Microalgal Biotechnology: Integration and Economy 9783110298321, 9783110298277

Table of contents :
Preface
List of contributing authors
1 Introduction – Integration in microalgal biotechnology
1.1 Integration on the process level
1.2 Integration on the metabolic level
1.3 Integration into environmental conditions
1.4 Adaptation to cultural realities
Integrated production processes
2 Products from microalgae: An overview
2.1 Microalgae: An introduction
2.2 Products
2.2.1 Use and production of algal biomass
2.2.2 Microalgae for human nutrition
2.2.2.1 Spirulina (Arthrospira)
2.2.2.2 Chlorella
2.2.2.3 Dunaliella salina
2.2.3 Microalgae for animal feed
2.2.4 Microalgae as natural fertilizer
2.2.5 Microalgae in cosmetics
2.2.6 Fine chemicals
2.2.6.1 PUFAs
2.2.6.2 Pigments
Pigments as antioxidants
Pigments as natural colorants
2.2.6.3 Polysaccharides
2.2.6.4 Recombinant proteins
2.2.6.5 Stable isotopes
2.2.7 Micro- and nanostructured particles
2.2.8 Bulk chemicals
2.2.9 Energy production from microalgae
2.2.9.1 Biodiesel
2.2.9.2 Bio-ethanol
2.2.9.3 Bio-hydrogen
2.2.9.4 Bio-gas
2.2.9.5 Biorefinery of microalgae
2.3 Conclusion
References
3 Spirulina production in volcano lakes: From natural resources to human welfare
3.1 Introduction
3.2 Natural Spirulina lakes in Myanmar
3.3 Environmental parameters of Myanmar Spirulina lakes
3.4 Spirulina production from natural lakes
3.4.1 Harvesting
3.4.2 Washing and dewatering
3.4.3 Extrusion and sun drying
3.4.4 Lake-side enhancement ponds
3.5 Sustainable Spirulina production from volcanic crater lakes
3.6 Myanmar Spirulina products
3.7 Spirulina as biofertilizer
3.8 Spirulina as a biogas enhancer
3.9 Spirulina as a source of biofuel
3.10 Myanmar and German cooperation in microalgae biotechnology
3.11 Discussion
3.12 Conclusion
Acknowledgments
References
4 Case study of a temperature-controlled outdoor PBR system in Bremen
Acknowledgments
References
5 Algae for aquaculture and animal feeds
5.1 Introduction
5.2 Microalgae use in aquaculture hatcheries
5.2.1 Microalgal strains used in aquaculture hatcheries
5.2.2 Methods of microalgae cultivation for aquaculture
5.2.3 Role of microalgae in aquaculture hatcheries
5.2.3.1 Microalgae as a feed source for filter-feeding aquaculture species
5.2.3.2 Microalgae as a feed source for zooplanktonic live prey
5.2.3.3 Benthic microalgae as a feed source for gastropod mollusks and echinoderms
5.2.3.4 Addition of microalgae to fish larval rearing tanks
5.2.3.5 Use of microalgal concentrates in aquaculture hatcheries
5.3 Use of algae in formulated feeds for aquaculture species and terrestrial livestock
5.3.1 Algae as a supplement to enhance the nutritional value of formulated feeds
5.3.1.1 Vitamins and minerals
5.3.1.2 Pigments
5.3.1.3 Fatty acids
5.3.2 Algae as a potential feed ingredient: source of protein and energy
5.4 Outlook
References
6 Algae as an approach to combat malnutrition in developing countries
6.1 Introduction
6.2 Algae in human food
6.3 Microalgae as a solution against malnutrition: meet Spirulina
6.4 Small-scale Spirulina production as a development tool
6.5 Spirulina as a business to combat malnutrition
6.6 Spirulina and its place in food aid and development policies
6.7 Evidence of Spirulina in malnutrition
6.8 Conclusion
Acknowledgements
References
7 Hydrogen production by natural and semiartificial systems
7.1 Biological hydrogen production of microorganisms
7.2 Photobiological hydrogen production by green algae
7.3 Photohydrogenproduction by cyanobacterial design cells
7.4 Photohydrogen production by a “biobattery”
7.5 Photobioreactor design for hydrogen production
7.6 Photobioreactor geometry
7.7 Process control
7.8 Upscaling strategies
References
8 The carotenoid astaxanthin from Haematococcus pluvialis
8.1 Introduction
8.2 Characteristics and biosynthesis
8.2.1 Chemical forms of astaxanthin
8.2.2 Astaxanthin biosynthesis
8.2.3 Function of astaxanthin
8.3 Haematococcus pluvialis
8.3.1 General characteristics
8.3.2 Factors responsible for ax accumulation
8.3.3 Industrial production of Haematococcus
8.4 Conclusions and outlook
References
9 Screening and development of antiviral compound candidates from phototrophic microorganisms
9.1 Introduction
9.2 Supply of natural compounds from microalgae
9.3 Sterilizable photobioreactors
9.4 Antiviral agents from microalgae
9.5 Antiviral screening
9.5.1 Primary target of screening
9.5.2 Smart screening approach
9.5.3 Basic process sequence
9.5.4 Antiviral activity and immunostimulating effects of Arthrospira platensis
9.5.5 Characterization of novel antiviral spirulan-like compounds
9.6 Conclusion
Acknowledgements
References
10 Natural product drug discovery from microalgae
10.1 Introduction
10.1.1 Eukaryotic microalgae
10.1.1.1 Dinoflagellates
10.1.1.2 Diatoms
10.1.2 Cyanobacteria
10.1.2.1 Proteinase inhibitors
10.1.2.2 Cytotoxic compounds
10.1.2.3 Antiviral substances
10.1.2.4 Antimicrobial metabolites
10.1.2.5 Miscellaneous bioactivities
10.1.3 Three examples of current microalgal drug research projects
10.1.3.1 Dolastatins as leads for anti-cancer drugs
10.1.3.2 Cryptophycins as leads for anti-cancer drugs
10.1.3.3 Microcystins as targeted anti-cancer drugs
10.1.4 Outlook
References
Socio-economic and environmental considerations
11 Biorefining of microalgae: Production of high-value products, bulk chemicals and biofuels
11.1 Introduction
11.2. Structural biorefining approach of microalgae
11.2.1 Approach
11.2.2 Cell disruption, fractionation and mild cell disruption of organelles
11.2.3 Extraction and fractionation of high-value components
11.2.4 Economically feasible continuous biorefining concept
11.3. Conclusions
References
12 Development of a microalgal pilot plant: A generic approach
12.1 Understanding the aims of the pilot plant
12.2 Pilot plant location and site selection
12.3 Develop the process flow diagram
12.4 Know what will be required to conduct experiments and measure the data
12.5 Sizing of the units
12.6 Plant layout
12.7 HAZOP study
12.8 Multidisciplinary review of the design
12.9 Tender for plant construction
12.10 Finalize the design
References
13 Finding the bottleneck: A research strategy for improved biomass production
13.1 Introduction: What do we expect from cell engineering?
13.1.1 The need for domestication of microalgae
13.1.2 Limitation of traditional approaches to strain improvement
13.2 Algal domestication through chloroplast genetic engineering
13.2.1 Chloroplast engineering in Chlamydomonas: progress and challenges
13.2.2 A synthetic biology approach to chloroplast metabolic engineering
13.2.3 Mitigating the risks and concerns of GM algae
13.3 Algal domestication through nucleus genetic engineering
13.3.1 Improving light to biomass conversion by regulation of the pigment optical density of algal cultures
13.4 Models for predicting growth in photobioreactors
13.4.1 PAM fluorimetry: a keyhole to look into the photosynthetic machinery
13.4.2 Microalgae cultivation in photobioreactors: the fluctuating light effects
13.4.3 Standard model for growth under an exponential light gradient
13.5 Cells’ response to changing environments: the example of nitrogen limitation
Acknowledgments
References
14 Trends driving microalgae-based fuels into economical production
14.1 Introduction
14.2 Leading trends
14.2.1 Microalgae biorefinery for food, feed, fertilizer and energy production
14.2.2 Biofuel production from low-cost microalgae grown in wastewater
14.2.3 Biogas upgrading with microalgae production for production of electricity
14.2.4 Hydrocarbon milking of modified Botryococcus microalgae strains
14.2.5 Hydrogen production combining direct and indirect microalgae biophotolysis
14.2.6 Direct ethanol production from autotrophic cyanobacteria
14.3 Production platforms
14.3.1 Ocean
14.3.2 Lakes
14.3.3 Raceways
14.3.4 Photobioreactors
14.3.5 Fermenters
14.4 Conclusions
References
15 Microalgal production systems: Global impact of industry scale-up
15.1 Microalgal biotechnology
15.2 Global challenges, production and demand
15.2.1 Global fuel production and demand
15.2.2 Global food production and demand
15.2.3 Solar irradiance and areal requirement
15.2.4 Global challenges
15.3 Potential production and limitations
15.3.1 Solar energy and geographic location
15.3.2 Potential productivity
15.3.3 Land resources
15.3.4 Carbon management and associated costs
15.3.4.1 CO2 requirements
15.3.4.2 CO2 utilization and sequestration
15.3.4.3 CO2 delivery
15.3.5 Nutrient management and associated costs
15.3.5.1 Phosphorus
15.3.5.2 Nitrogen
15.3.5.3 Nutrient recycling
15.3.6 Water management and associated costs
15.4 Global impact of scale-up
15.4.1 Addressing world production
15.4.2 Economics of large-scale microalgal production systems
15.4.3 Techno-economic analysis of microalgal production systems
15.4.3.1 Cultivation systems
15.4.3.2 Impact of capital costs
15.4.3.3 Downstream processing
15.4.3.4 Harvesting and dewatering
15.4.4 Dedicated versus integrated production models
15.4.5 Business models
15.4.6 Pathways to commercialization
15.5 Conclusion
References
Index

Citation preview

Microalgal Biotechnology: Integration and Economy Clemens Posten and Christian Walter (Eds.)

Microalgal Biotechnology: Integration and Economy Editors Clemens Posten and Christian Walter

DE GRUYTER

Editors Prof. Dr. Clemens Posten Institute for Life Science Engineering Bioprocess Engineering Karlsruhe Institute of Technology (KIT) Karlsruhe, Germany [email protected]

Dr. Christian Walter Institute of Bioprocess Engineering Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen-Nürnberg, Germany [email protected]

The book has 83 figures and 28 tables.

isbn 978-3-11-029827-7 e-isbn 978-3-11-029832-1 Library of Congress Cataloging-in-Publication Data A CIP catalog record for this book has been applied for at the Library of Congress. Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the internet at http://dnb.dnb.de. © 2012 by Walter de Gruyter GmbH, Berlin/Boston. The publisher, together with the authors and editors, has taken great pains to ensure that all information presented in this work (programs, applications, amounts, dosages, etc.) reflects the standard of knowledge at the time of publication. Despite careful manuscript preparation and proof correction, errors can nevertheless occur. Authors, editors and publisher disclaim all responsibility and for any errors or omissions or liability for the results obtained from use of the information, or parts thereof, contained in this work. Typesetting: Meta Systems GmbH, Wustermark Printing and binding: Hubert & Co., Göttingen ∞ Printed on acid-free paper. ○ Printed in Germany. www.degruyter.com

Preface Life-forms arise and decay. With the ability for photosynthesis and the generation of oxygen, cyanobacteria as pioneers of the group of microalgae provided the basis for life on earth as we know today. The phototrophic microorganisms have outlived a large number of various species and asserted themselves for 3.5 billion years. Every second oxygen molecule today derives from microalgae and the enzyme RuBisCO, which is responsible for the process of CO2-fixation, is presumably the most abundant protein on earth. In the face of scarcity of resources, increasing problems of overpopulation and demographic aging, as well as the trend of health and well-being in the industrial countries, microalgae are increasingly becoming the subject of debate as part of a sustainable solution. But, is it feasible to make the enormous biodiversity of the tiny coloured cells technically useful? And is the potential with respect to energetic use or agricultural, feed, food and medical applications fit for use in a good economic sense and ecologically worthwhile? What can we expect for the future? The weather forecasting as a statement about the way things will happen in the future is a game of probabilities. And the same is valid in the case of technological predictions. Short-term predictions are too optimistic, because of euphoria and ignorance of the inherent problems. Long-range foresights turn out more pessimistic on the basis of current knowledge and lack of imagination at the time the prediction is made. The truth lies in the middle. The perils of technological predictions are shown by various examples in history. Aviation pioneer Wilbur Wright predicted in 1901: “Don't think men will fly for a thousand years”. In 1886, the engineer Gottlieb Daimler expressed that: “The global demand for automobiles will not surpass one million, … due to a lack of available chauffeurs”. In 1920 the engineer Carl Benz remarked that: “The car is finally developed.” The former chairman and CEO of IBM Thomas Watson stated in 1943: “I think there is a world market for maybe five computers.” And Bill Gates can be cited from 1995 with a statement that: “The internet is just a hype”. Something similar is often attributed to microalgal biotechnology: does not work in (near) future and in a competitive way, too many technical limitations, very small market, just a hype. For microalgae, currently there is a phase of consolidation with the step from vision to pragmatism. Frame conditions are changing and in general the volume of new information is generated with increasing acceleration. Already today there are significant applications of microalgae in different fields, new markets are burgeoning and the question is not whether, but to what extent algae will be used in the future. This book will be of assistance to enhance the prediction accuracy of how to go forward with microalgae. And the reader is invited to get an idea of fields of application and technical implementation of microalgal biotechnology in the

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Preface

future. As in weather forecasts and the history of technical predictions – for sure – we can expect surprises. In view of this, I wish you stimulating and beneficial reading! Burghausen, December 2012

Christian Walter

Contents Preface v List of contributing authors

1 1.1 1.2 1.3 1.4

2 2.1 2.2 2.2.1 2.2.2 2.2.2.1 2.2.2.2 2.2.2.3 2.2.3 2.2.4 2.2.5 2.2.6 2.2.6.1 2.2.6.2

2.2.6.3 2.2.6.4 2.2.6.5 2.2.7 2.2.8 2.2.9 2.2.9.1 2.2.9.2 2.2.9.3 2.2.9.4 2.2.9.5

xv

Clemens Posten Introduction – Integration in microalgal biotechnology Integration on the process level 2 Integration on the metabolic level 4 Integration into environmental conditions 5 6 Adaptation to cultural realities Integrated production processes Rosa Rosello Sastre 13 Products from microalgae: An overview Microalgae: An introduction 13 Products 15 15 Use and production of algal biomass Microalgae for human nutrition 18 Spirulina (Arthrospira) 19 20 Chlorella 21 Dunaliella salina 21 Microalgae for animal feed 22 Microalgae as natural fertilizer 22 Microalgae in cosmetics 23 Fine chemicals 23 PUFAs 26 Pigments 26 Pigments as antioxidants 28 Pigments as natural colorants 29 Polysaccharides 31 Recombinant proteins 31 Stable isotopes 31 Micro- and nanostructured particles 33 Bulk chemicals 35 Energy production from microalgae 35 Biodiesel 40 Bio-ethanol 41 Bio-hydrogen 42 Bio-gas 43 Biorefinery of microalgae

1

viii 2.3

3 3.1 3.2 3.3 3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12

4

Contents

Conclusion References

44 44

Min Thein, Toe Aung, Khin Pyone Lwin, May Yu Khaing and Otto Pulz Spirulina production in volcano lakes: From natural resources to human welfare 51 Introduction 51 Natural Spirulina lakes in Myanmar 52 Environmental parameters of Myanmar Spirulina lakes 54 Spirulina production from natural lakes 57 Harvesting 57 Washing and dewatering 58 Extrusion and sun drying 59 Lake-side enhancement ponds 61 Sustainable Spirulina production from volcanic crater lakes 62 63 Myanmar Spirulina products 64 Spirulina as biofertilizer 67 Spirulina as a biogas enhancer 67 Spirulina as a source of biofuel 67 Myanmar and German cooperation in microalgae biotechnology 68 Discussion 68 Conclusion 69 Acknowledgments 69 References C. Thomsen, S. Rill and L. Thomsen Case study of a temperature-controlled outdoor PBR system in Bremen 73 77 Acknowledgments 77 References

Robin Shields and Ingrid Lupatsch 5 Algae for aquaculture and animal feeds 79 79 5.1 Introduction 79 5.2 Microalgae use in aquaculture hatcheries 80 5.2.1 Microalgal strains used in aquaculture hatcheries 82 5.2.2 Methods of microalgae cultivation for aquaculture 82 5.2.3 Role of microalgae in aquaculture hatcheries 5.2.3.1 Microalgae as a feed source for filter-feeding aquaculture 82 species 83 5.2.3.2 Microalgae as a feed source for zooplanktonic live prey 5.2.3.3 Benthic microalgae as a feed source for gastropod mollusks and 84 echinoderms

Contents

5.2.3.4 5.2.3.5 5.3 5.3.1 5.3.1.1 5.3.1.2 5.3.1.3 5.3.2 5.4

6 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8

7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8

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85 Addition of microalgae to fish larval rearing tanks Use of microalgal concentrates in aquaculture hatcheries 87 Use of algae in formulated feeds for aquaculture species and terrestrial livestock 88 Algae as a supplement to enhance the nutritional value of formulated feeds 88 Vitamins and minerals 88 Pigments 89 Fatty acids 90 Algae as a potential feed ingredient: source of protein and energy 90 Outlook 95 References 96 Christophe Hug and Denis von der Weid Algae as an approach to combat malnutrition in developing 101 countries Introduction 101 Algae in human food 101 Microalgae as a solution against malnutrition: meet Spirulina 102 Small-scale Spirulina production as a development tool 103 Spirulina as a business to combat malnutrition 104 Spirulina and its place in food aid and development policies 106 Evidence of Spirulina in malnutrition 107 Conclusion 109 Acknowledgements 109 References 109 Thomas Happe, Camilla Lambertz, Jong-Hee Kwon, Sascha Rexroth and Matthias Rögner 111 Hydrogen production by natural and semiartificial systems Biological hydrogen production of microorganisms 111 Photobiological hydrogen production by green algae 115 Photohydrogenproduction by cyanobacterial design cells 117 Photohydrogen production by a “biobattery” 119 Photobioreactor design for hydrogen production 120 Photobioreactor geometry 121 Process control 122 Upscaling strategies 123 References 124

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8 8.1 8.2 8.2.1 8.2.2 8.2.3 8.3 8.3.1 8.3.2 8.3.3 8.4

Claudia B. Grewe and Carola Griehl The carotenoid astaxanthin from Haematococcus pluvialis Introduction 129 Characteristics and biosynthesis 130 Chemical forms of astaxanthin 130 Astaxanthin biosynthesis 131 Function of astaxanthin 133 Haematococcus pluvialis 133 General characteristics 133 Factors responsible for ax accumulation 135 Industrial production of Haematococcus 138 Conclusions and outlook 140 References 140

9 9.1 9.2 9.3 9.4 9.5 9.5.1 9.5.2 9.5.3 9.5.4 9.5.5 9.6

129

Christian Walter Screening and development of antiviral compound candidates from phototrophic microorganisms 145 Introduction 145 146 Supply of natural compounds from microalgae 147 Sterilizable photobioreactors 150 Antiviral agents from microalgae 153 Antiviral screening 153 Primary target of screening 153 Smart screening approach 154 Basic process sequence Antiviral activity and immunostimulating effects of Arthrospira 156 platensis 157 Characterization of novel antiviral spirulan-like compounds 161 Conclusion 162 Acknowledgements 162 References

Timo Niedermeyer and Mark Brönstrup 10 Natural product drug discovery from microalgae 169 10.1 Introduction 170 10.1.1 Eukaryotic microalgae 170 10.1.1.1 Dinoflagellates 172 10.1.1.2 Diatoms 172 10.1.2 Cyanobacteria 174 10.1.2.1 Proteinase inhibitors 175 10.1.2.2 Cytotoxic compounds 179 10.1.2.3 Antiviral substances

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10.1.2.4 10.1.2.5 10.1.3 10.1.3.1 10.1.3.2 10.1.3.3 10.1.4

11 11.1 11.2. 11.2.1 11.2.2 11.2.3 11.2.4 11.3.

179 Antimicrobial metabolites Miscellaneous bioactivities 180 Three examples of current microalgal drug research projects Dolastatins as leads for anti-cancer drugs 183 Cryptophycins as leads for anti-cancer drugs 186 Microcystins as targeted anti-cancer drugs 187 Outlook 187 References 189

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183

Socio-economic and environmental considerations Michel H. M. Eppink, Maria J. Barbosa and Rene H. Wijffels Biorefining of microalgae: Production of high-value products, bulk chemicals and biofuels 203 Introduction 203 Structural biorefining approach of microalgae 205 Approach 205 Cell disruption, fractionation and mild cell disruption of organelles 208 Extraction and fractionation of high-value components 210 Economically feasible continuous biorefining concept 211 Conclusions 212 References 213

Garry Henderson 215 Development of a microalgal pilot plant: A generic approach Understanding the aims of the pilot plant 215 Pilot plant location and site selection 216 Develop the process flow diagram 216 Know what will be required to conduct experiments and measure the data 217 12.5 Sizing of the units 217 12.6 Plant layout 219 12.7 HAZOP study 221 12.8 Multidisciplinary review of the design 224 12.9 Tender for plant construction 224 12.10 Finalize the design 225 References 225 12 12.1 12.2 12.3 12.4

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13 13.1 13.1.1 13.1.2 13.2 13.2.1 13.2.2 13.2.3 13.3 13.3.1 13.4 13.4.1 13.4.2 13.4.3 13.5

Contents

Roberto Bassi, Pierre Cardol, Yves Choquet, Thomas de Marchin, Chloe Economou, Fabrice Franck, Michel Goldschmidt-Clermont, Anna Jacobi, Karen Loizeau, Gregory Mathy, Charlotte Plancke, Clemens Posten, Saul Purton, Claire Remacle, Carsten Vejrazka, Lili Wei and Francis-André Wollman Finding the bottleneck: A research strategy for improved biomass production 227 Introduction: What do we expect from cell engineering? 227 The need for domestication of microalgae 227 Limitation of traditional approaches to strain improvement 228 Algal domestication through chloroplast genetic engineering 229 Chloroplast engineering in Chlamydomonas: progress and challenges 229 A synthetic biology approach to chloroplast metabolic engineering 232 Mitigating the risks and concerns of GM algae 234 Algal domestication through nucleus genetic engineering 235 Improving light to biomass conversion by regulation of the pigment optical density of algal cultures 235 Models for predicting growth in photobioreactors 237 PAM fluorimetry: a keyhole to look into the photosynthetic machinery 237 Microalgae cultivation in photobioreactors: the fluctuating light 240 effects Standard model for growth under an exponential light 244 gradient Cells’ response to changing environments: the example of nitrogen 247 limitation 249 Acknowledgments 249 References

Vítor Verdelho, Ana P. Carvalho, Diana Fonseca and João Navalho Trends driving microalgae-based fuels into economical 253 production 253 14.1 Introduction 254 14.2 Leading trends 14.2.1 Microalgae biorefinery for food, feed, fertilizer and energy 254 production 14.2.2 Biofuel production from low-cost microalgae grown in 255 wastewater 14.2.3 Biogas upgrading with microalgae production for production of 257 electricity 14

Contents

14.2.4 14.2.5 14.2.6 14.3 14.3.1 14.3.2 14.3.3 14.3.4 14.3.5 14.4

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Hydrocarbon milking of modified Botryococcus microalgae strains 257 Hydrogen production combining direct and indirect microalgae biophotolysis 258 Direct ethanol production from autotrophic cyanobacteria 259 Production platforms 262 Ocean 262 Lakes 263 Raceways 263 Photobioreactors 263 Fermenters 263 Conclusions 263 References 264

Evan Stephens, Liam Wagner, Ian L. Ross and Ben Hankamer Microalgal production systems: Global impact of industry scale-up 267 15.1 Microalgal biotechnology 267 15.2 Global challenges, production and demand 268 15.2.1 Global fuel production and demand 268 15.2.2 Global food production and demand 269 15.2.3 Solar irradiance and areal requirement 270 271 15.2.4 Global challenges 272 15.3 Potential production and limitations 272 15.3.1 Solar energy and geographic location 274 15.3.2 Potential productivity 276 15.3.3 Land resources 278 15.3.4 Carbon management and associated costs 278 15.3.4.1 CO2 requirements 279 15.3.4.2 CO2 utilization and sequestration 280 15.3.4.3 CO2 delivery 281 15.3.5 Nutrient management and associated costs 282 15.3.5.1 Phosphorus 282 15.3.5.2 Nitrogen 282 15.3.5.3 Nutrient recycling 283 15.3.6 Water management and associated costs 285 15.4 Global impact of scale-up 285 15.4.1 Addressing world production 287 15.4.2 Economics of large-scale microalgal production systems 288 15.4.3 Techno-economic analysis of microalgal production systems 288 15.4.3.1 Cultivation systems 289 15.4.3.2 Impact of capital costs 15

xiv 15.4.3.3 15.4.3.4 15.4.4 15.4.5 15.4.6 15.5

Index

Contents

Downstream processing 290 290 Harvesting and dewatering Dedicated versus integrated production models 294 Business models 296 Pathways to commercialization 299 Conclusion 301 References 307

292

List of contributing authors Toe Aung Ministry of Industry, Myanmar Pharmaceutical Factory, Sagaing, Myanmar Chapter 3 Roberto Bassi Department of Biotechnology, University of Verona, Verona, Italy Chapter 13 Maria Barbosa Food & Bioprocess Engineering Group, Wageningen University, Wageningen, The Netherlands e-mail: [email protected] Chapter 11 Mark Brönstrup Biomarker & Diagnostics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany e-mail: [email protected] Chapter 10 Pierre Cardol Genetics of microorganisms, University of Liège, Liège, Belgium Chapter 13 Ana P. Carvalho Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Chapter 14 Yves Choquet Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris, France Chapter 13 Chloe Economou Algal Research Group, Department of Biology, University College London, London, United Kingdom Chapter 13

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List of contributing authors

Michel Eppink Food & Bioprocess Engineering Group, Wageningen University, Wageningen, The Netherlands e-mail: [email protected] Chapter 11 Diana Fonseca Alga Fuel, S.A., Belamandil, Olhão, Portugal Chapter 14 Fabrice Franck Laboratory of bioenergetics, University of Liège, Liège, Belgium Chapter 13 Michel Goldschmidt-Clermont Department of Molecular Biology, University of Geneva, Genève, Switzerland Chapter 13 Claudia Grewe Astaxa GmbH, Milz, Germany e-mail: [email protected] Chapter 8 Carola Griehl Dept. of Applied Biosciences and Process Technology, Anhalt University of Applied Sciences, Köthen, Germany e-mail: [email protected] Chapter 8 Ben Hankamer Institute of Molecular Bioscience, The University of Queensland, St Lucia, Australia e-mail: [email protected] Chapter 15 Thomas Happe Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität Bochum, Bochum, Germany e-mail: [email protected] Chapter 7 Garry Henderson Brisbane, Australia e-mail: [email protected] Chapter 12

List of contributing authors

xvii

Christophe Hug Fondation Antenna Technologies, Genève, Switzerland e-mail: [email protected] Chapter 6 Anna Jacobi Institute of Process Engineering in Life Sciences, Bioprocess Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany, Marcel Janssens, Bioprocess Engineering, Wageningen University and Research Center, Wageningen, The Netherlands Chapter 13 May Yu Khaing Ministry of Industry, Myanmar Pharmaceutical Factory, Sagaing, Myanmar Chapter 3 Jong-Hee Kwon Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität Bochum, Bochum, Germany e-mail: [email protected] Chapter 7 Camilla Lambertz Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität Bochum, Bochum, Germany e-mail: [email protected] Chapter 7 Karen Loizeau Department of Molecular Biology, University of Geneva, Genève, Switzerland Chapter 13 Ingrid Lupatsch Centre for Sustainable Aquatic Research, Department of Biosciences, College of Science, Swansea University, Swansea, UK e-mail: [email protected] Chapter 5 Khin Pyone Lwin Ministry of Industry, Myanmar Pharmaceutical Factory, Sagaing, Myanmar Chapter 3

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List of contributing authors

Thomas de Marchin Laboratory of Bioenergetics, University of Liège, Liège, Belgium Chapter 13 Gregory Mathy Laboratory of Bioenergetics, University of Liège, Liège, Belgium Chapter 13 João Navalho Alga Fuel, S.A., Belamandil, Olhão, Portugal Chapter 14 Timo Niedermeyer Head of Natural Product Chemistry, Cyano Biotech GmbH, Berlin, Germany e-mail: [email protected] Chapter 10 Charlotte Plancke Genetics of microorganisms, University of Liège, Liège, Belgium Chapter 13 Clemens Posten Institute for Life Science Engineering Bioprocess Engineering Karlsruhe Institute of Technology (KIT) Karlsruhe, Germany e-mail: [email protected] Chapter 1, 13 Otto Pulz IGV GmbH, Nuthetal, Germany Chapter 3 Saul Purton Algal Research Group, Department of Biology, University College London, London, United Kingdom Chapter 13 Claire Remacle Department of Life Sciences, Institute of Botany, University of Liège, Liège, Belgium e-mail: [email protected] Chapter 13

List of contributing authors

Sascha Rexroth Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität Bochum, Bochum, Germany e-mail: [email protected] Chapter 7 Stefan Rill Phytolutions GmbH, Bremen, Germany Chapter 4 Matthias Rögner Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität Bochum, Bochum, Germany e-mail: [email protected] Chapter 7 Rosa Rosello Institut für Bio- und Lebensmittel- technik, Bereich III: Bioverfahrenstechnik, Geb. 30.44, Karlsruher Institut für Technologie – KIT, Karlsruhe, Germany e-mail: [email protected] Chapter 2 Ian Ross Institute of Molecular Bioscience, The University of Queensland, St Lucia, Australia e-mail: [email protected] Chapter 15 Robin Shields Centre for Sustainable Aquatic Research, Department of Biosciences, College of Science, Swansea University, Swansea, UK e-mail: [email protected] Chapter 5 Evan Stephens Institute of Molecular Bioscience, The University of Queensland, St Lucia, Australia e-mail: [email protected] Chapter 15

xix

xx

List of contributing authors

Min Thein Ministry of Industry, Myanmar Pharmaceutical Factory, Sagaing, Myanmar e-mail: [email protected] Chapter 3 Claudia Thomsen Phytolutions GmbH, Bremen, Germany e-mail: [email protected] Chapter 4 Laurenz Thomsen Jacobs University Bremen, Bremen, Germany Chapter 4 Vítor Verdelho Alga Fuel, S.A., Belamandil, Olhão, Portugal e-mail: [email protected] Chapter 14 Liam Wagner Institute of Molecular Bioscience, The University of Queensland, St Lucia, Australia e-mail: [email protected] Chapter 15 Christian Walter Institute of Bioprocess Engineering, Friedrich-AlexanderUniversität Erlangen-Nürnberg, Erlangen, Germany e-mail: [email protected] Chapter 9 Lili Wei Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie Paris, France Chapter 13 Denis von der Weid Fondation Antenna Technologies, Genève, Switzerland Chapter 6

List of contributing authors

xxi

René Wijffels Food & Bioprocess Engineering Group, Wageningen University, Wageningen, The Netherlands e-mail: [email protected] Chapter 11 Francis-André Wollman Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris, France Chapter 13

Clemens Posten

1 Introduction – Integration in microalgal biotechnology The broad palette of microalgal products fascinates scientists worldwide. These include valuable oils, proteins and carbohydrates, as well as vitamins and minerals. People concerned about the future welfare of our world discuss the contribution of microalgae to fuel and feed a rapidly growing population. Anticipated applications are found in food, feed or pharma-industry on the one hand, and in fine or bulk chemicals and fuels on the other, see figure 1.1. But to move from the biological potential to sustainable and economically viable production processes, the integration of microalgae into processes, into the environment and into our society is required. Process development comprises the selection of strains and media in an upstream stage, the cultivation in photobioreactors in a bioreaction stage and product recovery and formulation in the downstream stage. In this direction a number of accomplishments have already been made, but more work is waiting to be done. In addition to the optimization of single process stages, the structure of entire processes has to be reviewed. But even more, behind the open technological questions, problems of integration of microalgal biotechnology into our society with market constraints and cultural

Fig. 1.1: Different products from microalgae and their possible fields of application.

2

1 Introduction – Integration in microalgal biotechnology

realities are lurking. Many small and middle sized companies have been founded but disappeared after the first failure either of their technology or their financial framework. But other enterprises appear to be having better success than their predecessors. That algae has definitely reached the public discussion in our society is finally clear from Obamas claim about “jet fuel that’s actually made from a plant-like substance – algae” (2012). Solutions are not found in single technologies but in an interplay of different fields of biology and engineering, as well as social and cultural sciences. Economically viable applications depend on local markets and the availability of human resources. Success stories and further anticipated developments are outlined in this book.

1.1 Integration on the process level The first step in integration lies in adjusting the different process steps to each other. Concerning a contribution of upstream operations to the performance of the cultivation, the focus could be more on process-oriented strain selection. This includes efficiency even at high irradiation rates, mechanical stability or lower sensitivity to temperature changes. Even seemingly simple things like medium composition have to be revisited. Many groups in research and industry work on renewed recipes to allow for higher cell densities, avoiding organic buffers and providing a completely balanced nutrient composition for easy medium recycling. A smart strain selection can even make downstream operations easier. Flocculation on command is often mentioned in this context. Even using filamentous algae is an option from this point of view. An alternative type of cultivation is the growth of algae in biofilms. This approach is less studied than suspended cultures, but could reduce auxiliary energy input and cell harvesting costs due to higher cell densities. This has already been shown to be of advantage for diatoms. Like many other microorganisms they tend to live attached to surfaces. Instead of forcing them to go into suspension, cultivation on plastic sheets or submersed strings allows easy harvesting by shedding. Further downstream in the process, operations like extraction or cell disruption can be optimized by looking at the cell wall of the production strain already during screening in the upstream stage. Adjusting cultivation and cell separation can be optimized by calculating a “window of operation”. For microalgae this means that an increasing cell density, eventually accompanied by a slightly lower efficiency, can be paid back by a more efficient separation step. To double the cell concentration from e.g. 3 g/L to 6 g/L means reducing the water flux through the centrifuge and therefore the energy demand for harvesting by 50 % per kg algae dry mass. A pre-concentration step can be helpful as well and must of course be adapted to cultivation on one hand and solid/liquid-separation on the other. Measuring and control techniques also have to be adapted to algae-specific demands. This is especially true in outdoor production facilities where rapidly

1.1 Integration on the process level

3

changing light intensities and temperatures are faced. Supplying carbon dioxide according to the light dependent photosynthetic activity is an option towards lower energy consumption and higher productivities. The regulation of mixing energy and carbon dioxide input in dependence on biomass concentration and activity can contribute to the reduction of auxiliary energy. This approach is often claimed but rarely implemented. Furthermore, in contrast to heterotrophic microorganisms, microalgae pass through a diurnal cycle synchronized by solar radiation. Over the day lipids are accumulated which are metabolized to proteins and other cell compartments during the night. If enough light is provided over the day, the cell division is also synchronized to the daily cycle. These cycles have to be considered for the choice of continuous or semi-continuous processes and for cell-harvesting cycles. For the realization of the above point good optical measurement techniques can be applied due to a clear media and specific optical characteristics of the cells. Light scattering techniques give information about morphological parameters of the cell and fluorescence measurements such as pulse amplitude modulation (PAM) allow for a suitable determination of the physiological activity. An even more intensive process operation is achieved by integration the single steps into each other, what is the case for in-situ product removal. This can save the cell harvesting step and can lead to a continuous product stream. One wellknown example, which has been applied to microalgae, is the extraction of products by direct contact of a solvent with the growing biomass. This “milking” has been done with a two phase system with water-immiscible solvents to extract carotinoids from Dunaliella or to extract hydrocarbons stored in the outer matrix of Botryococcus braunii cell colonies. In the best case the product is volatile or gaseous, thus leaving the reactor without any mechanical cell separation steps. That is e.g. the case for hydrogen production. For the cultivation it means operation in a steady-state modus avoiding cellular energy expenditure for cell mass formation. For the time being, an energy neutral production of microalgal biomass and therefore mass cultivation only for production of chemical energy carriers is difficult. Here the idea is, to use the residual biomass from the production of high and middle value products for biofuel production. This idea of splitting an educt into several products of different chemical composition and value is called the biorefinery. The easiest way for a technical realization of this principle is to first extract the product and then secondly to convert the biomass in a biogas plant to methane. But the biorefinery can be more specific. Oil can be fractionated into high value polyunsaturated fatty acids as food supplements, into middle value chemical precursors and into biodiesel using a low value fraction with saturated lipids. In principle, proteins are highly structured molecules, into which the cell invested quite some metabolic energy. So they are far too valuable for burning and should be used for food or feed. By the way, proteins can also be used as precursors for plastics production (e.g. polyamides). Finally carbohydrates can easily be degraded

4

1 Introduction – Integration in microalgal biotechnology

in the biogas plant. This idea of the biorefinery is subject to recent research. On the cellular level, the question of how far cell stoichiometry can be shifted towards the desired product stands in the foreground. As nothing is for free, it has to be determined, on what energetic costs that is possible. Oil production to high contents is for example done by the cell with a lower energetic efficiency than the formation of carbohydrates, it takes longer and it is accompanied by cell stress. It is a matter of optimization, which oil content is finally most profitable, it is not necessarily the highest possible. On the process level, different ways of separation, extraction and fractionation are investigated. The specific problem here is that the biomass is quite wet and the different steps have to be cheap and of low energy consumption. On the market level it can be asked, whether there are enough high value products to contribute to the world’s fuel supply remarkably with the residual biomass. Maybe food and aquaculture can solve the problem without further chemical separation steps.

