all aquatic habitats (as plankton) such as rivers, ponds (Muthukumar et al., 2007; Vijayan et al., 2014), lakes, water tanks, paddy fields, all types of marine environments including sea water, hypersaline salt pans (Thajuddin and Subramanian, 1992; Nagasathya and Thajuddin, 2008c), brackish waters (Sudha et al., 2007), soda lakes, all types of soils, deserts, cave walls, hot springs, polar regions (Singh et al., 2008), on tree barks, on leaf surfaces, rocks, in sewage, industrial effluents (Vijayakumar et al., 2007) and other extreme environments. Probably their gelatinous sheath (at least in part) contributes to their ability to withstand long periods of desiccation. The compactness of protein molecules and their bonds in the protoplasm also aid cells in withstanding extremes. Cyanobacteria also exist as symbionts in a variety of 155

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organisms (Thajuddin et al., 2014) such as angiosperms (Gunnera); gymnosperms (Cycads) (Praveenkumar et al., 2007; Thajuddin et al., 2010); pteridophytes (Azolla) (Subramanian and Malliga, 1988; Rai, 1990); bryophytes (Anthoceros); algae (Diatom – Rhizosolenia and green alga – Geosiphon); lichens (Shyamkumar and Thajuddin, 2009; Shyamkumar et al., 2010, 2011) and even animals (Pectinatella, Amoeba, Corals). In addition to their widespread distribution, cyanobacteria probably had a role throughout the biological history of the earth. Cyanobacteria have been a tool in examining the endosymbiotic origin of eukaryotic chloroplasts (Castenholz, 2001). The genome size of cyanobacteria, representative of all major taxonomic groups, lies in the range of 1.6 × 109 to 8.6 × 109 daltons. The majority of unicellular cyanobacteria contain genomes of 1.6 × 109 daltons, comparable in size to those of other bacteria (1.0 to 3.6 × 109 daltons) (Herdman et al., 1979a,b).

MORPHOLOGY Cyanobacteria are traditionally classified on the basis of their morphology into five orders representing Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales and Stigonematales as given in Bergey’s Manual (Holt et al., 1994). There are two major morphological types of cyanobacteria: coccoid and filamentous forms. Coccoid species range from single cells (Synechococcus; Figure 14.1, b) to colonies (Gloeocapsa and Chroococcus; Figure 14.1, c and e) or masses of various shapes (Microcystis; Figure 14.1, a). In some, cells are arranged in rows resulting in a flat plate (Merismopedia; Figure 14.1, f), or they may be radially arranged in spherical colonies (Gomphospharia; Figure 14.1, d). Filamentous forms produce a row of cells referred to as a trichome, a result of cell division in one plane and failure of the cells to secrete sheath material between the cells or in the plane of division. Trichomes may be simple straight (Oscillatoria; Figure 14.1, h) or in the form of aggregated bundles (Trichodesmium; Figure 14.1, k) and/or permanently spirally coiled (Spirulina; Figure 14.1, g). The trichome with the enclosing sheath is referred to as a filament (Lyngbya and Phormidium; Figure 14.1, i and j). Some filamentous species are characterized by true cell differentiation and form special cells called ‘heterocysts’ which, unlike normal vegetative cells, lack an oxygenic photosystem, biliprotein pigments and carboxysomes but possess an extraordinarily thick cell wall that is impervious to oxygen (Anabaena; Figure 14.1, m). The number and distribution of these cells is often of taxonomic importance. These are considered sites of nitrogen fixation,

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providing vegetative cells with combined nitrogen but are not viable when disconnected from the trichome. Many heterocystous cyanobacteria also form a second cell type, ‘akinetes’, which can germinate into trichomes when conditions are suitable for growth. The filaments are either unbranched (Nodularia; Figure 14.1, l) or branched (Mastigocoleus; Figure 14.1, q) or even with false branching (Scytonema; Figure 14.1, n). The forms with heterocysts can have them as intercalary heterocysts (Hormothomnion; Figure 14.1, p) or basal heterocysts (Calothrix; Figure 14.1, o).

MARINE CYANOBACTERIA Of the total area of 150 million km2 of the earth about 70.68% is occupied by oceans, but of the total photosynthetic productivity of 555.2 billion tons of dry weight/ year only 34.4% is contributed by the oceans (Bassham, 1975). India has a vast coastline of over 7,500 km; in addition to many freshwater lakes, ponds, puddles and backwater areas with a tropical climate that results in an abundance of natural populations of varied organisms. The ability of some organisms to grow in seawater is presumably related to a preference for alkaline conditions and an ability to tolerate high salt concentrations. The resistance, which many species show towards osmotic shock, extremes of temperature and reducing conditions, suits their existence in a variety of intertidal habitats. Desikachary (1959) suggested that probably 20% of all known cyanobacteria occur in saline conditions and most of them are truly marine. Considerable work has been carried out to understand the cyanobacterial biodiversity of marine environments of India (Thajuddin and Subramanian, 1990, 1991, 1992, 1994, 1995, 2002, 2005; Thajuddin, 1991; Subramanian and Thajuddin, 1995; Thajuddin et al., 2002; Sudha et al., 2007; Nagasathya and Thajuddin, 2008c; Suresh et al., 2012). In addition, a number of sporadic and casual reports on the occurrence of cyanobacteria in marine environments from India are noted in Table 14.1. Apart from the paucity of detailed surveys of marine cyanobacteria from India, even globally most of the reports are restricted not only in area but also in the number of species recorded (Table 14.2). In addition there are several similar reports with smaller numbers of species from different parts of the world including brines (Hof and Fremy, 1933; Stewart and Pugh, 1963; Whitton, 1968; Petrov, 1974; John and Lawson, 1977; Ralph, 1977; Aleem, 1978, 1980; Sage and Sullivan, 1978; Basson, 1979; McCarthy and Carpenter, 1979). As the basic requirement for initiating marine cyanobacterial biotechnology is to first enumerate the

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FIGURE 14.1  Cyanobacterial colonies. (a) Microcystis—colonies with irregularly spread cells, (b) Synechococcus—simple coccoid cyanobacterium, (c) Gloeocapsa—Spherical cells surrounded by a vesicular sheath, (d) Gomphosphaeria—radially arranged cordate cells in a hollow spherical mucilaginous matrix, (e) Chroococcus—cells with distinct two- and fourcelled dividing stage, (f) Merismopedia—flat colonies with regularly arranged cells, (g) Spirulina—spirally twisted trichome, (h)  Oscillatoria—simple trachomatous form, (i) Lyngbya—a filamentous form with lamellated sheath, (j) Phormidium— trichomes enclosed in a delicate sheath, (k) Trichodesmium—many trichomes aggregated into bundles (l) Nodularia—heterocystus filamentous form (m) Anabaena—filament with intercalary heterocys surrounded with akenites, (n) Scytonema— heterocystous filament with false branching, (o) Calothrix—filaments with basal heterocysts, (p) Hormothamnion—epilithic intercalary heterocystus filamentous form with hard mucilage sheath and (q) Mastigocoleus—heterocystus true branched filamentous form. (Figures a, d, h, l and m—dark field microscopic view; c, f, g, i, j, k, n, o and q—bright field microscopic view; b, e and p—confocal microscopic view).

available cyanobacterial wealth and understand their ecobiological properties, a detailed survey was made on the diversity and distribution of marine cyanobacteria of a continuous stretch in south India of over 2m660 km of the coastline from Tirakol of Goa state to Bhimunipattanam of Andhra Pradesh, encompassing the coastal regions Kerala, Karnataka and Tamilnadu

including Andaman, Nicobar and the Lakshwadeep group of islands (Figure 14.2). This survey included coverage of not only the shore and deeper sea but also stagnant seawater ponds and puddles (Figure 14.3, 7 and 11), backwater (Figure 14.3, 5 and 6) and saltpans (Figure 14.3, 9 and 10). The nature of shores in south India varied markedly in different

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TABLE 14.1 Records on Indian Marine Cyanobacteria Author Name

Record

Cleve Biswas Iyengar Biswas Boergesson Dixit Gopala Iyer and Sankara Menon Venkataraman Anand Ramachandran Anand and Venkatesan Anand et al. Santra et al. Shah et al. Ritika et al. Sugumar et al.

Reported on planktons of Indian Ocean Reporting 85 taxa from saline environments Reported Trichodesmium from Kurusadai Island Brackish water algal flora of India and Burma Record of five species from the coast of Bombay Reported ten species from seawater near Bombay Reported 32 marine species from Gulf of Mannar Reporting 19 species from Cape Comorin Reporting nine taxa from Madras coast Reported ten taxa from Port Novo Reported 17 species of seven genera from Madras coast Reported 25 taxa from Madras and south Arcot District Recorded the 67 blue green algal taxa from various saline habitats of West Bengal, India Recorded the cyanobacteria present in the Hausiar pond of Bhuji, India Reported ten marine cyanobacteria from Nagapattinam coast Reported 18 saltpan marine cyanobacteria from Cape Comorin Coast

Year 1901 1926 1927 1949 1935 1936 1936 1957 1982 1982 1985 1986 1988 2000 2011 2011

TABLE 14.2 Reports on Marine Cyanobacteria From Other Countries Author(s) Name Umezaki Webber Zaneveld Oah Moore Pulz Gonzalez and Parra Polderman Potts Krempin and Sullivan Marshall Hallfors Kass Khoja Hoffman Skulbery et al. Hussain and Khoja Nagarkar Hoffmann Calvo et al. Silva and Pienaar Lopez-Cortes et al.

Record Reported marine blue green algae of Japan Studied the blue green alga from a Massachusetts salt marsh, Pennsylvania Recorded 27 species from American coast Found 25% of the total flora of the saline waters in Chad area to be comprised of cyanobacteria Reported on the epilithic and epiphytic forms from Southern Baffin Island Studied the marine littoral algae in the Gulf of Baabo Reported 161 species of cyanobacteria belonging to 41 genera from the coast of Chile Reported diversity and seasonal aspects of salt marshes on the south west coasts of England Studied the distribution and taxonomic diversity of cyanobacteria from marine costal environments of Sinai peninsula Studied the seasonal abundance, microbial biomass of Chrococcoid cyanobacteria in southern California coastal waters Reported 16 species of cyanobacteria belonging to ten genera off the south eastern coast of United States Reported filamentous rock pool algae in the Tvarminne archipelago, south coast of Finland. Reported blue green algae of Danish Wadden Sea Reported 19 species of cyanobacteria from Red Sea coast of Saudi Arabia Recorded 62 genera of marine cyanobacteria in Papua New Guinea Studied the taxonomy of the toxic cyanophyceae (cyanobacteria) from Norway Reported blur green algal mats from Red Sea, Saudi Arabia Reported new records of marine cyanobacteria from rocky shores of Hong Kong Reported diversity and ecology of marine cyanobacteria in tropical regions of Belgium R NW Spaineported benthic algae of salt marshes Some benthic marine cyanophyceae of Mauritius Reported cyanobacterial diversity in extreme environments of Mexico

Year 1961 1967 1972 1973 1974 1975 1975 1975, 1978 1980 1981 1981 1984 1985 1987 1992 1993 1993 1998 1999 1999 2000 2001

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FIGURE 14.2  Areas of marine cyanobacterial survey.

regions. Except the rocky spots of Kovalam, Vizhigam, Edeva, Varkala (Kerala state), Dona Paula (Goa state), the shores of the Arabian Sea region (west coast) are completely sandy, while in the east coast of India, the shores are of different types. The shores of the Bay of Bengal region are mostly sandy (Figure 14.3, 1) with few extensive rocky spots at Bhimunipatnam, Vishakapatnam, Pudimadaka (Andhra Pradesh), Kovalam and Mahabalipuram (Tamil Nadu). The shores of the Palk Strait region exhibit greater variety. Along the great lagoons the shores are clayey (Figure 14.3, 5 and 6) and particularly in Adhirampatnamarea of Tamil Nadu, they are swampy and inaccessible (Figure 14.3, 2). The shores towards the Palk Bay from Palk Strait are of coarse sand and rich in organic matter due to the deposition of dense layers of sea grass over vast stretches (Figure 14.3, 12). However, most of the Palk Bay area has a sandy shore with scattered boulders and masses of coral rocks. The shores of the Gulf of Mannar region vary from place to place. On the south coast of Mandapam the shores are

rocky due to extensive sand stones (Figure 14.3, 3), while in Rameswaram island the shore is slightly rocky in some areas but mostly sandy. About 6–10 km away from the Mandapam sea shore there are several small islands viz. Shingle, Muyal, Kurusadai, Pulli, Pullivasal, etc. On Kurusadai island, a portion of the shore is of coral reefs (Figure 14.3, 4). The area between Mandapam and Kurusadai Island is shallow. The shores towards Cape Comorin (Tiruchendur, Idinthakarai, etc.) are sandy with intermittent rocky spots, while at Cape Comorin it is partly rocky and partly sandy. Most of the shores in Andaman (south, middle, north and little Andamans) and Nicobar (Car Nicobar, Katchal Island) and Lakswadeep Islands (Kavarathi, Agatti, Amini and Kadamath) are sandy with rocky and vast coral stretches in some areas. A total of 225 species of 58 genera belonging to 14 families of cyanobacteria were recorded from in the survey, of which 35 species were heterocystous and 190 species were non-heterocystous with the members of the family Oscillatoriaceae being predominant in the marine

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FIGURE 14.3  Pictures of survey-locations (1–12). 1. Sandy sea shore of Nagore (Bay of Bengal). 2. Muddy sea shore of Adhirampattinam (Palk straight). 3. Rocky shore of Mandapam (Gulf of Mannar). 4. Coral reef of Kurusadai island (Gulf of Mannar). 5. Muthupettai lagoon region (Palk Strait). 6. Epiphytic cyanobacteria on the pneumatophores of mangroves. 7. Stagnant sea water puddle with cyanobacterial blooms. 8. A portion of bloom of Trichodesmium erythreum (Mandapam region, Gulf of Mannar). 9. A salt pan with dense cyanobacterial growth (Palk Bay). 10. Bloom of cyanobacteria in unused salt pan. 11. Cyanobacterial mat in stagnant sea water in Nagapattinam (Bay of Bengal). 12. Collection of cyanobacteria on the drifted sea grasses at Devipattinam shore (Palk Bay).

habitats. It is interesting to note that, of the 225 species recorded in the entire main land coastal area, 189 species were from the east coast and 110 species were from the west coast. Among the various planktonic cyanobacteria, Trichodesmium erythraeum is common phytoplankter in tropical and subtropical seas, often forming extensive blooms and is important as a primary producer and as a source of new nitrogen via nitrogen fixation. Trichodesmium usually constitutes a minor component of the marine plankton, but in certain seasons it becomes extremely abundant and far exceeds any other plant life in the sea (Qasim, 1972). This phenomenon of forming ‘Red Tides’ takes its name from the red color of a pigment known as phycoerythrin produced in the cells. Thajuddin (1991) and Thajuddin and Subramanian (1990, 2002) studied the ecobiology of plankton in general and Trichodesmiurn in particular in the Gulf of Mannar area between Mandapam. Rameswaram and Kurusadal Island (Figure 14.1, k, and Figure 14.3, 8). Jeyaraj et al. (2015) also reported Trichodesmium erythraeum bloom along the Pudhumadam coast in the Gulf

of Mannar during the pre-monsoon season, and it did not cause any unusual mortality to the marine large fish and shellfish or disrupt the marine environment. As many as 101 species and 90 species of cyanobacteria were found in the backwaters of east and west coasts respectively. Ramachandran (1982) pointed out that the mangrove environment supported an abundant growth of benthic cyanobacteria which showed richness in diversity of the species. In total 80 species from the east coast and 67 species from the west coast were recorded from the salt pans (Figure 14.2). Several workers had earlier reported the occurrence of cyanobacteria in salt pans and salt lakes with very high salinity (Biswas, 1926; Woronichin, 1929; Aggarwal, 1951; Subbaramaiah, 1972; Anand and Venkatesan, 1985; Anand et al., 1986; Thajuddin, 1991; Thajuddin and Subramanian, 1992). It was interesting to note that nonheterocystous forms in general and species belonging to the family Oscillatoriaceae were dominant in these environments and were found growing even in 340% salinity. A striking feature of cyanobacteria in hypersaline

Diversity and Utilization of Marine Cyanobacteria

environments was the absence of heterocystous forms. Explanations for the absence of heterocystous forms in hypersaline environments have been offered in terms of high sulfide content and its toxicity to heterocystous forms (Howsley and Pearson, 1979) which was subsequently disputed, and the anaerobic conditions prevailing in the dark in these environments were believed to exclude the heterocystous forms (Oren and Shilo, 1979; Stal et al., 1985). No satisfactory explanation supported with experimental evidence has so far been made. The remarkable adaptability of cyanobacteria is, however, well known (Hof and Fremy, 1933; Van Baalen, 1962; Fogg et al., 1973; Carr and Whitton, 1982). Phormidium valderianum BDU 30501 was shown to grow in salinities ranging from 0 to 99% (Prabaharan, 1988). Thajuddin and Subramanian (1992) reported in marine waters as many as 75 of the species belonging to 30 genera which originally were reported from freshwater sources by Geitlar (1932) and Desikachary (1959). It therefore becomes clear that it is difficult to strictly segregate many of the cyanobacteria into marine and freshwater species as can be done with other algal forms. This survey also resulted in recording eight species, viz., Siphononema polonicum, Rodaisia violaceae, Dichothrix spiralis (Thajuddin and Subramanian, 1991), Pseudoncobrysa fluminansis, Oscillatoria quttulata, O. borneti, O. grossegranulata and Ammatoidea sp. (Thajuddin and Subramanian, 1995) for the first time from the Indian coasts. Six species, viz., Gloeocapsa gigantia, Johannesbaptistia mannarensis, Oscillatoria moniliformoides, Lyngbya venkataraamani, Desygloea marina and Microcoleus undulatus were totally new (Thajuddin, 1991). This survey also resulted in the establishment of a marine cyanobacterial germplasm collection. Identification of several species new to India and several suspected new species are under investigation. To explore the potential of marine cyanobacterial for the benefit of mankind, a unique National Facility for Marine Cyanobacteria (NFMC) with 300 strains of marine cyanobacterial germplasm was established at Bharathidasan University with the financial support from the Department of Biotechnology, Govt. of India during the year 1991.

POSSIBLE APPLICATIONS IN BIOTECHNOLOGY Cyanobacteria are one of the potential organisms which can be useful to mankind in various ways (Table 14.3). They constitute a potential resource in varied areas such as mariculture (Mitsui, 1975; Mitsui et al., 1981), food,

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feed (Mitsui, 1979; Venketaraman, 1983; Venketaraman and Becker, 1985; Subramanian et al., 1994; Uma et al., 1998), fuel (Mitsui, 1978; Subramanian and Prabakaran, 1994; Prabakaran and Subramanian, 1995, 1996), fertilizer (Venkataraman, 1964, 1977; Bardach et al., 1972; Venkatraman and Kaushik, 1980), medicine (Mynderse et al., 1977; Gerwick et al., 1989; Gustafson et al., 1989; Sivonen et al., 1989; Sundararaman et al., 1996; Gopalakrishnan et al., 1998), industry (Mitsui, 1978; Venkataraman, 1983; Gabbay and Tel-Or, 1985; Tredici et al., 1986) and combating pollution (Subramanian et al., 1994; Subramanian and Uma, 1996; Karna et al., 1999; Kalavathi et al., 2001; MubarakAli et al., 2011a), as the usefulness of cyanobacteria for these purposes has been reported (Figure 14.4) Cyanobacterial protein in food either as a supplement or as an alternative source has received worldwide attention. Some strains of Anabaena and Nostoc are consumed as human food in Chile, Mexico, Peru and the Philippines. N. commune with a high amount of fiber and moderate protein is of use as a new dietary fiber source and can play an important physiological and nutritional role in human diet. Among several cyanobacterial species, Spirulina is used as a food supplement due to its excellent nutrient composition and better digestibility due to a delicate cell wall. Spirulina contains very high protein (60–71%), 20% carbohydrate, 5% lipids, 7% minerals and 6% moisture. It is also a rich source of betacarotene, thiamine and riboflavin and is one of the richest sources of vitamin B12. It is commercially available in the market in the form of powder, granules or flakes and as tablets and capsules (Thajuddin and Subramanian, 2005; Thajuddin et al., 2018). Priyadarshani et al. (2012) studied the tolerance capacity of four cyanobacteria of Odisha coast to different concentration of NaCl and found with the increase of NaCl concentration in the culture media, growth, pigment and protein content were decreased in all test species. However, the influence of aeration and light intensity in four species of marine cyanobacteria namely Oscillatoria boryana, O. pseudogeminata, Phormidium tenue and L. majuscula isolated from Odisha coast resulted in significant increases of biomass and protein content of all the test species compared with non-aerated cultures. The use of algae and cyanobacteria in waste treatment proved beneficial in different ways by bringing about oxygenation and mineralization, in addition to serving as a food source for aquatic species. Using the marine cyanobacteria Oscillatoria sp. BDU10742 and Aphanocapsa sp. BDU16 and a halophilic bacterium Halobacterium US 101, Uma and Subramanian (1990) could treat ossein factory effluent and reduce calcium and chloride levels

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TABLE 14.3 List of Various Biotechnological Potentials of Marine Cyanobacteria as Reported Elsewhere over the Past Few Decades Organism Phormidium valderianum Phormidium tenue KMD 33 Oscillatoria salina, Plectonema terebrans and Aphanocapsa sp. Tolypothrix tenuis Gloeocapsa alpicola Aphanothece halophyletica, Dactyolococcopsis salina, Halothece sp., Oscillatoria sp. and Synechocystis sp. Nostoc muscorum Cyanothece sp. Phormidium persicinum Synechocystis sp. Phormidium tenue Phormidium valderianum Phormidium sp., Oscillatoria sp. and Anabaena azollae Phormidium valderianum Lyngbya majuscula

Oscillatoria willei Oscillatoria formosa NTDM02 Oscillatoria sp. and Phormidium sp. Oscillatoria sp. and Phormidium sp. Oscillatoria sp., Synechococcus sp., Nodularia sp., Nostoc sp. and Cyanothece sp. Microcoleus chthonoplastes, Spirulina subsalsa, Johannesbaptistia pellucida, Chroococcidiopsis sp., Aphanocapsa sp., Chroococcus sp., Gloeocapsa sp., Schizothrix sp. and Leptolyngbya sp. Pseudanabaena sp. Phormidium persicinum NTDP01 Lyngbya majuscula and Spirulina subsalsa Cyanobium, Synechocystis and Synechococcus, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria. Lyngbya sp. Oscillatoria willei Phormidium tenue NTDM05 Trichodesmium

Potentials

References

Textile dye removal and hydrogen production Decolorization of Paper mill effluent and antioxidant property Degradation of crude oil

Shah et al. (2001)

Raghukumar et al. (2001)

Rice callus regeneration Phytohormones Hydrogen production Oil compounds degradation

Storni de Cano et al. (2003); Gayathri et al (2017); Santhoshkumar et al. (2016) Antal and Lindblad (2005) Abed and Koster (2005)

Poly Hydroxy Alkalonates production Exopolysaccharide production

Sharma et al. (2007) Chuandong et al. (2007); Baldev et al. (2015) Lavania et al. (2016) Ribeiro et al. (2008)

Nagasathya and Thajuddin (2008a, 2008b)

Removal of Pb, Ni and Zn from contaminated sea water Degradation of aromatic hydrocarbons Pesticide-metabolizing enzymes Biodegradation of coir waste

Kumar et al. (2009) Palanisami et al. (2009) Anbuselvi and Rebecca (2009)

Tannic acid degradation Quorum quenching activity Antibiofilm activity Biological control agents Ligninolytic and antioxidative enzymes Dye degradation Biosorption of Pb (II) Biosorption of Cr (VI) Bioremediation of industrial effluents

Palanisami et al. (2011) Dobretsov et al. (2011) Santhakumari et al. (2016); LewisOscar et al. (2018); Sumathy et al. (2015) Saha et al. (2010) MubarakAli et al. (2011a) Kumar et al. (2011) Rajeshwari et al. (2012). Dubey et al. (2011)

Sunscreen pigments

Abed et al. (2011)

c-phycoerythrin production

Mishra et al. (2011); Pandiaraj et al. (2017)

Lead accumulation Anticancer compounds

Chakraborty et al. (2011) Costa et al. (2014); Swain et al. (2015)

Silver nanoparticles synthesis CdS nanoparticle synthesis Toxins

MubarakAli et al. (2011b, 2012) Shunmugam et al. (2017); Reehana et al. (2018)

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FIGURE 14.4  Potential applications of marine cyanobacteria (Thajuddin et al., 2018).

significantly at field levels to enable 100% survival and multiplication of Tilapia fish with only the cyanobacteria as the feed source. Shashirekha et al. (1997) found that another marine cyanobacterium, Phormidium valderianum BDU30501, was able to tolerate and grow at a phenol concentration of 50 mg/l and remove 38 mg/l within a retention period of seven days. This result opens the possibility of treating a variety of phenol containing effluents. The same organism was used to study the optimal conditions for adsorption/desorption of heavy metal ions (Cd2−, Co2−) for use in treating effluents containing heavy metals using immobilization techniques (Karna et al., 1999). Immobilized forms of two marine cyanobacteria, Oscillatoria sp. NTMS01 and Phormidium sp. NTMS02, were demonstrated for their efficient sorption for the removal of chromium (VI) ions in lower concentration (Rajeshwari et al., 2012). Another work examined the Pb2+ removal ability of the immobilized and free filaments of marine cyanobacteria Oscillatoria sp. NTMS01 and Phormidium sp. NTMS02 in batch experiments. The maximum removal of lead was observed with immobilized Oscillatoria sp. NTMS01 and Phormidium sp. NTMS02. (Kumar et al., 2011). Recently the efficiency of Chroococcus sp. to effectively remove Cr(VI) from aqueous solutions was reported. The ability of the organism to survive under such toxic conditions was also analyzed, and it was reported that this organism

undergoes special defense mechanisms to overcome the stress (Satheshkumar et al., 2012). Another marine cyanobacterium Oscillatoria boryana BDU92181 was found to effectively degrade and metabolize the recalcitrant melanoidin pigment which is the source of dark brown color in distillery effluents (Kalavathi et al., 2001). Subramanian and Uma (1996) have identified suitable cyanobacteria for treating several noxious effluents containing organophosphorus pesticides, detergents, antibiotics, etc. and even degradation of solid wastes like coir pith by the lignolytic action of certain cyanobacteria (Malliga et al., 1996). Kumar et al. (2009) have demonstrated the ability of the hypersaline cyanobacterium Phormidium tenue in the bioconversion of Anthracene to 8 hydroxy- anthracene 1,2 dione and 10 hydroxy- anthracene 1,2 dione, whereas Napthalene is converted into [1,2] Napthoquinone and Napthalene 1,2 diol. This strain was proposed to be most suitable for use in the bioremediation of polycyclic aromatic hydrocarbon pollution on seashores. A marine cyanobacterium Oscillatoria formosa NTDM02 demonstrated for the decolorization of amido black dye. The same organism was also shown to effectively decolorize the textile effluent efficiently in a short period of time and it was proposed that it can be used for the bioremediation of dye effluents (Mubarakali et al., 2011a). Hypersaline cyanobacterium Phormidium tenue

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KMD33 was reported as an efficient candidate to effectively breakdown the dyes from the effluent samples collected from different locations of the paper mill industry (Nagasathya and Thajuddin, 2008a). Many marine cyanobacteria fixing nitrogen have been tested for their nutritional value with the hybrid Tilapia fish fry, and many were accepted as single ingredient feed. Very high growth rates of Tilapia fish using marine cyanobacteria with in-door and out-door cultures were also reported (Mitsui et al., 1983). In studies conducted in NFMC laboratory, the marine cyanobacterium Phormidium valderianum BDU30501 was chosen as a complete aquaculture feed source, based on the nutritional qualities and non-toxic nature with animal model experiments (Uma et al., 1998). Several cyanobacteria are known to be rich sources of vitamins, and many are even known to excrete them into the milieu (Borowitzka, 1988). The carotenoids and phycobiliproteins of cyanobacteria have high commercial value. They are used as natural food colorants (Emodi, 1978), as food additives to enhance the color of the flesh of Salmonid fish (Schiedt et al., 1985) and to improve the health and fertility of cattle (Jackson, 1981). Many marine cyanobacteria, especially the feed grade Phormidium valderianum, have been found to be excellent sources of phycocyanin, a blue natural colorant useful as a phycofluor in diagnostics (Sekar and Subramanian, 1998) (Table 14.4). Cyanobacteria, being photoautotrophs, can photosynthetically transform simple, labeled compounds such as 14CO2, 13CO2, 33H2O and 15NO3 into complex organic compounds. Isotopically labeled cyanobacterial metabolites such as sugars, lipids and amino acids are commercially available (Subramanian and Shanmugasundaram, 1986a; Patterson, 1996). Several important enzymes are known to be produced in high enough amounts by cyanobacteria to be useful for commercial ventures. The fact that several marine cyanobacteria could be useful for large-scale production of enzymes such as beta lactamase, protease and lipase has been identified and characterized (Prabakaran et al., 1994). Several common as well as unique sequencespecific endonucleases are known from cyanobacteria such as Anabaena cylindrica (Acy I), Anabaena flosaquae (Afl I and Afl III), Anabaena variabilis (AvaI and Ava II), Anabaena variabilis UW (Avr II), Nostoc sp. PCC 7524 (Nsp C I) and Microcoleus sp. UFEX 2220 (Mst II), which can be made available in the market at lesser cost since cyanobacterial biomass production is much less expensive than bacteria (Elhai and Wolk, 1988; Patterson, 1996). Extra cellular phosphatases from cyanobacterial isolates have also been reported.

Handbook of Algal Technologies and Phytochemicals

They have the capacity to mineralize organic phosphorous by alkaline phosphotase activity (Giraudet et al., 1997). Several studies have reported the possibility of producing enzymes such as chitinase, L-asparaginase, L-glutaminase, amylase, protease, lipase, cellulase, urease and superoxide dismutase (Burja et al., 2001; Tan, 2007; Abed et al., 2009). Photoproduction of ammonia and amino acids in large amounts by cyanobacteria, which can be harvested continuously over longer periods, has been investigated (Musgrave et al., 1982; Mitsui et al., 1983; Subramanian and Shanmugasundaram, 1986b; Kerby et al., 1989). Analysis of extracellular growth promoting substances liberated by Nostoc muscorum and Hapalosiphon fontinalis in the external medium found them to be rich in several amino acids like sereine, arginine, glycine, aspartic acid, threonine, glutamic acid, cystine, proline, valine, ornithine, lysine, histidine and iso-lucine (Misra and Kaushik, 1989). In addition, cyanobacteria can be rich sources of several polyols, polysaccharides, lipids, fatty acids and halogenated compounds, etc. with varied properties employable as flocculants, surfactants or for other uses (Fattom and Shilo, 1984; De-Philippis and Vincenzini, 1998). Cyanobacteria in general and marine forms in particular are the richest sources of known and novel bioactive compounds including toxins with wide pharmaceutical applications (Carmichael, 1992; Becker, 1994; Patterson, 1996). Gustafson et al. (1989) reported anti-HIV activity of some marine cyanobacterial compounds from Lyngbyalager heimii and Phormidium tenue. A massive program of screening the extracts from the large culture collection of marine cyanobacteria in NFMC for anti-viral, anti-bacterial, anti-fungal and immuno-modulatory activities has been going on with great success. A compound has been purified from marine Oscillatoria laete-virians BDU20801 showing anti-candidal activity (Deth, 1999). A compound with immunopotentiating and male anti-fertility properties, without being toxic to other systems in a mice model, was reported to occur in the crude and partially purified extracts of Oscillatoria willei BDU130511 (Raghavan et al., 2002). Medically important gamma linolenic acid (GLA) is relatively rich in the cyanobacteria Spirulina platensis and Arthrospira sp. (Watenabe and Yamamoto, 1972; Cohen et al., 1987). This is converted into arachidionic acid and into prostaglandin E2 in the human body (Euler and Eliassen, 1967). `Prostoglandin E2 has a lowering action on blood pressure and the contracting function of smooth muscles. It plays a very important role in lipid metabolism. Cyanobionts namely Aphanocapsa sp. NTK28 and Nostoc sp. NTK29 isolated from cyanolichens were

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TABLE 14.4 List of Bioactive Metabolites Reported from Marine Cyanobacteria Source

Compound

Activity

Reference

Order Chroococcales Microcystis aeruginosa

Microviridin Toxin BE-4, Siatoxin

Antibiotic, anticancer

Synechococcus sp. Synechocystis trididemni

Lipopeptide Didemnin

Antibiotic Anticancer, antiviral

Arment and Carmichael (1996); Domingos et al. (1999); Shi et al. (1999) Ohta et al. (1994) Rinehart et al. (1981); Chun et al. (1986)

Order Pleurocapsales Hyella caespitosa

Carazostatin

Antifungal

Lyngbya polychroa Oscillatoria acutissima Oscillatoria nigroviridis Phromidium ectocarpi Phormidium tenue

Order Oscillatoriales Sulfolipid Anti-HIV activity Sulfolipid amide (bromo, chlor and pyrrole) Anti-HIV, anticancer, fatty acid (chloro sulfo thiazoline) Antifungal, antimicrobial lipopeptides Antimalarial, Dragomabin antiproliferative Dragonamide A, B Carmabin A, B Dragonamide C, D Anticancer Acutiphycin Anticancer Oscillatoxin Anticancer Hierridin, 2, 4-dimethoxy-6-heptadecyl-phenol Antiplasmodial, antibiotic Sulfolipid Anti-HIV activity

Schizothrix calcicola

E-1-chlorotridec-1 ene-6, 8 diol

Antibiotic, anticancer

Spirulina platensis

Calcium spirulan

Anticancer, anti-HIV activity

Anabaena flos-aquae

Lipopeptide, alkaloid

Antibiotic, anticancer

Anabaena variabilis Aphanizomenon flos-aquae Aulosira fertilisima Calothrix sp. Cylindrospermum licheniforme Hormothomnion enteromorphoides Nostoc sp.

Lipopeptide Aphanorphine, Siatoxin

Antibiotic Antibiotic, anticancer

Lyngbya lagerheimii Lyngbya majuscula

Cardellina et al. (1979) Gustafson et al. (1989) Gerwick et al. (1994); Luesch et al. (2001); Mynderse et al. (1988); Mitchell et al. (2000); Milligan et al. (2000); McPhail et al. (2007) Gunasekera et al. (2008) Barchi et al. (1984) Moore et al. (1984) Murakami et al. (1991b) Gustafson et al. (1989); Murakami et al. (1991a) Harrigan et al. (1999); Mynderse and Moore (1978) Yang et al. (1997); Campbell et al. (1982); Bhat and Madyastha (2000)

Order Nostacales

Nostoc commune

Aulosirazole Calothrixin A, B Cylindrocyclophane Hormothomnin Cryptophycins, Nostophycin, Nostocarboline

Nostodione, Microsporin

Anticancer Antimalarial, anticancer Anticancer Cytotoxic, antibiotic Anticancer, cytotoxic, antifungal, antibiotic, antimalarial, antileishmaniasis, cholinesterase inhibitor Antifungal, antibiotic

Matsunaga et al. (1989); Ma and Led (2000) Carmichel et al. (1975) Moore (1978) Stratmann et al. (1994) Issa (1999) Moore et al. (1990) Gerwick (1990) Trimurtulu et al. (1994); Yang and Shimizu (1993); Barbaras et al. (2008)

Böhm et al. (1995); Jaki et al. (2001) (Continued)

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TABLE 14.4 (CONTINUED) List of Bioactive Metabolites Reported from Marine Cyanobacteria Source

Compound

Activity

Reference

Nostoc elipsosporum Nostoc sphaericum Nostoc spongiaeforme Scytonema pseudohofmanni Tolypothrix nodosa Tolypothrix tenuis

Cyanovirin Staurosporine Nostocine A Scytophycine

Anti-HIV, antiviral Antiviral Antibiotic, antialgal Antifungal, antiviral

Tolyporphin Toyocamycin

Antibiotic Antifungal

Fischerella muscicola Haplosiphon fontinalis

Fischerellin Alkaloids, Indole

Antifungal, herbicidal Antibiotic, antifungal

Srivastava et al. (1998, 1999) Moore et al. (1987)

Haplosiphon welwitschii

Hapalosin, Welwisthtin

Anticancer, antibiotic

Stigonema dendroideum

Dendroamide

Zhang and Smith (1996); Smith et al. (1995) Ogino et al. (1996)

Dey et al. (2000) Wipf and Venkatraman (1996) Hirata et al. (1996) Moore et al. (1986b, 1996) Prinsep et al. (1992) Moore (1982)

Order Stigonematales

reported to have good antibacterial activity against clinical isolates such as Pseudomonas sp., Escherischia coli, Klebsiella sp. and Staphylococcus sp. (Shyamkumar et al., 2010). Apart from this, Nagasathya and Thajuddin (2008b) reported antioxidant property and increased activity of antioxidative enzymes such as catalase, superoxide dismutase and glutathione peroxidase from the hypersaline cyanobacterium Phormidiun tenue KMD33. Of late, extended-spectrum β-lactamase (ESBL) producing pathogenic bacteria pose a big challenge in clinical practice, warranting a new therapeutic strategy. In our study, methanolic extract of the marine cyanobacterium Oscillatoria acuminata NTAPC05 was fractionated, and the fractions were tested against ESBL producing bacteria Escherichia coli U655, Stenotrophomonas maltophilia B929 and Enterobacter asburiae B938. The fractions were effective against these bacteria at a concentration of 100 μg ml−1 while Cephalosporin was needed at a minimal concentration of > 125 μg ml−1 to be effective. For the first time monogalactosyldiacylglycerol containing palmitoyl was reported as the active fraction, and its bactericidal property against ESBL producers was demonstrated (Parveez Ahamed et al., 2017). A good amount of work has been carried out to bring out the nanobiotechnological potential of cyanobacteria. For instance, extracellular biosynthesis of silver nanoparticles using the marine cyanobacterium Oscillatoria willei NTDM01 was reported (MubarakAli and Thajuddin, 2009; MubarakAli et al., 2011a). Detailed characterization through UV-vis, FTIR, SEM and EDS spectral analysis revealed that the silver ions were reduced, and a secreted protein stabilizes the silver nanoparticles. Apart

Antibiotic

from eco-friendliness and easy availability, the low cost of production will also be advantageous when compared to other classes of microorganisms (Mubarakali et al., 2011b). Apart from cyanobacteria as biomass, their accessory pigment C-phycoerythrin (C-PE) was also found useful to synthesize nanoparticles. C-phycoerythrin from Phormidium tenue NTDM05 was used to synthesize CdS nanoparticles; the size of such nanoparticles was found to be about 5 nm. Essentially, the pigment stabilized the CdS nanoparticles. The pigment-labeled CdS nanoparticles could be applied as biolabel (MubarakAli et al., 2012).

CONCLUSION AND RECOMMENDATIONS Perusal of extensive literature indicates several lacunae in knowledge despite many marine cyanobacterial application reports. Most of them remain as feasibility reports without reaching the logical end of utilization. There are still several uncultivable cyanobacteria whose diversity is left untapped. Development of new culture media as well as methods with necessary supplements to grow them in culture are necessary, for which knowledge of ecobiology of such marine cyanobacteria is essential. In addition, metagenomic approaches must be used to reveal the actual diversity. Although several preservation methods such as lyopholization, liquid nitrogen storage, permanent slide preservation, etc. are available for other groups of microorganisms, there is no standardized protocol for the preservation of cyanobacteria in good condition. Hypersaline cyanobacteria are considered suitable for bioremediation of different kinds of effluents. Hence

Diversity and Utilization of Marine Cyanobacteria

development of separate germplasm of hypersaline forms would prove useful. Due to rapid industrialization and improper release of huge volumes of untreated effluents and sewage into coastal environments, sudden blooming of certain pollution indicator species of cyanobacteria has been reported which dominate and destroy native forms. Hence the native ecosystems are disturbed, and useful species could disappear. Ex situ conservation of the coastal environment is necessary to conserve the native flora and fauna including marine cyanobacteria. In spite of increased taxonomic research the world over, classification and ecobiological understanding of the existing biodiversity of microorganisms including cyanobacteria is inadequate. Knowledge of the existing biodiversity is incomplete due to declining numbers of taxonomists and the general lack of interest in taxonomy among emerging scientists. In order to develop interest in taxonomy, the students need to understand and develop interest on biodiversity and taxonomy. This is possible only with a good curriculum. Funding agencies should allocate enough funds for taxonomy and biodiversity research. Annual workshops should be conducted on morphological, biochemical and molecular taxonomy of different microbes by specialists in reputed institutions. Cyanobacteria have long been considered either as organisms of academic curiosity or as organisms of nuisance value. Pioneering work in India and other countries has raised the status of these microbes to the level of biotechnologically most useful organisms. Therefore, in tropical countries like India where biodiversity is rich, it is essential not only to understand and preserve the biodiversity of cyanobacteria found in all habitats but also to gainfully employ them for different biotechnological applications including pollution abatement.

ACKNOWLEDGMENT The authors are grateful to the initial funding from the Department of Ocean Development (Ministry of Earth Sciences) and Department of Biotechnology, Government of India, for their constant funding support enabling the type of work reported.

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Open Cultivation Systems and Closed Photobioreactors for Microalgal Cultivation and Biomass Production C.K. Madhubalaji, Ajam Shekh, P.V. Sijil, Sandeep Mudliar, Vikas Singh Chauhan, R. Sarada, Ambati Ranga Rao, and Gokare A. Ravishankar

CONTENTS Abbreviations���������������������������������������������������������������������������������������������������������������������������������������������������������� 179 Introduction to Microalgae Cultivation System������������������������������������������������������������������������������������������������������179 Open Cultivation Systems���������������������������������������������������������������������������������������������������������������������������������������180 Circular Ponds����������������������������������������������������������������������������������������������������������������������������������������������������180 Raceway Pond����������������������������������������������������������������������������������������������������������������������������������������������������180 Closed Photobioreactors�����������������������������������������������������������������������������������������������������������������������������������������181 Vertical Tubular Photobioreactor������������������������������������������������������������������������������������������������������������������������181 Bubble Column Photobioreactor��������������������������������������������������������������������������������������������������������������������182 Airlift Photobioreactor�����������������������������������������������������������������������������������������������������������������������������������183 Horizontal Tubular Photobioreactor�������������������������������������������������������������������������������������������������������������������184 Flat Panel Photobioreactor����������������������������������������������������������������������������������������������������������������������������������184 Helical Type Photobioreactor�����������������������������������������������������������������������������������������������������������������������������186 Stirred Tank Photobioreactor������������������������������������������������������������������������������������������������������������������������������186 Soft-Frame Photobioreactor�������������������������������������������������������������������������������������������������������������������������������187 Hybrid Type Photobioreactor�����������������������������������������������������������������������������������������������������������������������������187 Comparative Account of the Performance of Various Bioreactors�������������������������������������������������������������������������188 Future Prospects������������������������������������������������������������������������������������������������������������������������������������������������������196 Acknowledgments���������������������������������������������������������������������������������������������������������������������������������������������������197 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 197

BOX 15.1  SALIENT FEATURES Microalgae are considered valuable photosynthetic microorganisms for various applications, from CO2 utilization, to production of nutraceuticals, to wastewater treatment. Irrespective of the final application of microalgae, the process of cultivation remains the same. However, the production capabilities vary depending on the choice of cultivation system. Various kinds of open and closed photobioreactors are used for high microalgal biomass production. However, the design of a suitable photobioreactor depends on light availability and/ or distribution, CO2 supply-mass transfer, environmental/cultivation conditions including temperature and solar radiation, and mixing. To provide

the necessities of microalgae cells, various photobioreactors have been proposed. We have reviewed these open cultivation systems and closed photobioreactors in this chapter.