1.2 Integration on the metabolic level To take benefit from the photosynthetic capacity of microalgae does not automatically mean that they are the best producers of a desired product. In such cases, the metabolic steps of several microorganisms can be integrated into one process. This process of “cross-feeding” is already known from natural symbioses. A primary organism such as an alga converts the primary substrate, in case of algae light and carbon dioxide, to a metabolite. This metabolite serves as a product for a heterotrophic culture. Primary products such as intracellular starch, extracellular polysaccharides or even cell wall compounds can be provided to feed subsequent heterotrophic steps. A co-cultivation between algae and bacteria is often proposed in this context, but that has its limits in mutual shading and different optimum values for the respective environmental parameters. An example of the pattern of a phototrophic stage followed by a heterotrophic stage is the conversion of microalgae-produced starch into ethanol, analog to the conversion of starch from corn. Here starch is produced phototrophically, hydrolyzed by extracellular amylases of Aspergillus and finally converted to bioethanol by yeasts. The principle advantage is that every organism has to do the things it does best, e.g. yeast is already, in contrast to microalgae, very ethanol tolerant. The ethanol can be used as a fuel or as a platform chemical. Another example is the production of isoprene from microalgae excreted oils. In general, photosynthetic products should be used as the substrate for cross-feeding, which require only little metabolic energy during its production in the cell. This principal has been implemented in a process, where a Chlamydomonas biofilm produces and excretes only glycolate, an early metabolite in the citric cycle, which is then fed to a heterotrophic stage. This is an anaerobic culture adapted to glycolate as the sole carbon substrate which converts it to biogas. In this way the idea of cross feeding

1.3 Integration into environmental conditions

5

and in-situ product removal is combined. In this continuous process only low amounts of energy are wasted in the growth of algae as such or in external separation steps. Microalgae are discussed as perfect photosynthetic organisms on the one hand, and as producers of high-value compounds on the other. But both functions can be separated, in principle, in the sense of cross feeding. The market leader for large volumes of polyunsaturated fatty acids produces these PUFAs with heterotrophically grown microalgae. This uses the ability of the algae to accumulate high oil concentrations. But as the algae are fed with sugar, the whole process is only as energetically effective as the production of sugar cane is. Here a first stage with another microalga producing carbohydrates as feed could potentially bring more energetic efficiency to the process. The next logical step is to integrate autotrophic and heterotrophic metabolic steps genetically into one alga strain. Then the desired product is directly produced by the algae. A company developed an ethanol process in which a transgenic alga directly converts sunlight energy and carbon dioxide into ethanol. The abovedescribed process using two organisms, first an autotrophic organism to produce starch which is then converted into ethanol in a heterotrophic fermentation, is lumped into one process step. Furthermore the ethanol produced is directly evaporated by the excess heat from sun irradiation. This allows for a continuous process without cell separation. Whether the anticipated areal productivities of more than 50 t ethanol / ha / y from a designed plant of several hectare in Mexico can be reached is not proven, but this process demonstrates the possibility of a highly integrated process design.

1.3 Integration into environmental conditions Already existing examples for sustainable processes using microalgae are based on the smart interconnection with existing environmental mass and energy fluxes. Supply with sea water to compensate evaporation is the most obvious solution for the water management problem. Near-costal grounds or even waters are available to allow for algae plants in square-km scale. But also open ponds in the middle of a continent using brackish ground water are described. In this context the usage of cheap industrial low temperature heat for temperature control of closed photobioreactors should also be mentioned. Locations in the middle latitudes which are sunny in early spring but too cold for algal cultivation could be opened for operation. Next to water supplies, nutrients could become a logistic and financial problem. While ammonia production is an energy efficient process, phosphate is anticipated to become scarce in the future. Of course it is a must to recycle nutrients and water directly at the microalgal production plant, wherever possible. After extraction of oil for example and using proteins for feed, phosphate could be recov-

6

1 Introduction – Integration in microalgal biotechnology

ered from the residual biomass. Municipal waste water is a possible nitrogen and phosphate source. Consequently, a lot of research in this direction has started in the last few years. The goal is to install a short-cut between buying expensive nitrogen compounds as proteins or fertilizers on the market and destroying them finally in the waste water plant. Here algae can make a valuable contribution, not only to sewage treatment but to recycling valuable nutrients. Flue gas from coal or gas combustion, off-gas streams from chemical plants or lime kilns as well as other technically available carbon dioxoide sources from industrial regions are usually produced remote from possible microalgal production sites. The deeper meaning of running microalgal pilot plants close to combustion plants does not lie in cleaning the flue gas but in supplying higher carbon dioxide concentrations to the algae. That is necessary to overcome the mass transfer limitations between gas phase and fluid phase and to elevate the dissolved carbon level to avoid carbon dioxide uptake limitations. Coupling of algal cultivations to off-gas from heterotrophic fermentations as during cross-feeding would allow for a partial carbon recycling directly on a production site. Carbon dioxide from air would, in principle, avoid all logistic problems. Here some work is going on to investigate the thermodynamic limitations of CO2-concentrating from the atmosphere. The next necessity after water, nutrients, and carbon dioxide is the demand for auxiliary energy. The installation of photovoltaic panels on 10 % of an algal cultivation area to produce electrical energy has already been proposed. That sounds at the first glance contradictory to the idea of sustainability of algae, but it makes sense in so far as auxiliary energy for the algae is needed mainly during sunny hours. Photovoltaic directly at the site can meet this demand better than other energy sources as it makes conduction and storage of the electrical energy superfluous. Furthermore non-effective wavelengths contained in the solar radiation can be used. Infra-red radiation cannot be converted by the photosystem of microalgae, but causes heating of the cultivation medium. Instead of only reflecting IR radiation to reduce overheating, it could be converted into electrical energy employing so called transparent photovoltaic panels. Even a modest efficiency of about 15 % could be enough to supply the energy demand of the peripheral devices (pumps, pressured air, sensors). The final goal is the operation of a “stand-alone” or “plug-free” microalgal plant.

1.4 Adaptation to cultural realities Scientific research is the gate between the biological potential of the algae and the supply of products in line with the market. In the last few years visibility of microalgal biotechnology to the general public was generated by press releases like “Venture Beat: $ 144m new investment in algae”, or “… a federation which will bring together all the players in the development of micro-algae in France. A

1.4 Adaptation to cultural realities

7

Fig. 1.2: Mini-plate photobioreactors to gain kinetic growth and production parameters; in this example strains growing in the same cell density but with different antenna sizes are compared.

budget of US$ 210 million for 10 years has been estimated …”. Indeed, the amount of money which is spent now for research on microalgae goes into roughly estimated hundred millions per year. While it may be more focused on large flagship initiatives in the US it is not much less in Asia or the EU. Large scientific consortia have been established to integrate different scientific fields of basic and applied sciences. Pilot and demonstration plants are emerging all over the world. In this book some of these activities are presented by the responsible actors themselves. A large European research consortium has summarized its vision on “debottlenecking” microalgal metabolism to go for a smarter and more efficient production process, see also figure 1.2. For the time being, money from microalgae is made mainly as healthy food and feed supplements in comparably small markets. A few years ago normal consumers and even scientists did not believe that algae products could be an ingredient of the daily diet in really large volumes. Probably the imagination was a greenish, strange smelling something on the plate that the typical American or European consumer would not like. This assessment came without considering that even now products from naturally growing macroalgae (polysaccharides like Alginate or Agar-Agar, E401–E406) are processed in standard foods. In the meantime, big food companies have already developed and patented processes to gain color, smell and tasteless protein extracts from microalgae. Even if the application of these technologies in large volumes will take some time to be applied in developed countries, microalgae will contribute remarkably to supplying healthy food to a growing and hungry world population. Bulk products like ethanol or alkanes as platform chemicals are a further need of our society. Around 5 %–10 % of oil is being used for chemical syntheses. This

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1 Introduction – Integration in microalgal biotechnology

Fig. 1.3: Picture taken from the first flight (Berlin) of an airplane using solely fuel from microalgae (copyright eads).

demand can be covered by microalgae as well, forming a new green chemistry. That will not go without cloning the respective genes into the algae (see above). Other targets for genetic engineering are sharpening of product profiles, truncated antenna pigments or pesticide resistance. Genetic engineering will be applied to microalgae as a normal tool like it is already done in other fields of industrial biotechnology and in parts of agriculture despite the un-tapped natural diversity. According to Craig Venter “algae can feed the world and fuel the planet, … , we just have to combine genes in a way that nature has not done before” (2011). However, integration of microalgal biotechnology into our society must balance risks, opportunities and ethical considerations in the different fields of applications. This book offers a contribution towards developing a active discussion in this direction. The current hype in microalgal biotechnology is driven by the need for sustainable energy. The main driver is the aircraft industry. This is due to the fact that airplanes cannot fly with hydrogen or electricity from batteries but need liquid fuels of high energy density. Show flights with different airplanes and algae fuels have impressed the public (see e.g. fig. 1.3) but are only the tip of the iceberg of current industrial activities. How long will it take until algal biofuel is commercially available? Several large microalgal companies claim they will achieve technology readiness in the near term. However, algal mass production will not emerge in one day. Research and application have to move forward over several stepping stones characterized by larger areas but lower costs. Today we produce high value products in only a few thousand tons volume. Market prices for algae reach 50 $/kg dry mass or more.

1.4 Adaptation to cultural realities

9

The next steps will be food, feed for cattle and aquaculture. Especially this last item is intensively addressed facing empty oceans and the need for fish meal from natural catch to get healthy fish in aquaculture. Bulk products are going to follow where the final price of the product may not exceed 1 or 2 $/kg. For a remarkable contribution thousands of square km of algae production facilities are necessary. Only by learning from these applications will it be possible to reach really large amounts for biofuel production, where the process has to be completely energy neutral and the costs of the final product like biodiesel can compete with other sources. This book will accompany the reader along these stepping stones towards a really beneficial future of microalgal biotechnology.

Integrated production processes

Rosa Rosello Sastre

2 Products from microalgae: An overview 2.1 Microalgae: An introduction Microalgae are expected to become a renewable source with enormous potential for a wide variety of products. This chapter will give an overview over the current technological and commercial situation regarding products from microalgae, ranging from well-established applications in the field of cosmetics, human health and fine chemicals, to the market development of new mass products for the food and feed industry, bulk chemicals or biofuels. The process to obtain high-value products from microalgae has already been established, but the implementation of microalgae as relevant producers in middle- to low-value product markets requires further technological developments to improve the economic efficiency of current technologies. The Latin word “alga” describes an organism containing chlorophyll a and a vegetation body (thallus), which is not divided into roots, a stem and leaves (Lee 1989). Initially, only macroscopic marine plants such as seaweeds and kelps were defined by this term, but microscopic phototrophic organisms (microalgae) are also now included. Most microalgae, such as the green, red, brown algae and diatoms, are eukaryotic. The blue-green algae (cyanobacteria) are prokaryotic. Consequently, the word “alga” describes a life form, not a biological group. Microalgae show a greater diversity of taxonomic groups than higher plants (see Friedl et al. 2012).

Microalgae

Production (t/yr)

Comments

Totally processed algae biomass (micro and macro) Total global production Microalgal biomass Spirulina

9,000,000

Harvested from natural habitats and aquacultures

Chlorella Dunaliella salina Nostoc Aphanizomenon flos-aquae Haematococcus pluvialis Crypthecodinium cohnii Schizochytrium Aquaculture species

>10,000 5,000 3,000 2,000 1,200 600 500 300 240 10 >1,000

Dry weight (>1500 as feed supplement, ≥ 1000 for human nutrition) Dry weight Dry weight Dry weight Dry weight Dry weight Docosahexaenoic acid (DHA) DHA

Tab. 2.1: Global production rates of the commercial most relevant microalgae (Pulz and Gross 2004; Spolaore 2006).

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2 Products from microalgae: An overview

Fig. 2.1: Overview of technical relevant algae species. From left to right: microscopic image of the multicellular filamentous cyanobacterium Spirulina. The filaments are composed of cylindrical cells arranged in unbranched, helicoidaltrichomes (Richmond 1986); different morphologies of the cells from the diatom Phaeodactylum tricornutum depending on the growth stage (Robert Dillschneider, Karlsruhe Institute of Technology KIT 2011); structure of silicate exoskeleton from the diatom Cyclotella (Nikolay Krumov, KIT 2009); image from the red macroalgae Porphyra tenera (FAO 2012); Dunaliella salina left: orange cells after accumulation of carotenoids due to growth under stressed conditions right: green cells during growth under not limiting conditions (Roland 2005); image of the green microalgae Chlamydomonas reinhardtii (Anna Jacobi, KIT 2011); spherical cells from the eukaryotic microalgae Chlorella vulgaris (Linda Oeschger, KIT 2011); image of the red microalgae Porphyridium purpureum (Rosa Rosello Sastre, University of Karlsruhe 2008).

According to estimations, the number of microalgal species on earth is estimated at between 200,000 and several million (Norton et al. 1996), of which between 40,000 and 60,000 species have been identified to date, but the chemical composition of only a few hundred species has been investigated. Furthermore, fewer than 15 strains are used for cultivation on an industrial scale (Tab. 2.1). Selected species are shown in Figure 2.1. Considering the large biological diversity and new developments in the field of genetic engineering, microalgae are expected to become one of the most promising sources for new products. Nevertheless, in order to evaluate the use of different microalgae species for technical processes, fundamental knowledge about kinetic and dynamic parameters is required. For example, the content of storage materials such as lipids or starch in the cells depends on nutrient limitation and illumination conditions. These data cannot be determined based on one isolated measurement in the natural habitat or during cultivation in shaking flasks. To date, only about a dozen alga species have been characterized to an extent that allows productivities in technical plants to be estimated.

2.2 Products

15

Microalgae are typically single-cell or filamentous microorganisms 3–10 µm in size. Using the process of photosynthesis, microalgae convert light energy into chemical energy, thereby storing inorganic carbon as sugar and other organic molecules. The process takes place in specific cell compartments, chloroplasts. Therefore, photosynthesis is a biochemically, temporally and spatially organized and highly complex process. The efficiency of such phototrophic processes reach at the present state of technology values of max. 5 % (value based on sunlight). This means that microalgae convert sunlight into biomass and further products five times more efficiently than terrestrial crops. Hence, microalgae are increasingly becoming the focus of interest in the field of renewable resources. In addition to higher productivities per footprint area, microalgae are suitable for the production of strongly reducing and lipophilic molecules such as carotenoids, due to the high stacking density on cell membranes in which carotenoids are stored. Thus, the intracellular concentration of carotenoids in microalgae can achieve levels not attainable with other potential production systems such as recombinant heterotrophic bacteria.

2.2 Products 2.2.1 Use and production of algal biomass The use of algal biomass for human and animal food or as a source for raw materials for chemical production is not a recent development. In fact, the use of the microalga Spirulina as human food dates back to the Aztec era in Mexico (Furst 1978). Macroalgal fishing for human consumption in Japan has been described in the literature since ad 274 (Chapman and Chapman 1980). The first techniques for the cultivation of the macroalga Porphyra were developed in the Imperial Algae Gardens in Japan and were transformed into regular farming practice in Tokyo Bay around 1624 (Richmond 1986). Nowadays, the use of Porphyra, better known as “nori”, constitutes an industry in Asia with an annual turnover of about US$ 1 × 109 (Pulz and Gross 2004). Other established products from macroalgae include polysaccharides such as agar (E406), alginates (E403–E405) or carrageenan (E407). They are used, due to their colloidal properties, as gelling agents and stabilizers in microbiology and the food, pharmaceutical and textile industries, representing a total market of approx. US$ 6 × 109/yr and a harvesting rate of 7.5 × 109 tonnes (t) of brown and red macroalgal biomass per year (Pulz and Gross 2004). In comparison to macroalgal biotechnology, the cultivation of microalgae is only a few decades old (Borowitzka 1995). The microalgal biotechnology is therefore the younger sector of the algae biotechnology. Until recently, the use of fossil microalgae (diatoms) for dynamite production was the most characteristic example

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2 Products from microalgae: An overview

of microalgal applications. Diatom shells were used by Alfred Nobel in the absorption of nitroglycerin. Nowadays, microalgae are used as a source for several products of commercial interest, intracellular molecules such proteins, including essential amino acids, high molecular polysaccharides, pigments, lipids, polyunsaturated fatty acids (PUFA), biological active molecules and the biomass itself. The microalgal biomass market produces ca. 5,000 t/yr of dry matter, generating a turnover of approx. US$ 2.3 × 108/yr. These rates relate only to the biomass as the commercial product; processed products are not included. Thus, the total global production of microalgal biomass would be rated between 8,000 and 10,000 tonnes (Pulz and Gross 2004; Enderle 2011). The most important commercial microalgae and their respective total yearly biomass production including processed products are listed in Table 2.1. Several other microalgal species not listed in Table 2.1 are also cultivated on a large scale but in limited quantities. To date, the most important application for microalgal biomass produced (around 75 %) is in the food sector, in which the biomass is marketed as a health food and nutritional supplement. Around onefifth is used for aquaculture and animal farming, with a strong increasing trend, and only a few high-value products from microalgae are commercially available.

Fig. 2.2: Schematic overview of the process for the production of microalgal biomass including the mass flows, process steps and application sector for the different products, commercially established (upper side) and non-established products (bottom side).

2.2 Products

17

Group

Species

Cultivation system and Product producer countries

Application areas

Market value (US$/kg)

Cyanobacteria

Spirulina platensis

Open pond: USA (California, Hawaii), China, Taiwan, India; natural lakes: Myanmar

Food supplement, animal feed Cosmetics Food colorant (Lina Blue), cosmetics

5–60

Lyngbya majuscule

Chlorophyta

Chlorella vulgaris

Dunaliella salina

Haematococcus pluvialis

Rhodophyta

Phycocyanin Immunomodulating substances

Pharmaceuticals, nutrition

Open pond: China, Japan, Taiwan; tubular PBR: Germany

Biomass

Food supplement, cosmetics Cosmetics

Open pond: Israel, Hawaii, India, China natural lakes: Australia

Biomass

Open pond: Hawaii, India, China, Japan; tubular PBR: Israel, India

Isochrysis galbana Bacillariophyta

Biomass

β-Glucan

β-Carotene

Food supplement, animal feed, aquaculture Food colorant, cosmetics 13 C-β-carotene

200 5–15/mg

5–60 2/g 300–3,000

Astaxanthin

Feed coloring 3,000– (salmon), food sup- 10,000 plement, cosmetics, pharmaceuticals

Fatty acids

Animal nutrition

Odontella aurita

open pond

Fatty acids

Pharmaceuticals, cosmetics, baby food

Phaeodactylum tricornutum

open pond, basin

Lipids, fatty acids

Nutrition, biofuels

Porphyridium sp.

Tubular PBR

Polysaccharides Phycoerythrin

Pharmaceuticals, cosmetic, nutrition Food colorant

3–25/mg

Tab. 2.2: Relevant microalgae species for biotechnological applications (Pulz and Gross 2004; Griehl and Bieler 2011).

The best established ones are β-carotene from Dunaliella salina, DHA (docosahexaenoic acid) from Crypthecodinium cohnii and astaxanthin from Haematococcus pluvialis (Spolaore 2006) (see Grewe and Griehl 2012). Nowadays, interest in microalgal biomass is strongly increasing towards their use as a potential source of

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2 Products from microalgae: An overview

renewable energy such as bio-fuels (production of bio-diesel, bio-ethanol, biomethane or bio-hydrogen), fertilizers, pesticides and bulk chemicals (bio-plastics, bio-lubricants) (Torzillo and Vonshak 2003; Enderle 2011). Figure 2.2 summarizes the main concept of microalgal biotechnology, with the most important material flows, for commercially established and non-established products. Microalgae are cultivated in open systems such as natural or artificial lakes, open ponds or closed systems called photobioreactors (PBR). All of these are used for cultivation on an industrial scale. The different types of cultivation systems for technically relevant microalgae species and the fields of application of the products including retail value estimations are listed in Table 2.2. Table 2.2 clearly shows that currently the global biomass is almost exclusively from open systems. Open systems are mostly operated as raceway ponds. This technology has been established due to the minimal technical requirements resulting from low demands for auxiliary energy and low costs when normalized to the pond volume. The disadvantages of raceway ponds are the low productivities per footprint area, due to unfavorable light conditions and insufficient mixing. Open facilities exhibit a high degree of water evaporation per footprint area (1–3 m3/m2 and year depending on the region) and a high risk of contamination. The expected maximum performance for open ponds is a photo-conversion efficiency (PCE) of around 0.5–1 %, thus comparable to that of terrestrial energy plants such as maize or sugarcane. Microalgal dry biomass concentrations usually do not exceed 2 g/L (see Chisti, 2012). Because of growing interest in high-value products from microalgae for the pharmaceutical and cosmetic industries, systems will need to meet Good Manufacturing Practice standards. This can be achieved only by using closed systems. The productivity of PBRs is an order of magnitude higher than those in open systems, due to higher surface-to-volume ratios and better mixing conditions. However, the energy demand of PBRs is much higher than for open systems (Pulz and Gross 2004). Economical biomass production using the more efficient closed systems for markets interested in low-price products such as the agriculture, aquaculture and energy sectors will be possible only if the investment and operation costs are reduced (see Acién Fernández et al. 2012).

2.2.2 Microalgae for human nutrition The use of microalgal biomass for human nutrition is the best established and most important market of microalgal biotechnology based on quantity of produced biomass and sales turnover (Tab. 2.2). The produced biomass is predominantly manufactured for the food market as a nutritional supplement and sold as powder or compressed tablets, capsules or pastilles (Fig. 2.3). The yearly turnover is higher than US$ 3 million and is increasing.

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Fig. 2.3: Overview of commercial available products containing microalgae. From left to right: Chlorella powder (PureRaw 2010); Chlorella tablets, Filinchen Wellness-ACTIVE crisp bread, Chlorella ribbon noodles (Bio) (Roquette Klötze GmbH & Co. KG 2012b); Spirulina powder, tablets and capsules (Bienenschwarmmm-Onlineshop 2012; DXN-Marketing 2012; floramed GmbH 2012; NaturallyGreen 2012).

Microalgal powder can also be incorporated into pasta, crisp bread or snack food such as “Vital Cuts”. Those food products containing Chlorella powder are manufactured, for example, in Germany and sold under the product name “Filinchen Wellness-ACTIVE crisp bread”, “Chlorella ribbon noodles (Bio)” and “Chlorella-Vital cuts du Dr Ritter (Bio)” respectively (Roquette Klötze GmbH & Co. KG 2012a). Similar developments concerning functional food products have been observed in France and the USA (Pulz and Gross 2004). Also, in Asia, algal powder is processed into pasta, snack food, candy and drinks (Yamaguchi 1996; Liang et al. 2004). To date, the consumption of microalgal biomass is restricted to a few genera, such as Spirulina (Arthrospira), Chlorella, Dunaliella, Nostoc and Aphanizomenon. Chlorella and Spirulina dominate the market (Tab. 2.2).

2.2.2.1 Spirulina (Arthrospira) The cyanobacterium Arthrospira, better known as Spirulina, is used in human food because of its high protein content, which is 60–71 % of the dry matter (Becker 2004). Its excellent nutrient value is based on the high content of iron and essential unsaturated fatty acids, especially γ-linoleic acid. The high amount of γ-linoleic acid is unique among microalgae studied so far. Spirulina has an easily digestible cell wall and is one of the richest natural plant sources of vitamin B12, with a whole spectrum of further B-complex vitamins and natural carotenes (Richmond 1986; Rangel-Yagui et al. 2004; Soletto et al. 2005). Furthermore, various healthpromoting effects have been observed, such as prebiotic stimulation of the growth

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of intestinal Lactobacillus acidophilus (Pulz and Gross 2004), attenuation of hyperlipidemia, suppression of hypertension, protection against renal failure and suppression of elevated serum glucose level (Yamaguchi 1996; Vílchez et al. 1997; Liang et al. 2004). The main producers of Spirulina are located in Asia and the USA (Tab. 2.2). More than 10 % of the world production is from China, operated by Hainan Simai Enterprising (Hanian, China). Hainan Simai Enterprising, now named Hainan-DIC microalgae Co., Ltd, belongs to the Japanese DIC Corporation (Dainippon Ink and Chemicals, Japan). They produce around 350 t of Spirulina per year (DIC Lifetec Co. Ltd. 2012). The world’s largest Arthrospira production plant is located at Calipatria in South California owned by Earthrise® Nutritionals with a total area of 440,000 m² using open ponds. The yearly Spirulina production yields 450 t (Earthrise Nutritionals LLC 2012). Further production sites are located in Yangon (Myanmar) where Spirulina grow in natural volcanic lakes, and in Kona (Hawaii). The plant in Kona is operated by Cyanotech, which produces Spirulina Pacifica® in open ponds (330,000 m²). The microalgal biomass obtained is further manufactured as pure powder pressed in tablets or filled in bottles (Cyanotech Corp. 2012) (see Thein 2012).

2.2.2.2 Chlorella The high nutrient value of the green microalga Chlorella vulgaris (chlorophyta) is based on the effective assimilation by the human body of several intracellular products naturally wrapped in amino acids. Another important substance in Chlorella is β-1,3-glucan, a polysaccharide, which stimulates the immune system, destroys free radicals and reduces blood lipids. Other health-promoting effects that have been identified after consuming Chlorella are the ability to reduce gastric ulcers, to cure wounds and to prevent constipation. Also, diseases such as atherosclerosis, hypercholesterolemia and cancer can be prevented (Yamaguchi 1996; Jong-Yuh 2005). Chlorella is produced by more than 70 companies worldwide. The largest producer is Taiwan Chlorella Manufacturing & Co. Ltd. in Taipei (Taiwan) with an annual biomass output of 330 t in Taiwan and a further 300 t in another production site located in Hai-Nan (Hainan, China) using open ponds (Taiwan Chlorella Manufacturing Co. Ltd. 2012). In Germany, Chlorella biomass is produced in closed PBRs by Roquette Klötze GmbH & Co. KG (Klötze, Germany). The production plant consists of several units of tubular PBRs using glass tubes (500 km long) with a total volume of 700 m³. The plant is operated in a greenhouse covering an area of 1.2 ha and is the largest microalgae production plant in Europe with an annual production of 100 t of dry biomass (Roquette Klötze GmbH & Co. KG 2012a).

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2.2.2.3 Dunaliella salina Dunaliella salina (chlorophyta) is third on the list of most cultivated microalgae on a commercial scale globally (Tab. 2.1). Dunaliella salina is produced due to its antioxidant activity and its high β-carotene content, which can reach 14 % of dry weight (Metting 1996). The cells accumulate high amounts of carotenoids during growth under stress conditions such as high salinity or high temperatures, and thereby algae change in color from green to orange (Fig. 2.1) (Torzillo and Vonshak 2003). In contrast to other members of the chlorophyta, Dunaliella lacks a rigid polysaccharide wall. Therefore, the dried biomass is easily digestible by animals and humans (Richmond 1986). Globally, the largest producer of D. salina is Cognis Australia, now part of BASF. The plants located in west Australia consist of natural salt lakes with an area of 400 ha. Dunaliella powder is sold as a dietary supplement for human nutrition and animal feed. Extracts from the microalgae, a mix of several carotenoids, are manufactured under the product name Betatene® (BASF SE 2012).

2.2.3 Microalgae for animal feed The use of microalgal biomass as animal feed represents the second largest area of application. Up to 30 % of the current algal production globally is sold and used as a supplement for feeding different animals such as fish in aquaculture, pets and farm animals. More than 50 % of the Spirulina produced is used for this purpose (Spolaore 2006). Also, biomass of Chlorella and Scenedemus is added to the feed for domestic and farm animals. The addition of 5–10 % of algal biomass to the feed has a positive effect on the physiology of the animals, mostly because of the high content of vitamins, minerals and essential fatty acids. The algae enhance the animals’ immune system and fertility, and produce healthy skin and a shiny coat (Becker 2004; Pulz and Gross 2004; Spolaore 2006). The fact that aquaculture is one of the most important areas of application for microalgal biomass is obvious: algae form the basis of the natural food chain in aquatic, especially in marine systems. On the one hand, the biomass is directly used as a food source for different types of mollusks (carpet shells, oysters, scallops or sea ears), zooplankton (rotifers, copepods) and fish larvae (Grewe 2009). On the other hand, the biomass is used as an additive to fish feed, which improves the coloring of farmed salmons and the induction of important biological processes such as enhanced growth, resistance against diseases, firmer flesh, better flavor and brighter skin. Fish flesh becomes healthier and tastier, due to the high protein and unsaturated fatty acids content provided by microalgal biomass (Yamaguchi 1996; Brown et al. 1997, 1999; Muller-Feuga 2000). Not every microalga is suitable for aquaculture. Each strain needs to meet several criteria: it must be easily cultured, not be toxic and must have an easily

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digestible cell wall, the correct shape and size to be digested, and a high nutritional value. As mentioned above, the nutritional value is determined by the content of proteins, unsaturated fatty acids and vitamins. To date, only Chlorella, Tetraselmis, Isochyris, Pavlova, Phaeodactylum, Spirulina, Scenedemus, Skeletonema, Chaetoceros, Nitzschia and Thalassiosira meet the above-mentioned criteria. Thus, these algae are most frequently employed from the 40 species used for aquaculture (Pulz and Gross 2004). The global market for aquaculture products such as fish and shellfish is estimated to be more than US$ 40–50 billion/yr (Pulz and Gross 2004) with an annual growth rate of 8 % since 1970. A similar trend is expected for the next 25 years (Grewe 2009). The estimated costs for the production of microalgae to be applied in aquaculture range between US$ 50 and US$ 150/kg dry biomass with maximum values of US$ 1,500/kg dry biomass (Pulz and Gross 2004). Thus, algal biomass is the most expensive component of aquaculture production. Production costs for microalgal biomass should be minimized in such a way that high-value meal and oils from microalgae can economically substitute current low-value fish oils and meal for feeding farmed fish. Nevertheless, the fish demand for human nutrition is growing, and the actual prices for fish oil and meal are increasing due to the increasing scarcity of natural fish resources. These facts give a positive impetus for the application of microalgae biotechnology for aquaculture (see Shields and Lupatsch 2012).

2.2.4 Microalgae as natural fertilizer Microalgae play an important role in the soil ecosystem by improving its quality and fertility. The microalgae produce polymers that adhere to particles and store water in soils. This is an important factor, especially in more arid regions. Algae also add nitrogen and bioactive compounds to the soil, thus improving the growth of higher plants (Borowitzka 1995; Metting 1996; Ördög et al. 1996). As Pulz postulates, “soil microalgae should be regarded by microalgal biotechnologists as a promising area to find new species with unexpected properties” (Pulz and Gross 2004). Cyanobacteria such as Anabaena and Nostoc have already been established as bio-fertilizer for rice production in tropical and subtropical agriculture due to their ability to perform air-nitrogen fixation. These bio-fertilizers reduce chemical nitrogen demands by 15 %, thereby improving the biologically fixed nitrogen in soil to 20–30 kg per season and hectare (Raja et al. 2008). The trend for microalgal biotechnology in this area seems to be the application of biologically active compounds against plant diseases caused by viruses or bacteria (Spolaore 2006).

2.2.5 Microalgae in cosmetics A wide range of cosmetic products containing algal extracts (mainly from Chlorella and Spirulina) have been commercialized. Applications include facial and skin care

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in anti-aging and moisturizing creams, shower gels or body lotions, and in sunscreen creams or hair-care products (Stolz and Obermayer 2005a). Commercially available products and their active properties, as reported by the companies, include: – Protulines®: protein-rich extracts from Spirulina against first wrinkles, with skin tightening and stretch-mark repairing effects (Exsymol SAM, Monaco); – Dermochlorella®: extract from Chlorella vulgaris that stimulates collagen production from dermis cells improving tissue regeneration and combating lines and wrinkles (Codif Recherche et Nature, France); – Aquaflor®: skin and hair-care system from Spirulina platensis and Ascophyllum nodosum with moisturizing and regenerative properties (IGV GmbH, Germany); – PEPHA®-TIGHT: two-in-one skin tightener product from Nannochloropsis occulata extracts, which provides a perceptible instant tightening effect on the skin, protecting the skin cells against oxidative stress and stimulating the collagen production (DSM Nutritional Products Ltd, Branch Pentapharm, Switzerland); – PEPHA®-ACTIVE: extract from Dunaliella salina with cell-regenerating properties and a positive influence on the cell metabolism (DSM Nutritional Products Ltd, Branch Pentapharm, Switzerland). PEPHA®-ACTIVE is added in combination with PEPHA®-TIGHT in cosmetics products from Danielle Laroche. 2.2.6 Fine chemicals An existing market for highly valuable molecules extracted from microalgae has already been established, as in the case of bulk microalgal biomass. Based on relevant production amounts, the most important products are PUFAs and different types of carotenoids. An example of this is the sales turnover in 2009 for the carotenoid astaxanthin from Haematococcus pluvialis of US$ 25 million (Grewe 2009). Further products include pigments as antioxidants or natural colorants, biological active molecules and stable isotopes. Intensive research is being carried out in this area in order to find further biochemicals from microalgae. Considering the large number of microalgal species that are yet to be identified, a wide range of new biologically active substances can be expected.

2.2.6.1 PUFAs Microalgae are primary sources of PUFAs in the food chain. As shown in Table 2.3 the most important PUFAs obtained from different microalgae species are γ-linolenic acid (GLA), arachidonic acid (AA), eicosapentaeonic acid (EPA) and docosahexaeonic acid (DHA).

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PUFA

Structure

Potential application

Microalgae

γ-Linolenic acid (GLA)

18:3 ω6, 9, 12

Spirulina

Arachidonic acid (AA)

20:4 ω6, 9, 12, 15

Eicosapentaenoic acid (EPA)

20:5 ω3, 6, 9, 12, 15

Infant formula for full-term infants; nutritional supplement Infant formula for full-term/ pre-term infants; nutritional supplement Nutritional supplement; aquaculture

Docosahexaenoic acid (DHA)

22:5 ω3, 6, 9, 12, 15, 18

Infant formula for full-term/ pre-term infants; nutritional supplement; Aquaculture

Porphyridium

Nannochloropsis Phaeodactylum Nitzschia Cryptecodinium Schizochytrium

Tab. 2.3: Particularly interesting polyunsaturated fatty acids from microalgae (Spolaore 2006).

Fig. 2.4: Chemical structure of PUFA from microalgae: (a) arachidonic acid (AA); (b) docosahexaenoic acid (DHA); (c) γ-linolenic acid (GLA); (d) eicosapentaenoic acid (EPA).