ABBREVIATIONS M W

Meters Watt

INTRODUCTION TO MICROALGAE CULTIVATION SYSTEM Microalgae is an indispensable resource for various metabolites (Astaxanthin, β-carotene, omega-3, omega-6 fatty acids, proteins, carbohydrates, vitamins, 179

180

and minerals) production with multiple benefits of its application in food, feedstock, medical, cosmetic, biofuel, and chemical industries (Fernandes et al. 2015; Ranga Rao et al. 2018a,b, 2014). This led to an increasing demand for the microalgal biomass. Growing microalgae in natural environments (pond, lake) cannot produce the quality of the biomass for intended uses. Various types of cultivation systems with different configurations are used for microalgal cultivation, such as simple open circular ponds, open raceway ponds, and complex enclosed systems referred to as closed photobioreactors. The open cultivation in contained reactor is widely used especially for growing extremophilic organisms such as Spirulina and Dunaliella free from contamination. Also this system is cheaper to construct and provides excellent utilization of solar light source especially in the equatorial regions of the globe. However there is constant innovation in design geometry of the open cultivation systems to maximize the solar energy distribution on the culture surface (Fernández et al. 2001; Sierra et al. 2008) to provide adequate light (Terry 1986; Grobbelaar 1994), fluid dynamics, and mass transfer to achieve high biomass productivities (Rubio et al. 1999; Posten 2009). Closed bioreactors of various designs are being developed and adapted for controlled cultivation of the biomass with higher precision in providing the adequate quantity and quality of light; mixing of nutrients and dosing; regulation of pH, temperature, in addition to programmed harvesting of the biomass. The configuration of the photobioreactor has a great influence on CO2 capture and biomass production by microalgae. Various closed photobioreactors (flat panel, airlift, bubble column, stirred tank reactors) are considered for microalgae cultivation (Ranga Rao et al. 2016). In order to utilize the advantages of both open cultivation and closed bioreactors, hybrid reactors are in use.

OPEN CULTIVATION SYSTEMS Open cultivation systems have been widely used for commercial production of microalgal biomass. In open ponds, microalgae grow under the natural environmental conditions of light, temperature, humidity, and other weather conditions. Despite being simple and economical for microalgae cultivation, open system possesses several drawbacks viz. contamination by other local microalgal species, predator attacks, evaporation losses, etc. Open pond cultivation of microalgae is of mainly two types viz. circular ponds, and raceway ponds. They are briefly presented in the following section.

Handbook of Algal Technologies and Phytochemicals

Circular Ponds Circular ponds have a rotating arm located at the center having a depth in the range of 20–30 cm and diameter of 40–50 cm, mostly used in wastewater treatment ponds (Ting et al. 2017). Circular ponds are used in pilot scale commercial productions of Chlorella sp. The circular pond design (Figure 15.1A, B) includes the rotating arm moving in an axial direction to mix the culture. The major disadvantage of this system includes no control over cultivation parameters like temperature or pH, which allows the other microalgal strains to grow. Scenedesmus obtusus and Botryococcus were cultivated in open circular ponds (Ranga Rao et al. 2012; Jethani et al. 2019). However, effective pond management protocols are adopted to avoid contamination and control insect infestation, etc.

Raceway Pond This is the most suitable cultivation system used in commercial scale productions of algae. Worldwide, various microalgae viz. Chlorella, Dunaliella, Spirulina, Nannochloropsis oculata, and Haematococcus are being commercially cultivated using open raceway ponds (Richmond and Cheng-Wu 2001; Jiménez et al. 2003; Moreno et al. 2003; Moheimani and Borowitzka 2007; Radmann et al. 2007; Sun et al. 2018b). Raceway ponds are constructed as a group of channels or single channel with a depth of 15–50 cm (Ting et al. 2017) and operated at low depth of 12–16 cm. The mixing is done by paddlewheels that ensure the proper mixing and recirculation of the suspended algal cells. In general, a single paddle wheel could be sufficient for mixing a large area of microalgal culture of single channel. To achieve uniform density distribution and to reduce generated eddies, turbulent mixing is recommended during algal cultivation. Turbulence enables proper mixing of nutrients, exchange of atmospheric CO2, and exposes culture to receive sufficient sunlight. In open raceway cultivation, the cultivation parameters cannot be controlled and the culture remains under the influence of daily climatic changes. For the construction of open raceway ponds (Figure 15.2A, B), low cost materials viz. concrete, plastic, or rubber sheet are normally used. The energy requirement for culture mixing was reported to be 4 Wm−3 (Jorquera et al. 2010). A large diameter paddle wheel (2.0 m in diameter) rotating at low speed (10 rpm) is usually recommended for higher biomass productivity. The open raceway pond also has similar disadvantages to the circular pond viz. likely contamination, lack of temperature control, water loss

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Microalgal Cultivation Systems for Biomass Production

FIGURE 15.1  (A) Schematic representation of circular pond. (B) Commercial cultivation of algae in circular pond. (Source: https://nutriphys.com/en/all-about-chlorella/cultivation-method.)

due to evaporation, and poor mass transfer phenomena (Richmond 2004; Carvalho et al. 2006; Posten 2009). Accumulation of dissolved oxygen (DO) remains another issue. It has been observed that 25–40 mg L−1 of DO is accumulated during Chlorella cultivation (Weissman et al. 1988). During cultivation of Spirulina platensis in a 450 m2 raceway pond, it has been observed that a DO of 10 mg L−1 accumulated in winters, 30 mg L−1 in summer. The DO accumulation of more than 25 mg L−1 is associated with decreased biomass productivity.

to maximize light capture and conversion has been one of the major challenges in high density commercial microalgae cultivation. Photobioreactors of various configurations have been in practice for high density cultivation of contamination free microalgae cultures. Various factors such as design, configurations, operational suitability, and maintenance cost and space requirements are considered for the selection of the photobioreactors. The various photobioreactors of varying design and type used for the microalgae cultivation are discussed below.

CLOSED PHOTOBIOREACTORS

Vertical Tubular Photobioreactor

Microalgae biomass yields are reported to be higher in closed photobioreactors compared to open ponds (Ugoala et al. 2012). However, photobioreactors design

These photobioreactors are made up of transparent vertical tubes (Figure 15.3) through which light penetrates inside the tubes. A sparger is placed at the bottom of

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FIGURE 15.2  (A) Schematic representation of raceway pond. (B) Commercial cultivation of algae in raceway pond at Algae PARC pilot facilities in Wageningen, UR, the Netherlands (de Vree et al. 2015; de Vree 2016; obtained with permission from Global Open Research Support Executive, BioMed Central, Springer Nature).

the reactor which allows overall mixing and converts sparged gas into tiny bubbles for better CO2 dissolution. It also helps to avoid oxygen build up inside the reactor during photosynthesis. Vertical tubular PBRs have sufficient contact time for mass transfer. Two types are commonly used viz. the bubble column reactor and the airlift photobioreactor. Bubble Column Photobioreactor These photobioreactors are of vertical column configuration and have height greater than the diameter (Figure 15.4). The culture is aerated using air or gas for mixing and to generate the suspension solution. It has advantages like it requires less capital cost, has a high surface area to volume ratio, no moving parts, and sufficient heat and mass transfer. Uniform mixing and dissolution of CO2 through enhanced mass transfer is achieved by gas bubbling using a sparger. Whereas, upon scaling up of the reactor, the plates kept horizontally inside the reactor tube are used to break the perforated bubbles so as to avoid the coalescence (Doran

1995). The inlet gas flow rate which in turn dictates the culture circulation pattern from the dark zone and light zone greatly affects the photosynthetic efficiency. It has been observed that the gas flow rate  0.05). In addition, there was no significant difference in organ coefficient and blood cytology (P > 0.05) between the algae group and the control. Overall, a total of 10% addition of algae biomass in the feed showed no detrimental effects during the 90-day feeding. However, the triacyl glycerides and cholesterol were significantly reduced in the algal groups compared to the control group (P  15%). Decanter centrifuges can achieve a concentration factor of 11 (Grima et al. 2003) and consequently, are associated with a primary concentration method such as flocculation, flotation or gravity-assisted settling. Another disadvantage is the energy consumption: 3kWh/kg biomass (Hattab 2015) Hydrocyclone: can operate continuously and has the lowest energy consumption (0.3 kWh/kg biomass (Sharma et al. 2013)) but the worst concentration factor of four (Grima et al. 2003). As well as the decanter centrifuge, its usage depends on a primary harvesting process. Spiral plate: This is a relatively new technology and not adopted in industrial scale. It consists of a threephase separator (liquid/liquid/solid) that differs from disk stack by the short settling distance. It results in a higher dry solid content than disk stack centrifuges and has similar energy efficiency. The disadvantages are the high capital cost and the necessity to frequently

Handbook of Algal Technologies and Phytochemicals

stop the operation to remove the accumulated biomass (Fasaei et al. 2018). Besides its great efficiency, centrifugation consumes a large quantity of energy and is not easily scalable, requiring high maintenance. Because microalgae harvesting by centrifugation accounts for 20–30% of the total biomass production cost, it is mostly used in the production of high value microalgal bioproducts. The fact that every industrial plant of microalgae is intended for the production of high value-added products (food supplement, pigment, cosmetics) is totally or partially a reflection of the biomass harvesting costs. The need to reduce production costs encourages the development/optimization of centrifugation equipment and other harvesting techniques.

FILTRATION Filtration is the process where the particles (cells) are mechanically retained by a porous barrier (filter medium) while the fluid passes. The major advantages of filtration are the high concentration factor, greater than centrifugation (reaching up to 250 times) (Grima et al. 2003), dismissing further dewatering processes; and the simplicity and low cost of the operation. But large-scale use of filtration faces problems related to filter binding, pressure changes, speed of operation (it is much slower than centrifugation) and cost of filter membranes. For harvesting microalgae, the main filtration processes used are vacuum and pressure filtration: Vacuum filtration: Vacuum is generated at the filtrate side. Operation cost varies from 2.49 to 0.50 kWh/kg biomass (depending on solids concentration) (Fasaei et al. 2018). This technique can be operated continuously until clogging or fouling. Pressure filtration: The pressure is created at the feed size, forcing the liquid to pass through the filter. It can work with higher solids concentration than vacuum filtration (22–27% versus 18–22%) and has lower energy consumption (0.19–0.96 kWh/kg biomass) (Fasaei et al. 2018). Filter presses operating under pressure are suitable for recovering medium to large cells but fail to retain small organisms. For small cells, a primary harvesting technique is required to promote the agglomeration of cells and consequently increase of particle size. At the same time, it results in cost increase since it is described that single-step filtration is more cost attractive than combined with other harvesting methods (Fasaei et al. 2018). One possible solution to the recovery of small microalgae is the use of a precoat rotary drum filtration. Precoating can be done with diatomaceous earth, perlite, cellulose and special ground wood. These materials

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work as a barrier to retain microalgae cells (but are wrongly called filter-aids). Because the cells accumulate within the precoating material, recovery using this technique results in contamination of biomass. For this reason, microalgae are more often harvested using microstrainers, which are rotary drums covered by a straining fabric, stainless steel or polyester, resulting in uncontaminated biomass (Show et al. 2015).

microalgal ultrafiltration cases. For process development, several conditions are tested and the transmembrane flux evaluated. With a flux of 100 L.m−2.h−1 and a moderate filtration area of 100 square meters, easily achievable with a few commercial modules, it is possible to process 10 m3.h−1 of microalgal suspension with a footprint of a few square meters. The cells processed must resist the shear stress in the centrifugal pumps of the system.

TANGENTIAL FILTRATION (MICROFILTRATION)

Further Dewatering

Tangential filtration is a process where the liquid flow is parallel to the separation medium. With that arrangement, the buildup of biomass over the filter is kept at a minimum, and the process can be continuous. Tangential filtration has become popular in water treatment (as reverse osmosis) and purification steps (as ultrafiltration). The difference in these processes is the membrane pore size. For microalgal separation, the pores must be smaller than the size of microalga but are much larger than those in ultrafiltration, and thus the process is termed microfiltration. Advantages of microfiltration are that there are no moving parts in the filter (the fluid is pumped by external, usually centrifugal or positive displacement pumps) and very large volumes can be processed in parallel modules, concentrating suspensions from a few grams to hundreds of grams per liter. Disadvantages are the relatively high energy input and the high-water content of the filtrate in comparison with classical filtration, which puts this operation somewhere between centrifugation and classical filtration in terms of cost. Microfiltration depends highly on the membrane material and the presence of extracellular organic matter that may increase fouling (Hung and Liu 2006) and needs specific tailoring and process development (Rios et al. 2011). Table 20.3 shows some

Drying is an energy-intensive operation. Therefore, prior dewatering is important for an economic process. Direct filtration, filter and decanter centrifuges give biomass with a water content adequate for further drying – potentially, almost pure biomass without extracellular water in the cake. Other processes, however, will give biomass with variable water content. The water content may range from a wet cake with 30–50% extracellular water for tubular centrifuges, to suspensions with 80% or more extracellular water from continuous disk-stack centrifuges and even more water for biomass separated by flotation or sedimentation. The dewatering process is simply another solid–liquid separation but processing concentrated biomass and thus working with considerably lower volumes. A process that concentrates 10 m3.h−1 of microalgal culture from 2 to 200 g/L – a concentration factor of 100 times – will have to process only 100 L.h−1. Decanter, tubular, filter or disk centrifuges with intermittent discharge can be used in this step, but filtration would be an option with (usually) lower cost/benefit.

DRYING Drying is the removal of water by means of thermal energy. The process is relatively straightforward but

TABLE 20.3 Microalgal Ultrafiltration Examples Pore size, μm

Filter area, m²

Flowrate, L.h−1

Flux, L.m−2. h−1

Transmembrane Pressure, Bar

Harvesting Efficiency, %

Dunaliella tertiolecta and Tetraselmis Sp.

0.45

0.02

5.00

250

0.25

84.7

Chlorella vulgaris Chlorella minutissima Scenedesmus sp.

0.2 0.1

0.014 7.6

5.56 521.34

381 68.6

0.98 1.95

62.5

0.1

3.8

266.00

70

1.95

Chlorella vulgaris

0.2

0.014

3.312

240

2.4

Microalgal Species

100

Concentration Factor

Final Concentration, g.L−1

Reference Kang et al. (2015)

1.25 200

100

Kim et al., (2015) (Gerardo et al. 2015)

100

150

Gerardo et al. (2014)

4.8

16.66

Elcik and Cakmakci (2017)

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depends on laboratory experimentation to define adequate processing temperatures and drying kinetics. There are several types of drying, ranging from sophisticated processes where the biomass can be freeze or spray dried, to relatively simple solar drying. In common, they have heat transferred by conduction and/or convection to the solid, while water evaporates to air. Drying also works for dessolventizing extracted biomass, using inert gases. Because describing all drying types and process details is out of the scope of this text, we will focus on what can be expected of drying operations and how to start an informed process development with common dryers for microalgal biomass. Tray and roller drying: Tray drying is adequate for relatively small batches of material, e.g. dewatered biomass cake that is distributed in trays and placed in a drying chamber. The moist solids lose water to the air, which should be circulated and renewed to enhance mass transfer. Because there is no agitation, the dry solids form a pellicle or mass that must be later pulverized, as in Halim et al. (2011); in roller dryers, the wet biomass is applied to the outside of a heated, rotating cylindrical surface and can be scraped at the end of the drying section, thus giving a somewhat disaggregated material; tunnel and rotary drum dryers are common equipment but pose problems for algal biomass processing. Fluid bed dryers: This equipment uses forced air to suspend particles in drying, thus creating an agitation similar to that observed in a fluid and promoting high efficiency in drying. These dryers can be suitable for large-scale processing and formulation of biomass products. The process is energy intensive but gives a powdery material that is easily separated using cyclones and bag filters. Spray drying: This popular drying technique sprays a microalgal suspension in a current of warm air, essentially drying the droplets before they reach the bottom of the equipment, giving a powdered solid. Elaborate nozzle geometries can create encapsulated powders. Freeze drying: This is a process similar to tray drying, but biomass is previously frozen, and the pressure is reduced to below 6 mbar, to allow direct sublimation of ice into water. The process requires vacuum pumps or ejectors and is more energy-intensive than other drying methods but can give high quality biomass, low denaturation and even maintenance of cell viability after hydration. The process is frequently used in research to provide a pristine biomass that can be analyzed for its lipidic content, as in Luangpipat and Chisti (2017). For intermediary drying – for further biomass processing – classical processes are more adequate, while fluid bed, spray and freeze drying are adequate

Handbook of Algal Technologies and Phytochemicals

when the biomass will have a high aggregated value, such as for nutraceuticals. Process development: Drying depends on heating: the water vapor pressure increases exponentially with temperature, and the higher the temperature, the faster is the process and less air is needed. Therefore, the first thing that must be defined in drying is the temperature that the biomass can withstand. If the biomass is intended for nutritional purposes, then denaturation is not a problem and higher temperatures may be used – 60 to 80°C may be adequate. For biomasses that will be used for further processing into products such as carotenoids or enzymes, temperatures should be lower. That is possible if dry, circulating air is used: one cubic meter of air at 60°C can carry up to 130 g of water. However, mass transfer is slower in lower temperatures. The second thing that must be defined in drying is the final water content – for most uses, it is not necessary to remove all the water. Water contents around 10–12% are adequate for biomass storage and commercialization; lower contents may be required for further processing such as solvent extraction. Drying biomass to lower-than-necessary water content is a waste of energy and time – because removing water below the critical water content is harder than unbound water. This leads to the third point to be defined: the kinetics of drying. The drying rate varies mainly with the process condition (type of dryer, temperature, biomass layer thickness) and on the biomass composition, which will affect diffusion-controlled drying rate, dominant at the end of the process. Drying curves can be easily obtained using tray driers and periodic weighing, and from the curve defined, insights into the large-scale process can be made. Finally, drying can accommodate specificities of the biomass to be processed: although air at 1 atm is the most common fluid used to carry out the water from the biomass, it is also possible to use flue gases – which is more energy-efficient than using indirect drying – or inert gases, which are adequate for avoiding product oxidation. Other possibilities such as vacuum drying exist; the conditions should be defined to meet process requirements.

CONCLUSION AND FUTURE PERSPECTIVES Despite the large amount of research that has been done with microalgae, the industrialization is recent and thus, the volume processed in most reports is relatively low. Therefore, there is relatively little experience with algal biomass processing, beyond a few commercial species, most notably from the genera Spirulina, Chlorella, Dunaliella and Haematococcus. If at one side the processing of novel strains can be done applying these same technologies, at the other the process intensification

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depends on the optimization for each strain. Especially in the case of biomasses produced for biofuel or feed, which require low-cost processing, it is essential to refine flocculation, solid–liquid separation and drying processes regarding low chemicals and energy input. In that sense, downstream affects the process from the screening to culture development, and the most likely points to address in bioprospection and new process development that will facilitate biomass separation and drying are on the culture development. 1) Isolation of fast-growing but large species – and this is not easy, because the fastest growing microalgae tend to be on the lower end of the size spectrum; possibility of autoflocculation is a plus. 2) The development of high concentration cultures, which will require smaller volumes to be processed. That is not to say that biomass separation and drying cannot be improved. First of all, concentration of microalgal cultures is essential, and therefore two areas of improvement are: 3) Development of non-toxic flocculants. 4) Enhancement of concentration technologies, such as sedimentation, flotation and even ultrasonic flocculation. Finally, after concentration, the processing is similar to that of other classical microorganisms, except that some microalgae may require disruption for further processing, and high-PUFA containing biomass requires gentle drying. There is room for improvement in disruption of cell slurries and dry biomass and fast drying. Energy conservation is essential for the sustainability of processes, especially those which are low value-added, hence the importance of: 5) Integration of microalgal processing into biorefineries, using not only the CO2 from flue gases but maybe part of the wasted thermal energy of processes. 6) The use of solar drying – usually not considered for industrial purposes because it is slow and this may allow putrefaction, but quite possible if enough space is available and it is properly designed, as in Sahoo et al. (2017). There is surprisingly little technical information regarding large-scale drying of microalgal biomass, perhaps because relatively large cultures (in research) such as

10,000 L-tanks will lead to a mere 20 kilograms of dry biomass. While research in flocculation, filtration and centrifugation can be done with such volumes, drying studies require dewatered biomass which is routinely produced industrially, but not easily obtained in smaller scales. At the other side, pilot- and lab-scale biomass can be easily stored and processed in small batch equipment, leading to a gap in the scientific literature for microalgae drying – a research opportunity, for sure.

ACKNOWLEDGMENT The authors thank CNPq – the National Council for Scientific and Technological Development – and CAPES – the Coordination for the Improvement of Higher Education Personnel – the Brazilian agencies that support the authors’ research in microalgae biotechnology.