Figure 2.4 shows the chemical structure. All are essential fatty acids, which means that they cannot be synthesized by humans and animals, and need to be supplied by food. Functional foods and new food supplements represent the fastest-growing application section within the omega-3 and omega-6 PUFA ingredients market. Seventy-eight percent of the fatty acids are used in dietary supplements and 13 % in functional foods (Enderle 2011). Sources of omega-3 and omega-6 fatty acids are usually fish and fish oil. These products characteristically have a fishy odor and unpleasant taste, being very sensitive against oxidation and difficult to purify. Fur-

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thermore, a lack of fish resources is expected in the future. In contrast, fatty acids from microalgae have a higher quality, no unpleasant odor, a reduced risk of chemical contamination, and a better purification and stability, since they are from a purely vegetarian source (Lebeau and Robert 2003). Therefore, the biotechnological potential for PUFA from microalgae as a nutritional supplement in the food and feed market is very promising. To date, only DHA from the microalgae Crypthecodinium and Schizochytrium has been commercialized. This omega-3 fatty acid is the major structural fatty acid in the gray matter of the brain and in the retina of the eye, and a key component of heart tissue. The supplementation of DHA in breast-milk substitutes and other baby foods has been recommended since 1999 by several health and nutrition organizations due to the key role of DHA for the healthy development of brain and eyes in infants. The total global turnover for infant food is about US$ 10 billion per year (Ward and Singh 2005) and is one of the most important application sectors for microalgal products. Globally, the major producers of DHA from microalgae are Martek Biosciences Corporation (Maryland, USA) and Lonza Ltd. (Basel, Switzerland). Both companies produce heterotrophic DHA from different microalgal species. Lonza has patented the fermentation process using Ulkenia sp. SAM179. After fermentation, the oil fraction containing high amounts of DHA (at least 40 %) is purified by extraction and refining processes very similar to those used in the production of conventional vegetable oils. The microalgal oil is marked as DHAid™ and approved for use as a food ingredient according to Novel Food Regulations in EU, USA, China, Australia and New Zealand (Lonza Group AG 2012). Martek developed and patented the process to obtain DHA oil from Crypthecodinium cohnii. The heterotrophic process for the production of Life’sDHA™ uses fermenters 80,000 to 260,000 liters in volume. When grown, the microalgae is harvested and processed to extract the clear, amber-colored oil, rich in DHA. The whole process meets all the requirements of current Good Manufacturing Practices and the US Food and Drug Administration regulations. In 2010, DSM (Heerden, Netherlands) acquired Martek. This was the first major acquisition by DSM after its successful transformation into a Life Sciences and Material Sciences company (Royal DSM N. V. 2012). A wide range of products containing Life’sDHA™ oil are sold in more than 60 countries globally. The oil is available in soft gel capsules as a food supplement for children and adults or added to beverages such as fruit juices, milk, milk products, cereal products and bread. Figure 2.5 provides an overview of products containing Life’sDHA™ (Martek Biosciences Corp./Royal DSM N. V. 2012). DHAgold™ is also a DHA source for animals marketed by Martek/DSM. The product line DHAgold™ consists of powder from dried microalgal whole cells added as an active ingredient to aquaculture feed, pet food (dogs, cats, horses) or feed for swine and poultry, which is an effective way to enrich meats and eggs with DHA (Martek Biosciences Corp./Royal DSM N. V. 2012a).

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Fig. 2.5: Products containing lifeDHA™ oil (Martek Biosciences Corp./Royal DSM N. V. 2012b).

2.2.6.2 Pigments The first step in photosynthesis is the absorption of light. Microalgae have pigments located in the chloroplasts, and chlorophyll a is the primary photosynthetic pigment that constitutes the reaction centers of the photosystem. The photosystem also includes the antenna pigments responsible for light collection. Depending on the microalgae species, different kinds of pigments build the antenna complexes. For example, chlorophyll a and b are found in chlorophyta, phycobilins in cyanobacteria and rhodophyta (red algae) for efficiently light absorption. Further accessory pigments protect the cells against damaging radiation and the formation of free radicals (carotenoids) (Lips and Avissar 1986; Häder 1999; Fig. 2.6).

Pigments as antioxidants Due to their specific molecular structure, carotenoids have free-radical-scavenging and neutralization properties. Free radicals such as superoxide anions (O−2 ) or hydroxyl radicals (·OH) are highly reactive, with a high oxidizing potency, causing irreversible damage to human fat tissue, genetic material and cell membranes. Because of the potential of carotenoids to prevent cell and tissue damage, they are also called antioxidants. Furthermore, they stimulate the immune system (Lorenz and Cysewski 2000). From the more than 400 different types of carotenoids known, only a few have been commercialized to date. The most common carotenoid is

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Fig. 2.6: Chemical structure of pigments: (a) β-carotene; (b) astaxanthin; (c) phycoerythrin.

β-carotene. Extracted from Dunaliella salina, it is mainly used in the food industry as a food colorant and as pro-vitamin A. The second most important carotenoid from microalgae is astaxanthin, from Haematococcus pluvialis. It is used in the aquaculture as a natural red colorant for farmed salmon flesh. Further established products containing astaxanthin in the pharmaceutical and nutritional sector include: – Zanthin®, from the company Valensa International (Florida, USA): a natural astaxanthin complex supporting neurological and eye health. It protects the cells from the effects of UV light and has been patented for use in eye-health formulas (US Patent 5,527,533) (Valensa International 2012b); – SpiruZan®, also from Valensa International: a complex containing astaxanthin and Spirulina that provides benefits for neurological, cardiovascular, immune, anti-inflammatory and eye-health support (Valensa International 2012a);

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Azyris, a medicament from Sifi S.p.A (Lavinaio, Italy) containing Zanthin® for use as a tear supplement in the therapy of age-related macular degeneration. This company is the Italian market leader in the eye-care sector (SIFI SpA 2012).

Less important in terms of quantities, but further commercially used carotenoids from microalgae, are lutein, zeaxanthin and canthaxanthin. They are added to animal feed to color chicken skin and for pharmaceutical purposes. In cosmetics, the use of carotenoids from microalgae as natural preservatives and UV protection factors is increasing rapidly. The reason for this is the higher consumer acceptance for these natural ingredients compared to synthetic ingredients. The same trend can be observed in other application sectors for carotenoids (nutrition, animal feed, pharmaceutical industry). Nevertheless, there is still strong competition for synthetic carotenoids, which dominate the market due to lower production costs (Pulz and Gross 2004; Spolaore 2006). In the carotenoid market for β-carotene and astaxanthin, 90 % is covered by synthetic β-carotene and 95 % by synthetic astaxanthin (Ecke 2010). The main producers for synthetic astaxanthin and β-carotene are BASF (Ludwigshafen, Germany) and DSM (Heerlen, Netherlands). The search for further antioxidants from microalgae continues to find natural plant-based molecules, which are able to replace those that can only be produced synthetically or by heterotrophic processes using yeasts. An example for this tendency is given by the research on the production of the co-enzyme Q10 with the red microalgae Porphyridium purpureum. The optimization of process parameters such as the influence of light intensity and addition of acetate, towards a high accumulation of co-enzyme Q10, are the focus of research to establish a competitive phototrophic production process (Klein et al. 2011).

Pigments as natural colorants The phycobiliproteins phycoerythrin (red pigment) and phycocyanin (blue pigment) are only present in microalgae. They are produced on a large scale from Spirulina and Porphyridium (Roman 2002; Viskari and Colyer 2003) and are used as natural colorants for food and cosmetic products. DIC LIFETEC Co. Ltd (Tokyo, Japan), which is a part of DIC Corporation, has developed the food colorant Linablue A and Linablue HGE, based on phycocyanin extracted from Spirulina. Linablue is used for blue coloring of chewing gums, ice sorbets, popsicles, candy, soft drinks, milk products and wasabi. Another type of phycocyanin is applied as a natural colorant in cosmetic products such as eye liner and lipstick (Yamaguchi 1996; Viskari and Colyer 2003). Phycobiliproteins are also used in clinical and research immunology laboratories as a fluorescent marker for antibodies, receptors and other biological molecu-

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les in immunolabeling experiments, fluorescence microscopy or diagnostics (Roman 2002). The price for phycobiliproteins ranges between US$ 3/mg and US$ 25/mg for native pigment to US$ 1,500/mg for antibody–pigment complexes (Spolaore 2006).

2.2.6.3 Polysaccharides The industrial production of polysaccharides from macroalgae such as alginates (E401–E405), agar (E406) and carrageenan (E407) has been established for some years now. With annual ranges of 11,000 t for agar, 16,000 t for carrageenan and 30,000 t for alginate (Griehl and Bieler 2011), they represent the economically most important product from macroalgae. Because of their colloidal properties, they are used as gelling agents and stabilizers in the microbiology, food, pharmaceutical and textile industries. A new spray-protective foil based on polysaccharides from algae has been developed by a German chemist. According to the inventor, the automobile industry wants to use this bio-based foil to protect new cars against damage during production and transport. The patent of this new product is in progress (Bonaeda Biotechnologie 2012). In recent years, an increase in research and development activities to obtain polysaccharides from microalgae has been observed. Microalgae can be used as an alternative source, to prevent problems such as raw-material shortage or pollution occurring during macroalgal production (Hoek 1993). Polysaccharides from microalgae demonstrate the same colloidal properties as those from macroalgae. Several species show interesting properties of pharmacological importance opening further fields of application: as carrier material in nasal or oral drug delivery, non-viral gene delivery and tissue engineering systems. Furthermore, many biological activities such as antiviral, antitumor and anticoagulant have been reported. Due to this unique combination of biological activity and physicochemical properties, microalgal polysaccharides have opened new ways for the development of more efficient or new medicaments (Smelcerovic 2008). Examples of microalgal polysaccharides with interesting biological activities include β-1,3-glucan from different algal species and sulfated polysaccharides (Fig. 2.7) from rhodophyta. β-1,3-Glucan is accumulated as a storage material in several microalgal species (Chlorella, Skeletonema, Nannochloropsis, Euglena, Cyclotella). In particular, diatoms such as Skeletonema can store high amounts of β-1,3-glucan during the stationary growth phase (Stolz and Obermayer 2005b). Under heterotrophically conditions, the flagellate Euglena gracilis can accumulate an extremely high content of β-1,3-glucan, which can constitute more than 80 % of the cell mass. The biologically active properties of β-1,3-glucan include antiviral, antitumor and immune stimulation. Scavenging of free radicals, cholesterol-lowering properties, regulation

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2 Products from microalgae: An overview

Fig. 2.7: Chemical structure of the sulfated polysaccharide fucoidin (
-fucose + sulfate).

of postprandial blood glucose and insulin response in humans are some of the further investigated effects of β-1,3-glucan (Santek et al. 2009). Commercial applications of β-1,3-glucan are found in the following products: – The Miho cosmetics series from the Japanese company Nikken Sohonsha Corp. incorporates β-1,3-glucan from Chlorella in anti-aging and eye creams, body lotions, soaps and facial masks, because of the moisturizing and regenerating qualities from β-glucan. Another product from this company is Chlostanin GOLD tablets, used as a food supplement (Chlostanin Nikken Nature Co. Limited 2012). – Paramylon (β-1,3-glucan from Euglena gracilis) is found in cosmetics such as day creams from Clarins (Crème Douceur Jour, Clarins Lift Anti-Rides Jour) because of its firming effects (Clarins GmbH 2012; Stiftung zur Förderung der Hautgesundheit 2012). – β-glucan from the yeast Saccharomyces cerevisiae, not from microalgae, is the leading product for use as an immune stimulant additive for the fish-meal market (Wilhelm et al. 2006; Santek et al. 2009). Sulfated polysaccharides (Fig. 2.7) form gels with a very similar viscosity to that of agar and carrageenan and a high stability over wide pH, temperature and salt concentration ranges (Medcalf et al. 1975; Percival and Foyle 1979; Arad and Richmond 2004). The sulfate groups impart the polymers’ antiviral, anti-inflammatory and anticancer activities, and stimulate the immune system. The polysaccharides from the red microalgae Porphyridium sp. contain sulfate half ester groups, which are good antioxidants (Santek et al. 2009), exhibit antiviral activity against Herpex simplex viruses (HSV 1, 2) and Varicella zoster viruses, and prevent colon cancer

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(Fabregas et al. 1999; Dvir et al. 2000; Geresh et al. 2002; Matsui et al. 2003). These polysaccharides have a high potential to substitute the production of carrageenan from macroalgae. Furthermore, the sulfated polysaccharides from Porphyridium exhibit better rheological properties than carrageenan, and the microalga produces them extracellularly. The downstream process of microalgae polysaccharides can therefore be simplified to a mechanical separation compared to the extraction steps needed for macroalgae.

2.2.6.4 Recombinant proteins Phototrophic microorganisms are very suitable for the extracellular production of recombinant proteins (glycoproteins). The process development using mosses (bryophytes), one of the oldest groups of land plants, is very advanced (Lucumi and Posten 2006). The company greenovation Biotech GmbH (Heilbronn, Germany) has developed a BryoTechnology that offers the possibility to produce different pharmaceutical proteins (growth factor (VEGF), serum proteins (HSA), peptide hormones, enzymes (Phosphatase), vaccines and a wide range of oncology mAbs (IgG1 and 4)) using different production systems such as the BryoSpeed® and the BryoMaster® system (greenovation Biotech GmbH 2012). In the case of genetically modified microalgae, the first successful experiments to obtain recombinant proteins have been reported (Desplancq et al. 2008). The volume turnover for this highly specific market sector is expected to be low (see Cadoret et al. 2012; Kirchmayr and Griesbeck 2012).

2.2.6.5 Stable isotopes Closed systems (PBRs) enable the cultivation of microalgae under strictly controlled conditions. Therefore, phototrophic microalgae are the ideal choice to incorporate isotopes such as 13C, 2H and 15N from inorganic molecules (13CO2, 15NO–3, 2H2O), being much cheaper than organic sources. The cells produce stable isotope-labeled molecules. These highly valuable biochemicals can be used to determine molecular structures and for metabolic studies or clinical trials such as examination of the gastrointestinal tract or breath tests (Radmer 1996). For example, 13C-labeled β-carotene from Dunaliella salina has been produced to investigate human carotenoid metabolism (Raja et al. 2008).

2.2.7 Micro- and nanostructured particles Biomineralization is a process functioning as a bridge between the organic and inorganic world, in which biological systems selectively accumulate elements from the environment and assemble new and sophisticated functional structures. In the

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2 Products from microalgae: An overview

Fig. 2.8: Scanning electron micrographs of diatoms: (a) Biddulphia reticulata; (b) Diploneis sp.; (c) Eupodiscus radiatus; (d) Melosira varians (Mary Ann Tiffany, San Diego State University).

diverse world of microalgae, there are two widespread classes of biomineralization relevance: the diatoms and the coccolithophores (haptophytes). Diatoms, present in both fresh and saltwater, form an amorphous silicate exoskeleton (Fig. 2.8). The incorporation of nitroglycerin in diatoms enabled the invention of dynamite by Alfred Nobel and is a historical example of the potential of microorganisms in producing spatially structured materials for technical uses. Diatoms are natural lithographers at the nanometer scale. Revealing the mechanisms that diatoms employ, to lay down micro lines of silica, and adopting in practice the proteins that diatoms use to direct silica deposition could be very useful to the semiconductor industry but also could lead to versatile, nature-inspired engineering solutions (Mock et al. 2008). Engineers are using diatoms as a tool to build extremely sensitive sensors based on micro-fluidic devices (Allison 2008; Hildebrand 2008). The information-processing technology alters from electronically to optically based hardware, allowing the transport and storage of more information. The application potential of diatoms in this field is based on the demand of optical systems for materials with regularly repeating structures with features below the micrometer size range (Brandbury 2004). The uniform nanoscale pore structure together with their biocompatibility and chemical inertia facilitates the use of diatom shells as delivery vehicles for drugs (Wee et al. 2005). In addition, diatoms can become a template for the fabrication of lightweight but strong materials for the aerospace and car industry (Mann 2001; Wee et al. 2005).

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Fig. 2.9: Scanning electron micrographs of coccolithophores (Harald Andruleit, University of Bremen and Stephanie Oder, University of Karlsruhe).

Compared to diatoms, the coccolithophores inhabit only saltwater, as a significant part of the phytoplankton. Their exoskeleton consists of many (between 15 and 120) small calcite plates, called coccoliths, which are very sophisticated and unique for every strain form and size (Fig. 2.9). One of the most abundant and best-studied coccolithophores species, Emiliania huxleyi (Fig. 2.9), has a unique ability to build increasing amounts of free coccoliths, and it is this property that is responsible for the formation of algal blooms spread over tens of thousands of square kilometers (Schwarz-Weig 2009). Approximately 50 % of natural calcium carbonate production is related to coccolithophores (Schwarz-Weig 2009). Calcium carbonate has applications in various industry branches, for example as a lime fertilizer in forestry, as a filler for the plastic and paint industries or as the main component for the coatings used in the production of fine paper for specialist applications. The uniform micrometer size and specific spatial structures of the coccoliths define them as a very attractive and promising ingredient for the production of special-feature cements.

2.2.8 Bulk chemicals Bulk products are defined by having high production volumes (from 10,000 t/yr to more than 1,000,000 t/yr) and low prices between approx. US$ 1 and less than US$ 10/kg. Examples of bulk products include organic chemicals produced pre-

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dominantly from petroleum. In recent years, the biotechnological production of bulk products has been gaining increasing interest. On the one hand, it can replace those chemicals based on fossil fuels; on the other hand, new chemicals with additional functionalities can be produced. With the exception of ethanol, most companies producing organic chemicals have not upscaled bio-based production to a large scale, due to higher production costs. Nevertheless, the substitution of polymers or chemical building blocks using bio-based products has increased continuously. Market potential studies prognosticate a market share on bio-based polymers of 3–6 % by 2020, assuming a total demand of 70,000,000 t/yr. The market share for bio-based chemical building blocks is expected to be 10 % by 2020 (Patel et al. 2006). This means that for the actual production rate of acetic acid from methanol with a turnover of 7 × 106 t/yr, a substitution of 10 % with biomethanol would correspond to a demand of 7 × 105 t/yr bio-methanol for acetic acid production. Both examples clearly show the large market-volume potential for bio-based bulk products. Established biotechnological processes to obtain bulk chemicals are heterotrophic, using genetically modified bacteria that grow on glucose. The raw material glucose is not available in nature in a pure form, so additional energy is required to obtain glucose. The use of microalgae allows the production of bulk chemicals directly from inorganic CO2 under phototrophic growth conditions. The joint venture of DuPont Tate & Lyle BioProducts LLC today has led to one of the largest renewable materials facilities in the world in Loudon, Tennessee, and the venture has produced an innovative new product made from corn sugar, Bio-PDO™1,3-propanediol (DuPont Tate & Lyle BioProducts 2012). Solazyme, Inc. (San Francisco, USA) is a renewable oil and bio-products company, cultivating microalgae to produce oils and biomaterials that can be used as oleochemicals, in cosmetics or in foods. The microalgae are cultivated heterotrophically on a large scale. The glucose needed is obtained by enzymatic degradation of forest residues: switchgrass, miscanthus, sugarcane, corn and stover (Solazyme Inc. 2012a). Since 2010, Solazyme has signed a research and development agreement with Unilever. The algal oils were incorporated successfully into Unilever personal-care products. The use of algal oils instead of palm oil makes it possible for this company to reduce its overall environmental impact while increasing the size of its business (Solazyme Inc. 2012c). Further interesting products that can be replaced by Solazyme’s algal oils include functional fluids such as airplane deicing fluids, which currently are obtained from petroleum or conventional oils. The Dow Chemical Company has also had a partnership with Solazyme since 2011 to develop algal oils for use as dielectric fluids in the transformer market (Solazyme Inc. 2012b). The phototrophic production of microalgal oils is actually too expensive for use in the fossil-fuel-dominated sector. In order to reduce costs, intensive research work has started in order to modify microalgae genetically for the phototrophic

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production of base chemicals such as ethanol, ethylene, butane, propane (Enke 2010) or isobutyraldehyde (Atsumi et al. 2009). All these products have low boiling points and high vapor pressures, and diffuse outside the cells during the cultivation, thus increasing the product-recovery efficiency. Most of the studies are at the developmental stage or have been developed only on a lab scale. Further work is needed to establish the technology for application on a large scale and to demonstrate its economical feasibility.

2.2.9 Energy production from microalgae Several microalgae strains are able to accumulate high amounts of cellulose, starch, glycerin and oils (starting materials for the biofuel production) depending on the cultivation conditions. Other microalgae species produce hydrogen under anaerobic conditions and methane can be recovered as fermentation product of the remaining biomass. Both gaseous products can be used for energetic purposes. Therefore, the potential of microalgae as renewable energy source is very promising. Major drawbacks of microalgal biotechnology for the energy sector are still the high investment costs and the high demand on auxiliary energy for biomass production and biofuel processing. Current reactor designs require indeed more than 100 % of the recovered chemical energy (stored as biomass) as electric auxiliary energy only for mixing and gas transfer (Lehr and Posten 2009; Morweiser et al. 2010).

2.2.9.1 Biodiesel A large amount of microalgae accumulate high quantities of oils and other lipids in their natural habitats, and for this reason, microalgae are of interest as a renewable-fuel supply in the future. Although the idea of using microalgae as a source for biodiesel was already described in an American study over 12 years ago, research has intensified in recent years, as a result of continuously increasing oil prices. The potential of microalgae for biodiesel production is very promising due to higher growth rates, combined with high efficiency in converting sunlight and ability to accumulate higher amounts of lipids (from 20 % to 80 % of dry weight) (Schenk et al. 2008) compared to conventional crops (not more than 5 % of dry weight) (Amaro et al. 2011); therefore the oil yield per hectare obtained from microalgae can greatly exceed the yield from oil plants such as rapeseed, palm or sunflower. Another advantage of microalgae over higher plants is their metabolic flexibility. This means that a variation in the biochemical composition of the biomass (towards higher lipid or carbohydrate accumulation) can be easily regulated by varying the cultivation conditions (e.g. nitrogen starvation) (Tredici 2010). PBRs can be located on non-arable land,

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2 Products from microalgae: An overview

Crop

Oil yield (L/ha)

Land area needed (M ha)a

Percentage of existing US cropping areaa

Corn Soybean Canola Jatropha Coconut Oil palm Microalgaeb Microalgaec

172 446 1,190 1,892 2,689 5,950 136,900 58,700

1,540 594 223 140 99 45 2 4.5

846 326 122 77 54 24 1.1 2.5

a

To meet 50 % of all transport fuel needs of the USA.

b

70 % oil (by weight) in biomass.

c

30 % oil (by weight) in biomass.

Tab. 2.4: Potential of the biodiesel production from microalgae (Chisti 2007).

and microalgae can grow in readily available seawater or brackish water. Therefore, there is no competition with classical agriculture for resources. Furthermore, in large-scale applications, production during the whole year is possible. This is achievable due to the straightforward employment of effective process-engineering tools in photobiotechnology, e.g. for inoculation, maintenance, harvesting and so forth. Table 2.4 shows the results of a recent study comparing the land requirements needed to obtain 50 % of fuels used in transport in the USA with different renewable sources. It clearly shows that a reasonable and significant contribution to overall energy consumption can only be achieved using microalgae (Chisti 2007), but an exact calculation from the data shown in Table 2.4 is only possible to a limited extent. The calculated oil yields are the result of a combination of high growth rates obtained in the lab and high lipid concentrations. These assumptions are thermodynamically not possible. Lipid accumulation leads to an increase in the heating value of the biomass from 20 MJ/kg for algae with low lipid contents (between 20 % and 30 % dry weight) to 30 MJ/kg for oil-rich algae (50 % dry weight; Morweiser et al. 2010). While the highest growth rates are attained, the oil content corresponds with that of the former algae (20–30 % dry weight). For constant PCE values (conversion efficiency of light into biomass), that necessarily leads to lower measured areal productivities in terms of dry biomass. Furthermore, the photon demand will increase if proteins and lipids are synthesized, reducing the PCE due to ineffective metabolic steps. The indicated values have never been reached on a pilot scale so far. Nevertheless, the potential is still enormous for realistic assumptions for PCE amounts of 2–3 % over the whole cultivation process with the current state of technology. Microalgae produce 5–20 % of glycerin-based membrane lipids (glycosylglycerides) based on dry weight under normal, not growth-limiting, conditions. These glycosylglycerides contain fatty acids with different chain lengths from C10 to >C20.

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Fuel property

Algal biodiesel

Petroleum diesel

EN14214-standard

Higher heating value (MJ/kg) Kinematic viscosity (mm²/s) 40°C Density (kg/L) Carbon (wt%) Hydrogen (wt%) Oxygen (wt%) Sulfur (wt%) Boiling point (°C) Flash point (°C) Cloud point (°C) Pour point (°C) Cetane number

41 5.2 0.864 76 ≤ 12.7 ≥ 11.3 0 – 115 – –12 –

45.9 1.2–3.5 0.83–0.84 87 13 0 0.05 max 180–340 60–80 –15 to 5 –35 to –15 51

– 3.5–5.2 0.86–0.90 – – – < 10 max. 0.02 – > 101 – – > 51

Tab. 2.5: Comparison of selected properties of algal bio-oil and typical conventional diesel with respect to the European norm for biodiesel (Brennan and Owende 2010).

Under stress or limiting conditions, such as nitrogen starvation, mainly neutral lipids are produced as an energy reserve, typically triacylglycerides (Hu et al. 2008). The triacylglycerides are stored as lipid bodies in the cytoplasm of the cells. Biodiesel is a mixture of fatty acids of diverse lengths esterified with an alcohol, typically methanol. The suitability of microalgal biomass as a biofuel feedstock is closely related to the length and degree of saturation of its fatty acids as specified by the four key components: iodine value, oxidation stability, cetane number and cold-filter plugging point. A high iodine number represents a high degree of unsaturation of fatty acids in biodiesel, which is unfavorable because fatty acids with a higher content of double bonds are prone to oxidative damage. Otherwise, unsaturated fatty acids are beneficial for flow properties, especially at lower temperatures, and therewith result in an advantageous cold-filter plugging point. Lipids rich in long-chain fatty acids with a low degree of saturation exhibit a high cetane number, indicating a short ignition delay time and high combustion quality, whereas an excessive degree of saturation might conflict with the requirement for a reasonable cold-filter plugging point by precipitation at low temperatures (Ramos 2009). Beside other quality standards, the feedstock needs to comply with target ranges defined by the European norm, which is fulfilled in the case of algal biodiesel as shown in Table 2.5. A comparison of fatty acid profiles of several selected algae strains and conventional biodiesel feedstock (Tab. 2.6) shows that there are fatty acids of comparable chain length extractable from all feedstocks, whereas algal oils can contain considerably higher amounts of polyunsaturated fatty acids. The resulting lower coldfilter plugging point makes biodiesel from microalgae suitable, for example, as fuel for aviation. Nevertheless, the fatty acid profile of microalgae and crops is not constant and varies depending on the culture conditions and growth stage of the culture when

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2 Products from microalgae: An overview

Fatty acid

Doublebond positiona

Rapeseed (Ramos 2009)

Sunflower (Ramos 2009)

Nannochloropsis salina (Huerlimann 2010)

Phaeodactylum tricornutum (Jiang and Gao 2004)

Botryococcus braunii (Zhila et al. 2011)

C12:0b C14:0 C15:0 C16:0 C16:1 C16:2 C16:3 C17:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C20:4 C20:5 C22:0 C22:1 C24:0 C24:1

– – – – 9 7,10 7,10,13 – – 9 9,12 9,12,15 – 11 – 5,8,11,14 5,8,11,14,17 – 13 – 15

– – – 4.9 – – – – 1.6 33.0 20.4 7.9 – 9.3 – – – – 23.0 – –

– – – 6.2 0.1 – – – 3.7 25.2 63.1 0.2 0.3 0.2 0.7 – – – 0.1 0.2 –

5.0 – 0.5 37.5 23.3 – – 0.4 0.9 11.9 1.5 – 0.1 – – 3.3 15.3 0.4 – – –

– 4.5 – 25.8 37.5 – – – 1.3 – 5.1 2.0 – – – 1.6 13.1 – – – –

0.7 0.8 0.5 21.0 2.0 6.5 15.2 0.1 2.9 3.2 13.6 33.0 0.2 – 0.1 – – – – 0.2 –

a

Double-bond position beginning from the carboxyl group.

b

Fatty acid with chain length of 12 carbon atoms and 0 double bonds. The same nomenclature applies for all other fatty acids.

Tab. 2.6: Fatty acid profiles of biodiesel feedstock (wt.%) determined from gas-chromatographic analysis.

harvested. Optimal culture conditions for the desired fatty acid profile can be more easily regulated during the microalgal growth in PBRs than by oil crops on the landfill but at the cost of higher energy and CO2 demand. Therefore, strain selection needs to be performed with a special focus not only on generally applied selection criteria, such as lipid content and areal lipid productivity, but also on the fatty acid profiles matching biodiesel requirements. Further selection criteria for microalgae need to be considered, as they mainly influence the ability to produce biomass on a large scale in the respective environments. Of these, strainspecific optimal temperature range, salinity of the cultivation medium and ability to be maintained for longer periods in non-axenic cultures need to be taken into account. Table 2.7 shows a selection of microalgae species with potential for use in the production of oils for biodiesel based on the accumulated oil content. Most of these species have been tested only on a lab scale. In order to evaluate these for

2.2 Products

Microalgae species

Lipid content (percentage dry weight)

Botryococcus braunii Nannochloropsis sp. Schizochytrium sp. Neochloris oleoabundans Nitzschia sp.

25–75 31–68 50–77 35–54 45–47

39

Tab. 2.7: Oil content of selected algae strains with potential for biodiesel production (Schenk et al. 2008).

commercialization for biodiesel production, they need to be cultivated at a large scale under country-specific environmental conditions. Although algal cultivation is by far the most productive system to obtain biomass, the prices for algal biomass production are still too high (ca. US$ 2–10 for open ponds and US$ 20–100 for closed PBRs) to obtain biodiesel (Raja et al. 2008). The biodiesel option for energy recovery of microalgae has priority in the USA. Between 2009 and 2011, more than US$ 300 million was invested in research and development activities. In particular, aircraft companies, which are not able to replace the need for liquid fuels, take this option seriously and have already organized show-flights using fuel from microalgae. The company Cellana LLC (California and Hawaii, USA) was founded in Hawaii 2004 as a separate joint venture between HR BioPetroleum Inc. and Royal Dutch Shell PLC with the aim to grow marine algae and produce vegetable oil for conversion into biofuel. Since then, they have developed a six-acre facility in Kona (Hawaii) based on the patented ALDUO™ technology, a combination of enclosed PBRs for continuous algae growth under controlled conditions producing the inoculum for large-scale cultivation in open ponds. Meanwhile, the plant has been producing in the third year. The company has reported the cost-efficient production of military jet fuel (JP-8) from crude algae oil and biodiesel after the extraction of high-value omega-3 lipids for human nutrition and proteins for animal feed. Subsequent years will need to see proof of the sustainability and success of the refinery concept applied by Cellana to produce biofuels from microalgae. Since January 2011, HR BioPetroleum Inc. has been the sole owner of Cellana LLC (Cellana LLC 2012). Sapphire Energy Inc. (San Diego, USA), founded in 2007 with the mission to develop domestic, renewable sources of energy, predicts commercial production of crude oil from microalgae, scalably and economically viably within the next five years. During the fifth Bundesalgenstammtisch, a round table for algae experts in Germany organized in March 2012, Cynthia (C.J.) Warner, president of Sapphire Energy, outlined the development efforts that have been undertaken to lower costs and increase the scale of production of Green Crude oil. The technological development is based on the “whole value chain approach” starting from the required improvements to algae biology, through the development of low-cost cultivation

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2 Products from microalgae: An overview

systems to improvements in harvesting and extraction technologies. A commercial demonstration facility is currently under construction in Luna County, New Mexican desert, which should demonstrate the viability of algae cultivation to produce Green Crude oil. This Green Crude can then be processed at a refinery just like traditional crude oil to make all three major distillates: gasoline, diesel, and jet fuel. The demonstration plant should be able to produce as much as 100 barrels of algae-derived crude oil per day by the end of 2014 (Sapphire Energy Inc. 2012). Also, in Europe, there are a wide range of projects, funded by the EU, being carried out to develop sustainable and cost-efficient technology for the use of microalgae as a renewable source of liquid fuels. In the next three to five years, reliable results on a large scale can be expected in order to estimate the viability and future of this technology.

2.2.9.2 Bio-ethanol Several microalgae species accumulate intracellular starch as a storage substance that can be used for ethanol production. The process includes hydrolysis from the polysaccharide into sugar monomers, which can be fermented into ethanol. The end-product of fermentation is a mixture of ethanol (10 %) and water (90 %). Water is removed at the end of the process in order to obtain a usable fuel (96 % bioethanol). Due to the high energy demand, high cost and low efficiency, ethanol production from microalgae could not be established. An alternative production system licensed as Direct to Ethanol® technology has been developed by Algenol Biofuels Inc. (Florida, USA), founded in 2006. The technology is based on the availability of cyanobacteria to convert sugar produced via photosynthesis into ethanol. The strain optimization to enhance the ethanologenic pathways of cyanobacteria was developed by Cyano Biofuels GmbH (Berlin, Germany) in 2007. This company is now a wholly owned subsidiary of Algenol. Ethanol is synthesized intracellularly and diffuses through the cell membrane into the saltwater culture medium. It evaporates during the day into the top of a specially designed sealed PBR. In the evening, when the temperature drops, the ethanol accumulated at the reactor headspace condenses, along with water, and is collected in special troughs inside the PBR. This technology obviates the need for the further processing steps in traditional bio-ethanol production such as starch extraction from cells, hydrolysis and fermentation. As a result, a highly efficient phototrophic process has been developed, which converts the CO2 almost directly into product. On the downstream site, the ethanol is concentrated via vapor compression steam stripping, an energy-efficient technology, as described by Algenol. The company has used distillation or membranes in order to upgrade the ethanol as a transportation fuel. The whole process should have a carbon footprint that is 80 % smaller than that of gasoline (Algenol Biofuels Inc. 2012b).

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A pilot facility of 10 ha, partially financed by governmental funding, is under construction in Fort Myers Florida, adjacent to the research laboratories of Algenol, to demonstrate the commercial viability of the Direct to Ethanol® technology. The technology has also been licensed to BioFields S.A.P.I. de C.V. in Mexico. The company is awaiting the necessary environmental approvals in 2012 to start construction of the production plant on an area the size of more than 55,000 acres of nonarable land in the Sonoran desert in Mexico (Algenol Biofuels Inc. 2012a). Further work on strain optimization will follow in order to develop cyanobacteria that are able to produce ethylene, butane and propane in the same way as ethanol (Enke 2010). This proposal could establish sustainable production of bulk chemicals such as alcohols and monomers for further synthesis.

2.2.9.3 Bio-hydrogen As far back as 1939, Hans Gaffron discovered the ability of microalgae to produce gaseous hydrogen in experiments working with the microalga Scenedemus obliquus (Gaffron 1939; Gaffron and Rubin 1942). The production of hydrogen took place, however, only within a few minutes at the end of an anaerobic cultivation in the dark. This effect was caused by the strong inhibition of oxygen, produced during photosynthesis, to the enzyme responsible for the hydrogen production in the cells, hydrogenase. Hydrogenases catalyze the recombination of H+ and e– into hydrogen with, particularly in microalgae, a high specific activity of around 1,000 U/mg proteins (Happe and Naber 1993; Florin et al. 2001). Therefore, microalgal hydrogenases exceed the activity of most other known hydrogenases by a factor of 100 (Adams 1990). Due to the oxygen sensibility of hydrogenase, a large amount of time was needed until hydrogen could be produced using microalgae over several days. A. Melis first reported in 2000 the development of a two-step process with the chlorophyta Chlamydomonas reinhardtii, which is able to produce hydrogen over several days (Melis et al. 2000). The process principle implies the division of the photosynthetic growth phase (producing oxygen) from the anaerobic phase (induced by sulfur limitation under light) responsible for the induction of hydrogen formation by a change of culture medium. Since then, research on this field has focused on the clarification of the metabolic steps involved in hydrogen production and the development of new algae species with modified hydrogen production rates. In 2005, a strain of Chlamydomonas reinhardtii was selected that was able to produce hydrogen with an unusually high PCE of 2 %, Chlamydomonas reinhardtii stm6 (with PCE values for the wild type ranging from 0.1 % to 0.2 %). This strain exhibits a higher starch content than the wild type and shows characteristic blocked cyclic electron transport (Kruse et al. 2005). Due to the low PCE for the wild type, less research on the technical use of microalgae for hydrogen production has been performed. The low conversion effi-

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2 Products from microalgae: An overview

ciency made the process seem uninteresting from an economic point of view. The successful strain development, as demonstrated in the case of Chlamydomonas reinhardtii stm6, with a PCE of 2 % and a fivefold-longer production phase (ca. 300 h), hence prompts the need for the development of adequate reactor systems and process control strategies. Only with all optimizations completed can the potential for the economic viability of the process be assessed. For this, the identification of process parameters and their influence on the hydrogen productivity using appropriate reactor systems working under strong controlled conditions are required (Hankamer et al. 2007). A new technical development has already been implemented successfully. As already mentioned, in most experiments on hydrogen production with Chlamydomonas, sulfur limitation has been realized by a change of the culture medium. This process step involves solid–liquid separation, which involves additional equipment and energy expense. In order to avoid this procedure, a self-limiting system has been established on a lab scale based on an exact dosage of sulfur in the culture medium. One major task was the reduction in process complexity by eliminating the elaborate and energy intensive solid–liquid separation step. The limiting sulfur concentration allowed for the establishment of an exactly balanced batch system that induces the required sulfur-deprived state only via sulfur consumption by the cells themselves. Consequently, a process strategy for large-scale production was developed from these lab-scale experiments. The aforementioned strategy has been applied to determine light-dependent biomass growth and hydrogen-production kinetics to assess the potential of hydrogen production with Chlamydomonas reinhardtii as a basis for scale-up and further process optimization (Lehr 2011). A viability study for the hydrogen production of Chlamydomonas reinhardtii published by the National Renewable Energy Laboratory produced negative results as the characteristics of the new strain, and contributions from more elaborate PBR design developments were not taken into account (Amos 2004). A recently published study from Australia have a positive evaluation for hydrogen production using microalgae in the future (IBMcom Pty Ltd. 2007). In the midterm, there is indeed a high potential for the economic implementation of the process based on further technological developments to improve the PCE.

2.2.9.4 Bio-gas Anaerobic fermentation is a biological process, in which organic matter is transformed into a mixture of CO2 and CH4 using methanobacteria. The proportion of the mixture depends very much on the composition of the substrate used. The anaerobic fermentation of residual biomass into bio-gas is an already-tested and well-established technology. In Germany alone, a decentralized infrastructure of such plants already exists. The energy efficiency of bio-gas plants is very high, with values of more than 90 %. Taking into account that fermentation of the yield

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of 1 ha of maize provides a continuous power of just 2 kW makes clear the benefits of fermenting microalgal biomass in bio-gas plants. The advantages of microalgal biomass can be found in favourable harvesting and transport conditions. The PBRs can be located next to a bio-gas plant. Furthermore, there is no need for high cell concentrations or further processing steps. Only a moderate solid–liquid separation is needed to feed the algal cells into the bio-gas fermenter. Moreover, nutrient recycling for CO2, nitrogen and phosphate salts between the bio-gas plant and the PBR is achievable. The possibilities of establishing this technology are under investigation in ongoing projects (AlgenBioGas, BMBF, financed from the program FHprofUnd, and SunBioPath, an EU-financed project).

2.2.9.5 Biorefinery of microalgae The main problems with the use of microalgal biomass for energy purposes are the high biomass production cost on the one hand, and the lack of a net-energybalance for the process on the other hand. The current state of the technology shows a higher energy demand to cultivate microalgae and to extract products than can be recovered from the microalgae. Further improvements are needed, particularly in the fields of reactor development and product processing (Morweiser et al. 2010). Efforts to develop sustainable processes will benefit from the added value of using by-products beside the primary energetic use of the biomass. This idea is captured by the so-called biorefinery concept for microalgae (see Eppink et al. 2012). For example, after the extraction of 5 % carotenoids, the remaining 95 % biomass could be used for energy purposes (Stephens et al. 2010). In this case, the high-value product is the main product. The major challenge for this concept is clearly the adjustment of market demand for high-value products from microalgae as well as the bulk biomass mainly addressing the energy sector. At present, the product demand between both sectors is not balanced for the possible combinations of both products. The market for high-value products is much smaller than the material flows needed for energy production. Product application areas with higher demands would be food, bio-plastics, bulk chemicals and materials for construction (see Section 2.2.8). Intensive research in this direction is being conducted. Recent examples include the following: – Paramylon, a β-1,3-glucan polymer, can be extracted in pure form from certain microalgae strains with a high potential as a nutraceutical. The properties and marketed products using paramylon are described in Section 2.2.6.3. A new refinery concept to obtain paramylon and α-tocopherol (vitamin E) from the microalga Euglena gracilis is under investigation in a cooperative project (BMBF financing, project number EGY 08/017) between the University of Bielefeld and an Egyptian research institute. – Grenol GmbH (Wülfrath, Germany) is working on an efficient hydrothermal process for direct liquefaction of algal biomass. The carbon efficiency

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2 Products from microalgae: An overview

achieved is 1, which means that all the C-atoms from the algae can be found again in the product. At the end of the process, several basic compounds are synthesized that serve as raw materials, additives and fillers for the chemical industry, for example to produce polyurethane foam (Sanders 2008).

2.3 Conclusion The biotechnology of microalgae is still at an early development stage. Currently, the major products are the bulk biomasses for the food and feed industry. The market launch of extracts for direct use in human food is increasing continuously. High growth has been observed in the feed industry. The positive effects of using microalgae, especially for aquaculture, are incontrovertible. Aquaculture is one of the most important application areas for algal biomass with a stably increasing expansion. This market can be considered as the basis for the development of cheaper products that are needed in higher amounts. In order to establish and to increase the use of microalgae in this and in further application sectors, the actual costs of biomass need to decrease from the current cost of between US$ 20 and US$ 100/kg to less than US$ 1/kg. So far, the most profitable sector remains the market for fine chemicals. The high sale prices of these products for the cosmetic and pharmaceutical industry justify the production costs and generate high profits, but the volumetric demand is low in this sector. The next step should be the sustainable, solar power-operated production of bulk chemicals, such as monomers for bio-plastics, corresponding to only a small percentage of the whole crude oil consumption. The need for CO2-neutral fuels will open the most substantial and widest market sector for microalgae. Nevertheless, the speed of introduction of fuels from microalgae was initially overestimated. To date, the investment costs per footprint area, the auxiliary power demand and the effort for product processing have been too high. Intensive technological development is needed and has already been initiated, in order to improve the productivity of PBRs and to increase the optimal utilization of the raw material (algal biomass) by developing optimized microalgae-biorefinery concepts. In this case, the combined use of high-value products together with the exploitation of the remaining biomass for energy purposes could establish an economic and renewable process.

References Acién Fernández, F. G., J. M. Fernández Sevilla and E. Molina Grima. 2012. Principles of photobioreactor design. In: (Posten, C., Walter, C. eds) Microalgal Biotechnology: Potential and Production. De Gruyter, Berlin. Adams, M. W. W. 1990. The Structure and Mechanism of Iron-Hydrogenases. Biochimica Et Biophysica Acta 1020: 115–145. Algenol Biofuels Inc. 2012a. www.algenol.com/commercialization/commercialization, accessed on 2012.04.17.

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Algenol Biofuels Inc. 2012b. www.algenol.com/direct-to-ethanol/direct-to-ethanol, accessed on 2012.04.17. Allison, D. P., Y. F. Dufrêne, M. J. Doktycz and M. Hildebrand. 2008. Biomineralization at the Nanoscale: Learning from Diatoms. Methods in Cell Biology: 61–86. Amaro, H. M., A. C. Guedes and F. X. Malcata. 2011. Advances and perspectives in using microalgae to produce biodiesel. Applied Energy 88: 3402–3410. Amos, W. A. 2004. Updated cost analysis of photobiological hydrogen production from Chlamydomonas reihardtii green algae. In Milestone Report. Golden, Colorado: National Renewable Energy Laboratory. Arad, S., Richmond, A. 2004. Industrial Production of Microalgal Cell-mass and Secondary Products – Species of High Potential – Porphyridium sp. In: (A. Richmond, ed.) Handbook of Microalgal Culture. Biotechnology and Applied Phycology. Blackwell Publishing, Oxford, Iowa, Victoria. pp. Atsumi, S., W. Higashide and C. L. Liao. 2009. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nature Biotechnology: 1177–1180. BASF SE. 2012. www.cognis.com, accessed on 2012.03.30. Becker, W. 2004. Microalgae in Human and Animal Nutrition. In: (A. Richmond, ed.) Handbook of Microalgal Culture. Biotechnology and Applied Phycology. Blackwell Publishing, Oxford, Iowa, Victoria. pp. Bienenschwarmmm-Onlineshop. 2012. http://www.bienenschwarmmm.de/catalog/ index.php?cPath=76_93, accessed on 2012.04.24. Bonaeda Biotechnologie. 2012. www.bonaeda-biotechnologie.com, accessed on 2012.05.10. Borowitzka, M. A. 1995. Microalgae as sources of pharmaceuticals and other biologically active compounds. J. Appl. Phycol.: 3–15. Brandbury, J. 2004. Nature’s nanotechnologists: Unveiling the secrets of diatoms. PLoSBiol 10: e306. Brennan, L. and P. Owende. 2010. Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products. Renewable and Sustainable Energy Reviews 14: 557–577. Brown, M. R., S. W. Jeffrey, J. K. Volkman and G. A. Dunstan. 1997. Nutritional properties of microalgae for mariculture. Aquaculture 151: 315–331. Brown, M. R., M. Mular, I. Miller, C. Farmer and C. Trenerry. 1999. The vitamin content of microalgae used in aquaculture. Journal of Applied Phycology 11: 247–255. Cadoret, J.-P., A. Lejeune, R. Michel and A. Carlier. 2012. Algenics: Providing microalgal technologies for biological drugs. In: (Posten, C., Walter, C. eds) Microalgal Biotechnology: Potential and Production. De Gruyter, Berlin. Cellana LLC. 2012. http://cellana.com, accessed on 2012.4.17. Chapman, V. J. and D. J. Chapman. 1980. Seaweeds and Their Uses. 3rd Edition. Chapman & Hall, London. pp. 334. Chisti, Y. 2007. Biodiesel from microalgae. Biotechnology Advances 25: 294–306. Chisti, Y. 2012. Raceways-based production of algal crude oil. In: (Posten, C., Walter, C. eds) Microalgal Biotechnology: Potential and Production. De Gruyter, Berlin. Chlostanin Nikken Nature Co. Limited. 2012. http://nikken-miho.com, accessed on 2012.03.01. Clarins GmbH. 2012. http://de.clarins.com, accessed on 2012.3.1. Cyanotech Corp. 2012. www.cyanotech.com/index.html, accessed on 2012.04.24. Desplancq, D., A. S. Rinaldi, H. Horzer, Y. Ho, H. Nierengarten, R. A. Atkinson, B. Kieffer and E. Weiss. 2008. Automated overexpression and isotopic labelling of biologically active oncoproteins in the cyanobacterium Anabaena sp PCC 7120. Biotechnology and Applied Biochemistry 51: 53–61. DIC Lifetec Co. Ltd. 2012. www.dlt-spl.co.jp/business/en/spirulina/ecology.html#company, accessed on 2012.03.21.

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DuPont Tate & Lyle BioProducts. 2012. www.duponttateandlyle.com/fact_06–2007_ sustainability.php, accessed on 2012.04.15. Dvir, I., R. Chayoth, U. Sod-Moriah, S. Shany, A. Nyska, A. H. Stark, Z. Madar and S. M. Arad. 2000. Soluble polysaccharide and biomass of red microalga Porphyridium sp alter intestinal morphology and reduce serum cholesterol in rats. British Journal of Nutrition 84: 469–476. DXN-Marketing. 2012. www.dxnmalaysia.com/product/spirulina_faq.php, accessed on 2012.03.07. Earthrise Nutritionals LLC. 2012. www.earthrise.com/, accessed on 2012.03.25. Ecke, M. 2010. Mikroalgenproduktion im industriellen Maßstab – Betriebserfahrungen mit der 12.000 m² Photobioreaktoranlage in Klötze/Deutschland. Tagung des DECHEMAArbeitskreises „Algenbiotechnologie“. Produkte aus Algen., Frankfurt a. M. Enderle, T. 2011. The potential of microalgae for fuel and non-fuel applications. KIC. Enke, H. 2010. Direct coupling of photosynthesis to ethanol production in cyanobacteria. 8. European Workshop Biotechnology of Microalgae, Nuthetal. Eppink, M., Barbosa, M. and Wijffels, R. 2012. Biorefinery of Microalgae: production of high-value products, bulk chemicals and biofuels. In: (Posten, C., Walter, C. eds) Microalgal Biotechnology: Integration and Economy. De Gruyter, Berlin. Fabregas, J., D. Garcia, M. Fernandez-Alonso, A. I. Rocha, P. Gomez-Puertas, J. M. Escribano, A. Otero and J. M. Coll. 1999. In vitro inhibition of the replication of haemorrhagic septicaemia virus (VHSV) and African swine fever virus (ASFV) by extracts from marine microalgae. Antiviral Research 44: 67–73. FAO. 2012. http://www.fao.org/fishery/species/2790/en, accessed on 2012.04.10. floramed GmbH. 2012. http://shop.floramed.de/index.php/default/catalogsearch/result/?q= spirulina, accessed on 2012.05.02. Florin, L., A. Tsokoglou and T. Happe. 2001. A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain. Journal of Biological Chemistry 276: 6125–6132. Friedl, T., N. Rybalka and A. Kryvenda. 2012. Phylogeny and systematics of microalgae: An overview. In: (Posten, C., Walter, C. eds) Microalgal Biotechnology: Potential and Production. De Gruyter, Berlin. Furst, P. T. 1978. Hum. Nature March. Gaffron, H. 1939. The reduction of carbon dioxide with molecular hydrogen in green algae. Nature (London, United Kingdom) 143: 204–205. Gaffron, H. and J. Rubin. 1942. Fermentative and photochemical production of hydrogen in algae. Journal of General Physiology 26: 219–240. Geresh, S., A. Mamontov and J. Weinstein. 2002. Sulfation of extracellular polysaccharides of red microalgae: preparation, characterization and properties. Journal of Biochemical and Biophysical Methods 50: 179–187. greenovation Biotech GmbH. 2012. www.greenovation.com/scalability.html, accessed on 2012.03.01. Grewe, C. 2009. Mikroalgenbiotechnologie im industriellen Maßstab – Produkte und Anwendungen. 5. Köthener BT-Kolloquium Algenbiotechnologie, Köthen. Grewe, C. and Griehl, C. 2012. The Carotenoid Astaxanthin from Haematococcus pluvialis. In: (Posten, C., Walter, C. eds) Microalgal Biotechnology: Integration and Economy. De Gruyter, Berlin. Griehl, C. and S. Bieler. 2011. Algen: Rohstoffe für Gesundheit, Schönheit und Energie. In Nachwachsende Rohstoffe. Häder, D.-P., ed. 1999. Photosynthese. Georg Thieme Verlag, Stuttgart. pp. 270. Hankamer, B., F. Lehr, J. Rupprecht, J. H. Mussgnug, C. Posten and O. Kruse. 2007. Photosynthetic biomass and H2 production by green algae: from bioengineering to bioreactor scale-up. Physiologia Plantarum 131: 10–21.

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Min Thein, Toe Aung, Khin Pyone Lwin, May Yu Khaing and Otto Pulz

3 Spirulina production in volcano lakes: From natural resources to human welfare 3.1 Introduction Natural Spirulina lakes have been reported from several places in the tropical regions around the world (Hills 1980; Fox 1996). However, prominent and permanent Spirulina lakes occur only in Africa, Mexico and Myanmar. Africa and Mexico have traditionally used Spirulina as an edible microorganism, but Myanmar definitely has no such tradition (Min Thein 1987). The importance of Spirulina as a nutritional source was rediscovered by Leonard (1966) near Lake Chad during “The 1964–65 Belgian Trans-Saharan Expedition”. Further studies by European and International scientists have been conducted extensively (Shelef and Soeder 1980; Nakamura 1982; Ciferri 1983; Becker and Venkataraman 1984; Borowitzka and Borowitzka 1988; Lembi and Waaland 1988; Henrikson 1989; Pulz 1992; Doumengue et al. 1993; Fox 1996; Vonshak 1997; Pulz 2001; Pulz and Gross 2004; Richmond 2004; Pulz and Mewes 2006; Gershwin and Belay 2008; Henrikson 2010). According to these published papers, Spirulina is found to be a highly nutritious food source. It is rich in protein (more than 60 %), vitamins and minerals. It is also free from toxic compounds. Health benefits and nutritional values are also recognized. Most of the Spirulina products are nutritional supplements. It is also used in cosmetic products, functional health foods and drinks. The first industrial production of Spirulina from natural lakes began in Lake Texcoco, Mexico (Durand-Chastel 1980), but production was discontinued due to economic reasons. The second and still ongoing industrial production began in Twyn Taung Lake, Myanmar in 1988 (Min Thein 1993). Spirulina lakes in Africa are still harvested traditionally on a limited cottage industrial scale for local consumption (Batello et al. 2004). Aphanizomenon flos-aquae, another blue-green alga (cyanobacteria), is harvested in Lake Klamath, Oregon, USA and has also been marketed as a nutritional supplement since 1983 (Simplexity Health 2012). Spirulina biomass is produced commercially mainly in shallow open raceway ponds. The largest producers are Earthrise (DIC) Farms in California, Cyanotech in Hawaii and DIC in Hainan. China is the major producer of Spirulina (Henrikson 2010). Long-term cooperation between Myanmar and German scientists resulted in a sustainable production and use of Spirulina with appropriate technology, quality control and quality-assurance system. Myanmar is now a major producer and con-

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3 Spirulina production in volcano lakes: From natural resources to human welfare

sumer of Spirulina nutritional-supplement tablets, functional food, drinks and cosmetics.

3.2 Natural Spirulina lakes in Myanmar In 1982, during a short visit of Jülich by Min Thein, who was then a Humboldt Foundation (AvH) postdoctoral research fellow in Helgoland, Professor Soeder (pers. comm.) pointed out that natural lakes of Spirulina might occur in Myanmar and mentioned his willingness to assist in artificial culture, even if there were no such lakes. Fortunately, with follow-up research in Myanmar, Twyn Taung Lake

Fig. 3.1: Twyn Taung Spirulina volcanic crater lake (22°21′33.12″N, 95°00′48.15″E).

Fig. 3.2: Twyn Ma Spirulina volcanic crater lake (22°16′51.98″N, 94°58′19.03″E).

3.2 Natural Spirulina lakes in Myanmar

53

Fig. 3.3: Taung Pyauk Spirulina volcanic crater lake (22°18′09.41″N, 94°58′53.93″E).

Fig. 3.4: Ye Kharr Spirulina surface lake (21°58′25.23″N, 95°59′13.67″E).

was discovered in 1984, and Taung Pyauk and Twyn Ma volcanic crater lakes across the Chindwin River were visited in 1985. Ye Khar is a surface lake of old sea-bed origin found near Sagaing Hills across the Irrawaddy river opposite Mandalay city. These four natural Spirulina lakes (Figs 3.1–3.4) have a total water surface area of about 220 ha. Consequently, a project proposal was prepared in 1985 for the R&D of Spirulina production and use in Myanmar, and submitted to the Ministry of Education. About 60 undergraduate and post-graduate students (May Yu Khine 1987; Khin Pyone Lwin 1988; Toe Aung 1992) of the Marine Biology Department, Mawlamyaing University took part in the project. In 1987, an UNIDO/UNDP project was granted to

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3 Spirulina production in volcano lakes: From natural resources to human welfare

study Spirulina production and use in The Netherlands, Mexico and USA (Min Thein 1987). A paper on pilot-scale production of Spirulina in Myanmar was presented 1993 in Monaco (Min Thein).

3.3 Environmental parameters of Myanmar Spirulina lakes All four Myanmar Spirulina lakes are situated at about 22° North latitude (Tab. 3.1). The water of the three volcanic crater lakes is carbonate-dominated, while the fourth surface lake is sulfate-dominated.

Lake Parameter Location

Twyn Taung

22°21′33.12″N 95°00′48.15″E Water surface area 80 (ha) Depth (m) 50 pH 9.5 Base -CO3 Salinity (ppt) 4 Water surface 18–36 temperature (min– max)/year (°C) Light (min–max) 0–1300 (μmol/m2/s)

Twyn Ma

Taung Pyauk

Ye Kharr

22°16′51.98″N 94°58′19.03″E 60

22°18′09.41″N 94°58′53.93″E 30

21°58′25.23″N 95°59′13.67″E 50

35 10 -CO3 10 18–36

25 10 -CO3 1 18–36

8 8.6 -SO4 16 16–36

0–1300

0–1300

0–1500

Tab. 3.1: Environmental parameters of Spirulina volcanic crater lakes in Myanmar.

Fig. 3.5: Monthly average temperature and OD560 variation at Twyn Taung Lake, 2009.

3.3 Environmental parameters of Myanmar Spirulina lakes

55

Fig. 3.6: Monthly average temperature and OD560 variation at Twyn Taung Lake, 2010.

Fig. 3.7: Monthly average temperature and OD560 variation at Twyn Taung Lake, 2011.

The pH is most stable (between 9.5 and 10.5) in Lake Twyn Taung and Twyn Ma, and there is a low risk of contamination due to the high pH. However, the pH is slightly variable in the other two lakes due to occasional enrichment by freshwater. The annual rainfall in the region ranges from 90 to 120 cm. Since 1988, daily records have been taken for temperature (air and surface), pH and standing crop (OD560) at 8:00 and 14:00. Microscopic examinations have been carried out daily, and regular records of light and nutrient levels have also been made. Thus, the lakes are carefully monitored and managed as necessary. Twyn Taung Crater Lake is completely surrounded by a 100–150 m high crater rim and is well protected from any source of pollution from outside. Each year, the

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3 Spirulina production in volcano lakes: From natural resources to human welfare

Fig. 3.8: Daily temperature and OD560 variation at Twyn Taung Lake in March, 2009.

Fig. 3.9: Daily temperature and OD560 variation at Twyn Taung Lake in March, 2010.

summer algal blooms occur around March, showing a peak monthly average OD (standing crop) when the thermocline layer is more distinct than in the other months (Figs 3.5–3.7). This is preceded by a sudden rise in surface temperature (spring) from ~20 °C to ~25 °C. The peak ends in April as the temperature rises above ~35 °C. A second smaller peak occurs in October when the temperature drops to ~30 °C. During March, the daily temperature ranges from 23 to 37 °C at 14:00. Growth is found to be retarded outside this range. Daily records of standing crop (OD560) show a pattern of increase and decrease every 2–3 days and probably indicate a regeneration rate during the blooming season (Figs 3.8–3.10).

3.4 Spirulina production from natural lakes

57

Fig. 3.10: Daily temperature and OD560 variation at Twyn Taung Lake in March, 2011.

Although at optimum levels during the blooming summer months, light and nutrients seem to have no apparent influence on the blooming biomass. However, temperature seems to play a decisive role in the regular occurrence of summer Spirulina blooms.

3.4 Spirulina production from natural lakes 3.4.1 Harvesting Twyn Taung Crater Lake is the main harvesting station of Myanmar Spirulina biomass production. In this lake, Spirulina microalga filaments, ranging from loose to tight spiral forms (Fig. 3.11), can be found uniformly from the surface down to a depth of about 1 m. During the year, the concentration of Spirulina ranges from about 0.1 to 0.3 g/L, but for about 60 days in the summer months (February–April), it can reach 0.6 g/L. In some areas of the lake, due to gentle wind action, the floating Spirulina biomass can become about 30 cm thick (Fig. 3.12) with a concentration of 60 g/L. The cutting edge of Myanmar Spirulina production is the seasonal occurrence of massive algal blooms, which make harvesting the biomass manually by boat very simple, easy and economical (Fig. 3.13). The harvested biomass (25–30 g/L) is transferred to storage ponds and processed further or kept overnight.

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Fig. 3.11: Photomicrograph of loose and tight forms of Spirulina from Twyn Taung Lake.

Fig. 3.12: Thick mat of floating Spirulina biomass in March at Twyn Taung Lake.

3.4.2 Washing and dewatering Horizontal hollow cylinders with sieve cloth (Fig. 3.14) are used to wash off the alkaline water (Win Naing Oo, pers. comm.). The washed biomass is put into dewatering bags and held under pressure (Fig. 3.15). After about 1 h, a dough-like biomass is obtained. In the early years, settling cones made of cloth were used for washing (Min Thein 1993). The simultaneous use of at least a hundred cone was necessary, and the process was labor-intensive, time-consuming and uneconomical. Hollow-sieve

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Fig. 3.13: Collecting Spirulina biomass by boat at Twyn Taung Lake.

Fig. 3.14: Horizontal hollow cylinders with sieve cloths for washing.

cylinders eliminate the use of settling cones and help greatly in washing the biomass.

3.4.3 Extrusion and sun drying The dough-like thick paste is extruded into noodles and spread over plastic trays for sun drying (Fig. 3.16). The most important step in manual production of Myanmar Spirulina without using standard drying equipment such as spray dryers, drum dryers, or oven dry-

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Fig. 3.15: Dewatering bags placed under pressure.

Fig. 3.16: Dough-like thick paste extruded into noodles and spread over plastic trays for sun drying.

ers, is the introduction of extrusion technology. At the beginning of the Myanmar Spirulina research project in 1984, there were no reports in literature of any extrusion method used for algae drying (Shelef and Soeder 1980). The method was adopted after observing the production of fresh rice noodles at local spaghetti food shops in Mawlamyine, Myanmar. The rice dough is extruded through hand-held wooden extruders (piston and cylinder with pores) into hot water and served fresh to the customers.

3.4 Spirulina production from natural lakes

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Fig. 3.17: Inefficient hand-held extruder for extrusion and spreading of noodles.

Extrusion through such a cylinder creates the latent heat in the biomass without overheating (encountered with screw type extruders) and enhances the sundrying process. Drying can be achieved within 3–4 h at a sunny day. Without extrusion and with simple spreading and combing, the drying can take 2–3 days. The acidulation method was tried at the Oceanic Institute of Hawaii to avoid biomass fouling while drying. Professor Eric Duerr (pers. comm.) was surprised to discover that Myanmar was using a simple but effective extrusion technology (Min Thein 1987), and extrusion of Spirulina biomass into noodles was neither known in the literature nor practiced before Myanmar first used the method in 1984. The first used handheld extruder (Fig. 3.17) was not efficient. It took about 5 min for two persons to fill a drying tray. The pressure felt on the hands was also very tiring, and operators needed to take alternate turns after about five trays. A major breakthrough came in 1966 with the innovative invention of the extrusion trolley by Nyo Sein (pers. comm.) (Fig. 3.16). It took only about 1 min to fill a tray, and operators were able to work continously for 2–3 h. After the introduction of the extrusion trolley, daily production during the peak season ranged from 2 to 4 tons, and a record production of 5 dry tons of Spirulina chips per day has been achieved. The potential of available biomass can be as high as 10 t/d. Daily production during the off-peak season is carried out using inclined screens. The dewatering and drying processes are the same as in the peak season. A glasshouse with exhaust fans is used for drying on rainy days.

3.4.4 Lake-side enhancement ponds Raceway ponds with paddle wheels were built on the lake side and operated to increase the growth rate of Spirulina by adjusting light, temperature, pH and nutri-

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Fig. 3.18: Lake-side ponds for growth enhancement of Spirulina.

ents. The lake water is pumped into the ponds and drained back into the lake after 3–4 days of growth enhancement. Spirulina is not directly harvested from the enhancement ponds as a rule, only the lake water is filtered to harvest the naturalized biomass. Lake-side ponds (Fig. 3.18) have a total water surface area of 2 ha and are absolutely essential for sustainable growth and production management of the algal biomass in the lake.

3.5 Sustainable Spirulina production from volcanic crater lakes The algae production system of natural Spirulina lakes in Myanmar is sustainable and eco-friendly, and harvesting has been carried out continuously since 1988 (Fig. 3.19). At present, on the basis of market demand, the annual production capacity is more than 200 tons of Spirulina biomass as dry chips. About 70 % of annual production is achieved from Twyn Taung Lake. Ye Khar is the second production station. Lake Twyn Ma and Taung Pyauk are under development.

3.6 Myanmar Spirulina products

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Fig. 3.19: Spirulina biomass (dry tons) production over 23 years (1988–2011).

3.6 Myanmar Spirulina products Further processing for finished products is carried out at the GMP-certified Myanmar Pharmaceutical Industry (MPI) at Sagaing (Fig. 3.20). About 14 different Spirulina-based products (Fig. 3.21) such as nutritional supplements and antiviral tablets, local cosmetics (Thanakha), shampoo, aloe vera drinks, beer (extract supplied to the brewery) and biofertilizer are currently produced and mainly sold on the local market. Nutritional supplements and antiviral tablets account for more than 80 % of Myanmar Spirulina products. At present, about 1 million bottles of 50 g net weight (equivalent amount) are produced and marketed annually. Since 1989, more than 10 million bottles have been sold on the Myanmar market. Thus, according to the

Fig. 3.20: GMP certified Spirulina processing factory building, MPI, Sagaing.

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Fig. 3.21: Myanmar Spirulina products.

massive information feedback over the last 20 years, Spirulina tablets are widely accepted as a good nutritional supplement to aid good health and well-being of consumers in Myanmar. Many people who have malnutrition, diabetes, heart problem, cancer, HIV, tuberculosis and malaria have reported improvements in their health conditions, and such health benefits can also be enjoyed in developed and developing countries worldwide. The production of Spirulina in Myanmar is regarded by the government and people as a model project that has transferred from the R&D stage at the university level to an agro-based industry beneficial in the health sector.

3.7 Spirulina as biofertilizer The use of microalgae as biofertilizer has been under extensive study in Europe, and regular conferences are held through a cooperation between IGV GmbH and Hungary (Ördög and Szigeti 1997; Ördög 1999). Myanmar followed the idea, and the use of Spirulina as a biofertilizer has been studied since 2000. A poster was presented at the 6th European Workshop in Microalgae Biotechnology, Nuthetal, Germany in 2005 (Fig. 3.22). A number of Ph.D. theses have been submitted to the Department of Botany, University of Mandalay with regard to the effect of Spirulina biofertilizer on various cereals and legumes (Fig. 3.23) (Wai Wai Mar 2007; Kyaw Soe Naing 2008; San Win 2008; Thet Naing Htwe 2008; Win Naing Oo 2008; Kyaw Kyaw San 2009; Khin Lay Nandar Aung 2011; Aye Mya Nyein 2012; Khaing Khaing 2012; Mar Lar 2012; San San Aye 2012; Tin Tin Maw 2012; Win Mar 2012). Higher plant growth and grain yields as well as an increase of the nodule formation have been achieved by the Spirulina treatment. A better disease resistance is found in legumes. The growth of soil microorganisms such as bacteria and fungi also

Fig. 3.22: Effect of Spirulina biofertilizer on various crops in Myanmar.

3.7 Spirulina as biofertilizer

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Fig. 3.23: Effect of Spirulina suspension on growth and yield of Glycine max (L.) Merrill. (soybean) in field experiment (Mar Lar 2012).

increases significantly. A biofertilizer formulation with Spirulina is now produced commercially, as the application rate is found to be economically feasible for agriculture in Myanmar. Several species of microalgae have been reported to produce plant hormones or hormone-like compounds as well as other bioactive compounds relevant to agriculture. Spirulina is a microalga or cyanobacterium that is unique in being able to promote the growth of Lactobacillus. Lactobacilli are anaerobic bacteria that are primary decomposers or digesters present in the guts of animals and in the soil. The application of Spirulina probably acts as a soil conditioner that promotes the growth of essential microbes, especially the primary decomposers such as Lactobacilli in the food web of soil microorganisms. Thus, in addition to the bioactive compounds relevant for plant growth, Spirulina can be regarded as a tonic for local soil microorganisms and not as one of the directly introduced essential microbes that are now well known to be unsustainable for soil fertility. Spirulina based biofertilizer or soil conditioner as prepared in Myanmar are now used in growing rice, legumes and cotton. As its use increases and if enough biomass can be produced at low cost and with the appropriate technology, it is hoped that not only Myanmar but also its neighboring countries and agriculture worldwide will benefit from using Spirulina based biofertilizer or soil conditioner to restore the soil fertility of highly disturbed croplands and forest lands on earth.

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3.8 Spirulina as a biogas enhancer The concept of Spirulina presented above as a tonic for primary decomposers such as Lactobacillus bacteria is applicable in enhancing biogas production as well. In experimental trials, the use of Spirulina biomass has been found to increase the biogas production rate by nearly twofold for a given amount of waste matter such as cow and sheep dung (Lwin Win, pers. comm.).

3.9 Spirulina as a source of biofuel The downstream processes of producing biofuels from Spirulina and other microalgae has been extensively reported in the literature. Although producing biodiesel or bioethanol as a single product is still not economically feasible, the use of residues as biofertilizer or soil conditioner may well pave the way for economic feasibility. With this concept at hand, future attempts at large-scale production of Spirulina or other microalgae, comparable to the scale of production of agricultural crops, need to be conducted so that the per-unit costs will become competitive with the fossil-fuel prizes.

3.10 Myanmar and German cooperation in microalgae biotechnology Myanmar and Germany have a long historical record of cooperation in microalgae biotechnology. Mr S. Kurz, a German forester working in Myanmar, sent some collections of microalgae for identification and records that were the earliest known publications on Myanmar algae (Martens von 1871b, 1871a; Zeller 1873). During the last 30 years of cooperation between the scientists on the Spirulina project (1982), significant achievements can be summarized as follows: – discovery of natural Spirulina lakes in Myanmar due to scientific prediction of Professor Carl Soeder (pers. comm. 1982); – continued Humboldt Foundation AvH Fellowship (1998, 2009) leading to long-term technical cooperation with Professor Otto Pulz, President of the European Society of Microalgal Biotechnology; – training of Myanmar graduate staff (eight persons, 4 months) at IGV GmbH, Germany on quality control and quality assurance system of Myanmar Spirulina products; – sharing up-to-date information and inviting Myanmar scientists to attend international workshops and meetings; – formulation of Spirulina products such as nutritional supplement and antiviral tablets, functional foods and drinks including Spirulina beer, cosmetics and biofertilizer;

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assisting in local and foreign market promotions; research cooperation on sustainable production and use of Spirulina from crater lakes and other useful microalgae in Myanmar.

3.11 Discussion The appropriate technology with quality assurance and quality control systems has been developed and practiced in the production of Spirulina cyanobacteria from volcanic crater lakes in Myanmar, mainly through a unique long-term scientific cooperation between Myanmar and Germany, achieved as a follow-up to the Alexander von Humboldt Foundation (AvH) program. The success of Myanmar Spirulina biotechnology is mainly a result of the following: – the regular occurrence of massive Spirulina blooms in the summer months; – the high crater rims protecting the lakes from outside pollution; – the simple innovation of extrusion technology since 1985 and the use of an extrusion trolley; – the use of horizontal sieve cylinders for washing; – the use of lake-side growth-enhancement ponds; – regular recording and monitoring of environmental parameters of the lakes; – the application of HACCP and GMP principles in the production processes. During the last 30 years of R&D, the nutritional value of Spirulina got widely accepted in Myanmar, where there was no traditional production or use before. Its efficacy is more pronounced, as few Myanmar people have sufficient knowledge of nutrition, although food is plentiful locally. Myanmar is a good example of a country practicing a kind of green economy with its own production and multiple uses of Spirulina.

3.12 Conclusion The sustainable production and use of Spirulina blue-green alga from natural volcanic lakes in Myanmar is now well established worldwide. It is hoped also that this sustainability can be maintained in future with careful lake management and monitoring of environmental changes that might lead to depletion and extinction of the Spirulina natural resource. As Spirulina has excellent nutritional and health benefits as well as bio-remediation potentials such as restoration of soil fertility. As Myanmar Spirulina biotechnology is simple, and its products immensely useful, it is hoped that other developing countries will adopt the practice for the production of food and feed and to enjoy its benefits for personal well-being.

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Acknowledgments Thanks are due to the Alexander von Humboldt Foundation of Germany and the German Embassy in Yangon for awarding three research fellowships during a 30-year long-term period to Min Thein in 1981, 1998 and 2009. Thanks are also due to the Myanmar Government, Ministry of Education and Ministry of Industry, for their permission and assistance in conducting the project in a life-time period. The photographs were taken by San Tun Aung and Myo Min Oo.

References Aye Mya Nyein. 2012. Effect of Spirulina on growth, yield and nutritive value of Phaseolus vulgaris L. (Bosape). Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Batello, C., M. Marzot and A. H. Touré. 2004. The future is in ancient lake – Traditional knowledge, biodiversity and genetic resources for food and agriculture in Lake Chad Basin ecosystems. FAO Inter-Departmental Working Group on Biological Diversity for Food and Agriculture, Rome. pp. 258–285. Becker, E. W. and L. V. Venkataraman. 1984. Production and Utilization of the Blue-Green-Alga Spirulina in India. Biomass 4: 105–125. Borowitzka, M. A. and L. J. Borowitzka. 1988. Micro-algal Biotechnology. Cambridge University Press, Cambridge. pp. 477. Ciferri, O. 1983. Spirulina, the Edible Microorganism. Microbiol Rev 47: 551–578. Doumengue, F., H. Durand-Chastel and A. Toulemont. 1993. Spiruline algue de vie. Musée Océanographique. Bulletin de ´lInstitut Océanographique. Numéro spécial 12, Monaco. pp. 222. Duerr, E. 1987. Personal Communication. Durand-Chastel, H. 1980. Production and use of Spirulina in Mexico. In: (G. Shelef and C. J. Soeder, eds) Algae Biomass. Elsevier/North Holland Biomedical Press, Amsterdam. pp. 51– 64. Fox, R. D. 1996. Spirulina. Production and Potential. Edisud, Aix-en-Provence. pp. 232. Gershwin, M. E. and A. Belay, eds. 2008. Spirulina in Human Nutrition and Health. CRC Press, Boca Raton. pp. 312. Henrikson, R. 1989. Earth Food Spirulina: How This Remarkable Blue-Green Algae Can Transform Your Health and Our Planet. Ronore Enterprises Inc., San Rafael. pp. 174. Henrikson, R. 2010. Spirulina World Food: How this micro algae can transform your health and our planet. Ronore Enterprises Inc, Hana. pp. 192. Hills, C. 1980. The Secrets of spirulina: Medical discoveries of Japanese doctors. University of the Trees Press in cooperation with Journal of Nutritional Microbiology, Boulder Creek. pp. 218. Simplexity Health. 2012. www.simplexityhealth.com, accessed on 2012.02.29. Khaing Khaing. 2012. Effect of Spirulina on growth, yield and nutritive value of Paseolus lunatus L. (Penigya). Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Khin Lay Nandar Aung. 2011. Effect of Spirulina on the germination, growth, yield and yield component character of Vigna radiata (L.) Wilezek (Green Gram). Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Khin Pyone Lwin. 1988. Seasonal variation of the density of Spirulina inrelation to some ecological parameters in Twin Taung lake series. M.Sc. Thesis. Dept. of Marine Biology, University of Mawlamyaing, Mawlamyine, Myanmar.