REFERENCES Bakpai, Karesh, Ales Prokop, and Mark Zappi. 2014. Algal Biorefineries: Cultivations of Cell and Production. Vol. 1. New York, NY: Science Publishers. Beach, Evan S., Matthew J. Eckelman, Zheng Cui, Laura Brentner, and Julie B. Zimmerman. 2012. ‘Preferential Technological and Life Cycle Environmental Performance of Chitosan Flocculation for Harvesting of the Green Algae Neochloris Oleoabundans’. Bioresource Technology 121: 445–49. Bohuslav, Dobias, and Stechemesser Hansjoachim. 2005. Coagulation and Flocculation. 2nd ed. Santa Barbara, CA: CRC Press. Bolto, Brian, and John Gregory. 2007. ‘Organic Polyelectrolytes in Water Treatment’. Water Research 41 (11): 2301–24. Borges, Lucelia, Joaquin A. Morón-Villarreyes, Marcelo G. Montes D’Oca, and Paulo Cesar Abreu. 2011. ‘Effects of Flocculants on Lipid Extraction and Fatty Acid Composition of the Microalgae Nannochloropsis Oculata and Thalassiosira Weissflogii’. Biomass and Bioenergy 35 (10): 4449–54. Brady, Patrick V., Phillip I. Pohl, and John C. Hewson. 2014. ‘A Coordination Chemistry Model of Algal Autoflocculation’. Algal Research 5: 226–30. Bratby, John. 2016. Coagulation and Flocculation in Water and Wastewater Treatment. 3th ed. London, UK: IWA Publishing. Elcik, Harun, and Mehmet Cakmakci. 2017. ‘Harvesting Microalgal Biomass Using Crossflow Membrane Filtration: Critical Flux, Filtration Performance, and Fouling Characterization’. Environmental Technology 38 (12): 1585–96. Fasaei, F., J. H. Bitter, P. M. Slegers, and A. J. B. van Boxtel. 2018. ‘Techno-Economic Evaluation of Microalgae Harvesting and Dewatering Systems’. Algal Research 31 (April): 347–62.

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Garg, Sourabh, Yan Li, Liguang Wang, and Peer M. Schenk. 2012. ‘Flotation of Marine Microalgae: Effect of Algal Hydrophobicity’. Bioresource Technology 121: 471–4. Gerardo, M. L., M. A. Zanain, and R. W. Lovitt. 2015. ‘Pilot-Scale Cross-Flow Microfiltration of Chlorella minutissima: A Theoretical Assessment of the Operational Parameters on Energy Consumption’. Chemical Engineering Journal 280: 505–13. Gerardo, Michael L., Darren L. Oatley-Radcliffe, and Robert W. Lovitt. 2014. ‘Minimizing the Energy Requirement of Dewatering Scenedesmus sp. by Microfiltration: Performance, Costs, and Feasibility’. Environmental Science and Technology 48 (1): 845–53. Gorin, Kirill V., Yana E. Sergeeva, Victor V. Butylin, Anastasiya V. Komova, Victor M. Pojidaev, Gulfiya U. Badranova, Anna A. Shapovalova, Irina A. Konova, and Pavel M. Gotovtsev. 2015. ‘Methods Coagulation/ Flocculation and Flocculation with Ballast Agent for Effective Harvesting of Microalgae’. Bioresource Technology 193: 178–84. Grima, E., E.-H. Molina, F. G. Belarbi, Acién Fernández, A. Robles Medina, and Yusuf Chisti. 2003. ‘Recovery of Microalgal Biomass and Metabolites: Process Options and Economics’. Biotechnology Advances 20 (7): 491–515. Grima, E., F. G. Molina, Acién Fernández, and A. Robles Medina. 2013. ‘Downstream Processing of Cell-Mass and Products’. In: Handbook of Microalgal Culture: Applied Phycology and Biotecnology, edited by Amos Richmond and Qiang Hu, Second edition, 215. Oxford, UK: John Wiley & Sons. Hadjoudja, S., V. Deluchat, and M. Baudu. 2010. ‘Cell Surface Characterisation of Microcystis aeruginosa and Chlorella vulgaris’. Journal of Colloid and Interface Science 342 (2): 293–9. Halim, Ronald, Brendan Gladman, Michael K. Danquah, and Paul A. Webley. 2011. ‘Oil Extraction from Microalgae for Biodiesel Production’. Bioresource Technology 102 (1): 178–85. Harun, Razif, Manjinder Singh, Gareth M. Forde, and Michael K. Danquah. 2010. ‘Bioprocess Engineering of Microalgae to Produce a Variety of Consumer Products’. Renewable and Sustainable Energy Reviews 14 (3): 1037–47. Hattab, Mariam Al. 2015. ‘Microalgae Harvesting Methods for Industrial Production of Biodiesel: Critical Review and Comparative Analysis’. Journal of Fundamentals of Renewable Energy and Applications 5 (2). Henderson, Rita K., Simon A. Parsons, and Bruce Jefferson. 2010. ‘The Impact of Differing Cell and Algogenic Organic Matter (AOM) Characteristics on the Coagulation and Flotation of Algae’. Water Research 44 (12): 3617–24. Hung, M. T., and J. C. Liu. 2006. ‘Microfiltration for Separation of Green Algae from Water’. Colloids and Surfaces B: Biointerfaces 51 (2): 157–64. Kang, Seongkyun, Sangwoo Kim, and Jinwon Lee. 2015. ‘Optimization of Cross Flow Filtration System for Dunaliella Tertiolecta and Tetraselmis Sp. Microalgae Harvest’. Korean Journal of Chemical Engineering 32 (7): 1377–80.

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Kim, Kyochan, Joo Young Jung, Jong Hee Kwon, and Ji Won Yang. 2015. ‘Dynamic Microfiltration with a Perforated Disk for Effective Harvesting of Microalgae’. Journal of Membrane Science 475: 252–8. Kirnev, P. C. S., J. C. de Carvalho, J. T. Miyaoka, L. C. Cartas, L. P. S. Vandenberghe, and C. R. Soccol. 2018. ‘Harvesting Neochloris Oleoabundans Using Commercial Organic Flocculants’. Journal of Applied Phycology 30 (4): 2317–24. Koley, Shankha, Satyapal Prasad, Sourav Kumar Bagchi, and Nirupama Mallick. 2017. ‘Development of a Harvesting Technique for Large-Scale Microalgal Harvesting for Biodiesel Production’. RSC Advances 7 (12): 7227–37. Laamanen, Corey A., Gregory M. Ross, and John A. Scott. 2016. ‘Flotation Harvesting of Microalgae’. Renewable and Sustainable Energy Reviews 58: 75–86. Luangpipat, Tiyaporn, and Yusuf Chisti. 2017. ‘Biomass and Oil Production by Chlorella vulgaris and Four Other Microalgae—Effects of Salinity and Other Factors’. Journal of Biotechnology 257: 47–57. Mikulec, Jozef, Gabriela Polakovičová, and Ján Cvengroš. 2015. ‘Flocculation Using Polyacrylamide Polymers for Fresh Microalgae’. Chemical Engineering and Technology 38 (4): 595–601. Milledge, John James, and Sonia Heaven. 2011. ‘Disc Stack Centrifugation Separation and Cell Disruption of Microalgae: A Technical Note’. Environment and Natural Resources Research 1 (1). Ndikubwimana, Theoneste, Jingyu Chang, Zongyuan Xiao, Wenyao Shao, Xianhai Zeng, I-Son Ng, and Yinghua Lu. 2016. ‘Flotation: A Promising Microalgae Harvesting and Dewatering Technology for Biofuels Production’. Biotechnology Journal 11 (3): 315–26. Pahl, Stephen L., Andrew K. Lee, Theo Kalaitzidis, Peter J. Ashman, Suraj Sathe, and David M. Lewis. 2013. ‘Harvesting, Thickening and Dewatering Microalgae Biomass’. In: Algae for Biofuels and Energy, 165–85. Dordrecht: Springer Netherlands. Ralston, John, Daniel Fornasiero, and Nataliya Mishchuk. 2001. ‘The Hydrophobic Force in Flotation-A Critique’. Colloids and Surfaces A: Physicochemical and Engineering Aspects 192 (1–3): 39–51. Rios, Sergio D., Ester Clavero, Joan Salvadó, Xavier Farriol, and Carles Torras. 2011. ‘Dynamic Microfiltration in Microalgae Harvesting for Biodiesel Production’. Industrial and Engineering Chemistry Research 50 (4): 2455–60. Roselet, Fabio, Dries Vandamme, Milene Roselet, Koenraad Muylaert, and Paulo Cesar Abreu. 2015. ‘Screening of Commercial Natural and Synthetic Cationic Polymers for Flocculation of Freshwater and Marine Microalgae and Effects of Molecular Weight and Charge Density’. Algal Research 10 (1): 183–8. Sahoo, Narendra Kumar, Sanjay Kumar Gupta, Ismail Rawat, Faiz Ahmad Ansari, Poonam Singh, Satya Narayan Naik, and Faizal Bux. 2017. ‘Sustainable Dewatering and Drying of Self-Flocculating Microalgae and Study of Cake Properties’. Journal of Cleaner Production 159: 248–56.

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Salim, S., M. H. Vermuë, and R. H. Wijffels. 2012. ‘Ratio between Autoflocculating and Target Microalgae Affects the Energy-Efficient Harvesting by Bio-Flocculation’. Bioresource Technology 118: 49–55. Schwarz, S., W. Jaeger, B. R. Paulke, S. Bratskaya, N. Smolka, and J. Bohrisch. 2007. ‘Cationic Flocculants Carrying Hydrophobic Functionalities: Applications for Solid/ Liquid Separation’. The Journal of Physical Chemistry B 111 (29): 8649–54. Sharma, Kalpesh K., Sourabh Garg, Yan Li, Ali Malekizadeh, and Peer M. Schenk. 2013. ‘Critical Analysis of Current Microalgae Dewatering Techniques’. Biofuels 4 (4): 397–407. Shelef, G., A. Sukenik, and M. Green. 1984. Microalgae Harvesting and Processing: A Literature Review. Haifa (Israel): Technion Research and Development Foundation Ltd. Show, Kuan-Yeow, Duu-Jong Lee, and Arun S. Mujumdar. 2015. ‘Advances and Challenges on Algae Harvesting and Drying’. Drying Technology 33 (4): 386–94. Sili, Claudio, Giuseppe Torzillo, and Avigad Vonshak. 2012. ‘Arthrospira (Spirulina)’. In: Ecology of Cyanobacteria II: Their Diversity in Space and Time, edited by B. A. Whitton, 677–705. Springer. T’Lam, G. P., M. H. Vermuë, G. Olivieri, L. A. M. van den Broek, M. J. Barbosa, M. H. M. Eppink, R. H. Wijffels, and D. M. M. Kleinegris. 2014. ‘Cationic Polymers for Successful Flocculation of Marine Microalgae’. Bioresource Technology 169: 804–7.

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Tredici, Mario R. 2010. ‘Photobiology of Microalgae Mass Cultures: Understanding the Tools for the Next Green Revolution’. Biofuels 1 (1): 143–62. Uduman, Nyomi, Ying Qi, Michael K. Danquah, Gareth M. Forde, and Andrew Hoadley. 2010. ‘Dewatering of Microalgal Cultures: A Major Bottleneck to AlgaeBased Fuels’. Journal of Renewable and Sustainable Energy 2 (1): 12701. Vandamme, D., I. Foubert, B. Meesschaert, and K. Muylaert. 2010. ‘Flocculation of Microalgae Using Cationic Starch’. Journal of Applied Phycology 22 (4): 525–30. Wan, Chun, Md. Asraful Alam, Xin-Qing Zhao, Xiao-Yue Zhang, Suo-Lian Guo, Shih-Hsin Ho, Jo-Shu Chang, and Feng-Wu Bai. 2014. ‘Current Progress and Future Prospect of Microalgal Biomass Harvest Using Various Flocculation Technologies’. Bioresource Technology 184: 251–7. Wijffels, René H., and Maria J. Barbosa. 2010. ‘An Outlook on Microalgal Biofuels’. Science 329 (5993): 796–99. Wu, Zechen, Yi Zhu, Weiya Huang, Chengwu Zhang, Tao Li, Yuanming Zhang, and Aifen Li. 2012. ‘Evaluation of Flocculation Induced by PH Increase for Harvesting Microalgae and Reuse of Flocculated Medium’. Bioresource Technology 110 (Suppl 2): 496–502.

Section V Production of Algal Biomass and Products Worldwide

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Micro- and Macroalgae Production in Thailand for Food, Feed and Other Applications Current Trends and Future Challenges Apiradee Hongsthong, Ratana Chaiklahan and Boosya Bunnag

CONTENTS Abbreviations����������������������������������������������������������������������������������������������������������������������������������������������������������253 Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������254 Macroalgae��������������������������������������������������������������������������������������������������������������������������������������������������������������255 Applications of Macroalgae������������������������������������������������������������������������������������������������������������������������������������256 Microalgae��������������������������������������������������������������������������������������������������������������������������������������������������������������257 Applications of Microalgae�������������������������������������������������������������������������������������������������������������������������������������259 Algal Biotechnology Research in Thailand������������������������������������������������������������������������������������������������������������261 Future Prospects in Algal Biotechnology���������������������������������������������������������������������������������������������������������������261 Conclusion��������������������������������������������������������������������������������������������������������������������������������������������������������������262 Acknowledgments���������������������������������������������������������������������������������������������������������������������������������������������������262 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 262

BOX 21.1  SALIENT FEATURES Microalgae and macroalgae are well recognized in the food market and in agricultural and industrial sectors. However, marine algae have more agricultural applications than freshwater algae, especially as feedstock for aquaculture. The map locating large population of marine-algal farms in Thailand is present in the chapter, and the reason could be the fact that Thailand is popularly oriented to fisheries and aquaculture industry. Originally, the main purpose of marine-algal cultivation in Thailand was for use as feedstock for the larvae-stage of fish, shrimp, blue-crab and abalone. However, now it is increasingly used to treat the seawater that maybe contaminated with fertilizer for use in aquaculture industry. Thus, the expansion of the market for aquaculture would provide impetus to the algae-based feeds to support this industry. The current situation of local algal-biomass production and its applications as well as the high-value biochemical compounds present in micro- and macroalgae, including their extractions and potential uses, are discussed in this chapter. Taken together, it leads to the trends of

algal research in Thailand, which are now marketdriven to translational and application aimed activities. High throughput technologies, e.g., OMICS and system biology, are applied in algal research for commercial production of a range of products in food, feed, health, biochemicals and energy sectors.

ABBREVIATIONS PC Phycocyanin APC Allophycocyanin CPC C-Phycocyanin HSV-1 Herpes simplex virus type 1 SQDG Sulphoquinovosyl diacylglycerol LC–MS/MS Liquid chromatography–Tandem mass spectrometry GMO Genetic modified organism KMUTT King Mongkut’s University of Technology Thonburi SpirPep A web-based tool for bioactive peptide prediction SpirPro A Spirulina proteome repository webbased tool 253

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INTRODUCTION Thailand is a country in Southeast Asia with a total area of 513,120 km2. The average temperature is approximately 30°C year-round, especially in the area along the gulf of Thailand, where the local marine-algal strains are mass cultivated. In contrast, the northern region of Thailand, where the average temperature is approximately 25°C, is suitable for mass cultivated or naturally grown local freshwater-algal strains. In Figure 21.1, the map of Thailand is illustrated with labeled provinces, where the local algal-farms are located, and the names of the strains are also indicated. The map clearly shows that the population of marinealgal farms in Thailand is significantly larger than that of freshwater-algae. The reason could be the fact that Thailand is popularly oriented to fisheries and aquaculture industry. Thailand’s revenue earning exceeds

FIGURE 21.1  Location of algal mass cultivation in Thailand.

Handbook of Algal Technologies and Phytochemicals

82 billion baht per year from the export of fish and aquaculture products (www4.fisheries.go.th/index.php/dof/ main). Originally, the main purpose of marine-algal cultivation in Thailand was to use as feedstock for the larvaestage of fish, shrimp, blue-crab and abalone. However now it is increasingly used to treat the seawater that may be contaminated with fertilizer for use in aquaculture industry. Micro- and macroalgae are found in many parts of Thailand due to favourable climatic conditions. Almost 21 million tons of seaweed are utilized worldwide. Approximately 800,000 tons of seaweed are harvested from the wild, and the remaining 94% is produced by aquaculture. Production is dominated by Indonesia, China and the Philippines (White and Wilson 2015). In the following sections, the locally grown strains of marine- and freshwater algae and their applications

Micro- and Macroalgae Production in Thailand

will be thoroughly discussed. The high-value biochemical compounds present in the algal cells, including their extractions and potential uses, are addressed. Finally, the current trends and future challenges of biomass production and its related products or applications have been dealt with.

MACROALGAE Marine macroalgae: The majority of edible marine macroalgae of the genera Caulerpa, Gracilaria, Porphyra and Sargassum (Figure 21.1) are found along the coastlines of the Gulf of Thailand and the Andaman Sea (Lewmanomont 1990, 2006). Caulerpa, a marine macroalgae, which are known as sea grapes or green caviar (Figure 21.2a), have become a famous ingredient for snack food in the last five years. Caulerpa can be consumed fresh with traditional Thai sauce or salad dressing. C. lentillifera and C. racemosa are the two most popular edible types. C. lentillifera is a species that is well adapted to open-pond/open-lagoon mass cultivation (Mahadevan 2015; McHugh 2003; Ratana-arporn and Chirapart 2006). Caulerpa, is also used to treat seawater that may be contaminated with the fertilizers and bacteria that are the cause of early mortality syndrome, or EMS, in Litopenaeus vannamei under farming conditions. The successful aquaculture model that combines Caulerpa and L. vannamei cultivation is called the “Phetchaburi model” (www.fisheries.co.th/cf-phetchaburi). The Aquaculture Research and Development Center at Phetchaburi Coastal transferred the strain and the cultivation know-how to the farmers in 2013; subsequently the market for green caviars as human food drastically expanded. At present, there are many Caulerpa farms in the Phetchaburi province. The current demand for C. lentillifera in Thailand is approximately 1 ton per month, and the selling price for the food grade is approximately 300–800 baht per kilogram, depending on the grade, i.e., the size, length and number of branches. The pictures of several recipes and local products made from Caulerpa are shown in Figure 21.3.

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Moreover, the strains of Ulva rigida, Halymenia durvillei (Figure 21.2) and Acanthophora spicifera, which are isolated in Thailand aquatics, can be cultivated in an open-pond, and, at present, A. spicifera is successfully used as feedstock for abalone (Figure 21.4). In addition to the popular Caulerpa, 18 species of Gracilaria, also known as “Pom-Nang” have been identified in Thailand. However, G. fisheri, G. tenuistipitata, G. changii and G. salicornia are the most abundant species. Approximately 1.9 million tons of Gracilaria, a red-alga, is produced worldwide, which is approximately 9% of the total world seaweed produced in 2012 (White and Wilson 2015). G. fisheri and G. tenuistipitata are commonly harvested in large quantities as a commercial source of carrageenan, and only a small volume was utilized as food, animal feed and fertilizer by the coastal villagers. Dried Gracilaria of more than 200 tons per year was exported from Thailand to Japan, Germany, Italy and Hong Kong for agar extraction; only a small portion was used locally as food, food additives in the form of carrageenan and animal feed (Lewmanomont 1990, 2006). Another red seaweed, Porphyra, is widely used as an ingredient in Chinese-style soup, and the same style of soup is also found in Thailand. The common species used is P. vietnamensis. This species grows naturally, and no cultivation system has been developed so far. The annual yield of the algae is approximately 500 kg fresh-weight of algae; however, the production is highly dependent on environmental conditions (Lewmanomont 1990). Brown seaweeds, Sargassum, i.e., S. polycystum, S. oligocystum and S. crassifolium, are commonly found in Thailand. Their fresh form is consumed as a vegetable by local people, whereas the tea made from dried Sargassum is boiled in water for use as an alternative medicine to cure goiter and relieve fever (Lewmanomont 2006). Freshwater macroalgae: In Thailand, freshwater macroalgae is much less popular than marine macroalgae. The two genera of Cladophora glomerata and Microapora floccose are commonly found in Thailand (Figure 21.5).

FIGURE 21.2  Macroalgae mass cultivation at Aquaculture Research and Development Center at Phetchaburi Coastal: (a) C. lentillifera, (b) Ulva regida and (c) Halymenia durvillei.

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FIGURE 21.3  Examples of local seaweed products: (a) C. lentillifera salad, (b) C. lentillifera soap, (c) dried Solieria robusta and (d) dried G. fisheri.

These algae can be used as indicators for water quality because they can grow well in clean and flowing water that ranges from oligotrophic to mesotrophic water. These species are usually attached to rock or cobble in the bed of shallow rivers at depths of 30–50 cm. (Amornlerdpison et al. 2011; Thiamdao et al. 2012). Cladophora sp. is naturally grown in the dry season from December to February in rivers in the northern part of Thailand, especially in the Nan River of Nan province, in the Mekong River of the Chiang Khong district of Chiang Rai province and in some provinces in the northeastern part, e.g., Ubon Ratchathani (Figure 21.1). The local name of the algae is “Kai,” and it has been used as a food ingredient in local food recipes, e.g., a Thaistyle omelet. Currently, Kai has become an increasingly popular local snack food that is marketed to tourists under the program One Tambon One Product (OTOP) (Figure 21.5). The quantity of Kai used for food processing is approximately 3.5 tons per month, with a price of 10–20 baht per kilogram.