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Kyaw Kyaw San. 2009. Germination of Myanmar tea plants and cultivation of Camellia sinensis (L.) Kuntze by using Spirulina bioferilizer. Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Kyaw Soe Naing. 2008. Effect of Spirulina on the germination and growth of three important oil crops. Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Lembi, C. A. and J. R. Waaland, eds. 1988. Algae and human affairs. Cambridge University Press, Cambridge. pp. 590. Leonard, J. 1966. The 1964–65 Belgian Trans-Saharan Expedition. Nature 209: 126–127. Lwin Win. Personal Communication. Mar Lar. 2012. Effect of Spirulina on growth, yield and nutritive value of Glycine max (L.) Merrill (Pe-boke). Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Martens von, G. 1871a. A fifth list of Bengal algae (determined by G. von Martens, communicated by S. Kurz). Proc. Asiat. Soc. Beng. 49: 170–173. Martens von, G. 1871b. List of algae collected by Mr. S. Kurz in Burma and adjacent islands. J. Asiat. Soc. Beng. 40: 461–469. May Yu Khine. 1987. Studies on laboratory culture of Spirulina. M.Sc. Thesis. Dept. of Marine Biology, University of Mawlamyaing, Mawlamyaing, Myanmar. Min Thein. 1987. Laboratory examination of Spirulina sample from Burma and a study of Spirulina production and use. UNIDO/UNDP/BUR 85–018 report: 119. Min Thein. 1993. Production of Spirulina in Myanmar. In: (F. Doumengue, H. Durand-Chastel and A. Toulemont, eds) Spiruline algue de vie. Musée Océanographique. Bulletin de l’Institut Océanographique. Numéro spécial 12, Monaco. pp. 175–178. Nakamura, H. 1982. Spirulina: Food for Hungry World: A Pioneer’s Story in Aquaculture. University of the Trees Press, California. pp. 217. Nyo Sein. 1966. Personal Communication. Ördög, V. 1999. Beneficial effect of microalgae and cyanobacteria in plant/soil system with special regard to their auxin and cytokinin-like activity. International workshop and training course on microalgal biology and biotechnology -UNESCO (International Cell Research Organization), Mosonmagyaróvár. pp. 43–44. Ördög, V. and J. Szigeti. 1997. Cyanobacteria and microalgae as biofertilizers. Abstract of the 3rd European Workshop on Biotechnology of Microalgae (Plenary lecture), Bergholz-Rehbrücke. Pulz, O. 1992. Cultivation techniques for microalgae in open and closed systems. Proceedings of the 1st European Workshop on Biotechnology of Microalgae, Bergholz-Rehbrücke. pp. 61–66. Pulz, O. 2001. Photobioreactors: production systems for phototrophic microorganisms. Appl Microbiol Biotechnol 57: 287–293. Pulz, O. and W. Gross. 2004. Valuable products from biotechnology of microalgae. Appl Microbiol Biotechnol 65: 635–648. Pulz, O. and P. J. Mewes. 2006. Unterschiedliche antivirale Aktivitäten von Extrakten aus Spirulina – Arthrospira platensis. OM & Ernährung 116: 37–39. Richmond, A., ed. 2004. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing, Oxford. pp. 566. San San Aye. 2012. Effect of Spirulina on growth, yield and nutritive value of Labla purpueus (L.) Sweet (pegyi). Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. San Win. 2008. Effect of Spirulina on the germination and growth of tobacco. Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Shelef, G. and C. J. Soeder, eds. 1980. Algae Biomass: production and use. Elsevier/NorthHolland Biomedical Press, Amsterdam; New York. pp. 852. Soeder, C. J. 1982. Personal Communication. Thet Naing Htwe. 2008. Effect of Spirulina on the germination and growth of chick pea, soybean and butter bean. Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar.

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Tin Tin Maw. 2012. Effect of Spirulina on growth, yield and nutritive value of Vigna mungo (L.) Hepper (Mat-pe). Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Toe Aung. 1992. Harvesting and processing of Spirulina production from Twyn Taung lake Myanmar. M.Sc. Thesis. Dept. of Marine Biology, University of Mawlamyaing, Mawlamyaing, Myanmar. Vonshak, A. 1997. Spirulina platensis (Arthrospira): Physiology, Cell-biology and Biotechnology. Taylor & Francis Ltd, London. pp. 233. Wai Wai Mar. 2007. Effect of Spirulina on the Growth and Yield of Onion, Tomato and Chili. Ph.D. Thesis. Dept. of Chemistry, Yangon University, Yangon, Myanmar. Win Mar. 2012. Effect of Spirulina on growth, yield and nutritive value of Vigna unguiculata (L.) Walp (Pelunphyu). Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Win Naing Oo. 2008. Effect of Spirulina on the germination and growth of rice and wheat. Ph.D. Thesis. Dept. of Botany, Mandalay University, Mandalay, Myanmar. Win Naing Oo. 2005. Personal Communication. Zeller, G. 1873. Algae collected by Mr. S. Kurz in Arracan and British Burma, determined and systematically arranged by Dr. G. Zeller. J. Asiatic. Soc. Bengal. 42: 175–193.

C. Thomsen, S. Rill and L. Thomsen

4 Case study of a temperature-controlled outdoor PBR system in Bremen This project is a follow-up of a discussion on a study carried out by University of Virginia in 2010 (Clarens et al. 2010), which concluded that algal farms require six times as much energy as growing land plants – and emit significantly greater quantities of greenhouse gases. The aim of this project was to use flue gas as a CO2 source, waste heat for temperature control and, to a greater extent, wastewater to offset most of the environmental burdens associated with algae. In addition, the carbon footprint of the algae farm should clearly be positive, so significantly less CO2 must be produced via energy consumption than is mitigated by the algae. New technologies for closed bioreactors and open-pond systems are emerging. One such platform for evaluation and defining objectives for algae biomass production is the European Algae Biomass Association, and examples of the technology are provided by Brennan and Owende (2010). The most important aim is to reduce the installation and maintenance costs for the PBRs. R&D at Phytolutions together with Jacobs University Bremen at several power plants in Europe led to the development of a new closed endless film bioreactor. For large algae farms (several hectares), the installation costs can currently be reduced to € 25/m2 with expected production rates of 80–100 t/ha in Germany. This has to include mixotrophy using additional carbon sources from wastewater during the final stages of production. Many studies indicate that only favorable solar radiation would make fuel production via algae feasible. Thus, low latitudes are perfectly suited to biomass production. This project emphasizes the concept that algae production sites should be decentralized and spread along latitudes to avoid the expensive transport logistics associated with (wet) biomass. In order to compensate for insulation, the temperature can be controlled, and in the ocean, high phytoplankton production can be found even around 60° south (Sarmiento et al. 2004). European Union legislation demands that no fossil CO2, only CO2 emitted via renewable carbon sources, should be allowed to produce biofuels. This topic needs further discussion, since, to date, fossil CO2 can already contribute up to 20 % of the atmospheric CO2 content in populated industrial areas (Levin et al. 2008). The project is part of the EU-EFRE program and is carried out at a small 30 MW power plant in Bremen, Northern Germany. The power plant uses waste material as the energy source. Since January 2010, newly developed PBRs called phytobags have been tested at installations of up to 500 m2 under varying environmental conditions at this industry site (Fig. 4.1). The PBR system allows all-year-round

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4 Case study of a temperature-controlled outdoor PBR system in Bremen

Fig. 4.1: 500 m2 outdoor PBR system at the Bremen power plant, located close to the river Weser.

outdoor algae production and can be connected to existing CO2 and nutrient sources. The phytobags are available in a variety of sizes, from lab- to hectare scale. This newly developed endless-chamber PBR is produced from multi-layer polyethylene film. A typical PBR module is 3–12 m long and consists of 120 cm high vertical tubes of 50 mm diameter which are connected via one horizontal tube at the basis of the PBR (Fig. 4.2a). Turbulence in each of the inter-connected PBRs is achieved through an airlift system of ambient air provided by a frequency-controlled side channel blower with a 100 μm air filter. The attached electronics allow the production process to be controlled and monitored continuously. Internet access allows immediate support if needed. The 500 m2 PBR module is equipped with a pH/temperature sensor. At a preset pH of around 8, the control unit automatically opens a valve, and flue gas from the power plant 200 m away is provided at a pressure of 0.2 bar (20 kPa) and subsequently mixed with the air from the compressor for the airlift system inside the PBRs. The flue gas is taken directly from the emitting chimney of the plant, and no further treatments are carried out. The average values are 180 mg/Nm3 for NOx and 8.0 mg/Nm3 for SO2. Superficial flue gas in the PBR is collected via an exhausttubing system, which connects all PBRs and passively transfers the air/flue gas mixture through a central exhaust tube into the atmosphere via a 10 m high chimney. Additional sensors can be added, and the control electronics can also be adapted to a newly developed dewatering system called a phytoharvester. The harvesting process involves the concentration of dilute microalgae suspensions, typically 0.03 % TSS (open ponds) to 0.3 % TSS (closed photo bioreactors) into a slurry or paste with concentrations of 2–30 % TSS. The concentration obtained in the harvesting steps is crucial to the overall process as it influences the subsequent drying process. This system pre-concentrates algae suspensions before any separator or flocculation process proceeds. Step 1 of this process of algae dewatering drastically reduce costs and energy demands. The average power consumption is

4 Case study of a temperature-controlled outdoor PBR system in Bremen

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Fig. 4.2a: Detailed photograph of a phytobag. Fig. 4.2b: Detailed view of the pipes transporting the waste heat from the cooling fluid of the power plant with the connecting pipes into the PBRs.

0.32 kWh/m3 feed flow. The air scour design minimizes power consumption and avoids localized dewatering of the algae sludge. Particles of >1 μm are removed. The system can also be used to recycle the algae-free effluent, to pre-treat the production water and to minimize contamination. Requirements for cleaning and maintenance are minimized. The feed flow biomass concentration from the PBR to the dewatering process is two- to fivefold higher than from a typical open pond. Thus, energy demands for dewatering are up to 20-fold lower. Temperature control was achieved through another tubing system, which connects all phytobags on each side of the installation (Figs 4.2b and 4.3). Water with a temperature range of 15–25°C and a reduced pressure of 1 bar (100 kPa) from the waste heat cooling system of the power plant runs through these pipes and is returned to the power-plant drainage cycle. This waste heat water provides efficient temperature conditions inside the PBR to allow year-long production at temperatures of 8–25°C within the algae suspension, while outside temperatures can range between –10 and +40°C. Production values of biomass for seasonal intervals are shown in Figure 4.4. The calculated carbon footprint via energy demand has been constantly improved. Currently, ca. 0.6 kg of CO2 is emitted per kilogram of algae biomass produced. The energy demand of the phytoharvester in combination with a centri-

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4 Case study of a temperature-controlled outdoor PBR system in Bremen

Fig. 4.3: Data on ambient and PBR suspension temperature during January 2012; the source of the cooling water of the plant is the river Weser. During the summer, the difference between the inside and outside is approximately 15°C.

Fig. 4.4: Seasonal biomass production via measurements of optical density (OD) at the outdoor installation in Bremen. OD was measured spectrophotometrically at 750 nm. Tests confirmed that the percentage of inorganic matter inside the PBR stayed below 15 % of total suspended matter. The dashed red line in spring represents production inside a shaded greenhouse at a lignite-fired power plant (RWE-project, Niederaussem, Germany).

fuge produces a carbon footprint of 0.13 kg of CO2 per kilogram of algae biomass produced. This assumes an emission of 1 kg of CO2 per kWh of produced electricity

References

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and a CO2 fixation rate of 1.8 kg CO2 per kilogram of algae biomass. The resulting carbon footprint is 60 % positive. Biomass is dried using waste heat from the power plant. An increase in the temperature of the algae suspension of 10°C via waste heat from the power plant requires approximately 5 MW of waste heat energy per hectare of algae production site. For nutrient supply wastewaters from aquaculture, agriculture and domestic (urine) sources have been tested in 200-liter setups and will be applied to the 500 m2 installation during the project. The results show that power plants can provide ideal conditions for the sustainable production of algae. The waste heat from the power plant can be used for temperature control, thus allowing algae farms to be deployed at higher latitudes. The high energy demand via waste heat for temperature control must be taken into consideration when the PBRs are deployed at higher latitudes. These results provide additional input to discussions of algae farms in the vicinity of industry and power plants (Benemann 2011). Clearly, algae biomass production cannot mitigate the enormous CO2 emissions to achieve zero emissions. Emissions are always several orders of magnitude too high, but waste heat concepts and flue gas such as CO2 source can provide perfect temperature control for production at higher latitudes, and large, unused, non-agricultural areas around these sites are well suited to the deployment of decentralized algae production sites.

Acknowledgments The project is supported by the European Union: Investing in your future, European Regional Development Fund (VE0094A), and the authors would like to thank the cooperation partner Brewa WTW GmbH Bremen.

References Benemann, J. 2011. Can we save the world with algae? Presentation at the University of British Columbia, Dept. of Chemical Engineering, Vancouver. Brennan, L. and P. Owende. 2010. Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sust Energ Rev 14: 557–577. Clarens, A. F., E. P. Resurreccion, M. A. White and L. M. Colosi. 2010. Environmental Life Cycle Comparison of Algae to Other Bioenergy Feedstocks. Environ Sci Technol 44: 1813–1819. Levin, I., S. Hammer, B. Kromer and F. Meinhardt. 2008. Radiocarbon observations in atmospheric CO2: Determining fossil fuel CO2 over Europe using Jungfraujoch observations as background. Sci Total Environ 391: 211–216. Sarmiento, J. L., N. Gruber, M. A. Brzezinski and J. P. Dunne. 2004. High-latitude controls of thermocline nutrients and low latitude biological productivity. Nature 427: 56–60.

Robin Shields and Ingrid Lupatsch

5 Algae for aquaculture and animal feeds 5.1 Introduction Cultivated microalgae have long been integral to the hatchery production of many farmed finfish, shellfish and other commercially important aquaculture species. By contrast, macroalgae are less widely used in aquaculture, although they do provide an important source of nutrition for certain farmed invertebrates, such as sea urchins and abalone. There is an extensive published literature on the suitability of different algal strains for use in aquaculture hatcheries, their cultivation techniques, methods of delivery and modes of operation (Muller-Feuga et al. 2003a, 2003b; Muller-Feuga 2004; Zmora and Richmond 2004; Tredici et al. 2009; Conceição et al. 2010; Guedes and Malcata 2012). Given that detailed reviews already exist, the purpose of the current article is to introduce the biotechnology reader to algae production and use in hatcheries, including recent industry trends and future outlook. Alongside these well-established applications for micro- and macroalgae in aquaculture hatcheries, there is currently a drive to exploit algae in formulated animal feeds, both for aquaculture species and for terrestrial livestock. To date, technological developments and commercial applications have mainly focused on algae as a micro-feed ingredient, imparting specific beneficial properties rather than gross nutrients to the recipient animal. However, finite supplies of premium raw materials (particularly fish meal and fish oil) and the promise of much higher available quantities of algal biomass in future (i.e. biofuels agenda) are prompting evaluation of algal biomass as a major ingredient in formulated animal feeds, especially for aquaculture. Recent scientific findings reviewed herein do indicate good potential for microalgal biomass as a bulk feedstuff for formulated aquaculture feeds, but the future commercial viability of this will depend on available quantity, quality (composition) and cost in relation to currently used commodity materials. There are currently major gaps in supply and price preventing such use of algal biomass, and we anticipate these can only be bridged within a major algal biorefinery-forbiofuels framework.

5.2 Microalgae use in aquaculture hatcheries Aquaculture hatcheries producing juvenile finfish and shellfish for food represent the most numerous microalgal production facilities worldwide. This abundant

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capacity reflects both the importance of aquaculture for global food production (e.g. accounting for more than 45 % of global food fish production in 2008, Anon 2010a) and the key role of microalgae as a preferred or obligatory feed source for many aquaculture species, particularly marine finfish and invertebrates (Tredici et al. 2009). In more traditional, extensive forms of aquaculture, adventitious populations of microalgae are bloomed in ponds or large tanks, which act as mesocosms in which the aquaculture species occupies the highest trophic level. By contrast, intensive aquaculture hatcheries cultivate individual strains of microalgae in separate reactors and administer these regularly to the farmed species. There is an extensive published literature on the suitability of different microalgal strains for aquaculture hatchery use, their cultivation techniques, methods of delivery and modes of operation (Muller-Feuga et al. 2003a, 2003b; Muller-Feuga 2004; Zmora and Richmond 2004; Tredici et al. 2009; Conceição et al. 2010; Guedes and Malcata 2012). Given that detailed reviews already exist, the purpose of the current article is to introduce the biotechnology reader to microalgae-in-aquaculture, including recent industry trends and future outlook. The role of microalgae in aquaculture hatcheries may be summarized as follows: — All developmental stages of bivalve mollusks are directly reliant on microalgae as a feed source. Bivalve hatcheries therefore cultivate a range of microalgal strains for broodstock conditioning, larval rearing and feeding of newly settled spat. — Farmed gastropod mollusks (e.g. abalone) and sea urchins require a diet of benthic diatoms when they first settle out from the plankton, prior to transferring to their juvenile diet of macroalgae. — The planktonic larval stages of commercially important crustaceans (e.g. penaeid shrimps) are initially fed on microalgae, followed by zooplanktonic live prey. — The small larvae of most marine finfish species and some freshwater fish species also initially receive live prey, usually in the presence of a background of microalgae. Depending on whether these microalgae are allowed to bloom within the fish larval rearing tanks, or are added from external cultures, this is referred to as the “green water” or “pseudo-green water” rearing technique. — The zooplanktonic live prey referred to above are microscopic filter feeders that are themselves commonly fed on microalgae, although inert formulated feeds have been developed as a more convenient diet form for use by hatcheries. 5.2.1 Microalgal strains used in aquaculture hatcheries As referred to in previous reviews, only a small number of microalgal strains are routinely cultured in aquaculture hatcheries, based on practical considerations of

5.2 Microalgae use in aquaculture hatcheries

Group

Genus

Species

Area of application

Cyanobacteria Chlorophyta

Arthrospira Tetraselmis Chlorella

platensis suecica, chui sp., vulgaris, minutissima, virginica, grossii sp., tertiolecta, salina pluvialis sp., oculata

FFI B, CL R, FFI

sp. sp. calcitrans, gracilis costatum pseudonana sp. sp. sp. lutheri galbana, add. galbana “Tahiti” (T-iso) cohnii

RAD RAD B, CL B, CL B, CL GU GU GU B B, GW

Eustigmatophyceae (Phylum Heterokontophyta) Labyrinthulea (Phylum Heterokonta) Bacillariophyta (diatoms)

Haptophyta

Dinophyta (dinoflagellates)

Dunaliella Haematococcus Nannochloropsis Schizochytrium Ulkenia Chaetoceros Skeletonema Thalassiosira Nitzschia Navicula Amphora Pavlova Isochrysis Crypthecodinium

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FFI FFI R, GW

RAD

FFI: formulated feed ingredient; B: bivalve molluscs (larvae/postlarvae/broodstock); C: crustacean larvae (shrimps, lobsters); R: rotifer live prey; RAD: rotifer and Artemia live prey (dry product form); GU: gastropod molluscs and sea urchins; GW: “green water” for finfish larvae Tab. 5.1: Groups, genera and species of major microalgal strains used in aquaculture and their areas of application.

strain availability, ease of culture, cell physical characteristics, nutritional composition, digestibility and absence of toxins or irritants (Muller-Feuga et al. 2003a, 2003b; Muller-Feuga 2004; Tredici et al. 2009; Anon. 2010b; Guedes and Malcata 2012). Table 5.1 provides a non-exhaustive list of the most commonly used strains and their typical areas of application in aquaculture. A comprehensive literature exists on the nutritional composition of these and other microalgal strains, and their efficacy as aquaculture hatchery feeds (Brown et al. 1997; Muller-Feuga et al. 2003a, 2003b; Becker 2004; Guedes and Malcata 2012). While scientific studies have demonstrated the ability to manipulate the nutritional composition of individual microalgal strains (e.g. n-3 HUFA content of Nannochloropsis sp.: Pal 2011), in practice hatchery operators focus on maintaining uninterrupted supplies of microalgae by avoiding system crashes or culture contamination. Delivery of a balanced diet to the aquaculture species is generally

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achieved by supplying a mixture of different microalgal strains, guided by typical published nutritional profiles for these strains (e.g. Brown et al. 1997).

5.2.2 Methods of microalgae cultivation for aquaculture In approximate order of engineering complexity and achievable culture density, the main types of microalgal cultivation system used in aquaculture, all of which are phototrophic, are: — open ponds or tanks, with or without aeration or stirring; — bubble or airlift columns, usually oriented vertically, or less frequently horizontally; — closed photobioreactors (PBRs), most commonly tubular in configuration or less commonly flat-panel PBRs. These methods of microalgal cultivation have been regularly reviewed from an aquaculture standpoint over the past 15 years (Borowitzka 1997; Duerr et al. 1998; Muller-Feuga et al. 2003a; Zmora and Richmond 2004; Tredici et al. 2009). During this period, no major technological step-changes are discernible, although there is a notable trend towards greater adoption of closed PBRs and for semi-continuous or continuous modes of operation, alongside more established batch cultivation techniques. The adoption by aquaculture hatcheries of heterotrophically grown microbial biomass and biomass extracts as partial replacements for live microalgae also represents a significant technological advance during this period (see Section 5.2.3.5). The types of microalgal production system adopted by aquaculture hatcheries often reflect regional aquaculture preferences, rather than differences in the particular strains or quantities of microalgae required by different aquaculture species. To illustrate, microalgae used to provide green water for marine finfish larviculture (see Section 5.2.3.4) are often produced extensively using outdoor ponds or tanks in South East Asia, whereas European aquaculture hatcheries typically cultivate individual microalgal strains intensively in bubble or airlift columns, or closed PBRs (Shields 2001).

5.2.3 Role of microalgae in aquaculture hatcheries 5.2.3.1 Microalgae as a feed source for filter-feeding aquaculture species It is a common reproductive strategy among marine invertebrates to broadcast high numbers of microscopic larvae into the water column, to ensure widespread distribution of offspring. These planktonic larvae are different in appearance and habit

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from later developmental stages and undergo a dramatic metamorphosis to the juvenile form. Key examples from aquaculture include bivalve mollusks, decapod crustaceans (shrimps, crabs and lobsters), sea urchins and polychaete worms. In many cases, the larvae are filter feeders, relying on microalgae throughout their planktonic phase (e.g. bivalve mollusks, some sea urchins and polychaete worms) or alternatively switching from filter feeding to predating on zooplankton during larval development (e.g. penaeid shrimps). These life-history strategies require the aquaculturist to supply microalgae during some, if not all, of the hatchery phase. Tredici et al. (2009) provide a recent overview of the typical microalgal strains and feeding strategies used for these groups of aquatic invertebrates. For bivalve mollusk production, the obligation to provide microalgae continues into the nursery phase, since bivalves are obligate filter feeders throughout their life history. Bivalve hatcheries therefore tend to possess among the highest microalgal production capacity of any form of food aquaculture, with particular attention being paid to hygiene status to avoid crashes or transfer of pathogenic organisms to the shellfish (Aji 2011). Combinations of Bacillariophyte and Prymnesiophyte microalgal strains are the most commonly used feed source for bivalves, both for hatchery/nursery rearing and for conditioning of broodstock (Helm et al. 2004).

5.2.3.2 Microalgae as a feed source for zooplanktonic live prey Where larvae of aquaculture species are predatory rather than filter-feeding (e.g. finfish larvae and decapod crustacean larvae) the most common husbandry strategy is to feed with zooplanktonic live prey rather than formulated inert diets. This reflects the technological challenge and high costs of providing nutritionally balanced, digestible feeds in the correct physical form for small planktonic larvae, whose digestive capacity is only partially developed (Conceição et al. 2010) Thanks to innovations begun in the 1960s, aquaculture hatcheries almost ubiquitously use rotifers (Brachionus sp.) followed by brine shrimp (Artemia sp.) as the key zooplanktonic live prey for larval finfish and decapods (Bengtson 2003). These zooplankton are not the natural prey of the aquaculture species and have suboptimal nutritional composition, but their ease of culture (rapid reproduction rates, high stocking densities) outweighs their nutritional shortcomings in most cases (Lubzens and Zmora 2003; Dhont 2003; Conceição et al. 2010). Extensive research and product development have gone into improving rotifer and brine shrimp nutritional quality by manipulating their diet (in particular, to enhance n-3 HUFA content), e.g. by microalgal strain selection or by incorporating dried microalgal biomass into formulated inert diets. Where the aquaculture species of interest are either too small to accept rotifers as a first prey (e.g. some tropical snappers and groupers) or are prone to nutritionrelated developmental abnormalities (e.g. Atlantic halibut), copepods offer a suitable alternative zooplankton (Conceição et al. 2010). However, the lower culture

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densities achievable for copepods compared to rotifers/Artemia impose practical limitations on supplying them at larger scales of intensive aquaculture production. Hatchery production of rotifers was initially based on feeding with live microalgae and/or baker’s yeast. Commonly used microalgal strains for this purpose are Nannochloropsis sp., Tetraselmis sp., Pavlova lutheri and Isochrysis galbana (Conceição et al. 2010). Commercial off-the-shelf formulations have been developed and are now widely used as alternatives to live microalgae and yeast. Depending on their specific formulation, these products are intended to optimize growth and reproduction of the rotifers and/or to enhance their final nutritional composition before feeding to larvae. This latter process is widely referred to as “enrichment”. Even where hatcheries have adopted such artificial feeds for mass rotifer cultivation, it is common to retain rotifer master cultures on live microalgae, as this simplifies hygiene maintenance and lessens the likelihood of the cultures crashing. The use of brine shrimp, Artemia sp., in aquaculture is based on supplies of resistant cysts that are commercially collected from hypersaline lakes (Van Stappen 2003). These cysts represent a convenient storable product for aquaculture hatcheries, from which planktonic nauplii can be hatched on demand. Hatcheries do not typically provide live microalgae to these early stages of Artemia, since formulated products have been developed to grow and enrich the nauplii (Dhont 2003). Where copepods are used as an initial prey organism, live microalgae remain the preferred diet for planktonic groups (orders Calanoida and Cyclopoida), whereas benthic copepods (order Harpacticoida) are more amenable to cultivating on inert feeds (Støttrup 2003). Among the products used as feed for aquaculture live prey are several heterotrophically grown marine micro-organisms (Tredici et al. 2009). The first such product to reach the aquaculture market was the DHA-rich fungal thraustochytrid, Schizochytrium, which was initially developed as a human nutritional supplement but is also now widely used for aquaculture live prey production/enrichment in powder form. The dinoflagellate, Crypthecodinium cohnii, has been similarly exploited owing to its high DHA content.

5.2.3.3 Benthic microalgae as a feed source for gastropod mollusks and echinoderms Unlike bivalve mollusks, the larvae of abalone (gastropoda) and some species of sea urchin (echinoidea) do not require microalgae during their planktonic phase, relying instead on internal yolk reserves for energy. This simplifies hatchery rearing procedures (no microalgae required), but abalone and urchins do initially graze on benthic microalgae (those living on surfaces) when they settle out from the plankton (Heasman and Savva 2007; Azad et al. 2010). Natural assemblages of benthic diatoms are typically encouraged to grow as a feed source, by pre-exposing artificial substrates or macroalgal germlings to unfil-

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tered seawater, upon which the microalgae grow (Heasman and Savva 2007). This natural colonization process becomes limiting at higher abalone stocking densities, where the rate of algal growth can be outpaced by grazing (Dyck et al. 2011). The addition of cultured diatoms, such as Navicula sp., Nitzschia sp. and Amphora sp. (de Viçose et al. 2012) offers greater control for intensive abalone nurseries, although challenges exist in optimizing their methods of cultivation and deployment. Comparatively few publications exist on appropriate cultivation systems for diatoms. Araya et al. (2010) reported the use of 20-liter polycarbonate carboys containing PVC filaments for culturing mixed benthic diatom strains, which were administered successfully to postlarval Haliotis rufescens. The same research group has also described a PBR design for diatoms, based on an aerated acrylic cylinder containing a bottle brush-like array of PVC “bristles” (Silva-Aciares and Riquelme 2008).

5.2.3.4 Addition of microalgae to fish larval rearing tanks The practice of rearing marine finfish larvae in the presence of microalgae is commonplace and is typically, although not exclusively, associated with higher survival and growth rates than when larvae are reared in clear water (Muller-Feuga et al. 2003b; Tredici et al. 2009; Conceição et al. 2010). In the so-called “green water” technique, microalgae and zooplankton are bloomed within ponds or large tanks, into which the fish larvae are stocked. This rearing method can be based on natural microalgal assemblages, which are encouraged to bloom by fertilizer addition (Shields 2001). Alternatively, cultured microalgal strains can be inoculated into rearing tanks for this purpose provided the system water has been pre-treated to exclude competing micro-organisms. The “pseudo-green water” rearing technique relies instead on regular addition of cultured microalgae to the fish larval rearing tanks, to replace that removed by live prey grazing and dilution (water exchange). This approach is required to sustain the higher larval stocking densities that are typical in most commercial marine fish hatcheries in Europe and North America. Commonly used microalgal strains for this purpose are Nannochloropsis sp., Isochrysis sp. and Tetraselmis sp. (Fig. 5.1). Given that few groups of fish are equipped to feed directly on microalgae (Muller-Feuga et al. 2003b), extensive research effort has been applied to understand the mechanisms by which microalgae enable superior rearing performance of larval fish and to optimize their delivery in aquaculture hatcheries (Conceição et al. 2010). This research encompasses the effects of microalgae on: — nutritional status of live prey and fish larvae; — fish larval behavior, particularly feeding behavior; — larval digestive function;

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— microbial community composition in the rearing water and the larval digestive tract.

Fig. 5.1: Addition of live microalgae to a fish larval rearing tank: “pseudo-green water” technique; Centre for Sustainable Aquatic Research, Swansea University.

A range of effects have been reported across different microalgal strains, fish species, experimental conditions and observational/analytical techniques (reviews by Muller-Feuga et al. 2003b; Conceição et al. 2010). These include evidence for: — improved chemical water quality in the presence of microalgae; — greater larval absorption of soluble organics from the rearing water; — direct ingestion of microalgal cells (passive and active) by larvae; — improved visual contrast between prey and background; — enhanced prey capture rates and greater gut fullness; — stimulation of larval digestive enzyme production; — more diverse microflora in rearing water and in the larval digestive tract. Recent research by Natrah et al. (2011) suggests that microbial conditioning by microalgae may extend to impeding cell-to-cell signaling (quorum sensing) by bacterial pathogens. In a laboratory screening study focused on microalgal strains commonly used in aquaculture, several of the tested strains interrupted signaling by pathogenic Vibrio harveyii, leading the authors to postulate that such microalgae offer potential as aquaculture biocontrol agents. A logical extension of this research would be to challenge fish larvae with pathogens in the presence/absence of those microalgae that showed bioactivity during screening.

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5.2.3.5 Use of microalgal concentrates in aquaculture hatcheries Commercially available concentrates offer a convenient source of microalgae for aquaculture hatcheries. This area of microalgal product development has recently been reviewed by Tredici et al. (2009), including the technologies involved in concentrating and stabilizing microalgae, and descriptions of a range of commercially available products, with prices. The practice of concentrating live microalgae originated for local use within individual hatcheries, typically using disk-stack centrifuges or membrane filters (Molina Grima 2003). This practice is still used in some large hatcheries, although commercial concentrates have become widely adopted. From an aquaculture hatchery perspective, the key desired attributes for microalgal concentrates are: — high cell concentration without damage to cells; — suitable nutritional composition; — acceptable shelf-life (maintain nutritional quality, avoid spoilage) using standard cold storage methods, avoiding the use of preservatives that would be harmful to live prey or larvae; — hygienic and free from pathogens; — avoidance of clumping and easy to suspend uniformly in water; — regularly available and affordable. Two main categories of product have emerged: first, concentrates of those microalgal strains that are particularly favored for aquaculture and, second, industrial biotechnology strains such as heterotrophically produced Chlorella sp., which are available at a higher volume/lower price but have a more limited scope of application, such as in the production of live prey. Many of the key microalgal strains referred to in Table 5.1 are now available as concentrates (Tredici et al. 2009). These are frequently marketed as total replacements for live microalgae, although in practice, they usually serve as a backup or supplement to live microalgae produced in-house. This reflects both the high purchase costs of the concentrates and generally inferior rearing performance when compared to live microalgae, as previously reported across diverse aquaculture species and areas of hatchery deployment (Muller-Feuga et al. 2003b; Tredici et al. 2009; Conceição et al. 2010). That is not to underplay the value of such concentrates in providing backup against crashes or out-of-season shortages and in enabling smaller enterprises to operate without an in-house microalgal production capacity. However, as referred to in previous reviews, a lower unit cost and performance more on a par with live microalgae will be needed to facilitate market expansion within the commercial aquaculture sector. The performance discrepancy between concentrated and live microalgae is less marked in the area of live prey production, where industrially produced Chlorella

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is now routinely used for rotifer production, competing in the market with other forms of dry feed (Tredici et al. 2009).

5.3 Use of algae in formulated feeds for aquaculture species and terrestrial livestock Quite a number of animal nutrition studies on the “super-food” status of algae have been publicized and reviewed, and the challenge remains to support the claims being made by scientifically based evidence. Results from experimental studies can be difficult to interpret, as several compounds in algae can have confounding effects. Even when used at small amounts in livestock and aquaculture feeds, algae have been credited with improving immune system (Turner et al. 2002), lipid metabolism (Nakagawa 1997; Güroy et al. 2011), antiviral and antibacterial action, improved gut function (Michiels et al. 2011) and stress resistance (Nath et al. 2012; Sheikhzadeh et al. 2012) besides providing a source of protein, amino acids, fatty acids, vitamins and minerals and other biologically active phytochemicals (Becker 2004; Pulz and Gross 2004; Gouveia et al. 2008). Nutritional studies evaluating algae as a major feed ingredient for farmed animals are currently fewer in number, due to the large amounts of biomass needed. Thus, in most studies to date, the algal biomass/extracts from algae are considered not as an essential feed source but rather as enhancing “standard” feed formulations.

5.3.1 Algae as a supplement to enhance the nutritional value of formulated feeds 5.3.1.1 Vitamins and minerals In the view of consumers, the concept of sustainable, “chemical free” and organic farming has become very appealing, including the use of natural forms of vitamins and minerals instead of the synthetically produced ones. Both micro- and macroalgae have potential as mineral additives to replace the inorganic mineral salts that are most commonly used in the animal feed industry. It has been suggested that the natural forms are more bio-available to the animal than the synthetic forms and can even be altered or manipulated via the process of bio-absorption (Doucha et al. 2009). Mineral-rich seaweed has been incorporated in commercial salmon feeds at 15 % in lieu of manufactured vitamin and mineral pre-mixes (Kraan and Mair 2010). Final tests suggested that salmon fed the “seaweed” feeds appeared to be healthier and more active; flavor and texture were improved, which may have been due to the bromophenolic compounds found in seaweeds. Elsewhere, Enteromor-

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89

pha prolifera and Cladophora sp., when added to the feeds of laying hens, positively influenced egg weight and egg-shell thickness (Michalak et al. 2011). The vitamin content of algal biomass can vary significantly among species. Ascorbic acid shows the greatest variability according to Brown and Miller (1992), although this may have been due to differences in processing, drying and storage of algae, as ascorbic acid is very sensitive to heat. This highlights the drawback of supplying essential micronutrients via natural sources, i.e. there is too much variability arising from the combined effects of different algal species, growing season, culture conditions and processing methods to reliably supply the required micronutrients in a pre-determined fashion. Accordingly, algal biomass mainly offers a supplementary source rather than a complete replacement for manufactured minerals or vitamins in animal feeds.

5.3.1.2 Pigments The carotenoids are a class of yellow, orange or red naturally occurring pigments, which are distributed everywhere in the living world. Only the micro-organisms, fungi, algae and higher plants are able to synthesize carotenoids de novo; therefore, animals rely on the pigment or closely related precursor being supplied in their diets, which in nature would have passed on through the food chain. In farmed salmonid fish, it is necessary to supplement the diet with astaxanthin to achieve the pink color of the fillet. Synthetic carotenoids are mainly used for this purpose in commercial aquaculture, although algae-derived carotenoids can also impart pigmentation effectively (Choubert and Heinrich 1993; Soler-Vila et al. 2009). Astaxanthin obtained from Haematococcus pluvialis has been approved as a color additive, NatuRose®, in salmon feeds and is typically used for organically certified salmon production. Aside from salmonids, most species of farmed fish display pigmentation of the skin rather than the flesh, which contributes to their attractive appearance and thus satisfies customer demand. H. pluvialis has been shown to be successful in enhancing the reddish skin coloration of red porgy, Pagrus pagrus (Chatzifotis et al. 2011) and also of the penaeid shrimp, Litopenaeus vannamei (Parisenti et al. 2011). Both natural and synthetic sources of carotenoids have been successfully used to augment the yellow skin coloration in gilthead sea bream (Gomes et al. 2002; Gouveia et al. 2002). Chlorella sp. and Spirulina sp. are commonly incorporated into feeds for ornamental fish, where coloration and healthy appearance are the main market criteria (Gouveia and Rema 2005; Sergejevová and Masojídek 2011; Zatkova et al. 2011). Seaweeds are the preferred feed of sea urchins in nature, and in an aquaculture setting, carotenoid-rich sources such as Ulva sp. and Gracilaria sp. are necessary to enhance the orange color of the gonads that consumers prefer (Shpigel et al. 2005).

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For pigmentation of broilers and egg yolk, formulated feeds traditionally contain dehydrated alfalfa meal and/or corn, both of which are rich sources of lutein and zeaxanthin. Seaweed biomass has been reported to increase the pigmentation of egg yolk when used at a dietary inclusion level of 15 % (Strand et al. 1998). Gouveia et al. (1996) reported that Chlorella vulgaris biomass produced yolk pigmentation comparable to other commercially used pigments. Chlorella is credited not only with improving the health status of laying hens but also with improving egg quality and pigmentation (Halle et al. 2009). According to Waldenstedt et al. (2003), H. pluvialis also has good potential as a natural pigment enhancer in broiler chickens.

5.3.1.3 Fatty acids Farmed fish and shellfish offer rich sources of long-chain, highly unsaturated fatty acids (HUFA), due to the inclusion of fish meal and fish oil in formulated aquafeeds. HUFA are crucial to human health and play an important role in the prevention and treatment of coronary heart disease, hypertension, diabetes, arthritis, and other inflammatory and autoimmune disorders. Due to the global shortage of fish oil and fish meal, researchers are looking increasingly into alternative sources of lipid, including from algal biomass. Unlike terrestrial crops, algae can directly produce HUFA such as arachidonic acid (AA, 20:4n-6) (Porphyridium), eicosapentaenoic acid (EPA, 20:5n-3) (Nannochloropsis, Phaeodactylum, Nitzschia, Isochrysis, Diacronema) and docosahexaenoic acid (DHA, 22:6n-3) (Crypthecodinium, Schizochytrium). While most of these algae are not suitable for direct human consumption, they might indirectly boost their nutritional value for humans if added to animal feeds. However, relatively few studies have been carried out to date to evaluate microalgal lipids in feeds for farmed fish (Atalah et al. 2007; Ganuza et al. 2008). Despite the typically low lipid content of seaweeds, Dantagnan et al. (2009) reported that Macrocystis pyrifera meal enhanced the level of PUFAs in trout flesh, when included in the diet at a level of 6 %. Micro- and macroalgae have similarly been tested as alternatives to fish oil and flax seed for boosting the HUFA content of hen’s eggs (Carrillo et al. 2008; Kassis et al. 2010).

5.3.2 Algae as a potential feed ingredient: source of protein and energy In both aquaculture and agriculture, producers commonly rely on formulated feeds to ensure optimal growth, health and quality of the farmed animal. Given the economic importance of feeds and feeding, nutritionists therefore need to develop nutritionally balanced diets using commonly available raw ingredients. Once there are reliable data on the nutrient and energy requirements of the target species for

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Poultry Pigs Cattle Salmon Sea bream Tilapia Shrimp a

Percentage crude protein

Percentage crude lipid

Percentage crude carbohydrate

Metabolizable energy (MJ/kg)

Feed conversion ratioa

21.0 16.0 12.0 37.0 45.0 35.0 35.0

5.0 5.0 4.0 32.0 20.0 6.0 6.0

60 60 65 15 20 40 40

13.0 12.5 10.1 21.0 19.1 13.5 13.5

2.2 3.0 5.8 1.0 1.6 1.5 2.0

Feed conversion ratio = feed consumed (dry)/live weight gain.

Tab. 5.2: Typical composition of formulated feeds for livestock and several species of commercial fish (on as fed basis) and feed/gain ratio.

a given production performance, specific feeds can be formulated and a feeding regimen established. Typical compositions of feed and feed/gain ratio are summarized in Table 5.2 for several farmed terrestrial and aquatic animal species. This table just provides an overview, as different feed formulations are used, depending on the production stage of the target species. Since protein is generally one of the most expensive feed ingredients, targeted rations are used, and the amounts of protein in the diet are reduced as the animals grow. As can be seen, feeds for aquatic animals are more energy- and nutrient-dense than those for terrestrial animals, and as a result, fish need to be fed less to support each unit of growth, as indicated by the lower feed conversion ratio. Traditionally, fish meal and fish oil have been substantial components of feeds at least in aquaculture, but these sources are finite. With fish meal and fish oil prices increasing, there has been growing interest in partial or complete replacement of fish meal by alternative protein sources of either animal or plant origin. Raw materials other than fish meal are selected for their nutritive value, balance of amino acids, digestibility of proteins, lipids and quality of fatty acids, absence of anti-nutritional factors, availability and cost, and lipid rich algae biomass is being considered as one of the alternative ingredients of the future (Lupatsch 2009). To help in assessing algae as a potential source of protein and energy in the form of carbohydrates and lipids, Table 5.3 compares the typical nutritional profiles of commercially available animal feed ingredients with several selected micro- and macroalgae. In addition to quantifying the gross composition of feed ingredients, knowledge of their digestibility is needed in order to assess the nutritional value. Digestibility trials are usually carried out in vivo by adding an indigestible marker to the

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Fish meal Poultry meal Corn gluten Soybean Wheat meal Spirulina Chlorella Tetraselmis Gracilaria sp.b Gracilaria sp.c Ulva lactucab Ulva lactucac Schizochytriumd

Percentage crude protein

Percentage crude lipid

Percentage crude carbohydratea

Percentage ash

Energy gross (MJ/kg)

63.0 58.0 62.0 44.0 12.2 58.0 52.0 27.2 34.0 10.0 37.4 12.5 12.5

11.0 11.3 5.0 2.2 2.9 11.6 7.5 14.0 1.5 0.9 2.8 1.0 40.2

– – 18.5 39.0 69.0 10.8 24.3 45.4 37.1 50.1 42.2 57.0 38.9

15.8 18.9 4.8 6.1 1.6 13.4 8.2 11.5 26.9 34.0 17.4 24.5 8.4

20.1 19.1 21.3 18.2 16.8 20.1 19.3 18.0 13.4 11.2 15.7 11.2 25.6

The analyses were performed by the authors. Carbohydrates calculated as the difference % DM – (% protein + % lipid + % ash). b Cultured in effluent of fish tanks. c Collected from natural habitat. d Commercial product, Martek Biosciences. a

Tab. 5.3: Typical composition of commercially available feed ingredients and algae species (per dry matter).

feed at a known amount, collecting fecal matter by a suitable method and determining the ratio between nutrient and marker in the fecal matter. Very few of the required digestibility trials have been completed with micro- or macroalgal biomass to date, partly due to the limited availability of material. A digestibility trial with carnivorous mink, a model used for salmon and other farmed monogastric species, was recently reported by Skrede et al. (2011). Three microalgae Nannochloropsis oceanica, Phaeodactylum tricornutum and Isochrysis galbana were included at graded levels up to 24 % (dry weight) in the feed. The protein digestibilities determined by linear regression for N. oceanica, P. tricornutum and I. galbana were found to be 35.5, 79.9 and 18.8 %, respectively. The algae used had been freeze-dried prior to the trial, and the authors hypothesized that the cell wall of the diatom P. tricornutum may more easily be broken down by digestive processes than the others. The potential effects of algal cell wall structure on digestibility to humans and non-ruminant animals has been raised by several authors, as reviewed by Becker (2004). Janczyk et al. (2007) tested the digestibility of Chlorella biomass in rats using three treatments such as spray-dried, spray-dried and electroporated, and spray-dried and ultrasonicated. Ultrasonication was found to increase the protein digestibility of Chlorella from 53 % (spray-dried) to 63 %.

5.3 Use of algae in formulated feeds for aquaculture species and terrestrial livestock

93

The digestibility coefficient of sun-dried Spirulina biomass has been tested for Arctic char and Atlantic salmon at 30 % dietary inclusion level (Burr et al. 2011). Protein digestibility ranged between 82 % and 84.7 % for the two fish species respectively. These relatively high digestibility coefficients compare favorably with terrestrial plant ingredients, confirming the high potential of Spirulina as a protein source for farmed fish. The digestibility of “DHA-Biomeal”, a by-product from DHA-rich Schizochytrium after de-lipidation, has been assessed for gilthead sea bream (Lupatsch, unpublished data). The composition of the dry DHA-Biomeal was determined as 6 % protein, 9 % lipid and approximately 70 % carbohydrates including fiber. At 6 %, protein content was too low to be considered as a significant source of amino acids, and the overall energy digestibility was found to be only 64 %, thus placing DHA-Biomeal in the same category as a carbohydrate-rich ingredient such as whole wheat or corn meal. A possible means of increasing the nutritional value of algal ingredients such as DHA-Biomeal would be to break down the cell wall fragments by mechanical treatment, or even by removal of most of the fiber, although such processing steps may be prohibitively expensive. In addition to digestibility measurements, in vivo trials need to be carried out in which the novel feed ingredient is supplied in sufficient amounts. Even with seemingly nutritionally adequate diets, poor weight gain may be encountered in practice, because of the low palatability of the test ingredient and therefore reduced feed intake. Coutinho et al. (2006) found that supplementing feeds for goldfish fry with freeze-dried biomass of Isochrysis galbana, as a substitute for fish meal protein, had a negative effect on growth and survival (Coutinho et al. 2006). Aside from the question of palatability, one of the reasons may have been that the feeds were not iso-nitrogenous: dietary protein levels decreased with increasing algae inclusion level, and it is known that protein is a limiting factor, especially in the small, fast-growing larval stages (Fig. 5.2). In contrast, Nandeesha et al. (2001) reported improved growth rates for Indian carp fry with increasing levels of Spirulina platensis in feeds. Palmegiano et al. (2005) reported that sturgeon fed Spirulina-based feeds even outperformed those receiving fish meal-based diets. Contradictory results were reported by OlveraNovoa et al. (1998), where Spirulina-supplemented feeds depressed growth performance of tilapia fry. A more recent study by Walker and Berlinsky (2011) tested the nutritional value of a Nannochloropsis sp. and Isochrysis sp. mix for juvenile Atlantic cod. The authors described decreased feed intake and subsequently reduced growth with increasing algae inclusion. They concluded that reduced palatability of the algal meal caused the deterioration in cod growth. Valente et al. (2006) recommended that macroalgae such as Gracilaria and Ulva can be incorporated up to 10 % in European sea bass feeds without affecting the performance of fish. Other studies using seaweed have suggested that kelp meal works as an excellent additive (attractant, agglutinant and binder) in pelleted

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Fig. 5.2: Experimental fish feeds containing different proportions of dried Chlorella as a substitute for fish meal; Centre for Sustainable Aquatic Research, Swansea University.

feeds for penaeid shrimps and thus improved feed utilization efficiency in this slow-feeding species (Cruz-Suarez et al. 2009; Silva-Neto et al. 2012). Based on the known structural and compositional characteristics of algal biomass, it should be expected that ruminants are among the most suitable recipients, since they ought to be able to break down even unprocessed algal cell walls due to their unique digestive system. Performance parameters of lambs when fed U. lactuca all indicated that seaweed could be categorized as a low-energy high-nitrogen feedstuff in ruminants (Arieli et al. 1993). Conversely, an earlier study by Hintz et al. (1966) concluded that ruminants are unable to digest the carbohydrate fraction of Chlorella sp. and Scenedesmus sp. efficiently, although this poor performance may have been due to the specific characteristics of these algal strains. As noted above, the costs of fish meal and fish oil are steadily increasing. Thus, if a source of protein-rich or lipid-rich algal meal came onto the market at an affordable price, the animal feed industry would certainly consider using it, based on existing evidence of the nutritional value of algal biomass. However, as shown in Table 5.4, all categories of algal products are currently much higher in cost than the commodity feedstuffs used in animal feeds. One also has to consider the unit price of protein or lipid. To illustrate, a protein source such as soybean meal contains only 45 % protein but also a significant amount of indigestible bulk. Soy protein concentrates with up to 70 % protein are available, but this process renders them more expensive. Of the algal products listed in Table 5.4, Gracilaria is lowest in price; however, the price per unit of protein is still excessively high when compared to existing commodity feedstuffs that contain a much higher protein content.

5.4 Outlook

Ingredient

Main use

Price 2011 E/ton dry

Fish meala Soybean meala Rapeseed oila Wheata Fish oilb Tetraselmisc Spirulina sp.d Chlorella sp.d Gracilaria sp.d Laminaria – kelp sundried4

Feed Feed Food Feed Feed Bivalve shellfish Health food Health food Agar, feed Food

1,091 254 941 212 985–1,360 190,000–270,000 7,500–14,000 34,000–45,000 378–756 1,590–1,890

95

a

www.indexmundi.com www.globefish.org c www.reed-mariculture.com d www.alibaba.com b

Tab. 5.4: Global prices of ingredients used in animal feeds compared to currently available algal products.

5.4 Outlook Microalgae cultivation has been integral to modern forms of aquaculture for more than 40 years, developing and expanding alongside the “microalgae-for-food” and “microalgae-for-fuels” sectors. During this period, aquaculturists have devised robust methods for culturing a diverse range of phototrophic microalgal strains with high nutritional value that are more susceptible to crashes and contamination than those extremophiles that are mass-cultured for other purposes in open ponds or raceways (e.g. Arthrospira sp., Dunaliella sp., Haematococcus sp.). This aquaculture skills base and associated technologies (e.g. affordable closed PBRs) for culturing “sensitive” microalgal strains add value to the current microalgal biotechnology agenda of biofuels and high value biomass extracts through integrated biorefinery. It is expected that benefits will return to the aquaculture sector through current biotechnology investments, in the form of more efficient microalgal production systems and greater availability of high-quality microalgal biomass and extracts for use as hatchery feeds, etc. This is already illustrated by the adoption of heterotrophically produced microbial biomass (Schizochytrium sp., Crypthecodinium cohnii) as hatchery feeds; more abundant and cheaper feed products can be expected in future, provided the current aspirations of microalgal biotechnologists are realized. Whether algal biomass will be adopted in future as a bulk feedstuff to supply protein and energy in animal feeds, or will remain only as a supplement, will depend on biomass availability, composition and cost. As referred to in Section

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3.2, there is currently a large discrepancy in the global supply and purchase cost of algal biomass versus existing commodity animal feedstuffs, even for those categories of algal products that are produced at the largest scale. We conclude that until supplies increase and costs decrease, algal biomass and biomass extracts will continue to occupy niche markets within the animal feed sector, such as sources of pigments. The current global drive to produce biofuels from algae offers a key opportunity to shift existing biomass supply and cost structures in favor of animal feeds, within an integrated biorefinery. Assuming sufficient quantities of algal biomass do become available at a suitable price, algae producers and animal feed manufacturers will still need to take into account the potentially large variations in proximate composition (proteins, lipids, fatty acids, minerals, etc.) and digestibility encountered among different algal strains and growing conditions. Effort is needed to ensure a more consistent composition of algal biomass, so that manufacturers can readily incorporate this new feedstuff alongside existing ingredients in formulated feeds. To improve their digestibility, some types of algal biomass may require additional processing steps (over and above those applied to conventional feedstuffs) that add further to their cost. Although there are examples of macroalgal species containing relatively high levels of protein or lipid, it seems likely that microalgae will provide the most suitable bulk feedstuffs for use in finfish diets, whereas macroalgae may be more suitable for use with terrestrial livestock and with lower-trophic-level aquaculture species.

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Soler-Vila, A., S. Coughlan, M. D. Guiry and S. Kraan. 2009. The red alga Porphyra dioica as a fish-feed ingredient for rainbow trout (Oncorhynchus mykiss): effects on growth, feed efficiency and carcass composition. J. Appl. Phycol. 21: 617–624. Støttrup, J. G. 2003. Production and nutritional value of copepods. In: (J. G. Støttrup and L. A. McEvoy eds) Live Feeds in Marine Aquaculture. Blackwell Science Ltd. Oxford, UK. pp. 145– 205. Strand, A., O. Herstad, S. Liaaen-Jensen. 1998. Fucoxanthin metabolites in egg yolks of laying hens. Comparative Biochemistry and Physiology 119: 963–974. Tredici, M. R., N. Biondi, E. Ponis and L. Rodolfi. 2009. Advances in microalgal culture for aquaculture feed and other uses. In: (G. Burnell and G. Allan eds) New technologies in aquaculture: Improving production efficiency, quality and environmental management. Woodhead Publishing, Cambridge, UK. pp. 611–676. Turner, J. L., S. S. Dritz, J. J. Higgins and J. E. Minton. 2002. Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium. J. Anim. Sci. 80: 1947–1953. Valente, L. M. P., A. Gouveia, P. Rema, J. Matos, E. F. Gomes and I. S. Pinto. 2006. Evaluation of three seaweeds Gracilaria bursa-pastoris, Ulva rigida and Gracilaria cornea as dietary ingredients in European sea bass (Dicentrarchus labrax) juveniles. Aquaculture 252: 85–91. Van Stappen, G. 2003. Production, harvest and processing of Artemia from natural lakes. In: (J. G. Støttrup and L. A. McEvoy eds) Live Feeds in Marine Aquaculture. Blackwell Science Ltd. Oxford, UK. pp. 122–144. Viçose de, G. C., M. P. Viera, S. Huchette and M. S. Izquierdo. 2012. Improving nursery performances of Haliotis tuberculata coccinea: Nutritional value of four species of benthic diatoms and green macroalgae germlings. Aquaculture 334–337: 124–131. Waldenstedt, L., J. Inborr, I. Hansson and K. Elwinger. 2003. Effects of astaxanthin-rich algal meal (Haematococcus pluvialis) on growth performance, caecal campylobacter and clostridial counts and tissue astaxanthin concentration of broiler chickens. Anim. Feed Sci. Technol. 108: 119–132. Walker, A. B. and D. L. Berlinsky. 2011. Effects of Partial Replacement of Fish Meal Protein by Microalgae on Growth, Feed Intake, and Body Composition of Atlantic Cod. North American Journal of Aquaculture 73: 76–83. Zatkova, I., M. Sergejevová, J. Urban, R. Vachta, D. Štys and J. Madojídek. 2011. Carotenoidenriched microalgal biomass as feed supplement for freshwater ornamentals: albinic form of wels catfish (Silurus glanis). Aquaculture Nutrition 17: 278–286. Zmora, O. and A. Richmond. 2004. Microalgae for Aquaculture: Microalgae Production for Aquaculture. In: (A. Richmond eds) Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Science Ltd. Oxford, UK. pp. 365–379.

Christophe Hug and Denis von der Weid

6 Algae as an approach to combat malnutrition in developing countries 6.1 Introduction Algae are increasingly common in our food today. However, this is not a new phenomenon, as some species have been beneficially consumed for centuries by diverse cultures. The wealth of nutrients of algae has also led research towards investigating new approaches to combat malnutrition in developing countries. One approach is based on small-scale production of the microalga Spirulina and has proved to have considerable potential as a tool for development. Significant work has been performed to develop its production and distribution in order to reach malnourished populations. However, although this approach has been successful, it is only weakly supported by large international organizations and is not included in their development efforts. This might nevertheless evolve as successful field trials are accumulating evidence.

6.2 Algae in human food For centuries, coastal populations have taken advantage of algae to complement their food requirements, also using it as fertilizer and as animal feed. Traces of algae have been found in the ashes of prehistoric dwellings that suggest that mankind turned early to algae for its sustenance. Algae have been a traditional and timeless food source in coastal regions of East Asia. In Japan, China, and Korea, for instance, they are firmly anchored in food habits, and daily consumption keeps the algae farms busy. In North America and Europe, consumption is more limited and recent. Production remains mainly in the field of extracted by-products such as alginates and agars. These are used as food additives for their gelling or thickening properties in a variety of food productions. However, research is rapidly progressing towards the use of algae for development of novel food products, taking advantage of their compounds with functionalities that go beyond their basic nutritive value (Cornish and Garbary 2010). The combined presence of major nutrients – such as vitamins, minerals, antioxidants, rare essential amino acids and fatty acids, among others – contributes to this trend (MacArtain et al. 2007; Marfaing et al. 2010). Microalgae have gained particular popularity as a dietary supplement in industrialized countries, where calorie intakes are high, but micronutrients levels tend to be low. Human consumption is today by far the largest commercial application

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of the microalgal biomass production (Tramoy 2011). Of the microalgae, Spirulina, Chlorella and Dunaliella belong to the most commonly found species. In addition to their nutritional interest, microalgae have many potential benefits for human health reflected in the impressive amount of research carried out on the pharmacological properties of their specific metabolites. Most of these benefits have yet to be proven clinically, but the fact remains that a simple natural food such as algae may well improve a good number of health conditions (Gershwin and Belay 2007).

6.3 Microalgae as a solution against malnutrition: meet Spirulina Among microalgae, Spirulina is the one that has gained the most interest as a food supplement. This microalga possesses an extraordinarily high protein content of 50–70 % and a wide range of micronutrients. It contains all important vitamins (with the exception of vitamin C), high levels of essential micronutrients such as β-carotene, vitamin B12 and iron, and trace minerals. Moreover, thanks to the absence of a cell wall (unlike other microalgae), it provides an easily digestible product, without the need for processing (Falquet and Hurni 2006). Spirulina is able to treat successfully some of the most severe forms of malnutrition, such as vitamin A, protein, iron and zinc deficiencies. This is also the reason why it has been traditionally consumed for centuries in several regions of the world, such as the population in the Kanembu region of Lake Chad, who have notably avoided malnutrition for centuries despite their poor diets, especially in times of famine. This singular food was rediscovered in Chad in the 1950s by a European scientific mission that described dried green cakes sold in markets and produced from the abundance of Spirulina growing naturally in the lake (Habib et al. 2008). The rationale for the use of Spirulina as a solution against malnutrition brings together several complementary perspectives. First, Spirulina, as a natural product, provides a comprehensive solution to malnutrition, as it contains most critical micronutrients. It has been demonstrated many times to be a truly effective solution. Just 1–2 g per day is sufficient to correct the malnutrition of a child within a few weeks. In addition, recent studies show that Spirulina not only improves children’s physical and cognitive development but also helps people affected by HIV/ AIDS to feel better in their daily life and gain weight. Spirulina is also a very cost-effective solution, considering the ratio of production costs compared with its nutritional value. It can be easily cultivated in pool systems with relatively little upfront investment. An installation costs between € 10 and € 20 per square metre, and a pool of 200 square metres produces enough Spirulina for 1200 children per year. Finally, just as its traditional use in Chad suggests, Spirulina also sets the foundation for an approach that can create employment and income, establish a sustainable supply chain and therefore have a long-term impact (Heierli and von der Weid 2007).

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6.4 Small-scale Spirulina production as a development tool It is clear that the nutritional virtues of Spirulina are not its only advantages. Producing it locally potentially makes it more appealing than an imported foodstuff. Besides including local resources, product development that is based on local needs will also enhance its acceptance among the target beneficiaries. As such, Spirulina farms provide a solution, not only for malnutrition, but also to counter unemployment. Teaching locals to produce Spirulina will benefit them in the eyes of their peers as they acquire expertise in production. Several organizations, such as Antenna Technologies, have created capacity for developing small-scale Spirulina production, acting as facilitators to encourage local production. This small-scale production is orientated towards easier distribution to cover both rural and urban communities where the staple diet is poor. Antenna Technologies has developed a method using ponds, which allows local people to grow their own microalga independently. The key lies in transmitting know-how to target populations so that they can grow and market the microalgae product themselves. Establishing a Spirulina farm implies ensuring that several criteria, relating to climate (temperatures, light intensity), access to water, competence of local staff, and availability of agricultural fertilizers and bicarbonate, are met. The production technology is rather simple and requires little upfront investment. Cultivation may be carried out in unlined ditches or in concrete ponds. Stirring may be provided by a simple device driven by wind energy or by hand. The mature Spirulina is harvested by simple cloth filtration. After washing the Spirulina in freshwater, it can be directly mixed with the staple diet, without processing or cooking. If needed, Spirulina can also be preserved by immediate drying, with no significant loss in quality or nutritional value (Jourdan 2006). The major challenge for small-scale Spirulina farms lies in reaching financial sustainability. Once on site, research is carried out to improve productivity, competiveness and quality. It relates in particular to replacing imported inputs with local products, using cheap construction materials, improving stirring and working conditions, using the recycling systems of the growth medium, and improving drying and packaging (Hug and von der Weid 2011); see Figure 6.1. Small-scale farms in developing countries are fully aware of Spirulina’s potential to improve the nutrition of the population, and most are also motivated to develop into commercial businesses. As a consequence, Spirulina farmers in developing countries have mastered the production methods, from sowing to packaging during the past decade. The technical problems encountered 10 years ago have almost entirely disappeared, and the quality of Spirulina in developing countries has greatly improved. In several African countries, Good Manufacturing Practices and official quality control systems have been established (Charpy et al. 2008; Birot 2009).

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Fig. 6.1: Small-scale Spirulina farms in developing countries. Spirulina farms were established with support from Antenna Technologies. Small-scale Spirulina production is a relatively simple and straightforward process. It essentially consists of managing water and fertilizer levels, stirring, harvesting and drying. Source: Antenna.

Establishing a Spirulina farm as a local business also requires the creation of an effective distribution network and the establishment of an appropriate strategy to teach the local population about the nutritional benefits of Spirulina. One of the most important features of local Spirulina production is involving local women in the production process and also in creating a business for them. These women not only distribute but also educate peers about its nutritional values, and thus provide a service to the community. Combining local production with its distribution is a key for an efficient and long-lasting impact. Hence, Spirulina emerges not only as a remedy for malnutrition but also as a true tool for development (Heierli and von der Weid 2007).

6.5 Spirulina as a business to combat malnutrition Having the beneficiaries to contribute as customers is part of the paradigm of a sustainable long-term strategy to combat malnutrition. This implies the necessity

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Fig. 6.2: Spirulina product aimed at children in India. Antenna’s Green Tongue candy contains 0.5 g of Spirulina, which corresponds to half the daily recommended intake for a child up to 5 years old. The green tongue candies are also a good example of a creative use of the effect of Spirulina giving a green tongue, thereby making it very popular among children. Source: Antenna.

of making it so affordable that even the poor can contribute to the production cost of Spirulina enriched products. As a public-health solution, Spirulina must be marketed as a tangible product as well as a service in the form of information about its usefulness. In this sense, the product has to include a strategy of outreach: the product should not only be easily consumed but also be distributed in such a way so that it reaches populations that need it most (Heierli and von der Weid 2007). The business model identifies children and lactating women as the primary target group. As malnutrition impacts young children most severely, it is important to reach this target group through their mothers, who can be relied on to convey the message of nutrition from peer to peer. Such an approach is especially relevant in countries where promotion through the conventional media is not suitable because of its low coverage and high illiteracy rates. Being part of the value chain, sales activities may contribute indirectly to the fight against malnutrition as the additional income can result in improved food intake by both women and their children. Women therefore not only represent sales channels but also are beneficiaries (Heierli and von der Weid 2007) Product development requires a deep understanding of the needs of the customers and the local circumstances in order to develop a functional solution that incorporates critical features into the design of the product. In its raw form, Spirulina may be sold only to those who are already convinced of its value. For a wider market, a more attractive product is needed. Product development in order to reach targets such as malnourished children is therefore an area that deserves much

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attention. The product needs to be easily chewable, distributable and produced locally with simple technology. There are several examples of specifically designed products meeting these requirements. The Spirulina-enriched “chikkies” found in India are an excellent example of such product development, based on local needs and local resources. A chikki is a kind of energy biscuit based on peanuts and belongs to the most popular snacks for children in India. Spirulina chikkies were hence very well accepted (Heierli and von der Weid 2007). Another recently marketed product in India takes the form of a candy. This product represents another example of such a link to successfully reach poor and malnourished children. Its retail price (1 INR = € 0.02) lies within the price range for a regular snack that poor children in India can afford. It is therefore competitive and affordable, and also represents a business opportunity for sales women (Antenna 2011); see Figure 6.2.

6.6 Spirulina and its place in food aid and development policies Small-scale Spirulina production as an approach to combat malnutrition is today mainly promoted by NGOs and local health institutions in developing countries. Their work is exhorted by numerous positive field experiences and endorsed by the growing evidence from clinical studies. In spite of this, large international organizations and UN agencies such as the WHO, the WFP or UNICEF still do not clearly recommend it or promote its use. To date, these agencies have taken only non-committal positions on the use of Spirulina in the fight against malnutrition. The fact that Spirulina in this context is neither a medicine nor a foodstuff may have represented a hurdle. Should Spirulina be assessed under the angle of malnutrition as a public-health matter (thus under the responsibility of the WHO) or a food security matter (FAO), or is it rather an issue about the condition of children (UNICEF)? In the end, none of these institutions truly takes a stand. This situation may soon develop, since several countries, such as Burkina Faso or Senegal, have implemented governmental plans to develop the cultivation of Spirulina. Moreover, in 2005, several African and South American countries put forward a draft resolution demanding a clear stance from the UN General Assembly in favour of the production and use of Spirulina. (The UN draft resolution on Spirulina submitted during the 60th session of the UN General Assembly (2005) about “The use of Spirulina to combat hunger and malnutrition and help achieve sustainable development” is available at: http://www.un.org/ga/60/second/ draftproposals.htm.) Following this, the FAO was mandated and prepared a review on this subject in 2008 (Habib et al. 2008). (The FAO Review on Spirulina (2008) can be downloaded from ftp://ftp.fao.org/docrep/fao/011/i0424e/i0424e00.pdf.) Although this document treats the issue of malnutrition only superficially, it nevertheless recognizes the potential of Spirulina in this field. In its report, the FAO made two recommendations in that regard (Habib et al. 2008, p. 26):

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“International organizations working with Spirulina should consider preparing a practical guide to small-scale Spirulina production. This small-scale production should be orientated towards: (i) providing nutritional supplements for widespread use in rural and urban communities where the staple diet is poor or inadequate; (ii) allowing diversification from traditional crops in cases where land or water resources are limited.” “There is a role for both national governments – as well as intergovernmental organizations – to re-evaluate the potential of Spirulina to fulfil both their own food security needs as well as a tool for their overseas development.”

The first recommendation is widely followed today, since NGOs working with Spirulina continue their efforts for its development and humanitarian promotion. In recent years, considerable progress has been achieved in this domain in dozens of countries. Antenna Technologies, for instance, is involved in Spirulina programmes in more than 10 African and Asian countries. Regarding the second FAO recommendation, a large amount of work still needs to be performed. In its latest guidelines regarding the choice of foods and ingredients for moderately malnourished children, the WHO concluded (for the first time) that Spirulina could play a role in treating children with moderate malnutrition and recommended further investigation on this subject (Michaelsen et al. 2009). (Conclusions and recommendations on microalgae and Spirulina in the WHO guidelines on foods and ingredients for moderately malnourished children from 6 months to 5 years of age: “Microalgae may be good sources of micronutrients and high-quality protein, but availability might be low due to the cellulose content. Spirulina, a cyanobacterium, seems to have protein and micronutrients with a better bioavailability and has a high content of n-6 PUFAs. Some studies suggest that Spirulina could have a role in treating children with moderate malnutrition, but this should be investigated further.” (Michaelsen et al. 2009, pp. S378–S379).) This represents a slow progression fed by the large number of successful field initiatives and the accumulating evidence.

6.7 Evidence of Spirulina in malnutrition The need to increase acceptance among policymakers calls for more systematic research with Spirulina in malnutrition contexts. Below is a survey of some of the most recent clinical trials conducted with Spirulina, in particular with children and vulnerable populations (such as HIV/AIDS individuals), that helps provide compelling evidence: – In India, a randomized clinical trial on 60 schoolgirls addressed not only the purely nutritional effects of a small intake of Spirulina (1 g/day), but also possible indirect effects on their cognitive development (Sachdeva et al. 2004). This study led to positive and statistically significant results on both the haematological condition of the pupils and their intellectual performance. It

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ends with a recommendation to the Indian government about the supply of free Spirulina in schools, particularly in deprived regions. In Burkina Faso, a comparative study on the nutritional recovery of 170 children (of whom half were HIV-positive) demonstrates the benefits of Spirulina in the treatment of child malnutrition, as well as its particularly positive impact on the nutritional rehabilitation of HIV-infected children (Simpore et al. 2005). Another work by the same authors compared the nutritional benefits of diets composed of Spirulina and/or of Misola (Simpore et al. 2006). (Misola is a common nutritional complement composed of millet (60 %), soy (20 %), peanuts (10 %), sugar (9 %) and salt (1 %) that is widely used for malnourished children in West Africa.) The study was on 550 severely malnourished children under the age of 5. An improvement in body weight was observed in all children, especially those whose diet was made of both Spirulina and Misola. The authors concluded that Spirulina and/or Misola added to traditional food are good diets for severely malnourished children. The diet with Spirulina and Misola yields the best results, because it combines Misola’s high caloric value with Spirulina’s high protein and micronutrient content. In the Central African Republic, a 6-month randomized trial was carried out on adults infected and affected by HIV (Yamani et al. 2009); 160 patients were divided into two groups. Patients in the first group received 10 g of Spirulina per day, while patients in the second group received a placebo. This study showed a significant improvement on the main end-points (weight, arm girth, number of infectious episodes, CD4 count, protidemia). However, no clear conclusions could be drawn from a clinical standpoint because of methodological problems reported by the authors. In Cameroon, a randomized single-blind study on 52 malnourished HIV adults compared a group supplemented with Spirulina with a group supplemented with soya beans (Azabji et al. 2011). This work showed significant improvements in anthropometric, biological and immunological parameters in HIV-positive malnourished patients receiving Spirulina. The authors concluded that there is sufficient evidence in providing Spirulina regularly for this type of patient.

These recent clinical trials carried out in malnutrition show that Spirulina can be associated with a positive impact on children’s nutritional status as well as on other critical health outcomes. However, these trials still suffer from the same weaknesses as earlier studies regarding their statistical power (due to their small study populations and short follow-up periods, among others) and are subject to some criticism regarding their methodology. A systematic review from 2008 analysing previous studies also came to this conclusion, recommending a large-sized double-blind clinical trial to provide more conclusive results (Halidou Doudou et

References

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al. 2008). Such a trial would obviously be desirable. But one must keep in mind the difficulty of conducting such studies in developing countries and even more in finding sponsors. Despite several attempts to raise funds for this purpose, nothing materialized. Nevertheless, there remains a great deal of positive results, combined with successful field experience over the years, which should start to provoke reactions from decision-makers.

6.8 Conclusion In developing countries, malnutrition is a heavy burden with devastating socioeconomic consequences. Although many strategies against malnutrition exist, in order to be sustainable and avoid an economy of dependence, the solution must be local. Against this background, an approach based on algae, involving the growing and processing of a simple micro-organism as a source of alternative food supplements, might represent one of the most promising approaches in the longterm. Producing Spirulina and distributing it by developing special products may not be cheaper than classic feeding approaches in malnutrition. However, it presents advantages in terms of local ownership, and microeconomics. Much work is involved in building up viable local production units. Similarly, the broad set of approaches required – from the marketing of Spirulina products to the involvement of women in feeding programmes – adds dimensions that are less emphasized in common feeding schemes. The aim to combat malnutrition, on the one hand, and trying to rely on social entrepreneurship to ensure sustainability, on the other, presents unique challenges, but in a world confronted by severe economic crises making funds for development more uncertain, such efforts seem more than ever necessary.