APPLICATIONS OF MACROALGAE Feed supplements: Apart from utility as a source of food ingredients the major applications of marine alga are for feedstock and feed additives in aquaculture. Thailand is the third largest shrimp exporter in the world. The applications of marine macroalgae as feedstocks and feed additives for shrimp and their larvae

are gaining popularity. Shrimp farmers claim that the macroalgae help to enhance the survival rate and growth rate of the larvae and shrimp. Marine and freshwater macroalgae are natural sources of carotenoids, polysaccharides and phenolic compounds, which exhibit antioxidant, antigastric ulcer, anti-inflammatory, analgesic and hypotensive activities, including anticancer and antimicrobial activities (Amornlerdpison et al. 2011; Fabrowska et al. 2016; Imjongjairak et al. 2016; Laungsuwon and Chulalaksananukul 2013, 2014; Peerapornpisal et al. 2006; Srimaroeng et al. 2015; Suanmali et al. 2017). Polysaccharides extracted from marine (G. fisheri) and freshwater (C. glomerata) algae contain sulfates, which are key factors in the regulation of immunomodulatory activities (Imjongjairak et al. 2016; Surayot et al. 2016a, 2016b). Moreover, high amounts of minerals and balanced amino acid profiles are present in macroalgae (Benjama and Masniyom 2012; Ratana-arporn and Chirapart 2006; Setthamongkol et al. 2015). Therefore, the bioactivity mentioned above may be involved in the increasing survival rate and growth rate of larvae and crustaceans. Sriprach et al. (2011) reported that the black tiger shrimp (Penaeus monodon) fed with 3% of G. fisheri for 60 days showed enhanced growth performance in terms of final weight, weight gain, length gain, specific growth rate and average daily gain, compared to control. Another study, which was conducted by Promya and Chitmanat (2011), corroborated these observations wherein productivity of African sharptooth catfish

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FIGURE 21.4  Examples of aquatic species fed by algae in Thailand: (a) SangChan fish or Milkfish or Chanos chanos, (b) Abalone or Haliotis asinina and (c) Blue crab or Portunus pelagicus.

(Clarias gariepinus) significantly increased when 5% Cladophora was provided in the diet. In term of pigments, Cladophora alga was rich in carotenoids. When 7.5% C. glomerata was fed to catfish and Nile tilapia (Oreochromis niloticus), the carotenoid content in the flesh of the catfish and Nile tilapia was significantly enhanced from 0.96 to 12.26 μg carotenoid/g fresh weight (Ruangsomboon and Choochate 2014). Water treatment and biosorption: The “Phetchaburi model,” as mentioned earlier, is the model in which the marine macroalgae, C. lentillifera, are used to reduce contamination of fertilizers and bacteria, in seawater, for use in aquaculture. There are many reports that show that this algal biomass could be employed as a biosorbent for positively charged contaminants in wastewater, such as heavy metal ions and dyes. However, the adsorption

capacity provided by C. lentillifera for the removal ions of Cu2+ and Pb2+ was still relatively low compared to other biosorbents, e.g., the Chlamydomonas reinhardtii (Madacha et al. 2006; Tüzün et al. 2005), Ecklonia maxima (Feng and Aldrich 2004) and Spirogyra species (Gupta et al. 2006). Recently, a research group from Thammasat University developed the alginate Cladophora or so called in Thai as “Kai-Kon” to use as biosorbents for heavy metals in rivers and as feed for ornamental fish that can also work as fish tank decolorant (www.sci.tu.ac.th).

MICROALGAE The mass cultivation of microalgae in Thailand is a lot less widespread than that of the macroalgae, even though

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FIGURE 21.5  Cladophora sp. or locally known as Kai: (a) Kai harvested in Nan River, (b) dried Kai and (c) local food products made from Kai.

microalgae are promising sources of food, feed, fuel and pharmaceutical ingredients. Marine microalgae: Three strains of marine microalgae, Chaetoceros, Thalassiosira and Skeletonema, are popular in Thailand in shrimp hatchery farms. The selection of algae species depends on the experience of farmers (Pechmanee 1997). In the eastern part of Thailand, Chaetoceros and Thalassiosira are favorite strains, whereas hatcheries in the southern part favor Skeletonema. The cultures are sold in the ready-to-use form, and hatcheries can directly use them to feed shrimp larvae.

The price of Chaetoceros is approximately 20–30 baht per liter. There are two Thai companies that cultivate Chaetoceros and Thalassiosira in commercial scale, namely, the Nathong farm in Chachoengsao Province and Algaeba Co. Ltd. in Bangkok (Figure 21.6). Other providers of Chaetoceros are small-scale production units run by local farmers. The cultivation of Chaetoceros in open-pond systems might be easily contaminated by other organisms, such as bacteria. Even protozoa cause devastating reduction in productivity. Therefore, researchers have attempted to develop the cultivation process in a closed system.

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FIGURE 21.6  Pictures of an algae farm, Algaeba Co. Ltd: (a) mass cultivation system and (b) the company products for shrimp hatcheries.

For example, Krichnavaruk et al. (2007) explored semicontinuous and continuous modes in airlift photobioreactors to improve the final cell concentration of C. calcitrans and to decrease the contamination. Whereas Ritcharoen et al. (2014) designed the internal and external loops for the airlift photobioreactors to solve the problem of light limitation, which resulted in an increased growth rate and final cell concentration. Moreover, the nutrition in the cells can be manipulated by changing the growth medium; for example, Pimorat et al. (2010) reported that adding sodium bicarbonate 0.05 g/L in a modified f/2 medium of C. gracilis culture resulted in a significant enhancement in the total lipid (18.71%) and carbohydrate (13.79%) contents. Freshwater microalgae: The only well-known freshwater microalga in Thailand is Spirulina (Arth­ rospira sp.), which is wildly used as a food supplement. Spirulina is the only cyanobacterium that can be mass cultivated in outdoor ponds without contamination problems due to the high alkalinity of the growth media. The largest Spirulina farm in Thailand and in Southeast Asia is Boonsom Farm, which is located in the Mae Wang district of Chiang Mai (Figure 21.1), and has a production area of over 60,000 square meters. However, there are some small producers, such as Royal Chitralada Projects and EnerGaia Co.Ltd. Interestingly, EnerGaia cultivates Spirulina on the rooftop of a hotel, in Novotel Bangkok Siam Square, downtown Bangkok (Figure 21.7). Moreover, the company also develops portable and easy-to-use cultivation

ponds for Spirulina cultivation and plans to commercialize this system.

APPLICATIONS OF MICROALGAE Food and feed supplements: In Thailand, microalgae are well known for two applications, namely, food supplements and feed additives for aquaculture. For food supplements, the size of the market share of Spirulina is the largest. Consumers recognize Spirulina as a superfood due to its high nutrition and lack of toxins. Until now, there were five Arthrospira sp. whose genome sequencing has been completed, and none of them contained a toxin gene. In terms of animal tests, Spirulina was found to have no effect on the food and water intake behaviors, growth or health status of all treated animals and the values in clinical chemistry that were monitored throughout the study period did not reveal significant differences between the control and treated groups (Hutadilok-Towatana et al. 2008, 2010). Microalgae are also used as feed stocks and feed additives to increase the survival and growth rates of aquaculture. The larvae of the tiger shrimp (P. monodon) and the Pacific white shrimp (Litopenaeus vannamei) are fed with these live foods for at least two weeks or up until two months. In the first protozoal stage (zoeal stage) of shrimp larvae, diatoms, i.e., Chaetoceros and Thalassiosira, are used as feed additives, as they contain essential nutrients and have the proper size for allowing a faster and healthier larval growth. The natural

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FIGURE 21.7  Pictures of Spirulina (Arthrospira) farms in Thailand. (a–c) Boonsom Farm and (d–f) EnerGaia Co., Ltd.

feedstock, such as diatom, causes less water pollution compared to artificial feedstock. Spirulina as feed additives are also wildly used on a commercial scale. Hemtanon et al. (2005) reported that a crude extract from S. platensis can inhibit the growth of the white spot syndrome virus and Vibrio harveyi in the black tiger shrimp. In the case of African sharptooth catfish, or Clarias gariepinus, the significant increase in weight gain, the specific growth rate, the average daily growth and the protein conversion rate were observed after it was fed with a 5% Spirulina supplemented diet for 60 days. The red and white blood cell counts, including the immunity of the catfish, were also enhanced (Promya and Chitmanat 2011). Similarly, the carotenoid content of the C. macrocephalus ovary was drastically increased from 1.22 to 3.0 mg/100 g of dry weight in the presence of 10% S. platensis in the diet (Chainapong and Traichaiyaporn 2013). Wastewater treatment: The advantage of microalgae is not only its use as feedstock for shrimp larvae but also its use for water quality control in shrimp culture-tanks. The nitrogenous compounds in the tanks were greatly reduced when S. platensis was co-cultured with P. monodon (Chuntapa et al. 2003). When the semi-continuous culture of S. platensis was harvested from the co-culture, the level of nitrate reduced from 18 to 4 mg/L, whereas the ammonium and nitrite level decreased from 0.6 to 0.15 mg/L.

Moreover, Spirulina has the ability to use several nutrients, viz., bicarbonate (HCO3−), nitrate (NO3−), nitrite (NO2−), ammonium (NH4+) and phosphate (PO43−), present in the wastewater from pig/poultry farms, for biomass production. Thus, using wastewater as a growth media for Spirulina cultivation could save the production cost while removing the environmental contaminants. The biomass productivity and cell compositions in terms of protein and pigments of Spirulina are summarized as shown in Table 21.1. Moreover, Spirulina was investigated to remove heavy metals in wastewater. Rangsayatorn et al. (2002, 2004) showed that S. platensis had a maximum capacity for cadmium (Cd) adsorption, with 98.04 mg Cd/g biomass. The cadmium uptake of S. platensis was not affected by the temperature, but it was influenced by the pH of the solution. At pH 7.0, the process of cadmium uptake was rapid, and 78% of the metal was removed from the solution within 5 min. After 30 min exposure, S. platensis removed 95.93% of Cd in the solution. The immobilized form of S. platensis on alginate gel and silica gel showed Cd adsorption capacities of 70.92 and 36.63 mg/g biomass, respectively. Bioactive compounds and extraction: Spirulina is well-known for biochemical compound extraction due to the fact that its contents have potential for nutraceutical application. PC, a major pigment in Spirulina cells, shows high antioxidant activity. Thai researchers found

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TABLE 21.1 Productivity and Cell Compositions of Spirulina (Arthrospira) platensis Cultivated in Various Wastewater Streams Types of Wastewater Starch factory Fermented Thai Rice noodle factory Cafeteria kitchen Pig farm Lac factory Cafeteria

Productivity (g/L)

Reference

(g/m2/d)

protein

phyco.

chla

caro.

12.5

52 59

5.96 14

1.13 0.236

0.26

Tanticharoen et al. (1993) Vetayasuporn (2004)

35.86 57.9 59.11 56.25

1.795 19.5 7.13 0.612

0.029

Promya et al. (2008) Chaiklahan et al. (2010) Pumas and Pumas (2016) Promya et al. (2018)

1.0 0.82 12.0 0.624 0.40

Cells Composition (% of dw.)

0.017 0.389

Note: dw = dry weight, phyco. = phycocyanin, chla = chlorophyll a, caro. = carotenoid.

that APC exhibits higher activity than CPC in scavenging peroxyl radicals, whereas CPC represents higher activity in scavenging hydroxyl radicals than APC (Cherdkiatikul and Suwanwong 2014). The process of PC extraction, separation and purification, as reported by the group from KMUTT, demonstrated over 80% recovery through a microfiltration membrane technique (Chaiklahan et al. 2011). Food grade PC with a purity of approximately 1.0 was obtained after the process of ultrafiltration. Although PC is commercially produced in many countries, Thailand’s efforts in the largest scale of PC extraction was carried out on a pilot scale of 100 L at KMUTT. Chaiklahan et al. (2012) reported the maximum stability of PC at 47°C at a pH range of 5.5–6.0. The presence of glucose, sucrose and sodium chloride in the solution can slow down the process of PC degradation. The lipid extraction from Spirulina using three rounds of ethanol extraction at 60°C showed that 85% of the total fatty acids can be recovered (Chaiklahan et al. 2008). The lipid extracts contain the SQDG which showed potent activity against HSV-1 in Vero cells, with an IC50 of 6.8 μg/mL. However, the synthetic compound, acyclovir, has an IC50 of 1.5 μg/mL and is used for HSV-1 inhibition (Chirasuwan et al. 2009). Moreover, the hot water polysaccharide extracts from Spirulina were also found to have anti-HSV-1 activity.

ALGAL BIOTECHNOLOGY RESEARCH IN THAILAND The current trend in algal biotechnology research in Thailand has mainly shifted to molecular biological studies. In the past, the majority of studies were on algal taxonomy and cultivation techniques. Therefore, multidisciplinary techniques, e.g., genome/proteome

analyses, genetic manipulation, biochemical extraction and mass cultivation, have been applied. However, in Thailand, GMOs are legally prohibited in the environment, thus limiting their utility. Spirulina has been an excellent choice for making GMOs since the mass cultivation system has been well developed; however, the gene modification, gene editing and gene transfer system of this organism are still under constant innovation. Research on transformation of Spirulina has been extensively carried out by the algal biotechnology group at KMUTT. The genome sequencing of the A. platensis strain C1 was completed, and the comparative proteome of the cells grown under optimal and stress conditions was analyzed to understand the stress response mechanisms (Cheevadhanarak et al. 2012; Kurdrid et al. 2011). Furthermore, the bioinformatics platform for proteomewide analysis of the cyanobacteria was developed, i.e., SpirPro and SpirPep (Anekthanakul et al. 2018; Senachak et al. 2015). In the case of SpirPep, the bioactive peptide databases gathered from all available public databases were integrated with an in silico protein digestion tool, which can handle the proteome level dataset. Thus, this type of tool assists in the discoveries of bioactive peptides from genome analyzed organisms, such as Spirulina.

FUTURE PROSPECTS IN ALGAL BIOTECHNOLOGY Currently, algal biotechnology has become tremendously important due to the various applications of algae, as mentioned above. As the demand for algal biomass has increased, the mass cultivation technology coupled to a high biomass production at low cost has been the need of the hour. One of the factors that has gained importance is

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TABLE 21.2 Summary of Algal Strains and Their Applications in Thailand Algal Strain

Application(s) Macroalgae

Marine algae:   Caulerpa   Gracilaria   Acanthophora   Sargassum Freshwater algae:  Cladophora

Local food products, water pre-treatment for aquaculture Local food products, carrageenan-extraction source, feed, fertilizer Feed for Abalone Local functional food Local food products Microalgae

Marine algae:   Chaetoceros   Thalassiosira   Skeletonema Freshwater algae:   Spirulina

Feed for shrimp hatcheries Feed for aquaculture Feed for aquaculture Food supplements, feed additives, source of phycocyanin, wastewater treatment

the enhancement of nutrient efficiency. Moreover, reuse of the growth media with periodic dosing of the nutrients based on stoichiometric conversion values will be scientific and also affords precise automation of inputs. Thus, a company in Thailand is aiming at a business of producing portable cultivation ponds that are easy/readyto-use and precise to operate. Additionally, the utilization of algae for carbon dioxide sequestration is gaining attention. At present, a synthetic biology approach to enhance the efficiency of CO2 utilization is being attempted (Giordano et al. 2005; Sayre 2010; Singh and Singh 2014). Future years will witness a range of biochemical products from algal sources which may include bioactive peptides, food and feed additives and bio-hydrogen production as alternate fuel.

CONCLUSION Currently, in Thailand, the applications of algae in the food market and in the agricultural and industrial sectors are well recognized (Table 21.2). Algae have been known as human food since ancient times. Algal metabolites as food and feed additives are the new trends in the market. Regarding the agricultural sector, both marine and freshwater strains of algae are widely used for feed additives for aquaculture. Thus, the expansion of the market for aquaculture would provide impetus to the algae-based feeds to support this industry. Moreover, in the industrial sector, carbon dioxide sequestration will be the technology of the immediate future. Research is expected to be mainly oriented to

translational and application aimed activities. Moreover, OMICS technology and synthetic biology are possible techniques that can drive the studies related to algal biotechnology in the coming years. An adoption of genetic engineering and gene editing technologies with controlled cultivation of the algal biomass will provide the needed thrust to the algal biotechnology for commercial production of a range of products in food, feed, health, chemicals and energy sectors.

ACKNOWLEDGMENTS The authors would like to thank the private sectors, Boonsom Farm, Algaeba Co. Ltd., EnerGaia Co. Ltd. and the Bann-Nongbua algal-product community enterprise group for their kind cooperation in providing useful information and pictures. We also thank Dr. Montakan Tamtin, director of Phetchaburi Coastal Aquaculture Research and Development Center, and her assistant, Mr. Prapat Kasawatpat, for providing details on macroalgae mass cultivation in Thailand. Moreover, we appreciate the help in manuscript preparation by Ms. Nattayaporn Chirasuwan, Ms. Petcharin Puckdee, Ms. Sirilak Saree and Ms. Phutnichar Phuengcharoen.

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Global Microalgal-Based Products for Industrial Applications Ambati Ranga Rao and Gokare A. Ravishankar

CONTENTS Abbreviations����������������������������������������������������������������������������������������������������������������������������������������������������������268 Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������268 Important Algal Species for Industrial Applications�����������������������������������������������������������������������������������������������268 Spirulina sp. �������������������������������������������������������������������������������������������������������������������������������������������������������268 Chlorella sp. ������������������������������������������������������������������������������������������������������������������������������������������������������268 Haematococcus pluvialis������������������������������������������������������������������������������������������������������������������������������������269 Dunaliella salina������������������������������������������������������������������������������������������������������������������������������������������������269 Isochrysis glaban������������������������������������������������������������������������������������������������������������������������������������������������269 Global Algal-Based Products for Food and Feed���������������������������������������������������������������������������������������������������269 Global Algal-Based Industries and Their Products�������������������������������������������������������������������������������������������������269 Sapphire Energy�������������������������������������������������������������������������������������������������������������������������������������������������269 Algenol���������������������������������������������������������������������������������������������������������������������������������������������������������������270 Aurora BioFuels�������������������������������������������������������������������������������������������������������������������������������������������������270 Solix Biofuels�����������������������������������������������������������������������������������������������������������������������������������������������������270 Solazyme������������������������������������������������������������������������������������������������������������������������������������������������������������270 Earthrise Nutritionals LLC���������������������������������������������������������������������������������������������������������������������������������271 Nutrex Hawaii�����������������������������������������������������������������������������������������������������������������������������������������������������272 Algapharma Biotech Corp (Far East Biotech Co., Ltd)�������������������������������������������������������������������������������������272 Taiwan Chlorella Manufacturing Company�������������������������������������������������������������������������������������������������������273 Fuji Chemical Industries Co., Ltd����������������������������������������������������������������������������������������������������������������������273 Parry Nutraceuticals�������������������������������������������������������������������������������������������������������������������������������������������274 Heliae Development LLC�����������������������������������������������������������������������������������������������������������������������������������274 BioProcess Algae LLC���������������������������������������������������������������������������������������������������������������������������������������274 Algaecan Biotech Ltd�����������������������������������������������������������������������������������������������������������������������������������������274 Market Value for Algal Products�����������������������������������������������������������������������������������������������������������������������������274 Safety Aspects of Algal Products����������������������������������������������������������������������������������������������������������������������������275 Future Prospects������������������������������������������������������������������������������������������������������������������������������������������������������275 Conclusion��������������������������������������������������������������������������������������������������������������������������������������������������������������276 Acknowledgments���������������������������������������������������������������������������������������������������������������������������������������������������276 References��������������������������������������������������������������������������������������������������������������������������������������������������������������� 276

BOX 22.1  SALIENT FEATURES Algae are classified as photosynthetic microorganisms found in both fresh and marine habitats. They have the ability to grow faster than conventional crops. Algae produce bioactive compounds such as lipids, fatty acids, pigments, carbohydrates, proteins, hydrocarbons, minerals, amino acids, etc. Microalgae are grown in photo-autotrophic,

heterotrophic and mixotrophic conditions using raceway and photobioreactors for biomass production for industrial applications. A few algal species such as Spirulina, Chlorella sp., Haematococcus pluvialis and Dunaliella salina are extensively cultivated for commercial products, food, feed, nutraceutical, pharmaceutical, cosmetic and fuel applications. The market for algal products is increasing day-by-day in the market and will be 267

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expected to reach $44.78M by 2023. Currently, most of the consumers are looking for alternative molecules from the natural resources rather than synthetic ones. In view of this the current book chapter has discussed an overview of most important algal species for commercial purpose; global algal biomass producing companies; and their products for biofuel, food, feed, nutraceutical, pharmaceutical and cosmetic applications.