Acknowledgements We would like to thank Christopher Morgan, Michael Briner and Fanny Lansaque for their valuable suggestions on the manuscript.

References Antenna Technologies, 2011: Green Tongue candies – a breakthrough in combating malnutrition?; http://www.antenna.ch/en/programs/Spirulina-programs/products (Download 21. 3. 2012) Azabji Kenfack M., Edie Dikosso S., Loni G., Onana A., Sobngwi E., Gbaguidi E., Nguefack T., Von der Weid D., Njoya O., Ngogang J., 2011. Potential of Spirulina platensis as a nutritional supplement in malnourished HIV-infected Adults in Sub-Saharan Africa: A randomised, single-blind study. Nutrition and Metabolic Insights 4 (2011): 29–37.

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Birot R., 2009. Promouvoir l’entrepreneuriat social et solidaire en adaptant une stratégie marketing à un projet de développement – Création d’un réseau de vente et de promotion de la spiruline en milieu rural, au Burkina-Faso. PURPAN, Toulouse. 121 p. Charpy L., Langlade M. J., Alliod R., 2008. La spiruline peut-elle être un atout pour la santé et le développement en Afrique ? Institut de Recherche pour le Développement, Marseille, 49 p. Cornish M., Garbary D., 2010. Antioxidants from macroalgae: potential applications in human health and nutrition. Algae 25/4 (2010): 155 – 171 Falquet J., Hurni J. P., 2006. Spiruline, Aspects Nutritionnels. Antenna Technologies, Genève. 41 p. Gershwin M. E., Belay A., 2007. Spirulina in Human Nutrition and Health. CRC Press, Boca Raton, 312 p. Habib M. A., Parvin M., Huntington T. C., Hasan M. R., 2008. A review on culture, production and use of Spirulina as food for humans and feeds for domestic animals and fish. FAO Fisheries and Aquaculture Circular No. 1034, Food and Agriculture Organization of the United Nations, Rome, 33 p. Halidou Doudou M., Degbey H., Daouda H., Leveque A., Donnen P., Hennart P., Dramaix-Wilmet M., 2008. The effect of spiruline during nutritional rehabilitation: Systematic review. In: Revue d’Épidémiologie et de Santé Publique 56 (2008): 425–431. Heierli U., von der Weid D., 2007. Sustainable Approaches to Combat Malnutrition: Small-Scale Production and Marketing of Spirulina. Antenna Technologies, Geneva, 67 p. Hug C., von der Weid D., 2011. Spirulina in the fight against malnutrition: Assessment and Prospects Antenna Technologies, Geneva, 28 p. Jourdan J. P., 2006. Grow your own Spirulina, Antenna Technologies, Geneva, 222 p.; http:// www.antenna.ch/en/research/malnutrition/guides (Download 21. 3. 2012) MacArtain P., Gill C., Brooks M., Campbell R., Rowland I., 2007. Nutritional value of edible seaweeds. Nutr Rev 65 (2007): 535–43. Marfaing H., Menguy A., Sassi J. F., Lerat Y., 2010: Les algues, des applications traditionnelles aux marchés émergents. Industries Alimentaires et Agricoles, mai/juin 2010, p. 29–30 Michaelsen K. F., Hoppe C., Roos N., Kaestel P., Stougaard M., Lauritzen L., Molgaard C., Girma T., Friis H., 2009: Choice of foods and ingredients for moderately malnourished children 6 months to 5 years of age. In: Food and Nutrition Bulletin 30 (2009): S343–S404. Sachdeva R., Kaur R., Sangha J. K., 2004: Effect of supplementation of Spirulina on the haematological profile and intellectual status of school girls (7–9 years). Journal of Human Ecology 15 (2004): 105–108. Simpore J., Zongo F., Kabore F., Dansou D., Bere A., Nikiema J. B., Pignatelli S., Biondi D., Ruberto G., Musumeci S., 2005 : Nutrition Rehabilitation of HIV-Infected and HIV-Negative Undernourished Children Utilizing Spirulina. Annals of Nutrition and Metabolism 49 (2005): 373–380. Simpore J., Kabore F., Zongo F., Dansou D., Bere A., Pignatelli S., Biondi D. M., Ruberto G., Musumeci S., 2006: Nutrition rehabilitation of undernourished children utilizing spiruline and Misola. Nutrition Journal 5 (2006): 3. Tramoy P., 2011: Microalgae Market and Application Outlook. CBDMT, Paris, 211 p. Yamani E., Kaba-Mebri J., Mouala C., Gresenguet G., Rey J. L., 2009: Use of Spirulina supplement for nutritional management of HIV-infected patients: Study in Bangui, Central African Republic. Médecine Tropicale 69 (2009): 66–70.

Thomas Happe, Camilla Lambertz, Jong-Hee Kwon, Sascha Rexroth and Matthias Rögner

7 Hydrogen production by natural and semiartificial systems 7.1 Biological hydrogen production of microorganisms Hydrogen metabolism is a widespread phenomenon in the world of microorganisms. Hydrogenase, the key enzyme of this particular metabolism, catalyzes the reversible and simple reaction of protons and electrons to molecular hydrogen (2 H+ + 2 e– → H2). Hydrogenases are classified within three distinct groups depending on the metal atoms of their active site. The active center of [NiFe] hydrogenases is characterized as binuclear complex with one Ni and one Fe atom, coordinated by several ligands, including one CO and two CN ligands (Frey 2002). [NiFe] hydrogenases are composed of at least two subunits accompanied by further [FeS] cluster. The mostly monomeric structure of [FeFe] hydrogenases enclose a well-conserved active center (H-cluster), composed of a [4Fe4S] subcluster and a [2Fe2S] subcluster connected by single cysteinyl sulfur (Peters et al. 1998) and, if existent, accessory [FeS] cluster (Calusinska et al. 2010). CO and CN ligands coordinate the [2Fe2S] cluster similar to [NiFe] hydrogenases. [Fe] hydrogenases differ from the first two groups. They catalyze CO2 reduction to methane by H2 oxidation (Vignais and Billoud 2007). Notably, any [FeS] or [NiFe] cluster is missing (Yang and Hall 2008), and the only Fe atom is not redox-active (Kim and Kim 2011). Whereas H2 production often serves as an electron valve for excess electrons to avoid over-reduction of various pathways, H2 consumption supplies electrons for metabolic reactions. Although the hydrogen metabolism is believed to play an important role for the energy balance of cells, extensive knowledge of these metabolisms is available only for few microorganisms, prokaryotes as well as eukaryotes, and may differ considerably from organism to organism. The strict anaerobic bacterium Clostridium pasteurianum produces H2 as a byproduct during fermentation of organic products to butyric acid (Calusinska et al. 2010). During the fermentative process, H2 production is mediated by an [FeFe] hydrogenase via oxidation of ferredoxin. Ferredoxin, on the other hand, is reduced either by pyruvate ferredoxin/flavodoxin oxidoreductase (PFOR), which transfers electrons to ferredoxin by pyruvate degradation or by NADH oxidation catalyzed by NADH ferredoxin oxidoreductase (NFOR) (Fig. 7.1a). In contrast, oxidation of molecular hydrogen plays a major role in the genus Ralstonia, a facultative chemolithoautotrophic proteobacterium. Ralstonia contains three types of [NiFe] hydrogenases, of which two are involved in energy balance:

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Fig. 7.1: Diversity of hydrogen mechanism of Clostridium (a), Ralstonia (b), Escherichia coli (c) and nitrogen-fixing cyanobacteria (d). (a) In the anaerobic growth of Clostridium, glucose is degraded to pyruvate during glycolysis (dashed line), and pyruvate is further converted to butyric acid and acetic acid. Hydrogen production is catalyzed by an [FeFe] hydrogenase (HydA) which receives electrons from ferredoxin (Fd). (b) Ralstonia oxidizes hydrogen via a membrane bound hydrogenase (MBH) or a soluble bidirectional hydrogenase (SH). Electrons are transferred from HoxG to HoxK and finally to cytochrome b (HoxZ) where the electrons are fed into the respiratory chain via plastochinone. NAD+ is directly reduced by the SH in the presence of hydrogen, and NADH is mainly used for carbon dioxide fixation (Calvin cycle; Rbc: RubisCo, ribulose-1,5-bisphosphate carboxylase/oxygenase). (c) Escherichia coli contains several [NiFe] hydrogenases. The membrane bound Hyd1 and Hyd2 take up hydrogen and transfer the electrons to the respiratory chain via plastochinone. The FHL (formate-hydrogen lyase)-complex is involved in hydrogen production. The formate dehydrogenase H (FDH-H) subunit cleaves formate to CO2 and protons. The released electrons are transferred within the complex via additional proteins (Hyc) to the hydrogen evolving protein Hyd3 (HycE). (d) Cyanobacterial hydrogen production is mediated, among others, by nitrogenase (Nif) activity. Nitrogenase forms a complex of dinitrogenase (NifDK) and dinitrogenase reductase (NifH). Molecular nitrogen is assimilated and converted to ammonium in an energy-con-

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Fig. 7.1: (continued) suming (16 ATP per N2) process that releases H2 as by-product. Hydrogen is recycled by a heterodimeric uptake [NiFe] hydrogenase (HupL, HupS), most likely linked to the respiratory chain.

an H2 uptake membrane-bound hydrogenase (MBH) and a bidirectional soluble hydrogenase (SH) located in the cytoplasm (Fig. 7.1b). The MBH is attached to the membrane via the hydrophobic C-terminal domain of the subunit HoxK and a diheme cytochrome b (HoxZ); plastochinone transports the electrons to the respiratory chain (Bernhard et al. 1997; Burgdorf et al. 2005). The NAD-dependent tetrameric hydrogenase (SH) shows high similarities to the peripheral Complex I part of the mitochondrial respiratory chain. In the presence of hydrogen, SH directly reduces NAD+ to NADH. The unique third hydrogenase is part of transcriptional regulation due to the ability of sensing hydrogen (Burgdorf et al. 2005).

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Escherichia coli as an H2 producer and consumer has an extraordinarily complex fermentative metabolism. Pyruvate formate lyase (PFL) as a key enzyme of the fermentative process cleaves pyruvate into acetyl-CoA and formate. Formate is oxidized by the FHL (formate hydrogen lyase) complex to CO2, and H2 production is catalyzed by a formate dehydrogenase and a hydrogenase (Rossmann et al. 1991; Sawers 2005) (Fig. 7.1c). Besides the hydrogenase as part of the FHL-complex (Hyd-3), E. coli contains at least three additional hydrogenases of the [NiFe]-type (Redwood et al. 2008). Purple non-sulfur bacteria, e.g. Rhodobacter sphaeroides and R. capsulatus, perform anoxygenic photosynthesis. In contrast to the photosynthesis of cyanobacteria, microalgae and higher plants, only one photosynthetic reaction center is involved. Although in this case sun light generates ATP, additional organic sources are required to generate reduction equivalents. Different from the H2 production by hydrogenases, Rhodobacter produces H2 only under nitrogen starvation. During nitrogen fixation, 16 ATP per N2 are needed for the reduction of nitrogen to ammonium with one H2 being generated as a by-product; H2 production rates up to 10.5 mmol [H2 l–1 h–1] have been determined for Rhodobacter sphaeroides (Miyake and Kawamura 1987). Normally, nitrogenases are accompanied by uptake [NiFe] hydrogenases to recycle both hydrogen and electrons for metabolic pathways. In order to increase the hydrogen production, different approaches have been taken, such as generation of mutant strains, which lack the uptake hydrogenase (Franchi et al. 2004; Öztürk et al. 2006) or immobilization of cells in gel droplets or on matrices (Tsygankov 2001). Although hydrogen production could be increased, H2 production still remains energetically expensive. Hydrogen production is also observed in many cyanobacterial strains under different growth conditions (Das and Veziroglu 2001; Dutta et al. 2005; Abed et al. 2009). Generally, cyanobacteria are divided into N2- and non N2-fixing strains: N2fixing cyanobacteria mainly produce H2 by nitrogenase activity accompanied by an uptake [NiFe] hydrogenase under nitrogen-starvation conditions (Fig. 7.1d). In contrast, non N2-fixing strains exhibit only bidirectional [NiFe] hydrogenases, similar to some N2-fixing strains (Schutz et al. 2004). Bidirectional hydrogenases interact with NAD(P)H as a redox partner and convert protons and electrons to H2 (Carrieri et al. 2011). Although N2 fixation and H2 production are not directly linked to the photosynthetic electron transport chain as in green algae (see Section 7.2), photosynthesis is still the primary energy source, as ATP and NAD(P)H are generated mainly by oxidation of carbohydrates produced during oxygenic photosynthesis. However, the high O2 sensitivity of nitrogenases prevents the simultaneous performance of N2 fixation and oxygenic photosynthesis. To overcome this problem, N2 fixation and photosynthesis are separated temporally or spatially within the cells. For instance, unicellular cyanobacteria strains fix N2 under anaerobic conditions, whereas filamentous strains form specific cells, heterocysts, with an anaerobic

7.2 Photobiological hydrogen production by green algae

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environment (Kumar et al. 2010). This is achieved by both reducing the O2 evolving PS2 activity and increasing the respiratory rate. Also, the cell wall of heterocysts is thicker than that of normal cells, which hampers O2 diffusion into the cell and enables microaerobic conditions.

7.2 Photobiological hydrogen production by green algae Photobiological production of hydrogen by photosynthetic microorganisms promises a clean carbon-free renewable energy from sunlight and water without the need for additional organic carbon and energy sources. One of the best-studied microalgae producing photobiological hydrogen with the highest efficiency is the unicellular green alga Chlamydomonas reinhardtii. Hydrogen production leads to photochemical efficiencies of 1.33 % (Laurinavichene et al. 2006) to 1.53 % (Kosourov and Seibert 2009) for immobilized and nutrient-deprived wild-type C. reinhardtii cells and even 3.22 % for a nutrient-deprived D1 protein mutant strain of C. reinhardtii (Scoma et al. 2012). In comparison, an uptake-hydrogenase-deficient mutant of the cyanobacteria Anabaena shows a solar efficiency of only 0.14 % (Lindblad et al. 2002; Brentner et al. 2010). Increasing attention has been focused on H2 production by microalgae (Hemschemeier et al. 2009; Kim and Kim 2011; Srirangan et al. 2011) since the 1930s. In 1939, the biochemist Hans Gaffron discovered for the first time a hydrogen metabolism of green microcalgae (Gaffron 1939). He observed anaerobic hydrogen uptake and formation of water with simultaneous carbon dioxide reduction in the light and, vice versa, hydrogen emission in the dark. H2 production could even be increased about 10-fold by illumination of anaerobic cultures in the absence of carbon dioxide and hydrogen (Gaffron and Rubin 1942). Over 50 years later, the first molecular evidences were published, when the proton-reducing [FeFe] hydrogenase of the green alga Chlamydomonas reinhardtii was isolated and identified as a small iron-containing protein of about 48 kDa, localized in the chloroplast stroma (Happe and Naber 1993; Happe et al. 1994). Genes encoding HYDA1 (Florin et al. 2001; Happe and Kaminski 2002) and a second hydrogenase HYDA2 were identified in C. reinhardtii (Forestier et al. 2003). Gene expression and protein activity were shown to be dependent on anaerobic conditions (Happe and Kaminski 2002; Stirnberg and Happe 2004). In fact, the hydrogenase is even irreversibly inactivated by oxygen within a second timescale (Stripp et al. 2009; Lambertz et al. 2011). Routinely, green algae cultures had been flushed with inert gas (Happe and Kaminski 2002) or adapted to the dark, resulting in very low in vivo H2-evolution rates in the dark (Gfeller and Gibbs 1984) and short-lived H2-production in the light (Ghirardi et al. 1997). A milestone for biohydrogen production of green algae in solving this problem was set by Melis and coworkers in 2000. They showed H2 production of sulfur-depleted, sealed and illuminated C. reinhardtii cultures for

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Fig. 7.2: Two-stage scheme of photosynthetic hydrogen production in sulfur-depleted C. reinhardtii cells. The decreasing sulfur concentration within the cells after depletion of sulfur is shown in yellow (triangle), and the accumulation of hydrogen gas within the culture is shown in blue (triangle), related to stage 1 and stage 2. Above, linear photosynthetic electron transport of stage 1 (left) and photohydrogen production (right) are indicated. Left, stage 1: Upon sulfur depletion, linear photosynthetic electron transport prevails, with electrons from water splitting at photosystem 2 (PS2) being transported to photosystem 1 (PS1) via plastoquinone (PQ), cytochrome b6/f complex (Cytb6/f) and plastocyanin (PC). PS1 transfers electrons to ferredoxin (PetF), which serves as an electron donor for the ferredoxin-NADP+ reductase (FNR). NADPH serves as a reductive equivalent in the Calvin cycle (RubisCo, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbc)) for CO2 fixation and triose-phosphate (Triose-P) generation. Right, stage 2: PS2 activity is strongly reduced (light color, dashed line), LHCII from PS2 migrates to PS1. The constant cell respiration leads to anaerobiosis. Electrons for H+ reduction originate both from a remaining PS2 activity and especially from degradation of starch that has been accumulated in stage 1. These electrons are fed into the electron transport chain via Nda2 (class-II type NAD(P) dehydrogenase) and finally transfered via PetF to the final electron acceptor, the [FeFe] hydrogenase.

several days starting one day after sulfur deprivation (Ghirardi et al. 2000; Melis et al. 2000). The absence of sulfur slows down photosynthesis, in particular PS2 activity and O2 production, to 25 % within 24 h (Wykoff et al. 1998; Melis et al. 2000; Antal et al. 2003). In contrast, O2 consumption by mitochondrial respiration remains constant (Melis et al. 2000; Hemschemeier et al. 2008) resulting in anoxic conditions, which is a precondition for hydrogenase activity and H2 evolution. Thus, sulfur deprivation leads to a temporally two-stage separation of O2 and H2 evolution (Fig. 7.2). In the first stage, normal photosynthesis, CO2 fixation, protein biosynthesis and cell growth continue, combined with significant starch accu-

7.3 Photohydrogen production by cyanobacterial design cells

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mulation in the cells (Grossman 2000; Fouchard et al. 2005). Thereafter, a lack of sulfur results in impaired protein biosynthesis and cell growth, affecting especially sulfur containing proteins with high de novo synthesis such as the D1 protein of the PS2. Photo-oxidation degrades these D1 proteins within hours (Wykoff et al. 1998); this causes decreased water-splitting activity, which finally leads to net O2 consumption (Melis 1999) and anaerobic conditions in sealed cultures. In order to maintain important pathways such as ATP synthesis, the metabolism changes into a complex variant of “anaerobic oxygenic photosynthesis” (Hemschemeier et al. 2009). Due to severely reduced CO2 assimilation, [FeFe] hydrogenase now takes over as electron sink, keeping photosynthetic electron transport and proton-gradient generation constant, which enables ATP synthesis. Electrons for H2 production are provided both by the remaining PS2 activity and by non-photochemical PQ reduction (Rumeau et al. 2007) by endogenous substrate oxidation, e.g. starch (Mus et al. 2005; Bernard et al. 2006), whereas common electron carriers are used for both pathways (Fig. 7.2). Nowadays, several hydrogenase genes and proteins of diverse algal species, e.g. Chlamydomonas moewusii, Scenedesmus obliquus and Chlorella fusca, have been identified showing a high degree of similarity (Schnackenberg et al. 1993; Florin et al. 2001; Wunschiers et al. 2001; Winkler et al. 2002a, 2002b; Kamp et al. 2008). In all unicellular green algal species analyzed so far, hydrogenases are coupled to the photosynthetic electron transport via ferredoxin PetF, which is reduced by PS1 and donates its electrons to the hydrogenase, as has been shown for C. reinhardtii (Winkler et al. 2009). Strategies used to enhance hydrogen production involve genetic engineering to achieve a better supply of organic substrates (Kruse et al. 2005) or better photosynthetic electron transfers (Mus et al. 2005). Mutants impaired in cyclic electron flow (CEF) around PS1, for instance, have a high potential for increased short- and long-term H2 photoproduction (Tolleter et al. 2011). In the wild type, anaerobiosis increases CEF, which is caused by state transitions of the light-harvesting complexes from PS2 to PS1 (Finazzi et al. 2002). This maintains the proton gradient over the thylakoid membrane and prevents over-acidification of the thylakoid lumen. CEF also limits electron supply to hydrogenases, thereby reducing hydrogen production. Loss of the CEF process seems to supply additional electrons to the hydrogenase, resulting in three- to fourfold-higher H2 production rates (Tolleter et al. 2011).

7.3 Photohydrogen production by cyanobacterial design cells In order to overcome the inherent limitations of biological hydrogen production and to achieve an economically competitive rate of produced hydrogen in the future, a design organism has to be constructed, which is stepwise-optimized towards the production of bioenergy instead of biomass. As the primary events of

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photosynthesis occur with a quantum efficiency near 99 %, and as all subsequent steps towards biomass formation result in a tremendous loss of energy – ending up with an average efficiency below 1 % in most cases – hydrogen production has to be coupled as close as possible with the primary events of photosynthesis (Esper et al. 2006). Cyanobacteria are ideal model organisms to engineer such a close coupling, as they are the most primitive organisms to perform oxygenic photosynthesis and as many strains are amenable to easy genetic transformation. Also, most of them are easy to cultivate in mass cultures, and strains can be isolated from extreme environments ranging from psychrophilic (below zero degree) to thermophilic (up to about 75°C) environments including fresh- and seawater species. Disadvantages of cyanobacteria that have to be “engineered” involve the following main points: 1. All cyanobacteria contain a [NiFe] hydrogenase of low activity and high oxygen sensitivity, which is also not directly coupled to PS1 via Ferredoxin as in the case of green algae (see previous paragraphs). 2. Cyanobacteria contain a large number of light-harvesting complexes, which can make up more than 50 % of their cellular mass. These “Phycobilisomes” are unfavorable for mass culture, as they are a waste of energy and also contribute considerably to the self-shading effect, which prevents the efficient use of light energy at high cell densities. 3. Electron transport is not balanced in favor of a linear transport, which is a prerequiste for efficient coupling between photosynthesis and hydrogen production; this is obvious from the PS2/PS1 ratio, which can be as low as 10 %. To overcome these problems, a strategy has to be developed to design a cell that, in contrast to existing “natural” cells, can be “milked” for energy. Starting from the well-characterized model-cyanobacterium Synechocystis PCC 6803, key enzymes for the light-triggered hydrogen production are stepwise-optimized for this purpose. This iterative process should finally yield a design cell with considerably higher linear electron transport, guiding electrons from water-splitting mainly to an imported high-turnover hydrogenase while reducing the electron flow for biomass formation to less than (estimated) 25 %. These cells should then be cultivated under mass culture conditions in special photobioreactors, which are designed for highly efficient light input, continuous culture conditions and low production price. If successful, this combined set-up should result in an H2 production per liter of cell culture approximately 100 times higher than the average rates achieved under anaerobic conditions with Chlamydomonas cells. The key characteristics of the design cell are as follows (Fig. 7.3): 1. reduced phycobilisome antenna: it can be shown that this, in addition to the aforementioned effects on mass cultivation, also results in a considerably higher PS2/PS1 ratio, increasing approximately from 0.1 to 0.4 (Bernat et al. 2009);

7.4 Photohydrogen production by a “biobattery”

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Fig. 7.3: Future “design cell” with components that have to be optimized for maximal H2-production. Phycobilisomes (PBS), photosystems (PS1 and PS2), ferredoxin (Fd), ferredoxin-NADP-oxidoreductase (FNR), hydrogenase (H2ase) and ATPase. For details, see text.

2.

partially uncoupled ATPase, resulting in a twofold-higher linear electron transport rate (Imashimizu et al. 2011); 3. considerably reduced CO2 fixation rate (down to about 25 %) in favor of H2 production via imported [FeFe] hydrogenase from green algae (up to 75 % of photosynthetic electrons seems realistic): this is achieved via an engineered reduced affinity between Fd and FNR (work in progress); 4. oxygen-tolerant [FeFe] hydrogenase from green algae, accepting electrons directly from Fd: ongoing research in various laboratories via directed and random mutagenesis, “directed evolution” approaches, etc. The combination of all these approaches in one future design cell shows the potential of natural cells for the production of renewable energy, in this case hydrogen, and the expectation of achieving the above-mentioned improvement by a factor of 100.

7.4 Photohydrogen production by a “biobattery” In parallel with the “design cell approach”, semiartificial systems can be used as proof of principle for the efficient coupling of water-splitting photosynthesis and hydrogen production. As the key elements for this process originate from various organisms, their interaction in the future design cell can be simulated if they are combined as isolated components in a semiartificial device. Due to the high oxygen sensitivity of presently available [FeFe] hydrogenases, this component is combined in a separate anaerobic compartment with isolated PS1, which is immobilized on

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Fig. 7.4: “Biobattery” as semiartificial minimal system, consisting of an oxygen evolving half cell (by water-splitting PS2, left) and a hydrogen-evolving half cell (with immobilized PS1 and hydrogenase, anaerobic, right). Os-polymers are used for optimal contact between gold electrodes and photosystems.

gold electrodes (Fig. 7.4, right-hand side). In contrast, isolated PS2 is immobilized on a gold electrode in an aerobic compartment (Fig. 7.4, left-hand side). The electric connection between both compartments allows the light-induced current between water splitting and hydrogen production to be quantified, provided both photosystems are efficiently connected to their electrode surfaces. Also, electron transfer between PS1 and hydrogenase can be optimized at the expense of FNR, for instance by evaluation of various Fd species. In summary, the “biobattery” approach is a very useful minimal model system for: 1. evaluation of the highest possible efficiency of this system (Badura et al. 2006, 2008, 2011a); 2. optimization of light-triggered electron transfer reactions in semiartificial and artificial devices (Badura et al. 2011b); 3. creating a blue print for combining the ideal partners in the natural design cell (Waschewski et al. 2010).

7.5 Photobioreactor design for hydrogen production Generally, mass cultivation of micro-algae appears promising as there is no competition for arable land which is needed for food production (Kheshgi et al. 2000; Stephens et al. 2010). Besides photosynthetic production of bio-diesel, bio-ethanol or biomass, photosynthetic hydrogen production is attractive, as it provides higher photochemical efficiencies for energy conversion by avoiding energy losses from carbon fixation and gluconeogenesis (Kheshgi et al. 2000). In addition, the effi-

7.6 Photobioreactor geometry

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cient separation of the gaseous product from the culture media is advantageous in the downstream processing. Although we focus here on a photobioreactor (PBR) design for direct biophotolysis in cyanobacterial organisms (see Fig. 7.3), there are general constraints for the photobiological production of biofuels (Hankamer et al. 2007; Morweiser et al. 2010). The most prominent requirements are a reduction in the investment costs and auxiliary energy demand.

7.6 Photobioreactor geometry While process engineering principles concerning light distribution, mass transfer and hydrodynamics have been established (Janssen et al. 2003; Lehr and Posten 2009), the design of PBRs has not yet converged and is mainly governed by economic constraints set by the product. For hydrogen production, a high surface/ volume ratio to obtain high cell densities (Pulz and Gross 2004) and an efficient product removal to overcome product inhibition are mandatory, while high growth rates will counteract high hydrogen production rates. Such high surface/volume ratios are realized by flat-plate reactors (FPRs), alveolar plate reactors and tubular reactors – in contrast to bubble columns, which are frequently found in lab-scale cultivation (Fig. 7.5; Posten 2009). FPRs especially provide considerable advantages for hydrogen production regarding the isolation of gaseous products and a low auxiliary energy demand for aeration and mixing. Due to their basic and robust design, they have been applied successfully for decades (Davis et al. 1953) for cultivation of cyanobacteria (Hu et al. 1996), green algae (Zhang and Melis 2002) and anoxygenic photosynthetic bacteria (Hoekema

Fig. 7.5: Bubble column (left) and flat-plate reactor (right), two of the most common designs of photobioreactors facilitating in situ isolation of the released hydrogen.

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et al. 2006). Also, the high surface/volume ratio of FPRs allows especially high photosynthetic productivities (Richmond et al. 2003). If the construction of FPRs is based on common semi-manufactured products available in industrial quantities, the investment cost can be reduced considerably (e.g. KSD, Hattingen).

7.7 Process control Although more difficult to realize, continuous cultivation has several advantages over batch cultures, such as constant production rates and elimination of down time for cleaning and sterilization. Especially for hydrogen production, extended growth is undesirable, while constant regulation of cell density and media composition is important, which can be achieved by either chemostat or turbidostat process control. In both cases, fresh media are constantly added while biomass and used media flow out. Under chemostatic control, a steady-state cell density results from a fixed dilution and a specific growth rate, while thermostatic conditions use a feedback between pump and cell-density measurement. As chemostat control is not limited by biofouling of online sensors, stable cultivation and production conditions can be extended for virtually unlimited periods (Fig. 7.6). In contrast, turbidostat process control is applied under varying light and under environmental conditions affecting growth. Due to direct correlation with the growth rate, the

Fig. 7.6: Cultivation of a slowly growing cyanobacterial strain with reduced light-harvesting phycobilisome antenna (PAL-mutant of Synechocystis PCC 6803). Chemostat process control (dilution rate of 0.012 h–1) enables stable cell densities without impairing viability or photosynthetic activity. Over a period of eight months, only limited biofouling is observed in zones with low flow velocities (Kwon et al., unpublished).

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dilution rate can be applied as an extremely useful parameter for process optimization (Kwon et al. 2012).

7.8 Upscaling strategies In order to produce biofuels in reasonable quantities, the design of large-scale photobioreactor systems is required. As light supply has the highest impact on volumetric productivity (Janssen et al. 2003), light penetration into the reactor is the most critical parameter for upscaling. In the case of flat-panel reactors, the thickness cannot be increased without impacting the volumetric productivity (Richmond et al. 2003). Also, height and width are restricted due to interference with the mixing behavior. Figure 7.7 presents the prototype of a 100-liter flat plate photobioreactor constructed according to these rules. For the outlined reasons, any further significant scale-up can be achieved only by increasing the number of such bioreactor units, which in turn increases the investment costs, as each unit requires its own set of control devices, aeration and media supply. In summary, an efficient solution for the control of these bioreactor units has to be found in order to achieve economic feasibility for large-scale photobiological processes, as well as efficient routes for up- and downstream processes.

Fig. 7.7: Prototype of a 100-liter flat-bed photobioreactor (KSD in collaboration with chair Plant Biochemistry).

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Janssen, M., J. Tramper, L. R. Mur and R. H. Wijffels. 2003. Enclosed outdoor photobioreactors: light regime, photosynthetic efficiency, scale-up, and future prospects. Biotechnol Bioeng 81(2): 193–210. Kamp, C., A. Silakov, M. Winkler, E. J. Reijerse, W. Lubitz and T. Happe. 2008. Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae. Biochim Biophys Acta 1777(5): 410–416. Kheshgi, H. S., R. C. Prince and G. Marland. 2000. The potential of biomass fuels in the context of global climate change: Focus on transportation fuels. Annual Review of Energy and the Environment 25: 199–244. Kim, D. H. and M. S. Kim. 2011. Hydrogenases for biological hydrogen production. Bioresour Technol 102(18): 8423–8431. Kosourov, S. N. and M. Seibert. 2009. Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions. Biotechnol Bioeng 102(1): 50–58. Kruse, O., J. Rupprecht, K. P. Bader, S. Thomas-Hall, P. M. Schenk, G. Finazzi and B. Hankamer. 2005. Improved photobiological H2 production in engineered green algal cells. J Biol Chem 280(40): 34170–34177. Kumar, K., R. A. Mella-Herrera and J. W. Golden. 2010. Cyanobacterial heterocysts. Cold Spring Harb Perspect Biol 2(4): a000315. Kwon, J. H., M. Rögner and S. Rexroth. 2012. Direct approach for bioprocess optimization in a continuous flat-bed photobioreactor system. J. Biotech.: (submitted). Lambertz, C., N. Leidel, K. G. Havelius, J. Noth, P. Chernev, M. Winkler, T. Happe and M. Haumann. 2011. O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase. J Biol Chem 286(47): 40614–40623. Laurinavichene, T., A. S. Fedorovm, M. L. Ghirardi, M. Seibert and A. Tsygankov. 2006. Demonstration of sustained hydrogen photoproduction by immobilized, sulfur-deprived Chlamydomonas reinhardtii cells. Int J Hydrogen Energy 31: 659–667. Lehr, F. and C. Posten. 2009. Closed photo-bioreactors as tools for biofuel production. Curr Opin Biotechnol 20(3): 280–285. Lindblad, P., K. Christensson, P. Lindberg, A. S. Fedorov, F. Pinto and A. Tsygankov. 2002. Photoproduction of H2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: from laboratory experiments to outdoor culture. Int J Hydrogen Energy 27: 1271– 1281. Melis, A. 1999. Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage ? Trends Plant Sci 4(4): 130–135. Melis, A., L. Zhang, M. Forestier, M. L. Ghirardi and M. Seibert. 2000. Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol 122(1): 127–136. Miyake, J. and S. Kawamura. 1987. Efficiency of light energy conversion to hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides. Int J Hydrogen Energy 3: 147–149. Morweiser, M., O. Kruse, B. Hankamer and C. Posten. 2010. Developments and perspectives of photobioreactors for biofuel production. Appl Microbiol Biotechnol 87(4): 1291–1301. Mus, F., L. Cournac, V. Cardettini, A. Caruana and G. Peltier. 2005. Inhibitor studies on nonphotochemical plastoquinone reduction and H(2) photoproduction in Chlamydomonas reinhardtii. Biochim Biophys Acta 1708(3): 322–332. Öztürk, Y., M. Yücel, F. Daldal, S. Mandaci, U. Gündüz, L. Türker and I. Eroglu. 2006. Hydrogen Production using Rhodobacter casulatus Mutants with Genetically Modified Electron Transfer Chain. Int J Hydrogen Energy 31: 1545–1552. Peters, J. W., W. N. Lanzilotta, B. J. Lemon and L. C. Seefeldt. 1998. X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282(5395): 1853–1858.

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Posten, C. 2009. Design principles of photo-bioreactors for cultivation of microalgae. Engineering in Life Sciences 9(3): 165–177. Pulz, O. and W. Gross. 2004. Valuable products from biotechnology of microalgae. Appl Microbiol Biotechnol 65(6): 635–648. Redwood, M. D., I. P. Mikheenko, F. Sargent and L. E. Macaskie. 2008. Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production. FEMS Microbiol Lett 278(1): 48–55. Richmond, A., Z. Cheng-Wu and Y. Zarmi. 2003. Efficient use of strong light for high photosynthetic productivity: interrelationships between the optical path, the optimal population density and cell-growth inhibition. Biomol Eng 20(4–6): 229–236. Rossmann, R., G. Sawers and A. Bock. 1991. Mechanism of regulation of the formatehydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon. Mol Microbiol 5(11): 2807–2814. Rumeau, D., G. Peltier and L. Cournac. 2007. Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. Plant Cell Environ 30(9): 1041–1051. Sawers, R. G. 2005. Formate and its role in hydrogen production in Escherichia coli. Biochem Soc Trans 33(Pt 1): 42–46. Schnackenberg, J., R. Schulz and H. Senger. 1993. Characterization and purification of a hydrogenase from the eukaryotic green alga Scenedesmus obliquus. FEBS Lett 327(1): 21–24. Schutz, K., T. Happe, O. Troshina, P. Lindblad, E. Leitao, P. Oliveira and P. Tamagnini. 2004. Cyanobacterial H(2) production – a comparative analysis. Planta 218(3): 350–359. Scoma, A., D. Krawietz, C. Faraloni, L. Giannelli, T. Happe and G. Torzillo. 2012. Sustained H production in a Chlamydomonas reinhardtii D1 protein mutant. J Biotechnol 157(4): 613–619. Srirangan, K., M. E. Pyne and C. Perry Chou. 2011. Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria. Bioresour Technol 102(18): 8589–8604. Stephens, E., I. L. Ross, J. H. Mussgnug, L. D. Wagner, M. A. Borowitzka, C. Posten, O. Kruse and B. Hankamer. 2010. Future prospects of microalgal biofuel production systems. Trends Plant Sci 15(10): 554–564. Stirnberg, M. and T. Happe. 2004. Identification of a cis-acting element controlling anaerobic expression of the hydA-gene from Chlamydomons reinhardtii. In: (J. Miyake Y. I., M. Rögner, eds) in Biohydrogen III. Elsevier Science, Oxford. pp. 117–127. Stripp, S. T., G. Goldet, C. Brandmayr, O. Sanganas, K. A. Vincent, M. Haumann, F. A. Armstrong and T. Happe. 2009. How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms. Proc Natl Acad Sci U S A 106(41): 17331–17336. Tolleter, D., B. Ghysels, J. Alric, D. Petroutsos, I. Tolstygina, D. Krawietz, T. Happe, P. Auroy, J. M. Adriano, A. Beyly, S. Cuine, J. Plet, I. M. Reiter, B. Genty, L. Cournac, M. Hippler and G. Peltier. 2011. Control of hydrogen photoproduction by the proton gradient generated by cyclic electron flow in Chlamydomonas reinhardtii. Plant Cell 23(7): 2619–2630. Tsygankov, A. A. 2001. Hydrogen photoproduction by purple bacteria: Immobilized vs. Suspension cultures. In: (J.Miyake T. M., A., eds) Biohydrogen II. San petro, Pergamon. pp. 229–243. Vignais, P. M. and B. Billoud. 2007. Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107(10): 4206–4272. Waschewski, N., G. Bernat and M. Rögner. 2010. Engineering photosynthesis for H2 production from H2O: Cyanobacteria as design organisms. In: (Vertes A., Qureshi, N., Yukawa, H., Blaschek, H. P., eds) Biomass to Biofuels – Strategies for Global Industries. John Wiley & Sons, Chichester, UK. pp. 387–401. Winkler, M., B. Heil and T. Happe. 2002a. Isolation and molecular characterization of the [Fe]hydrogenase from the unicellular green alga Chlorella fusca. Biochim Biophys Acta 1576(3): 330–334. Winkler, M., S. Kuhlgert, M. Hippler and T. Happe. 2009. Characterization of the key step for light-driven hydrogen evolution in green algae. J Biol Chem 284(52): 36620–36627.