ABBREVIATIONS WHO USFDA GRAS EPA DHA FSSC FEBICO ECR EFSA GMO

World Health Organization United States Food and Drug Administration Generally Recognized as Safe Eicosapentaenoic acid Docosahexaenoic acid Food Safety System Certification Far East Biotech Co LTD European Community Regulation European Food Safety Authority Genetically Modified Organisms

INTRODUCTION The current food industry has led to an increase in healthier, cheaper and more economic products. The use of bioactive compounds such as pigments, proteins, carbohydrates, polyunsaturated fatty acids, etc., exhibits high functional properties, which play a major role in reducing various diseases. Algae are photosynthetic micro-organisms, found in both marine and freshwater water bodies (Duong et al. 2012). Algae are photosynthetically efficient, similar to terrestrial plants (Duong et al. 2012). Algae are also efficient in converting solar energy into biomass due to their efficient cellular structure (Quintana et al. 2011). Algae are classified into Cyanophyceae, Chlorophyceae, Bacillariophyceae and Chrysophyceae (Anemaet et al. 2010), and this classification was based on features such as pigments produced, chemical storage, photosynthetic membranes and other morphological characteristics (Hoek et al. 1995). Algae produces various bioactive compounds with high commercial value, such as proteins, lipids, fatty acids, minerals, hydrocarbons, polysaccharides, amino acids, nutrients, vitamins, etc. (Wells et al. 2016). These bioactive molecules play a major role in functional food applications for health benefits (Wells et al. 2016). A few algal species—Spirulina sp., Chlorella sp., Dunaliella salina and Haematococcus pluvialis—have received

great attention as nutritional supplements (Wells et al. 2016). Algal growth, biomass yields and metabolites production vary based on growth conditions such as media, nutrients, temperature, pH, light intensity, minerals, carbon dioxide, salinity, initial density and physiology (Sarada et al. 2012). Metabolites from algae have realized applications as food, pharmaceutical, nutraceutical and cosmeceutical ingredients (Cardozo et al. 2007; Liu et al. 2016; Sarada et al. 2012). This chapter provides evidence of continued importance of algae as nutritional supplements to functional food applications for health benefits. The most important algae species such as Spirulina sp. Chlorella sp., Dunaliella salina, Haematococcus pluvialis and Isochrysis glaban are discussed. Furthermore, algae-based industries and their products are provided.

IMPORTANT ALGAL SPECIES FOR INDUSTRIAL APPLICATIONS Spirulina sp. Spirulina sp. is a cyanobacteria largely considered as cyanophycean algae used as food source by local populations of Africa and Mexico for centuries (Habib et al. 2008). Spirulina sp. is now used as a feed supplement to animals due to its high content of protein (Capelli and Cysewski 2010). However, it also produces other molecules beneficial to health, such as linolenic acid, β-carotene and phycocyanin (Papadaki et al. 2017; Ward and Singh 2005). Spirulina sp. has shown biological properties in in vitro and in vivo models such as antihypertension, anti-hyperlipidemia, anti-cancer property, protection against renal failure, antioxidant activity, suppression of serum glucose levels and growth promotion of the intestinal tract (Capelli and Cysewski 2010)

Chlorella sp. Chlorella sp. is a fresh water microalga used as a feed and food supplement in the United States, Europe, Japan and China (Moejes and Moejes 2017). It is one of the potential sources of nutrients such as vitamins, minerals and pigments and is promoted in health food markets as well as animal feed by the aquaculture industry (Salati et al. 2017; Tang and Suter 2011). Chlorella sp. showed various health promoting effects on a range of diseases such as cancer, diabetes, ulcers, wounds, anemia, hypertension, infants’ malnutrition and preventive action against atherosclerosis. It also accumulates β-1,3-glucan, which is an active immuno-stimulator, a reducer of blood lipids and a free radical scavenger (Reyes Suarez et al. 2008).

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Haematococcus pluvialis H. pluvialis is a unicellular fresh water microalga and accumulates high amounts of carotenoids especially astaxanthin and its esters under various stress conditions (Ranga Rao et al. 2013). Astaxanthin and its esters showed various biological activities in in vitro and in vivo models (Ranga Rao et al. 2014). H. pluvialis is considered the best source of astaxanthin for commercial applications (Shah et al. 2016). Astaxanthin is widely used as colorant in foods and pigment sources in the aquaculture industry (Shah et al. 2016). Due to high market demand for astaxanthin there is great interest in establishing efficient large-scale production systems through industrial production of biomass of H. pluvialis.

Dunaliella salina D. salina is a green microalga grown in high salt media, and it is able to accumulate high quantities of β-carotene, which is used as food colorant and also pro-vitamin A molecule (Bonnefond et al. 2017). D. salina contains high carotenoid content up to 10–14% (Ben-Amotz and Avron 1983). Carotenoids are accumulated in D. Salina alga in various stress factors (Bonefond et al. 2017; Da Silva Vaz et al. 2016). In addition to β-carotene, D. salina produces lipids, fatty acids and glycerol, etc. (Da Silva Vaz et al. 2016). β-carotene from D. salina finds use as a dietary supplement, nutraceutical and pharmaceutical agent (Murthy et al. 2005).

Isochrysis glaban I. glaban is a marine microalga with the ability to produce polyunsaturated fatty acids, docosahexaenoic acid and eicosapentaenoic acid which are stored in its lipid bodies (Fidalgo et al. 1998; Sukenik and Wahnon 1991). I. glaban species are used as feed for aquatic animals such as larvae and juvenile mollusks, crustaceans and as fish feed in commercial farms (Lemahieu et al. 2015). This alga was shown to be the most suitable source of nutrition for aquatic animals due to its promotion of rapid growth of aquatic animals (Huerlimann et al. 2014). I. glaban also produces sterols, tocopherols and pigments used as nutraceuticals in food applications (Sun et al. 2014). Based on the current literature, algae produce various natural molecules including chlorophylls, carotenoids, fatty acids and other bioactive constituents, which are shown in Tables 22.1 and 22.2. These molecules have diverse chemical frameworks and demonstrate various biological activities in algal cells, such as enhancing the efficiency of light energy

utilization, protecting against solar radiation and related effects and antioxidant effect. In addition to this, extracted algal pigments can be used as colorant in food and as potent antioxidants in food, nutraceuticals and pharmaceutical applications for improving human health. Therefore, microalgae are considered a source of functional foods, due to their efficient and sustainability characteristics based on the renewable nature of biomass production.

GLOBAL ALGAL-BASED PRODUCTS FOR FOOD AND FEED Most of the companies are stepping into carotenoids, astaxanthin, phycocyanin, EPA and DHA to capture the markets. Products from algae are identified in different stages of development based on the survey. Products in the advanced development stage include fucoxanthin, proteins, polysaccharide and phycoerythrin. Another product in the early development stages is lutein, but most products in this stage are non-food/feed applications (Table 22.3). Most of the global companies from the US and Europe dominate the algal products market through skincare products (Table 22.4).

GLOBAL ALGAL-BASED INDUSTRIES AND THEIR PRODUCTS Globally, algal companies such as Sapphire, Algenol, Aurora, Solix, Solazyme, Earthrise Nutritionals LLC, Nutrex Hawaii, Algapharma Biotech Corp (Far East Biotech Co., Ltd), Taiwan Chlorella Manufacturing Company, Fuji Chemical Industries Co., Ltd, Parry Nutraceuticals, Heliae Development LLC, BioProcess Algae LLC and Algaecan Biotech Ltd are working on cultivation of various algal species and also algal-based products such as biofuels (bio-oil, bio-gas, bio-ethanol, bio-hydrogen, bio-diesel, bio-methane, bio-fertilizers and bio-butanol), food supplement, feed, nutraceuticals and cosmetic applications (Figure 22.1). A brief outline of these companies is provided here, however, the status of several companies keeps changing and hence may vary at any point of time. The information is mainly provided for a general appreciation by the readers on the importance of the topic of algae rather than giving insight into the companies.

Sapphire Energy Sapphire Energy has a very advanced algal production facility at Columbus, New Mexico (www.sapphireenergy.com). This company has the world’s first

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TABLE 22.1 High Value Components in Microalgae Species for Health Food Applications Microalgal Species Spirulina sp.

High Value Component

H. pluvialis

Biomass protein, phycocyanin, β-carotene, γ-linolenic acid Biomass, lipids, pigments, β-glucan Astaxanthin and its esters

D. salina

Biomass, β-carotene

Isochrysis glaban

Fatty acids, carotenoids

Nannochloropsis sp.

Pigments, fatty acids

Porphyridum sp.

Arachidonic acid, polysaccharides Pigments, lutein, β-carotene

Chlorella sp.

Scenedesmus sp. Botryococcus braunii

Application

References

Food colorant, fluorescent markers

Capelli and Cysewski (2010); Papadaki et al. (2017)

Health food, pharmaceutical, nutraceutical Food colorant, feed additive, health food, food supplement, cosmetics, antioxidant Food supplement, food colorant, feed additive, cosmetics, antioxidant Food supplement, brain development for child, cardiovascular health Food supplement, brain development for child, cardiovascular health, cosmetics Health food, cosmetics, nutrition

Qi and Kim (2017); Reyes Suarez et al. (2008) Ranga Rao et al. (2013, 2014, 2018) Tang and Suter, (2011); Murthy et al. (2005) Lemahieu et al. (2015)

Nutraceutical, pharmaceutical, nutrition, cosmetics

Liu et al. (2016); Kilian et al. (2011) Kim et al. (2017); Lutzu et al. (2017) Custódio et al. (2014); Ranga Rao et al. (2018a)

commercially demonstrated algae farm from production to extraction. It produces green crude algal oil with fossil fuel properties. This product contains 100% renewable crude oil with reduced carbon footprint compared to other petroleum-based products.

and nutraceutical industries. A2 Feed™ is rich in protein and used for animal and aquaculture feed for healthy animals. A2 Fuel™ contains a high amount of biodiesel from algal biomass for alternative energy purpose as transportation fuel.

Algenol

Solix Biofuels

Algenol is an algae-based company, located in Fort Myers, Florida, founded in 2006. Algenol is an industrial biotechnology company, and it produces ethanol from algae. Algenol produces four major fuels such as gasoline, diesel, jet and ethanol. They have a proprietary process which can convert more than 85% of its carbon dioxide feedstock into the four fuels. Algenol tested their algae for non-toxicity and non-invasiveness in natural sources. Algenol has established its new unit in Southwest Florida which is developing viable fuels from algae (http://algenol.com/corporate-highlights/).

Solix Biofuels is located in Fort Collins, Colorado (www.solixbiofuels.com). It is focusing on specific R&D projects and providing technical and analytical services. This company plays a major role in cultivation of algae. Solix brought various products in the market such as Solasta® Astaxanthin, Solmega® DHA omega-3 and other natural ingredients. Solasta® Astaxanthin is a natural astaxanthin extract prepared from Haematococcus pluvialis. This product is a rich source of astaxanthin as personal care and dietary supplements.

Aurora BioFuels Aurora BioFuels is one of the bioenergy companies based on algae which is located in California (https:// openei.org/wiki/Aurora_BioFuels_Inc). This company produces various manufactured products such as A2 Omega-3™; A2 Feed™; A2 Fuel™ and A2 protein™ for nutrition, pharmaceutical and fuel purposes. A2 Omega-3™ contains a high amount of omega-3 oils which replaces fish oil and fermented products for pharmacy

Solazyme Solazyme is one of the biotechnology-based companies located in the United States (/www.solazyme.com/). It supplies algae-based ingredients with various brands such as Utz Quality Foods Inc., Enjoy Life Foods, Thrive Algae Oil brand and Hormel Food Corporation (www. solazyme.com). Solazyme developed a food ingredient named AlgaVia which was launched and received GRAS Certification. The protein rich algal powder is offering an allergen and gluten free source of vegan protein,

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TABLE 22.2 Products from Algal Species by Global Companies for Food and Feed Applications Microalgae Species H. pluvialis

Spirulina

Chlorella Chlorella Porphyridum

Manufacturers Cyanotech (USA), EID Parry (India), Mera Pharma (USA), BioReal (Sweden), Nutra (USA), Parry Nutraceuticals (India), AlgaTech (Israel), Blue Biotech (Germany), Fuji Chemicals (Japan), Mera Pharma (USA), BioReal (Sweden) Cyanotech (USA), Earthrise (USA), Dainippon (Japan), EID Parry (India), Nutra (USA), Blue Biotech (Germany), Inner Mongolia Biomedical Eng (Mongolia), Panmol (Australia), Spirulina Mexicana (Mexico), Siam Alga Co (Thailand), Nippon Spirulina (Japan), Koor Foods Co (Israel), Nan Pao Resins Chemicals (China), Hainan Simai Pharmacy (China), Myanmar Spirulina (Myanmar) RoquetteKloetze (Germany), Blue Biotech (Germany), Earthrise (USA), Dainippon (Japan), Chlorella Co. (Taiwan), Necton (Portugal) Phycom (Netherlands) InnovalG (France)

Nannochloropsis Schizochrytium Chrypthecodinium Nannochloropsis

Ocean’s Alive (USA), Flora Health (USA)

Odontella Schizochrytum Ulkenia D. salina

Innoval G (France) Xiamen Huison Biotech Co. (China) Lonza (Switzerland) EID Parry (India), Cognis Australia (Australia), BASF (Germany), Nature Beta Technologies (Australia) Tianjin Lantai Laboratory (China), Aqua Carotene Ltd (Australia), Pro Algen Biotech (India), Shaanxi Sciphar Biotechnology Co. Limited (China), DSM (Germany) Blue Biotech (German), SandaKing (Japan), DIC Lifetec (Japan) Innovative Aqua (Canada), Blue Biotech (Germany)

Spirulina Nannochloropsis Isochrysis Pavlova, Phaeodactylum, Chaetoceros, Skelotenma Thalassiosira Tetraselmis Nannochloropsis; Isochrysis Nannochloropsis Isoschrysis

DSM (USA) Blue Biotech (Germany)

Necton (Portugal)

Innovative Aqua (Canada)

Products Astaxanthin as dietary supplement, food colorant and food ingredient Spirulina as dietary supplement

Chlorella as dietary supplement Chlorella as food ingredient Porphyridum as dietary supplement EPA and DHA as dietary supplement EPA and DHA as food ingredient EPA and DHA as food ingredient EPA and DHA as food ingredient EPA and DHA as food ingredient EPA and DHA as food ingredient β-carotene, food additive and food color

Phycocyanin as food colorant Algae as paste; living algae as feed for fish Biomass for aquaculture; living algae as feed for fish in aquariums

Algae paste

Source: Christien et al. (2014).

whereas the lipid rich algal powder offers to replace need of eggs in recipes. AlgaWise Ultra Omega-9 contains 90% of mono-unsaturated fatty acids and 4% saturated fatty acids. AlgaWise high stability algae oil is projected to resist oxidation and reduce the need for preservatives and extra stabilizing ingredients. TerraVia offered Thrive algal oil which was marketed as the best oil for the heart because of its high content of monounsaturated

fatty acids. TerraVia supplies other products such as aquaculture feed, AlgaPrime DHA, AlgaPur Algae oil and Encapso for food, feed and personal care products.

Earthrise Nutritionals LLC Earthrise Nutritionals LLC is one of the top-quality Spirulina producers, having generally recognized as safe

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TABLE 22.3 Different Stages of Algae Products Products

Different Stages

EPA/DHA, proteins, astaxanthin, carotenoids, phycocyanin; whole biomass; fucoxanthin, soil amendments, vaccines, beta-glucan; polysaccharides, fatty acids Aquaculture feed, antioxidants; anti-fungal biomass; bioreactors; phycobiliproteins; oils, lutein, enzymes, cosmetics, antimicrobial, terpene, probiotic, carbohydrates, waxes and resins Omega-6 oils for nutritional applications, animal feed, fuel, cosmetics, noodles with whole algae; phycoerythrin

Advanced development

Early development

Commercialized

Source: Christien et al. (2014).

TABLE 22.4 Cosmetics Products from Algae Algae

Producers

Cosmetics

Spirulina Chlorella Alguronic acid in algae culture Skeletonema costatum Spirulina Nannochloropsis Dunaliella Salina Porphyridium cruentum Chlorella Phaeodactylum tricornutum Dysmorphococcus Globosus

Soliance (France) LVMH (France) Algenist Solazyme (California, USA) Soliance (France) Exsymol S.A. M. (Monaco) Pentapham (Switzerland)

Personal care skin products Personal care skin products Personal care skin products Anti-aging skin product Anti-aging skin product Anti-aging skin product

Soliance, Codif (France)

Hydrating skin product

Soliance Soliance

Anti-inflammation Slimming products

Source: Christien et al. (2014).

(GRAS) status. This company is located in California (http://earthrise.com/about/origins). Spirulina is manufactured with good manufacturing practices as stipulated by the United States of Food and Drug Administration. It has the world’s largest Spirulina farm in the Sonoran Desert of southeastern California. Earthrise produces high quality products in the form of powder (whole cell algal biomass), capsules, recipes and gels, etc., for food, feed, nutraceutical and pharmaceutical applications. The quality control system includes safety standards certified by international standards such as ISO 9001:2015, Food safety system certification (FSSC) 22000:2011, ISO 22000:2005 and ISO/TS 22002-1:2009.

Nutrex Hawaii Nutrex Hawaii uniquely delivers the biomass of Spirulina, cultivated in a Biosecure zone free from

herbicides, pesticides and pollutants in Kona, Hawaii (www.nutrex-hawaii.com). Spirulina pond is fed with drinking water from aquifers and infused with pure, deep, ocean water containing beneficial trace minerals. This company supplies with two brands, Bioastin® and Hawaiian Spirulina®. Hawaiian Spirulina® is in the form of tablets which supports the human immune system and cardiovascular health. Whereas Bioastin® contains astaxanthin in the form of gel caps as a source of powerful antioxidant and provides a wide range of impressive health benefits.

Algapharma Biotech Corp (Far East Biotech Co., Ltd) Algapharma Biotech Corp manufactures organic Chlorella and organic Spirulina (www.febico.com). It offers various products with different brands such as

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FIGURE 22.1  Algal-based products for biofuel, food, feed, nutraceutical, pharmaceutical and cosmetic applications. (Obtained from global algal companies.)

FEBICO Sorokina®, Biophyto® Premium Spiruina, Apogen®, Apomivir®, Flogen®, Apogina® phyco-radiance powder, Narogen® energy collagen mask and Apogen® children granule for dietary supplements and nutraceutical and pharmaceutical applications. FEBICO Sorokina® is obtained from Chlorella Sorokiniana, and this product is used for dietary supplements. Biophyto® Premium Spirulina is another product derived from organic algae and used for nutraceuticals. Apogen® is extracted from algae using special extraction methods. It is shown to offer protection against viral infections; evidently Apomivir® is approved by USFDA and Taiwan FDA for the treatment and alleviation of influenza associated syndrome. Flogen® is FEBICO’s brand name for selling phycobiliprotein products such as Flogen® Phycobiliprotein, Phycobiliprotein, LycoFlogen® ® LyoFlogen Recombinant protein, Flogen® Conjugate and Flogen® Conjugate Service. Apogina® Phycoradiance powder boosts the metabolic rate of skin and also eliminates the excess keratin thereby enhancing complexion with shiny skin. Narogen® Energy Collagen Mask is extracted from algae and provides moisture to skin cells.

Taiwan Chlorella Manufacturing Company Taiwan Chlorella Manufacturing Company was the first company to produce Chlorella biomass in Taiwan,

established in 1964 (www.taiwanchlorella.com), and the world’s largest producer. Chlorella can be used in foods such as pasta, cookies and also made in the form of tablets and capsules, enhancing the nutritional quality of the diet. This company made various products with Chlorella in the form of tablets, powder, extracts and noodles. This company has maintained best quality products with quality assured certificate from ISO 9001 (2008). It has the best quality control processing unit which validates the quality of the products through examination of microbial safety, nutritional parameters, color inspection, volume verification and also purity tests.

Fuji Chemical Industries Co., Ltd Fuji Chemical Industries is developing various health food supplements using natural astaxanthin from Haematococcus pluvialis (www.fujichemical.co.jp). This company has a very high reputation worldwide on health care products, specifically those made from astaxanthin obtained from algal biomass. Fuji Chemicals is located in Toyama, Japan. It has several other subsidiary branches in various countries such as AstaReal Co., Ltd., Astavita Inc. and AstaReal, Inc. in the United States; AstaReal AB in Europe; AstaReal Pte. Ltd. in Singapore; AstaReal Pty Ltd. in Australia and New Zealand and AstaReal Pvt Ltd. in Middle East and India.

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The Fuji Chemical Industries group started with astaxanthin work in 1994 and have diversified astaxanthin products from raw materials to finished products. Fuji Chemical Industries have made various products in the market with brand names AstaREAL® and AstaTROL® for health, food, cosmetics and pharmaceutical applications. AstaReal brands comprise of algal oil extract, powder, water soluble pigments, beadlets, biomass and soft gel capsules for nutraceutical, cosmetic and food applications.

Parry Nutraceuticals Parry Nutraceuticals is a very large company located in Chennai, India, and is one of the world leaders in producing algal based products (http://www.parrynutraceuticals.com). This company is of E.I.D Parry (I) Ltd. and a part of the USD 4.3 billion Murugappa group. This company is certified by International Food and Safety Standards, and products have charted constant growth in all the markets, worldwide. This company supplies various products with major brands in more than 38 countries, the main markets being in North America, Europe and South East Asia. It is offering organic Spirulina (Arthrospira platensis) biomass, organic Chlorella vulgaris biomass and astaxanthin from Haematococcus pluvialis with approved quality certifications through a well-developed scientific process, and also as the best and safest Spirulina in the globe. This company brought various products with brand names Organic Spirulina, Zanthin™, Organic Chlorella, Spiruzan™, natural mixed carotenoids, lycopene and Xanmax® for health food applications in protecting the body against cell and tissue damage by scavenging free radicals. These products are available in the market in the form of powder, capsules, tablets and gels which help in improving immune response and boosting of health in different ways.

Handbook of Algal Technologies and Phytochemicals

in phototrophic, mixotrophic and heterotrophic culture conditions. It has the ability to integrate multiple algal cultivation systems with control conditions. It has capacity to cultivate both natural and genetically modified organisms (GMOs) for various high value molecules for health food applications. Phycoterra® has shown improved root development to early germination and increased plant fresh weight and fruit yield when it is tested in various agricultural crops in the United States and abroad (http://phycoterra.com/news-and-results/ trial-results/).

BioProcess Algae LLC BioProcess Algae LLC is focused on providing feedstock for animal feeds, nutritionals and transportation fuels industries from microalgal cultivation in a cost effective manner including favorable carbon balances (www. bioprocessalgae.com). It is based in Omaha, Nebraska. It is currently running a demonstration plant at the Green Plains Inc. ethanol plant in Shenandoah, Iowa. Bioreactors installed in Shenandoah are tied directly to the CO2 exhaust gas and have been operating continuously since 2009.

Algaecan Biotech Ltd Algaecan Biotech Ltd is a Canadian company which has developed novel systems for the production of astaxanthin from Haematococcus culture for nutraceutical industry. It is producing efficient algae-derived products for pharmaceuticals, cosmetics and animal feed using algal photobioreactor cultivation. Also, the company consistently produces a higher yield of products than competing products obtained from open raceway ponds. This company was founded by James Irwin and Shane Lander in 2009 (http://algaecan.com).

Heliae Development LLC

MARKET VALUE FOR ALGAL PRODUCTS

Heliae Development LLC is one of the algal technology companies which is located in Gilbert, Arizona. It is working on applied life science and technology focusing on developing microalgal commercial system for useful products. It is developing nutraceutical ingredients for health food applications and also products for agriculture and aquaculture. Technology includes environmental remediation, providing sustainable low-cost sources of nutrition for a growing world population. Currently it has made two products, Phycoterra® Agricultural Product and Phycoterra® Organic Ag Product, both algal based. Heliae has expertise in cultivation of algae

Most consumers prefer to consume functional foods and nutraceuticals containing natural ingredients for preventing various disorders such as cancer, ulcer, diabetics, neuro disorder, etc. The consumers are trying to incorporate functional foods and nutraceuticals into their daily diet to improve health conditions. Functional foods are well-defined as foods that are consumed as part of a regular diet, contributing to improving health conditions (Hasler 2002; Ozen et al. 2012). Nutraceuticals are administered in the form of tablets, capsules, powders and syrups which are identified to benefit the health of the consumers by increasing the body response to

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Global Microalgal-Based Products

various disorders (Augustin and Sanguansri 2015; Gul et al. 2016). An increasing demand for health food applications in the global market can be satisfied by exploring algae as one of the possible sources of functional food and nutraceutical ingredients and also novel bioactive compounds for human health benefits. The global algal market is estimated to be worth in the range of $1.1bn by 2024. The total production volume and market size of algae in general are relatively small. Algal production and the global production volumes of microalgae were estimated at 1,000 tones dry wt in 1999. This increased to 5,000 tones dry wt representing €1 billion by 2004 (Spolaore et al. 2006). In 2011, the total production raised to 9,000 tons dry wt (Acien et al. 2018). The value of the global marine biotechnology market in 2011 with microalgae as its main component was estimated at €2.4 billion with an expected yearly growth of 10% (Guedes et al. 2011). Over 75% of the algal production was used in the health food applications as dietary supplement (Chacon-Lee and Gonzalez-Marino 2010). The algal-based food ingredients such as DHA and EPA represent a growing market. Algal-based DHA is used in baby food in the United States and also other companies such as Unilever, Dow Chemical and BASF. Market value for microalgae-based products are presented in Table 22.5. Spirulina and Chlorella still are by far the largest production volumes. But they also point to the large potential markets for high-value products such as DHA/EPA, β-carotene and astaxanthin. In general, the production is quite concentrated on a small number of players, except for Chlorella production.

SAFETY ASPECTS OF ALGAL PRODUCTS Food safety is important for the food products made from various microbial sources. Algae are used as whole cell biomass and its extracts are used in various nutraceutical and pharmaceutical products (Chacon-Lee and Gonzalez-Marino 2010). Food products using whole cell algal biomass or extracts from algae are follow food safety regulations. In 2002, the European Community Regulation (ECR, EC 178/2002) reported on food safety which provides a wide range of improvement to food products. It gives the outline of research areas not covered by specific harmonized rules. It provides principles and obligations of all areas of food production. It establishes ethics and responsibilities which mean to provide a robust science base, well-organized arrangements and procedures to support in making a decision on food safety related issues. It holds the general principle governing food in food safety specific to a national level. The regulation relates to numerous stages of food processing, food production and food products distribution. These food safety regulations have been established by the European Food Safety Authority (EFSA). Food safety is important to algal biotechnology and bioprocess as it requires precise consideration when microalgae are grown in open and closed systems for biomass and other products of interest due to the possible contamination with other competitive microorganisms.

FUTURE PROSPECTS Numerous key steps have to be considered from microalgal cultivation to final products in the market, including

TABLE 22.5 Market Value for Microalgae-Based Products

Algae Spirulina Chlorella Astaxanthin from Haematococcus Phycobiliproteins EPA/DHA from Chrypthecodinium β-carotene from Dunaliella Salina, Schizochrytium and Nannochloropsis

Companies More than 15 companies (Cyanotech and Earthrise) More than 70 companies (Cyanotech and Earthrise) More than eight companies (Fuji chemicals and Cyanotech) More than two companies More than four companies (Martek/DSM) More than ten companies (Cognis/BASF)

(Lammens 2012; Milledge 2012; Spolaore et al. 2006); NA, Not available.

Biomass Production (Tons/Year dry wt)

Biomass Production (Yearly Turnover)

5,000 tons/year

$40 M

2,000 tons/year

$38 M

300 tons/year

$10 M

NA 240 tons/year 1,200 tons/year

$300 M NA

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screening, isolation, selection, production and applications of the microalgae and their bioactive components in addition to production cost and environmental issues. There are some important questions that have to be addressed: how to make or design mass culture systems for efficient biomass production to reduce bioprocess cost and timeline? How to develop sustainable technologies for commercial process of a cost-effective manner? How to increase the production of high-quality bioactive molecules from algae for human health benefits for the global market? Some of the integrated technologies are required to be developed for health food applications. Novel technologies are required for cell harvesting, cell disruption and downstream processing of algal metabolites. It is required to have more sophisticated cultivation systems and also to establish downstream processing units for harvesting biomass and also to develop various extraction methods for recovery of bioactive compounds in a cost-effective manner. These technologies are very challenging and also crucial to the algal cultivation process. If we overcome the above problems it may be easy to get the bioactive molecules efficiently from algae for health food applications.

CONCLUSION Worldwide, many consumers are looking toward nutraceutical products as a way to make a better lifestyle. Many consumers are looking for food ingredients from natural sources in their diets. Synthetic ingredients are opposed by consumers in the global market due to potential adverse effects on health. A functional food helps to fight and prevent various disorders, enhance human wellness and energy and help to make a healthier life. In view of this, food scientists can explore microalgae as a possible source of food ingredients for human health food applications. An alga produces potential bioactive compounds that can be marked as nutraceutical products in the market for health food applications. Spirulina sp., Chlorella sp., D. salina, H. pluvialis and I. glaban have been receiving great attention globally, because they produce protein rich biomass and various bioactive molecules such as pigments, essential fatty acids, proteins, etc. The use of algal biomass or algal extracts has led to the advancement of food products. A few algal products have performed well in the food sector across the globe. However, there are still opportunities to be unlocked in this specific area. The major challenges to be optimized are cost-effective production systems to obtain efficient biomass and also functional molecules for food applications. The use of microalgae as a source of food ingredients in functional foods and nutraceuticals poses

Handbook of Algal Technologies and Phytochemicals

challenges to engineers and scientists to bring in innovations to realize the potential of algal products for the benefit of society and market needs.

ACKNOWLEDGMENTS The first author acknowledges Vignan’s Foundation for Science, Technology and Research University for providing financial support and research facility for this work and also the World Academy of Science for the Award of Young Affiliate for the year 2014–2018.

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Da Silva Vaz, B., Moreira, J.B., de Morais, M.G., and Costa, J.A.V. Microalgae as a new source of bioactive compounds in food supplements. Curr Opin Food Sci 2016. 7: 73–77. Duong, V.T., Li, Y., Nowak, E., and Schenk, P.M. Microalgae isolation and selection for prospective. Energies 2012. 5: 1835–1849. Fidalgo, J.P., Cid, A., Torres, E., Sukenik, A., and Herrero, C. Effects of nitrogen source and growth phase on proximate biochemical composition, lipid classes and fatty acid profile of the marine microalga Isochrysis galbana. Aquaculture 1998. 166: 105–116. Guedes, A.C., Amaro, H.M., and Malcata, F.X. Microalgae as source of carotenoids. Mar Drugs 2011. 9: 625–644. Gul, K., Singh, A.K., and Jabeen, R. Nutraceuticals and functional foods: The foods for the future world. Crit Rev Food Sci Nutr 2016. 56: 2617–2627. Habib, M.A.B., Parvin, M., Huntington, T.C., and Hasan, M.R. A Review on Culture, Production and Use of Spirulina as Food for Humans and Feeds for Domestic Animals and Fish. FAO Fisheries and Aquaculture Circular. No. 1034. FAO, Rome. 2008. p 33. Hasler, C.M. Functional foods: Benefits, concerns and challenges-a position paper from the American Council on Science and Health. J Nutr 2002. 132: 3772–3781. Hoek, C., Mann, D., and Jahns, H.M. Algae: An Introduction to Phycology. Cambridge University Press. 1995. Huerlimann, R., Steinig, E.J., Loxton, H., Zenger, K.R., Jerry, D.R., and Heimann, K. Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO). Gene 2014. 545: 36–44. Kilian, O., Benemann, C.S., Niyogi, K.K., and Vick, B. Highefficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc Natl Acad Sci U S A 2011. 108: 21265–21269. Kim, H.M., Oh, C.H., and Bae, H.J. Comparison of red microalgae (Porphyridium cruentum) culture conditions for bioethanol production. Bioresour Technol 2017. 233: 44–50. Lammens, T.M., Franssen, M.C.R., Scott, E.L., and Sanders, J.P.M. Availability of protein derived amino acids as feed stock for the production of bio-based chemicals. Biomass Bioenerg 2012. 44: 168–181. Lemahieu, C., Bruneel, C., Ryckebosch, E., Muylaert, K., Buyse, J., and Foubert, I. Impact of different omega-3 polyunsaturated fatty acid (n-3 PUFA) sources (flaxseed, Isochrysis galbana, fish oil and DHA gold) on n-3 LC-PUFA enrichment (efficiency) in the egg yolk. J Funct Foods 2015. 19: 821–827. Liu, L., Pohnert, G., and Wei, D. Extracellular metabolites from industrial microalgae and their biotechnological potential. Mar Drugs 2016. 14: 191. Lutzu, G.A., Zhang, L., Zhang, Z., and Liu, T. Feasibility of attached cultivation for polysaccharides production by Porphyridium cruentum. Bioprocess Biosyst Eng 2017. 40: 73–83. Milledge, J. Microalgae – Commercial potential for fuel, food and feed. Food Sci Technol 2012. 26: 28–30.

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Moejes, F.W., and Moejes, K.B. Algae for Africa: Microalgae as a source of food feed and fuel in Kenya. Afr J Biotechnol 2017. 16: 288–301. Murthy, K.N., Vanitha, A., Rajesha, J., Swamy, M.M., Sowmya, P.R., and Ravishankar, G.A. In vivo antioxidant activity of carotenoids from Dunaliella salina a green microalga. Life Sci 2005. 76: 1381–1390. Ozen, A.E., Pons, A., and Tur, J.A. Worldwide consumption of functional foods: A systematic review. Nutr Rev 2012. 70: 472–481. Papadaki, S., Kyriakopoulou, K., Tzovenis, I., and Krokida, M. Environmental impact of phycocyanin recovery from Spirulina platensis cyanobacterium. Innov Food Sci Emerg Technol 2017. 44: 217–223. Qi, J., and Kim, S.M. Characterization and immune-modulatory activities of polysaccharides extracted from green alga Chlorella ellipsoidea. Int J Biol Macromol 2017. 95: 106–114. Quintana, N., Van der Kooy, F., Van de Rhee, M.D., Voshol, G.P., and Verpoorte, R. Renewable energy from cyanobacteria: Energy production optimization by metabolic pathway engineering. Appl Microbiol Biotechnol 2011. 91: 471–490. Ranga Rao, A., Deepika, G., Ravishankar, G.A., Sarada, R., Narasimha Rao, B. P., Lei, B., and Su, Y. Industrial potential of carotenoid pigments from microalgae: Current trends and future prospects. Crit Rev Food Sci Nutr 2018a Jan 25. 1–22. Ranga Rao, A., Deepika, G., Ravishankar, G. A., Sarada, R., Narasimha Rao, B. P., Su, Y., and Lei, B. Botryococcus as an alternative source of carotenoids and its possible applications – An overview. Crit Rev Biotechnol 2018b. 38: 541–558. Ranga Rao, A., Phang, S.M., Sarada, R., and Ravishankar, G.A. Astaxanthin: Sources, extraction, stability, biological activities and its commercial applications: A review. Mar Drugs 2014. 12: 128–152. Ranga Rao, A., Sindhuja, H.N., Dharmesh, S.M., Sankar, K.U., Sarada, R., and Ravishankar, G.A. Effective inhibition of skin cancer, tyrosinase, and antioxidant properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis. J Agric Food Chem 2013. 61: 3842–3851. Reyes Suárez, E., Bugden, S.M., Kai, F.B., Kralovec, J.A., Noseda, M.D., Barrow, C.J., and Grindley, T.B. First isolation and structural determination of cyclic β-(1,2)glucans from an alga, Chlorella pyrenoidosa. Carbohydr Res 2008. 343: 2623–2633. Salati, S., D’Imporzano, G., Menin, B., Veronesi, D., Scaglia, B., Abbruscato, P., Mariani, P., and Adani, F. Mixotrophic cultivation of Chlorella for local protein production using agro-food by-products. Bioresour Technol 2017. 230: 82–89. Sarada, R., Ranga Rao, A., Sandesh, B.K., Dayananda, C., Anila, N., Chauhan, V.S., and Ravishankar, G.A. Influence of different culture conditions on yield of biomass and value added products in microalgae. Dyn Biochem Proc Biotechnol Mol Biol 2012. 6: 77–85.

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Macro and Micro Algal Impact on Marine Ecosystem A Global Perspective Sarban Sengupta and Ruma Pal

CONTENTS Abbreviations����������������������������������������������������������������������������������������������������������������������������������������������������������279 Introduction�������������������������������������������������������������������������������������������������������������������������������������������������������������279 Algae as the Basis of the Oceanic Food Web�����������������������������������������������������������������������������������������������������280 The Biological Pump������������������������������������������������������������������������������������������������������������������������������������������281 Harmful Algal Blooms (HABs)��������������������������������������������������������������������������������������������������������������������������283 Algae and Global Change in the Marine Environment��������������������������������������������������������������������������������������283 Summary and Conclusion���������������������������������������������������������������������������������������������������������������������������������������284 Acknowledgment����������������������������������������������������������������������������������������������������������������������������������������������������284 References���������������������������������������������������������������������������������������������������������������������������������������������������������������284

BOX 23.1  SALIENT FEATURES The oceans constitute the world’s largest ecosystem. In this enormous and dynamic ecosystem, both micro and macro algae play a variety of roles that can be both positive and negative. Primary production by macro and micro algae forms the basis of the food web in most of the oceans and is also responsible for the phenomenon known as the “Biological Pump”—the biological pumping of carbon to the depths where it is either sequestered from the environment or respired back to the inorganic form. The Biological Pump transports carbon in the form of Particulate Organic Carbon (POC) as well as Particulate Inorganic Carbon (PIC) from the surface to the depths below. Not all algae however have positive effects upon the oceanic environment and its inhabitants. Some produce noxious blooms that by depriving the water column of Dissolved Oxygen (DO) kill all marine life whilst others produce potent toxins that reach humans as they make their way up the food chain. Still others include species that are non-toxic to humans but cause damage to fish and invertebrates especially by clogging or damaging their respiratory devices. Anthropogenic activities have radically altered the biogeochemical cycling of elements especially

carbon and nitrogen and in their wake also altered the structure and functioning of numerous ecosystems. Marine ecosystems are no exception as human activities have also altered the abundance and distribution of several algae (both macro and micro algae).

ABBREVIATIONS CO2 C NEP OC DOC

Carbon Dioxide Carbon Net Ecosystem Productivity Organic Carbon Dissolved Organic Carbon

INTRODUCTION A diverse array of photoautotrophic organisms ranging from microscopic cyanobacteria to huge eukaryotic macro algae fall under the general term algae. Although cyanobacteria are now classified separately as cyanoprokaryotes, in this chapter, the roles they play in the Earth’s oceans will be discussed together under the banner of algae as their activities are comparable to eukaryotic algae.

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Algae as the Basis of the Oceanic Food Web Almost three-quarters of the Earth’s surface is covered by the oceans where algae form the base of the food chain. Phytoplanktons account for nearly half of global primary production (~500 × 1015 g of carbon fixed/year) (Falkowski 1994), although they constitute less than 1% of the world’s photosynthetic biomass (Falkowski 2000, Chrisholm et al. 2001). Enormous variation in size exists in phytoplankton communities. Seiburth’s (1979) method of for classifying phytoplankton based on their size as per logarithmic scale is widely accepted. Plankton whose size exceeds 200 µm are classified as mesoplankton, between 20–100 µm as microplankton. Smaller than the microplankton comes the nanoplanktons (2–20 µm) and picoplankton (0.2–2 µm). Of these various types, the “Classical” phytoplankton is constituted by the microplankton—majorly the Dinophytes and Bacillariophytes. A variety of pigmented flagellates including Prasinophytes, Chrysophytes, Cryptomonads, Haptophytes as well as a few Dinoflagellates, Euglenoids, tiny Bacillariophytes and Chlorophytes constitute the nanophytoplankton. Prokaryotes are the principal constituents of the picophytoplankton although a few eukaryotes especially the Chlorophytes are small enough to be included in this group. Previously, the open ocean ecosystem was considered to be the most appropriate example of ecosystem functioning and was used as a standard for defining the concepts of food chain and food web. A typical illustration would be a pyramid with phytoplankton forming the base, two horizontal layers representing zooplankton and fishes upwards respectively and a shark or whale representing the tip of the pyramid. However, it has now been widely recognized that a larger number of trophic levels are present than hitherto suspected and that a large amount of primary production is directed as dead organic matter through the activities of decomposers before it becomes available for phagotrophs (Fenchel 1988). Besides phytoplankton, macro algae also play an important role as primary producers although their role is confined to coastal areas which comprise ∼7% of the total ocean area (Borges et al. 2005). However, it is also worthwhile to note that marine macro algal beds have been recorded to possess higher Net Ecosystem Productivity (NEP) as compared to other marine vegetation types such as seagrass beds (Kim et al. 2015). In particular, on rocky coastlines in cold water areas, macro algae form underwater forests called kelp forests that harbor diverse marine biota. Mainly Phaeophytes form kelp forests. Three basic morphological groups or “guilds” of kelp are recognized

Handbook of Algal Technologies and Phytochemicals

on the basis of the canopy height of their fronds (Dayton 1985). Foremost among them is the giant kelp Macrocystis spp. (Figure 23.1) which dominates kelp forests along the west coasts of South and North America and at several scattered locations in the southern Pacific Ocean including Southern Australia, New Zealand, South Africa and several sub Antarctic islands (Steneck et al. 2002) and grows up to 45 m (Abott and Hollenerg 1976). Smaller canopy kelps (which can reach up to 10 m) include Alaria fistulosa which grows in the Pacific coast of Asia and Alaska, Ecklonia maxima in South Africa and its Northern Hemisphere counterpart, Nereocystis leutkeana, which occurs from Central California to Alaska (Dayton 1985, Steneck et al. 2002). Other species of kelp include Laminaria spp. (which form forests in the North Pacific from Japan across coastal Alaska to Northern California), Eisenia spp., Pleurophycus spp. Thalassiophyllum spp., Pterygophora spp., etc. Together, these producers support diverse communities of marine fishes, mammals, crustaceans, mollusks, other algae and epibiota that make them one of the most productive and diverse ecosystems on the Earth (Mann 1973). A variety of economically important polysaccharide products are obtained from macro algae. They include agar-agar and agarose from Rhodophytes (like Gracilaria sp., Gelidium sp., etc.), alginates from Phaeophytes (particularly Laminaria sp., Macrocystis sp. and Ascophyllum sp) and carrageenans from Rhodophytes (from Eucheuma cottonii, E. spinosum, Chondrus crispus). In marine environments, certain algal toxins are secreted which often reach humans in potent concentrations as they make their way up the food chain. Dinoflagellates such as Gambidiercus toxicus are mainly responsible for producing the toxins (ciguatoxin and maitotoxin) that accumulate in the flesh of over 400 species of marine predatory fish and cause ciguatera fish poisoning. Humans are affected when they consume fish whose flesh is laden with ciguatoxin (Figure 23.1) or maitotoxin. Affected individuals suffer from a host of gastrointestinal, cardiovascular and neurological disorders (Friedman et al. 2017). Paralytic shellfish poisoning is another instance of seafood-based poisoning caused by dinoflagellates like Alexandrium spp. (Van Dolah 2000). Bivalve mollusks accumulate these toxins in their tissue during filter feeding. Subsequently they are passed up the food chain and result in fatalities in humans, birds (Nisbet 1983) and humpback whales (Geraci 1989). Saxitoxin (Figure 23.1) exerts its effect by inhibiting the channel conductance of voltage gated sodium channels (Van Dolah 2000). Diarrhetic shellfish poisoning is caused by Dinophysis fortii and manifests itself with severe diarrhea and vomiting. The compounds responsible for exerting these effects

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FIGURE 23.1  1. Padina dubia. 2. Padina gymnospora. 3. Padina boryana. 4. Tricleocarpa fragilis. 5. Sargassum cristaefolium. 6. Amphiroa fragilissima.

are Okadaic acid (Figure 23.1), Dinophysis Toxin (DTX1) and DTX-2. Neurotoxic shellfish poisoning is caused by brevitoxin produced by blooms of Gymnodinium breve. Symptoms include severe muscle pain, numbness and tingling of the perioral area, nausea and loss of motor control. Amnesic shellfish poisoning is the only known case of shellfish poisoning which is caused by domoic (Figure 23.1) acid from a Bacillariophyte (Pseudonitzschia multiseries) which results in amnesia (either permanent or temporary) and gastro-intestinal effects like nausea, vomiting, diarrhea, etc.

The Biological Pump Among the biogeochemical cycles operating in nature, the carbon (C) cycle has attracted a particularly great

deal of attention from scientists. The global carbon cycle encompasses five distinct pools—the Atmospheric, Biotic, Pedologic, Geologic and the Oceanic pools. The pools are interconnected, and the C flux between these pools is extremely prone to human disturbances (Lal 2008). Anthropogenic activities like conversion of forest to agricultural land and combustion of fossil fuels have created dire imbalances in the biogeochemical cycling of carbon by selectively enriching the atmospheric pool with carbon dioxide (CO2). This selective enrichment of the atmospheric C pool has caused the atmospheric level of CO2 to rise from the pre-industrial level of 280 ppm to 399 ppm in 2015. Currently, it is rising at a rate of 2.2 ppm/year (Sengupta et al. 2017), causing global warming and associated climate change. Of the five interconnected pools, it is the Oceanic pool that is

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responsible for the absorption of the greatest amount of the emitted CO2. In the upper oceans, the photosynthetic activities of algae result in the fixation of inorganic CO2 to the organic carbon (OC) of biomass. Part of this organic matter is respired back to CO2 by the photoautotrophs, part of it is secreted as dissolved organic carbon (DOC), and part of it is consumed by herbivores through which it passes up the food chain. But a fraction of this OC escapes and sinks down to the bottom as particulate organic carbon (POC: a complex mix of dead cells, excretory products and amorphous aggregates). Thus, C fixed by photoautotrophs in the sunlit upper waters is transported to the depths below (Volk and Hoeffert 1985, Martin 1990, Falkowski 2000). Nearly one-fourth of the C fixed by the action of photoautotrophs sinks to the depths below (Falkowski and Wilson 1992, Falkowski et al. 2000, Sengupta et al. 2017). In the deep ocean, part of the C fixed is re-mineralized back to DIC through the activity of decomposers and detritivores, but part of it is buried by the sediments where it proceeds to form fossil fuels (Chrisholm et al. 2001). This transport of carbon to the depths by phytoplankton is termed as “the Biological Pump”. At any given moment, there are two different carbon cycles operating in nature: the slow cycle and the fast cycle. The former is tectonically driven and operates on time scales of millions of years (Berner 1990) consisting of the volcanic outgassing of CO2 coupled with the weathering of silicate rocks. The latter involves the biofixation of carbon into biomass and its return to the inorganic form by the subsequent oxidation of this organic carbon through respiration. The biological pump transfers carbon from the fast biologically driven cycle to the slow geologically driven cycle (Chrisholm et al. 2001). On the other hand, anthropogenic combustion of fossil fuels brings C that has been sequestered away from the Earth’s atmosphere in the slow cycle back into circulation in the fast cycle. The Biological Pump consists of two discrete but interconnected processes: the “Calcium Carbonate Pump” which transports C as calcium carbonate and the “Organic Carbon Pump” which transports C as organic carbon. The ratio between the former and the latter is known as “Rain Ratio” (~0.1) (Rost and Riebesell 2004). In oceanic environments, coccoloithophores (Haptophyta)—a group of planktonic microalgae that produce calcite skeletons—are by far the most common calcifying algae (Morse and Mackenzie 1990, Rost and Riebesell 2004) (e.g. Plate 23.1). The reactions involved in biogenic calcification are as follows:

Ca 2 + + 2HCO3-  CaCO3 + CO2 + H 2 O (23.1)



Ca 2 + + CO32 -  CaCO3 (23.2)

As can be deduced from the above reactions, biogenic calcification actually elevates the atmospheric partial pressure of CO2. This process is also known as the carbonate counter pump. Hence, the sinking of Coccolithophore skeletons is considered to be a smaller avenue for CO2 drawdown as compared to other phytoplankton species such as Chlorophytes, Bacillariophytes, etc. However, as because the ratio of calcareous to non-calcareous primary production has a strong impact on the relative strengths of the two pumps, calcium carbonate has been proposed to act as a ballast mineral that potentially amplifies the potency of the Biological Pump in totality (Armstrong et al. 2001). Also, coccoliths can potentially increase the sedimentation and burial of certain sections of POC when they are used as food and subsequently excreted as fecal pellets (Buitenhuis et al. 1996). In oceanic ecosystems, cyanobacteria have been recognized as key players in the precipitation of calcium carbonate. Cyano-prokaryotes both produce extracellular polysaccharides which act as binding sites for calcium and carbonate ions as well as elevate the pH of the water body through photosynthesis. Both processes have been known to impact the mineralogy and morphology of the carbonate minerals (Dittrich and Sibler 2010). Studies on cyanobacterial extracellular polymeric substances indicate that polysaccharides secreted by cyanobacteria possess a strong capacity to exchange protons with the surrounding environments. Lab scale studies with Synechococcus sp. demonstrated that extracellular polysaccharides were able to precipitate calcium carbonate. Examples of cyanobacterial calcification include Stromatolites and whiting events. Whiting events are characterized by the large-scale precipitation of calcium carbonate crystals in conjunction with the production of organic compounds like polysaccharides by cyanobacterial cells (Thompson et al. 1997, Yates and Robbins 2001). Calcifying macroalgae like Padina sp. (Phaeophyta), Tricleocarpa sp. and Amphiroa sp. (Figure 23.1) (Rhodphyta) also play an important role in this regard. Although ocean acidification arising from increasing CO2 levels (Doney et al. 2009) is a matter of concern for the future of calcifying algae, in situ studies on Padina sp. have documented greater abundance of these species in near CO2 rich areas (Johnson et al. 2012). This happens because photosynthesis and calcification are closely related (Okazaki et al. 1986), and higher photosynthetic rates created by higher CO2 concentrations at least partially offset increased dissolution of CaCO3 at low pH (Johnson et al. 2012). Thus, although ocean acidification results in deposition of less CaCO3

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in the tissues of Padina sp., greater profusion of these algae in the intertidal zone can result in greater CaCO3 deposition and subsequently greater C sequestration (Sengupta et al. 2017). According to Engel et al. (2004), not all organic particles in the ocean originate from cellular debris. In recent years, extracellular polysaccharide particles described as Transparent Exopolymeric Substances (TEP) have gained a lot of attention in the field of limnology (Passow 2002). TEP possess a surface reactive nature and hence support coagulation processes that increase the formation of large aggregates (marine snow) (Engel 2000, Passow 2002). This in turn enhances carbon pumping to the deep ocean (Asper et al. 1992).

Harmful Algal Blooms (HABs) Amongst the 5,000 recorded extant species of marine phytoplankton, around 300 species have been found to occur in numbers so great that they discolor the surface of the sea (Hallegraf 1993). HABs are harmful in three basic ways: i. Certain species which normally produce only harmless discolorations of the water can under certain circumstances produce blooms so dense that by creating anoxic conditions can kill off all marine life. Examples include Dinophytes such as Noctiluca scintillans and Gonyaulax polygramma and the cyanobacterium Trichodesmium erythraeum. ii. The second category includes species which produce potent toxins that reach humans through the marine food chain thereby causing a variety of disorders as discussed previously. iii. The third category includes those species which are non-toxic to humans but cause damage to fish and invertebrates especially by clogging or damaging their respiratory devices. Examples include Chrysophytes such as Chrysochromulina leadbeteri, C. polylepis, Prymnesium patelliferum, P. parvum, Raphidophydtes such as Chattonella antiqua, Heterostigma akashiwo (Figure 23.1), etc. Although HAB formation is a natural phenomenon occurring throughout recorded history with references as early as the Old Testament of the Bible, their occurrence and abundance has increased over the past few decades due to a combination of anthropogenic activities. Firstly, an overexploitation of fishery stocks worldwide is leading to a shift to intensive aquaculture of shellfish and finfish. Reminiscent of “sensitive bioassays” for harmful

algal organisms, these intensive aquaculture systems often bring to light the presence of problematic organisms in waters where they were not thought to occur before. In finfish systems, algal species can potentially damage the gills of finfish. Dense concentrations of the diatoms Chaetocaeros concavicornis and C. convolutes (~5,000 cells/liter) have been held responsible for the deaths of commercial fishes like lingcod, coho, sockeye, Chinook and pink salmon. The diatom possesses long narrow spines (setae) equipped with smaller barbs along their length. The setae have the tendency to break off and puncture the gill membranes of the fish. Subsequently capillary hemorrhage sets in along with dysfunction of gas exchange at the gills and mucus overproduction. Together with secondary infections these ultimately kill the fish (Bell 1961, Rensel 1991). A more widespread problem for fish farmers is the production of hemolysins: fatty acids or galactolipids that destroy RBCs and are produced by diverse groups of algae such as Prymnesiophytes like Prymensium parvum and Chrysochromulina polylepis and Raphidophytes like Chattonella antiqua, Heterostigma akashiwo, etc. Over the past few decades, anthropogenic increase in the pumping of nutrients especially nitrogen into lakes and coastal ecosystems via riverine discharge have contributed to notably higher algal production in coastal areas (Smith 2003). For example, during the period 1976–1986 the number of red tides increased eight-fold in the Hong Kong Bay area corresponding to a 2.5-fold increase in nutrient loading and a six-fold increase in the human population.

Algae and Global Change in the Marine Environment Over the past few decades human activities have profoundly altered the structure and functioning of ecosystems either directly or indirectly, including marine ecosystems. Generally marine biotic communities are believed to be more intensely regulated by top-down controls than terrestrial communities (Shurin 2006) and therefore more prone to climate driven modulation of interactions between consumers and their prey. The intensity of herbivory in marine environments can be visualized when we take into account the fact that globally, herbivores consume 70% of benthic primary production (Poore et al. 2012). Examples of phase shifts caused by herbivory include kelp forests where an increase in herbivory by sea urchins leads to deforested barrens (Steneck et al. 2002) and coral reefs where a decrease in herbivory leads to a shift from coral to algal dominated reefs (Hughes et al. 2010). In both the cases,

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ocean warming has been implicated as a cause (Steneck et al. 2002, Ling 2008). Anthropogenic activities also exacerbate the introduction of non-indigenous species into ecosystems where they were not originally present. According to Siguan (2002), 97 species of algae (63 Rhodophytes, 20 Phaeophytes, 11 Chlorophytes and three Dinophytes) have been inadvertently introduced into the Mediterranean Sea. Several of these species are invasive and have a negative impact on the economy and ecology of the region. Of these Caulerpa taxifofia (Figure 23.1) is especially noteworthy as it displaces native climax communities of Posidonia oceanica (Phaeophyta) (de Villele and Verlaque 1995). However, the biggest interaction of algae with humans perhaps comes from interferences with the functioning of the Biological Pump by enhancing primary productivity by increasing the inflow of nutrients into aquatic ecosystems (Purvaja and Ramesh 2000). The response of different algal species to elevated CO2 levels varies, but an increase in growth caused by an increase in photosynthesis is observed in all species (Sengupta et al. 2017). However, human activities also contribute to enhanced greenhouse effect and global warming which can reduce the supply of nutrients to primary producers especially in deep water ecosystems. Subsequently, primary production and ultimately carbon sequestration are reduced (Bopp et al. 2001, Gregg et al. 2003, Sarmiento et al. 2004, Polovina et al. 2008). Proposals are even on to fertilize the High Nutrient Low Chlorophyll (HNLC) regions of the oceans with micronutrients to enhance carbon export into the deep ocean. If implemented, they would alter the structure and functioning of the world’s largest ecosystem on a grand scale (Chrisholm 2001).

SUMMARY AND CONCLUSION Overall, it can be concluded that both micro and macro algae play vital roles in the Earth’s oceans. They form the basis of the food web and support other marine flora and fauna and are responsible for the biological sequestration of carbon. Annual growth and decay of the phytoplankton community of the marine ecosystem support the micro-fauna by providing food and oxygen on one hand, and at the end of the growing season mass deposition of biomass is the effective process of carbon burial on the other. However, some produce noxious blooms and secrete toxins killing marine life, which affect the total economy of coastal countries. The huge kelp forests at ocean beds not only maintain the oxygen level at deep sea but are also used as a source of various

Handbook of Algal Technologies and Phytochemicals

economically important chemicals, including agar-agar. Anthropogenic activities are responsible for modifying the marine environment around us. The response of algae to these changes is significant. The eutrophication of water bodies resulting from anthropogenic nutrient enrichment favor bloom forming species whereas slow growing species are weeded out. Non-indigenous species are also introduced into habitats where they were previously not present. The future of marine algae in this ever-changing environment remains to be seen.

ACKNOWLEDGMENT The authors acknowledge the University of Calcutta for access to the articles and the University Grants Commission for fellowship to Sarban Sengupta.

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Index β-carotene, 205, 269 β-glucan, 124, 126 β-glucosidases, 43 1,4-pentanediol (PenDO), 135, 136 2,5-furandicarboxylic acid (FDCA), 135, 136 3-hydroxyisobutyryl-CoA hydrolase (HIBCH), 81 5-hydroxymethylfurfural (HMF), 136 A2 FeedTM, 270 A2 FuelTM, 270 A2 Omega-3TM, 270 AA, see Arachidonic acid (AA) Abscisic acid (ABA), 115 ACCase, see Acetyl-CoA carboxylase (ACCase) Acetyl-CoA, 55, 70 Acetyl-CoA carboxylase (ACCase), 78, 81 Acid hydrolysis, 42 Activated sludge process, 5 Acute toxicity and mutagenicity test, 218 Acyl-ACP thioesterases (FATs), 55 Adenosine diphosphate (ADP), 8 Adenosine triphosphate (ATP), 8, 68 ADP, see Adenosine diphosphate (ADP) Advanced filtration, 236 Advanced oxidative systems (AOS), see Algal ponds AFI, see Aquaculture Foundation of India (AFI) Agar, 43 AI, see Artificial intelligence (AI) Air circulation, 14–15 Airlift photobioreactor, 183–184 Alexandrium minutum, 15 Algae, 123–128; see also Algae cultivation; Algae production; Algal biomass; Algal systems in aquaculture, 124 attributes, 124 bivalves feed, 126–127 crustaceans feed, 127–128 fish feed, 124–126 overview, 123–124 Algaecan Biotech Ltd, 274 Algaecides, 15 Algae cultivation, 91–96 growth and productivities, 92–93 culture parameters and nutrients, 92–93 selection, 92 industrial feasibility, 95 innovations in downstream processing, 93–95

lipid production and productivities, 93 overview, 91–92 Algae production, in Thailand, 253–262 biotechnology future prospects, 261–262 research, 261 macroalgae, 255–256 applications, 256–257 microalgae, 257–259 applications, 259–261 overview, 254–255 Algae–water separation technologies, 235 Algal biomass, 241–247 centrifugation, 244 drying, 245–246 filtration, 244–245 flocculation, 242–243 flotation, 243–244 overview, 241–242 tangential filtration, 245 dewatering, 245 Algal harvesting techniques, 52 Algal ponds, 25–26, 29–30 Algal systems, 231–237 advanced filtration, 236 algae–water separation technologies, 235 centrifugation, 235 dissolved air flotation, 235–236 electrolytic methods, 236 feed and food, 236–237 life cycle analyses, 233–234 nutrient recycle, 234–235 open pond algaculture, 234 overview, 232 production, 232–233 sustainability, 233 sustainable water use and recovery, 234 technoeconomic analysis, 233 use of wastewater, 234 wastewater remediation, 234 Algapharma Biotech Corp, 272–273 AlgaVia, 270 AlgaWise Ultra Omega-9, 271 Algenol, 270 Alginate, 45 Alginic acid, 126 Amylase, 43 Anaerobic ponds, 26 ANN model, see Artificial neural network model (ANN model) Apogina®, 273 Apomivir®, 273 Aquaculture, algae in, 124 attributes, 124 bivalves feed, 126–127

crustaceans feed, 127–128 fish feed, 124–126 Aquaculture Foundation of India (AFI), 145, 147, 150, 151 Aquaculture Research and Development Center, 255 Arachidonic acid (AA), 128 Areal loading rate model, 26–27 Arsenic, 80 Arsenoribosides, 80 Arthrospira platensis, 241 Artificial intelligence (AI), 59 Artificial neural network model (ANN model), 59 Ascophyllum esculentum, 117 Ascophyllum nodosum, 115, 118 Assimilation process, 5 AstaREAL®, 274 AstaTROL®, 274 Astaxanthin, 205, 244, 269 ATP, see Adenosine triphosphate (ATP) Aurora Biofuels, 270 Autoflocculation, 57 Auxenochlorella protothecoides, 235 Auxins, 113 Bacillariophytes, 280 Barley straw, 15 BCAA, see Branched-chain amino acid (BCAA) BES, see Bioelectrochemical systems (BES) Betaine lipids (BLs), 79 Betaines, 115 Bioactive compounds and extraction, 260–261 Biochemical oxygen demand (BOD), 27, 28, 29 Bio-crude, 58 Biodiesel production, 95 from microalgae, 51–60 challenges, 59 closed cultivation systems, 54–55 cultivation, 54 harvesting, 55–56 lipid extraction, 56–57 lipid to biodiesel, 57–59 lipid/triacylglycerol biosynthesis, 55 metabolic engineering, 55 open cultivation system, 54 overview, 51–52 scale-up strategies, 59 Bioelectrochemical system (BES), 101, 103–104 coupling photobioreactor and, 104–105 harvested algae biomass in, 103–104

287

288 Bioethanol, 41, 43–44 Bio-fertilizer, 112–113 Bioflocculation, 57 Biofuel, 45–46 Biological pump, 281–283 Bioprocess Algae LLC, 274 Biorefinery, 30, 59–60 Bioremediation algae’s role in, 30 Bio-remediation of wetlands by planting (BWP), 14 Biotechnology and marine cyanobacteria, 161, 163–164, 166 Bligh and Dyer method, 57 BLs, see Betaine lipids (BLs) BOD, see Biochemical oxygen demand (BOD) Botryococcus braunii, 54, 135 Botryococcus sp., 7 Branched-chain amino acid (BCAA), 81 Bubble column photobioreactor, 182–183 BWP, see Bio-remediation of wetlands by planting (BWP) CA, see Carbonic anhydrases (CA) Calcium Carbonate Pump, 282 Calvin cycle, 68 CAPEX, see Capital expenditures (CAPEX) Capital cost, 30 Capital expenditures (CAPEX), 135–136, 137, 138, 140, 141, 142 Carbohydrate, 207–208 metabolism, 68–70 glycolysis and gluconeogenesis, 68–69 lipid metabolism, 70 protein metabolism, 69–70 TCA cycle and oxidative phosphorylation, 69 Carbonate counter pump, 282 Carbon concentrating mechanism (CCM), 63, 66–67, 68 functions of carbonic anhydrase, 67 types of carbonic anhydrases, 66–67 Carbon dioxide (CO2) sequestration, 63–71 Calvin cycle, 68 carbohydrate metabolism, 68–70 glycolysis and gluconeogenesis, 68–69 lipid metabolism, 70 protein metabolism, 69–70 TCA cycle and oxidative phosphorylation, 69 carbon concentrating mechanism (CCM), 66–67, 68 functions of carbonic anhydrase, 67 types of carbonic anhydrases, 66–67 factors affecting utilization, 64–66 light, 64–65 mixing, 65–66 pH, 65

Index temperature, 65 importance of microalgae, 64 industrial application, 70–71 microalgae cultivation systems, 64 ribulose-1, 5-bisphosphate carboxylase oxygenase (RuBisCO), 67–68 accumulation of HCO3 –, 68 location, 67 Carbonic anhydrases (CA), 66–67 Carotenoids, 124, 205 Carrageenan, 43 Catalytic hydrothermal gasification, 236 Caulerpa lentillifera, 255, 257 Cell disruption, 57, 217 Cellulase, 43 Cellulose, 43 Central Salt and Marine Chemicals Research Institute (CSMCRI), 145, 147 Centrifugation, 94, 235, 244 CFD, see Computational fluid dynamics (CFD) Chaetoceros, 258–259 Chaetoceros gracilis, 92 Chara globularis, 114 Chemical flocculants, 57 Chemicals and polymers production, 133–142 methodology, 135–136 design considerations, 135–136 investigated scenarios, 135 photo-bioreactors design, 135 overview, 134–135 scenarios description, 136–137 S1, 136 S2, 136 S3 and S4, 136–137 techno-economic analysis, 137–142 comparison of scenarios, 137–138 effect of plant capacity, 138–139 scenario-S1a, 139–140 sensitivity analysis, 140–141 Chlamydomonas, 30, 85 Chlamydomonas reinhardtii, 55, 67 Chlorella saccharophila, 67 Chlorella sp., 30, 59, 125, 268 Chlorella vulgaris, 61, 17, 241 Chondrus crispus, 126 Cladophora glomerata, 255, 257 Closed cultivation systems, 83, 54–55 Closed photobioreactors