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Winkler, M., C. Maeurer, A. Hemschemeier and T. Happe. 2002b. The isolation of green algal strains with outstanding H2-productivity. In: (Miyake J. I. Y., Roegner M., eds) Biohydrogen III. Elsevier Science, Oxford. pp. 103–115. Wunschiers, R., K. Stangier, H. Senger and R. Schulz. 2001. Molecular evidence for a Fehydrogenase in the green alga Scenedesmus obliquus. Curr Microbiol 42(5): 353–360. Wykoff, D. D., J. P. Davies, A. Melis and A. R. Grossman. 1998. The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii. Plant Physiol 117(1): 129–139. Yang, X. and M. B. Hall. 2008. Trigger mechanism for the catalytic hydrogen activation by monoiron (iron-sulfur cluster-free) hydrogenase. J Am Chem Soc 130(43): 14036–14037. Zhang, L. and A. Melis. 2002. Probing green algal hydrogen production. Philos Trans R Soc Lond B Biol Sci 357(1426):1499–1507; discussion 1507–1411.

Claudia B. Grewe and Carola Griehl

8 The carotenoid astaxanthin from Haematococcus pluvialis 8.1 Introduction Haematococcus pluvialis (Chlorophyceae, Volvocales) is a unicellular freshwater alga distributed in many habitats worldwide. The green alga is currently the natural source with the highest contents of astaxanthin (3,3′-dihydroxy-β,β-carotene4,4′-dione, ax) and the main producing organism for its commercial production worldwide. The presence of ax in H. pluvialis was initially reported by Wollenweber (1908), re-discovered for its use in microalgal biotechnology in 1991 (Borowitzka et al. 1991; Boussiba and Vonshak 1991) and is still subject of intensive research. Ax is a red secondary carotenoid (SC) with 13 conjugated double bonds and thus strong coloration properties. It is synthesized de novo in some algae, plants, yeast and bacteria, and enters the human food chain via fish or marine invertebrates. Insufficient nutrient uptake in animals leads to their discoloration, since animals are generally not capable of de novo ax synthesis. This is the main reason why ax is widely used for the coloration of farmed salmonids, e.g. Atlantic salmon (Salmo salar), which accumulates the colorant in contents of 3–11 ppm causing the characteristic reddish flesh. Moreover, ax possesses strong antioxidant and anti-inflammatory effects. It is widely accepted that oxidative stress and inflammation play an important role in the development of many chronic diseases that are widespread, especially in Western civilizations, such as atherosclerosis, diabetes, hypertension and some types of cancer. The health-stabilizing properties of ax as an antioxidant were investigated in a large number of studies, pre- and clinical trials; its potential for the prevention and treatment of various diseases and beneficial effects on cardiovascular health, the immune system, metabolic syndrome, ocular, liver, skin, gastrointestinal and neurological health in humans and animals appears to be overwhelming (Yuan et al. 2011; Fassett and Coombes 2012). Nevertheless, there is no EFSA (European Food Safety Authority) authorized health claim for the EU or a therapeutic application so far. Ax is approved as a food additive (food dye E161j) in the EU and the main application of natural ax from H. pluvialis is as dietary supplement and in cosmetics. Supercritical CO2 extracts from H. pluvialis gained “novel food“ status, FDA (Food and Drug Administration) granted ax from H. pluvialis “GRAS” status (Generally Recognized As Safe). Ever since, natural ax has been a multi-million-dollar market with a growing number of products and applications. In the global turnover of carotenoids, ax had the greatest share in 2009 (above 25 % of over US$ 1 billion) (Mortensen 2009). More than 90 % of total ax sales

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8 The carotenoid astaxanthin from Haematococcus pluvialis

are currently derived from a complex chemosynthesis. The purchase price of the chemosynthetic product is about US$ 600 to 800 per kg, while that of natural ax from H. pluvialis is in the range of US$ 4,000–6,000. For human consumption or cosmetics, this large difference is acceptable, and sales are fortified by a strong consumer tendency towards green, sustainable products.

8.2 Characteristics and biosynthesis 8.2.1 Chemical forms of astaxanthin Non-photosynthetic ax accumulates in algal cells in cytoplasmatic lipid vesicles after being exposed to certain environmental stress conditions. Ax occurs mainly in the form of fatty acid acyl esters: 77 % monoester, 16 % diester and a small fraction of unesterified ax (Grewe and Griehl 2008). The esterification of ax with fatty acids of different chain length (mainly oleic acid) causes significant changes in the molecular properties in terms of hydrophobicity, bioavailability and bioactivity (Mercke Odeberg et al. 2003): solubility and storage capacity in lipid vesicles as well as its antioxidant capacity are enhanced (Ceron et al. 2007), and the molecule becomes more stable against oxidative degradation. Configuration and constitution of ax are also significantly involved in its chemical properties as well as in its biological activity, e.g. antioxidant capacity and absorption characteristics. A proportion of over 97 % ax occurs in all-E form, the thermodynamically most stable conformation, while 1.4 % 9-Z-ax and 0.7 % 13-Z-ax were identified (Holtin et al. 2009). In addition to mono-Z forms, an additional 3.6 % of total ax was found to be a di-Z form in a H. pluvialis extract. The Z-conformation causes an additional absorption maximum, which may play a role in the avoidance of UV-irradiance stress for the algal cell and thus serves an important biological function. It seems likely that the total amount of Z-isomers is influenced by the environmental conditions to which the algae strains have been exposed. Furthermore, stereoisomers exhibit different biological activity; Liu and Osawa (2007) reported that Z-isomers of ax, especially 9-Z-ax, have a much higher antioxidant capacity in vitro than all-E-isomers. Due to its two stereogenic centers at the C3 and C3′ position of the β-ionone rings, ax possesses four possible configurations: (3S, 3′S), (3R, 3′R), (3R, 3′S) and (3S, 3′R). The (3S, 3′S) and (3R, 3′R) isomers are enantiomeric forms. In all-E configuration the optically inactive mesoforms (3S, 3′R) and (3R, 3′S) are identical in their spatial structure. On the contrary, a Z-configuration (e.g. 9-Z) leads to four ax stereoisomers. Since the industrial chemosynthesis for the production of ax is not stereoselective, the widely used ax product contains a stereoisomeric mixture of (3S, 3′S)-ax, (3R, 3′S)-ax and (3R, 3′R)-ax in the ratio 1 : 2 : 1. In living organisms, ax is synthesized in high enantiomeric purity in either the (R, R) or (S, S) form. In

8.2 Characteristics and biosynthesis

131

H. pluvialis ax is present in the (3S, 3′S) form, while the yeast Xanthophyllomyces dendrorhous synthesizes (3R, 3′R)-ax (Grewe et al. 2007).

8.2.2 Astaxanthin biosynthesis The basic building blocks of ax are the active isoprene molecules isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are formed via the plastidal methylerythritol phosphate pathway (MEP) in H. pluvialis (Fraser and Bramley 2004). Subsequent to an elimination of a diphosphate residue, IPP and DMAPP condense (“head-to-tail”) through a nucleophilic substitution to the C10body of geranyl diphosphate (GPP). Isopentenyl diphosphate isomerase (IDI) appears to be a rate-limiting enzyme of the carotenoid biosynthesis. Two more IPP additions lead to the formation of diterpene geranylgeranyl diphosphate (GGPP, C20), the direct precursor of all carotenoids, via farsenyl diphosphat (FPP). These steps are catalyzed by a prenyl transferase. The tail-to-tail addition of two molecules of GGPP leads to prephytoene pyrophosphate (PPPP), from which the first colorless carotenoid phytoene (C40) is formed. The reaction is considered as the entrystep reaction to carotenoid biosynthesis (Tran et al. 2009). The formation of phytoene is catalyzed by the phytoene synthase (PSY). The system of conjugated double bonds is prolonged by a successive dehydrogenation in four steps, forming the intermediates ξ-carotene, proneurosporin and prolycopene, and resulting in redcolored lycopene, catalyzed by the phytoene desaturase (PDS) and the ξ-carotene desaturase (ZDS). Through subsequent cyclizations at the periphery of the lycopene molecule, β,β-carotene (β-carotene) and β,ε-carotene (α-carotene) are formed. In H. pluvialis, the biosynthesis of SC starts from β-carotene, via hydroxylation and/ or introduction of keto groups in two different ways: initial introduction of keto groups to form canthaxanthin (cx) and subsequent hydroxylation or via zeaxanthin with initial hydroxylation and subsequent oxidation. The formation of ax is catalyzed by the carotenogenic enzymes β-carotene ketolase (BKT) (Lotan and Hirschberg 1995) and β-carotene hydroxylase (CRTZ) (Linden 1999). In H. pluvialis, ax biosynthesis occurs mainly linear via cx (see Fig. 8.1). Both the presence of intermediates such as adonixanthin (Orosa et al. 2001; Grewe and Griehl 2008) and the bi-functionality of CRTZ and BKT indicate that other pathways to ax are active, too. In H. pluvialis, BKT is present both in the chloroplast and the cytoplasmatic lipid vesicles, but its activity is limited to the lipid vesicles (Grünewald and Hagen 2001). Since the PDS is exclusively localized in the chloroplast, it seems likely that β-carotene is transported across the chloroplast envelope to cytosolic lipid bodies, where the introduction of keto groups takes place. Collins et al. (2011) located β-carotene in the chloroplast as well as in the cytosol. Yet no clear indication of a transit sequence has been found in one of the three bkt genes identified in H. pluvialis so far. Triacyl glycerols (TAGs) serve

Fig. 8.1: Biosynthesis of primary (PC) and secondary carotenoids (SC) in H. pluvialis.

132 8 The carotenoid astaxanthin from Haematococcus pluvialis

8.3 Haematococcus pluvialis

133

as a reservoir of oleate and other fatty acids for the esterification of ax that consequently takes place on the interface of the globule (Zhekisheva et al. 2002).

8.2.3 Function of astaxanthin The function of ax in the red cysts of H. pluvialis is discussed controversially. In general, one may distinguish between the direct and indirect functionality of ax. Directly, ax acts as sunscreen and protects the cell against light-induced oxidative damage by absorbing excessive radiation, mainly at blue wavelengths (Wang et al. 2003). Excessive radiation leads to an over-excitation on PSII (photosystem II) and an accompanying increased oxygen and ROS (reactive oxygen species) formation. The photoprotective action of ax was demonstrated in red flagellates exhibiting a lower blue-light-induced decrease in PS II efficiency compared to red light (Hagen et al. 2000). The carotenoid may moreover act as a physico-chemical barrier, scavenging ROS. The high antioxidant capacity of ax was measured both in vitro and in vivo (Kamath et al. 2008). Nevertheless, the direct protection of cytosolic ax against ROS can be questioned due to the spatial separation between the ROS generation side (chloroplast) and the accumulation side (lipid droplets in the cytosol). The biosynthesis of ax also has some indirect effects: molecular oxygen is incorporated in its molecular structure; Li et al. (2008) measured an O2 consumption of up to 9.94 % of total evolved oxygen. Further oxygen is consumed by its reduction to water, catalyzed by PTOX (plastid terminal oxidase), which uses electrons from the four desaturation steps during phytoene formation. For those pathways, ax is the end product of a detoxification as well as a protecting agent itself. All the suggested functions lead to the detoxification of oxygen and thus to the reduction in oxidative stress in the algal cell. Finally, the carotenoid formation presents a suitable way to store energy and carbon under nutrient-limitation conditions.

8.3 Haematococcus pluvialis 8.3.1 General characteristics In its green stage, the visible characteristics of H. pluvialis are two isokont flagella, a cup-shaped chloroplast, pyrenoids and an anterior eye-spot. Its cytoplasm is surrounded by a gelatinous layer nerved by cytoplasmatic strands. The cells divide via sporangium formation: flagellates are released from zoosporangia, while nonmotile cells originate from aplanosporangia (Lee 2008). In a later growth stage, cells lose their motility and form green palmelloid aplanospores. Besides this vegetative growth, H. pluvialis also possesses a sexual-reproduction life cycle that is

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8 The carotenoid astaxanthin from Haematococcus pluvialis

Fig. 8.2: Microscopic image of H. pluvialis during stress exposure: (a) vegetative stage, (b1) Palmella-stage, (b2) Ax-accumulating flagellates, (c) transition to Aplanospore-form and (d) ax-rich aplanospores. Scale bar: 5 μm.

induced by varying cultivation conditions and of which very little is known (Pröschold 1997). During exposure to adverse environmental conditions the cell morphology changes considerably, the diameter increases up to 50 μm, and a thick cell wall is formed. It consists of an outer primary wall, a trilaminar sheath, a secondary wall and a tertiary wall that contains an acetolysis-resistant and aliphatic bio-polymer, called algeanan (Damiani et al. 2006). The aplanospores accumulate ax with average contents of 20–40 mg/g of dry weight. The formation of ax starts in perinuclear lipid globules, but ax accumulation is not restricted to aplanospores (see Fig. 8.2). As the light stress persists, the SC-containing globules migrate to the peripherical region and finally form a closed lipid layer beneath the cell wall. Table 8.1 provides an overview on the gross biochemical composition of H. pluvialis biomass both in green (vegetative) and in red (aplanospore) stage. Together with ax content, the TAG and the carbohydrate content increase in H. pluvialis. According to the lipid composition of the globules, they are derived from cytoplasmic membranes (Grünewald et al. 2001); the total fatty acid content can account for up to 30 % of total dry weight (Miao et al. 2008). The phospholipid content does not change, while the glycolipid fraction nearly doubles (Damiani et al. 2010). The ax formation is accompanied by the reduction in photosynthetic activity of PSII and the loss of cytochrome f, which leads to a reduced electron transport and a high respiration rate (Boussiba 2000). Both the chlorophyll and the protein content drop; their de novo synthesis is not possible under N-depleted conditions. The total carotenoid content is markedly enhanced in the red stage, and the characteristic PC pattern of vegetative stage is replaced by SC, mainly ax (80–99 % of total carotenoids). The ratio of carotenoids to chlorophylls is about 0.2 in the green stage and increases in the red stage by an order of magnitude (2–9). The exact chemical composition of the biomass clearly depends on environmental con-

8.3 Haematococcus pluvialis

Composition Content (percentage of dry weight)

Green stage

Red stage

Proteins Lipids (% of total): Neutral lipids Phospholipids Glycolipids Carbohydrates Carotenoids (% of total): Neoxanthin Violaxanthin β-Carotene Lutein Zeaxanthin Astaxanthin (incl. esters) Adonixanthin Adonirubin Canthaxanthin Echinenone Chlorophylls

29–45 20–25

17–25 32–37

59 23.7 11.5 15–17 0.5

51.9–53.5 20.6–21.1 25.7–26.5 36–40 2–5

8.3 12.5 16.7 56.3 6.3 n.d. n.d. n.d. n.d. n.d. 1.5–2

n.d. n.d. 1.0 0.5 n.d. 81.2 0.4 0.6 5.1 0.2 0

135

Tab. 8.1: Typical composition of H. pluvialis biomass (nutritional profile red stage MSDS AlgaTechnologies; carotenoids (Grewe and Griehl 2008), green stage (unpublished data), lipid composition (Damiani et al. 2010)).

ditions, including the source of nutrients and the mode of nutrition. The ax biosynthesis process is reversible: if conditions become growth-favorable again a transition from red to green morphotype occurs, ax is degraded, and sporangium formation is induced.

8.3.2 Factors responsible for ax accumulation The accumulation of ax in H. pluvialis is induced by different environmental conditions, which are generally not favorable for growth, and are often referred to as “stress”. They actuate an imbalance in the metabolism, which requires metabolic adaptive responses. The stress conditions that trigger ax accumulation in H. pluvialis are manifold but can be grouped as: nutrient deficiency (nitrate, phosphate and sulfate), high light intensities, drought stress, high salinity, high temperatures, ROS and acetate (high C/N ratio). It is commonly agreed upon that light is the most important inductive carotenogenic factor. With respect to light intensity, the terms “low light” and “high light” are frequently being used. Low light can be considered below 100 μE/(m2*s), an optimum for growth in the range of 50–60 μE/(m2*s) (Harker et al. 1995). Light

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8 The carotenoid astaxanthin from Haematococcus pluvialis

saturation of H. pluvialis was measured by Fan et al. (1994) at 90 μE/(m2*s) and photoinhibition above 500 μE/(m2*s) (Wang et al. 2003). For autotrophic induction of ax accumulation, a light enhancement is used, e.g. by a factor of 4.7 to 350 μE/ (m2*s) (Wang et al. 2003). A trigger value of 170 μE/(m2*s) was reported by Boussiba and Vonshak (1991), while higher ax contents were achieved using light intensities of 1,550–1,650 μE/(m2*s) (Harker et al. 1995). García-Malea et al. (2009) did not measure photoinhibition up to 2,500 μE/(m2*s). With respect to light quality, it was reported that the tolerance of mature cyst cells to UV-B was sixfold higher than that of immature cyst cells (Kobayashi 2003). Steinbrenner and Linden (2003) showed that blue light was significantly more effective in terms of final ax content than red light. The combination of high light intensity and blue light (λ = 470 nm) results in a higher final ax concentration and per-cell ax content (Park and Lee 2001). The development of LEDs facilitated investigations on light quality on ax formation in H. pluvialis, since emitting wavelength can be influenced very precisely. Moreover, energy consumption is markedly reduced. LEDs of short wavelengths (380–470 nm) were found to yield ax contents of up to 5–6 % of dry weight (Katsuda et al. 2004). Therefore, a new strategy of cultivating H. pluvialis was suggested: using red LEDs for green phase and switching to illumination with blue LEDs at a high light intensity to induce ax accumulation in a second stage. Flashing light has been investigated as an alternative illumination source, since it was reported that the utilization efficiency of light received by the algae tends to be greater in intermittent light than that of steady light of equal intensity (Kim et al. 2006). Although the relative ax yield per photon was higher for flashing light, continuous light at 200 μE/(m2*s) yielded higher ax productivities (16 mg/(L*d). On the contrary, in mixotrophic culture of H. pluvialis, flashing blue LEDs at 18 μE/(m2*s) and flashing frequencies of 25–200 Hz equal or higher final ax concentrations were measured compared to continuous light (Katsuda et al. 2008). The combination of flashing light and blue light LEDs reduced the energy use for the same final ax yield by 33 % (Katsuda et al. 2006). Table 8.2 summarizes a selection of publications dealing with productivity of ax in H. pluvialis (different strains), grouped according to their mode of nutrition. The reported ax productivity in red stage ranges from 0.1 to maximal 20.8 mg/(L*d), with final ax contents in the biomass between 0.5 and 6 %. The very high ax content of Dominguez-Bocanegra et al. (2004) should be questioned, as ax contents of 5–6 % are usually not exceeded. Average values in lab scale in terms of ax content and ax productivities can be set to 2–4 % or 3–10 mg/(L*d), respectively. Based on the published results, autotrophic cultivations are more productive than mixo- or heterotrophic ones. The use of heterotroph-autotroph cycles enhances the ax productivity to values above the mixotrophic cultivations. The more recently presented one-step production method shows very high ax productivities (20.8 mg/(L*d)) but is associated with much lower ax contents in the bio-

8.3 Haematococcus pluvialis

137

Mode

Volume (litres)

PBR type

Ax content (%)

Ax productivity [mg/(L*d)]

Reference

Autotrophic, batch

0.25

6.0

16.6*

0.5

Erlenmeyer flasks Bubble column

4.0

11.5

0.5

Bubble column

n.g.

8–14*

55 55 25,000 0.4

Airlift Bubble column Tubular PBR n.g.

2.0 0.5 2.5 2.3

4.4 0.12 2.2 2.7

0.25

2.26

3.6*

0.55

Aerated Erlenmeyer flasks n.g.

n.g.

2.8–3.5*

1.8

Bubble column

1.1

20.8

1.8

Bubble column

0.6

2.2

50

Tubular PBR

1.34

8.0

1

Flat plate

4.8*

14

0.2/1

Erlenmeyer flask/bubble column Fermenter/PBR

n.g.

17*

Kang et al. (2007) Aflalo et al. (2007) Wang et al. (2003) Lopez et al. (2006) Olaizola (2000) Orosa et al. (2001) Kang et al. (2005) Katsuda et al. (2008) Del Rio et al. (2008) García-Malea et al. (2009) García-Malea et al. (2009) Kang et al. (2010) Ranjbar et al. (2008)

1.6*

4.4

Mixotrophic, batch

Chemostat, autotrophic

Fed batch, autotrophic Heterotrophicautotrophic, two stage Autotrophicheterotrophic sequences

2.3/1

Hata et al. (2001)

n.g.: not given * calculated from given values Tab. 8.2: Overview of ax productivity in H. pluvialis grown under various conditions.

mass (Del Rio et al. 2008). In order to realize chemostat processes, a much higher degree of automatization is needed. Aflalo et al. (2007) reported that a two-stage system performs better (by a factor of 2.5–5) than the one-stage system. Nevertheless, the single research results are hardly comparable, because the applied intensity, direction, spectral composition, geometry and volume of the photobioreactor PBR, culture density and the measurement itself influence the referenced value. In order to eliminate the changing light regime during growth, chemostat or turbidostat experiments or lumostatic operation of PBRs by increasing the light intensity based on the specific growth should be performed. This method was described for H. pluvialis in bubble column PBRs by Choi et al. (2003) and Lee et al. (2006), and appears to be a good strategy for increasing the cell growth of H. pluvialis compared to cultivations with a constant supply of light energy.

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8.3.3 Industrial production of Haematococcus The current methodology for the production of ax from H. pluvialis on an industrial scale follows a two-stage approach: vegetative biomass is grown under best-possible growth conditions (green stage) followed by subsequent production of ax under permanent stress conditions (red stage). If tubular systems are used, autotrophic nutrition and low layer thickness dominate, while closed PBRs work mixotrophically and use artificial illumination. Here, sunny production locations in general encounter temperature problems that are not well tolerated at that stage. These problems are confined by using evaporation cooling. The second ax producing stage uses deprivation of nutrients, higher light and temperatures as well as salt addition to the medium. Often dilution steps are carried out in order to achieve a higher light input per cell. In general, the production methods have to be distinguished by PBR type: open ponds and tubular PBRs dominate the process. They both rely on a high global solar irradiation, the reason why the production facilities are located at sunny locations. While closed PBRs produce an ax-containing powder of 4–5 %, open systems yield biomass with much lower contents, typically of 1.5–2 %. Process disturbances can result in even lower values. Table 8.3 provides an overview of production sites of H. pluvialis worldwide. Cyanotech Corp., based in Hawaii, has been in operation since 1984 and has extended production from Arthrospira to H. pluvialis. A combination of closed PBRs and raceway ponds are employed for the production of ax. For growth enhancement, CO2 is injected on demand, and cold ocean water (10 °C) cools the suspension via heat exchangers. The induction period is in the range of 5–8 days, and cells are harvested by settlement and centrifugation, while medium is being recycled. H. pluvialis cells are dried and subsequently milled for disintegration (Cysewski and Lorenz 2004). Parry Nutraceuticals has also developed an open pond cultivation system for the production of ax in India. Here, a patented dilution system is applied, and short induction cycles are used. After harvesting, the biomass is spray-dried. A recently founded company, Atacama Bio Natural Products, is now

Company

Location

PBR type

Cultivation conditions

Status

Cyanotech Algatechnologies Fuji Chemicals

Hawaii, USA Israel Sweden, Hawaii Japan India Hawaii

Open ponds Tubular PBRs Fermenters Biodome Flat panel PBRs Open ponds Tubular PBRs/ open ponds Open ponds

Autotrophic Autotrophic Mixotrophic Autotrophic n.a. Autotrophic Autotrophic

Expanding Expanding Operating Closed Closed 2010 Operating Closed

Autotrophic

Starting

Yamaha Parry Nutraceutical Aquasearch/Mera Pharmaceuticals Atacama Bio Natural Products

Chile

Tab. 8.3: Current and closed production sites of H. pluvialis.

8.3 Haematococcus pluvialis

139

Fig. 8.3: Production facility of H. pluvialis of Algatechnologies Ltd, located in the Desert of Negev, Israel.

producing H. pluvialis in the Atacama Desert of Chile taking advantage of high solar irradiation including UV. Algatech, founded in 1999, was the first to use tubular PBRs for the commercial production of ax. The plant operates in a kibbutz in the Negev Desert, Israel. Both horizontally and vertically arranged glass tubes about 10 cm in diameter are employed (see Fig. 8.3), producing about 3 tons of biomass per month. In Sweden, Fuji Chemicals operates fully closed bioreactors, employing a mixotrophic production on the basis of acetate and artificial illumination. Aquasearch, which was also based on Hawaii, used a newly developed tubular PBR (Aquasearch Growth Module, AGM) in combination with a red cycle of 5 days in open ponds. The average depth was 15 cm, and temperatures between 16 and 35 °C were measured during a day cycle (Olaizola 2000). During operation, 9–13 g/(m2*d) have been reached, with ax contents of 1.5–2.5 %. While the ax content in the individual products can be measured, and quality can be compared directly, data on ax productivity are not easily available. Biomass productivities of 1 g/(L*d) in autotrophic cultures were published several times; these values appear hardly to be within reach of average outdoor production values. Average biomass productivities of 0.4–0.7 g/(L*d) appear to be more reliable. H. pluvialis cultures are very sensitive to contamination by other algae. Susceptibility to environmental conditions depends upon the growth stage; while the red stage withstands high temperatures, etc., the green stage is sensitive to high temperatures. Problems in mass cultures are dominated by: – high temperatures in the green phase; – contamination with faster-growing green algae (e.g. Scenedesmus spp.); – predators (amoeba and protozoa) (Cysewski and Lorenz 2004); – chytrid parasites (Hoffman et al. 2008).

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Closed PBRs appear to represent the most successful system to grow this microalga, since the resulting problems can be controlled more easily, and productivity is higher by at least an order of magnitude.

8.4 Conclusions and outlook Astaxanthin from H. pluvialis can be seen as the main application of microalgal biotechnology in closed PBRs, while biomass production in open ponds (Arthrospira, Chlorella and Dunaliella) can be classified as “better agriculture”. The growing demand for natural ax for food, supplements and cosmetics has resulted in a steadily rising market. In the case of ax, this means moreover a shift from petrochemistry-based chemosynthetics to a biotechnological product, which means advances in terms of coloring effects, bioavailability, chirality and configuration of the final product. Research is still focusing on optimization of ax content or cultivation conditions, including new designs of PBRs and investigations on the regulation of its biosynthesis including molecular biotechnology methods. New cultivation approaches that make use of closed material cycles will gain more importance. Future developments in the field will concentrate on breeding and genetic engineering of H. pluvialis to improve both the growth rate and the ax content. Here it has to withstand concurrence of other organisms, especially that of higher plants and E. coli, where genetic tools are readily available. Hence, an integrated genomics, proteomics and metabolomics approach will be required in order to better understand ax biosynthesis and its regulation. This will undoubtedly lead to advances in microalgal biotechnology in general. Within the next decade, the development of new functional products, especially in the food and supplement sector, but also in cosmetics and pharmaceutical products, will facilitate market growth, which in turn will stimulate scientific work.

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Hoffman, Y., C. Aflalo, A. Zarka, J. Gutman, T. Y. James and S. Boussiba. 2008. Isolation and characterization of a novel chytrid species (phylum Blastocladiomycota), parasitic on the green alga Haematococcus. Mycol Res 112: 70–81. Holtin, K., M. Kuehnle, J. Rehbein, P. Schuler, G. Nicholson and K. Albert. 2009. Determination of astaxanthin and astaxanthin esters in the microalgae Haematococcus pluvialis by LC-(APCI) MS and characterization of predominant carotenoid isomers by NMR spectroscopy. Analytical and Bioanalytical Chemistry 395: 1–10. Kamath, B. S., B. M. Srikanta, S. M. Dharmesh, R. Sarada and G. A. Ravishankar. 2008. Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis. European J Pharmacol 590: 387–395. Kang, C. D., S. J. Han, S. P. Choi and S. J. Sim. 2010. Fed-batch culture of astaxanthin-rich Haematococcus pluvialis by exponential nutrient feeding and stepwise light supplementation. Bioprocess and Biosystems Engineering 33: 133–139. Kang, C. D., J. S. Lee, T. H. Park and S. J. Sim. 2005. Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis. Appl Microbiol Biotechnol 68: 237–241. Kang, C. D., J. S. Lee, T. H. Park and S. J. Sim. 2007. Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of Haematococcus pluvialis: C/N ratio and light intensity. Appl Microbiol Biotechnol 74: 987–994. Katsuda, T., A. Lababpour, K. Shimahara and S. Katoh. 2004. Astaxanthin production by Haematococcus pluvialis under illumination with LEDs. Enzyme Microbial Technol 35: 81–86. Katsuda, T., K. Shimahara, H. Shiraishi, K. Yamagami, R. Ranjbar and S. Katoh. 2006. Effect of flashing light from blue light emitting diodes on cell growth and astaxanthin production of Haematococcus pluvialis. J Biosci Bioeng 102: 442–446. Katsuda, T., H. Shiraishi, N. Ishizu, R. Ranjbar and S. Katoh. 2008. Effect of light intensity and frequency of flashing light from blue light emitting diodes on astaxanthin production by Haematococcus pluvialis. J Biosci Bioeng 105: 216–220. Kim, Z., S. Kim, H. Lee and C. Lee. 2006. Enhanced production of astaxanthin by flashing light using Haematococcus pluvalis. Enzyme and Microbial Technology 39: 414–419. Kobayashi, M. 2003. Astaxanthin biosynthesis enhanced by reactive oxygen species in the green alga Haematococcus pluvialis. Biotechnol Bioprocess Eng 8: 322–330. Lee, H., M. Seo, Z. Kim and C. Lee. 2006. Determining the best specific light uptake rates for the lumostatic cultures in bubble column photobioreactors. Enzyme Microbial Technol 39: 447– 452. Lee, R. E. 2008. Phycology. 4th Edition. Cambridge University Press, Cambridge. pp. 560. Li, Y., M. Sommerfeld, F. Chen and Q. Hu. 2008. Consumption of oxygen by astaxanthin biosynthesis: A protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae). J Plant Physiol 165: 1783–1797. Linden, H. 1999. Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation. Biochim Biophys Acta 1446: 203–212. Liu, X. and T. Osawa. 2007. Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer. Biochemical and Biophysical Research Communications 357: 187–193. Lopez, M. C., R. Sanchez Edel, J. L. Lopez, F. G. Fernandez, J. M. Sevilla, J. Rivas, M. G. Guerrero and E. M. Grima. 2006. Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors. J Biotechnol 123: 329–342. Lotan, T. and J. Hirschberg. 1995. Cloning and expression in Escherichia coli of the gene encoding beta-C-4-oxygenase, that converts beta-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. FEBS Lett 364: 125–128.

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Mercke Odeberg, J., A. Lignell, A. Pettersson and P. Hoglund. 2003. Oral bioavailability of the antioxidant astaxanthin in humans is enhanced by incorporation of lipid based formulations. Eur J Pharm Sci 19: 299–304. Miao, F., D. Lu, C. Zhang, J. Zuo, Y. Geng, H. Hu and Y. Li. 2008. The synthesis of astaxanthin esters, independent of the formation of cysts, highly correlates with the synthesis of fatty acids in Haematococcus pluvialis. Sci China Ser C-Life Sci 51: 1094–1100. Mortensen, A. 2009. Supplements. In: (G Britton, S. Liaaen-Jensen and H. Pfander, eds) Carotenoids. Vol.5 Nutrition & Health. Birkhäuser, Basel. pp. 67–82. Olaizola, M. 2000. Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors. J Appl Phycol 12: 499–506. Orosa, M., J. F. Valero, C. Herrero and J. Abalde. 2001. Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N-tarvation and high light conditions. Biotechnol Lett 23: 1079–1085. Park, E. K. and C. G. Lee. 2001. Astaxanthin production by Haematococcus pluvialis under various light intensities and wavelengths. J Microbiol Biotechnol 11: 1024–1030. Pröschold, T. 1997. Gametogenese in Grünalgen. Thesis. University of Göttingen. Ranjbar, R., R. Inoue, H. Shiraishi, T. Katsuda and S. Katoh. 2008. High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor. Biochem Eng J 39: 575–580. Steinbrenner, J. and H. Linden. 2003. Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol Biol 52: 343–356. Tran, D., J. Haven, W. G. Qiu and J. E. W. Polle. 2009. An update on carotenoid biosynthesis in algae: phylogenetic evidence for the existence of two classes of phytoene synthase. Planta 229: 723–729. Wang, B., A. Zarka, A. Trebst and S. Boussiba. 2003. Astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae) as an active photoprotective process under high irridiance. J Phycol 39: 1116. Wollenweber, W. 1908. Untersuchungen über die Algengattung Haematococcus. Ber Deutsch bot Ges 26: 238–298. Yuan, J. P., J. Peng, K. Yin and J. H. Wang. 2011. Potential health-promoting effects of astaxanthin: A high-value carotenoid mostly from microalgae. Molecular nutrition & food research 55: 150–165. Zhekisheva, M., S. Boussiba, I. Khozin-Goldberg, A. Zarka and Z. Cohen. 2002. Accumulation of oleic acid in Haematococcus pluvialis (Chlorophyceae) under nitrogen starvation or high light is correlated with that of astaxanthin esters. J Phycol 38: 325–331.

Christian Walter

9 Screening and development of antiviral compound candidates from phototrophic microorganisms 9.1 Introduction In the pharmaceutical industry, there is an urgent need to identify novel, active compounds as lead substances for effective drug development in many therapeutic areas (Shu 1998). Since there is an increased acquired resistance of different pathogens against existing pharmaceuticals and a demand for treatment for diseases with so far unsatisfactory therapy approaches, the development of innovative, antiinfective drugs and substances with anti-tumour activity is becoming more and more important. Natural products play a crucial role in drug discovery with an inestimable molecular diversity. A recent review depicted that although combinatorial chemistry has now been used for approximately 70 % as a discovery source, e.g. in the area of cancer, 50 % of today’s drugs are either natural compounds or directly derived therefrom, whereas 25 % of new drugs are synthetic in origin (Newman and Cragg 2012). Currently, there is a substantial decrease in the absolute number of novel approved drugs, and the loss of patent protection for important medicines is forthcoming (Kraljevic et al. 2004; Li and Vedera 2009). It is likely temporary that the current industry model for drug discovery does not favour natural products, as the potential for new discoveries in the longer term is enormous (Li and Vedera 2009). The finding of appropriate substances from conventional natural sources as terrestrial microorganisms, here especially fungi and actinomycetes, is declining worldwide (Magarvey et al. 2004; Sato et al. 2009). Thus, there is a need for unconventional and widely untapped resources, such as unusual plants or myxobacteria, and the inestimable biodiversity of the marine habitat can be used as a source of new biomolecules. A total of over 70 % of the Earth’s surface consists of water. The proportion of the oceans to the biosphere is 95 % and only a minor part of the habitat has been explored and specified. As a matter of fact, the aquatic area is one of the last mega-diverse natural sources for drug development. Within this high diversity of the marine habitat, the phototrophic microorganisms, that means microalgae, including cyanobacteria, have a leading part as primary producers of oxygen and are at the bottom of the food chain. In addition to the classical fields of application, such as the production of β-carotene or polyunsaturated fatty acids (PUFAs) as well as in the use as health food or aquaculture feed, there is increasing interest

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in microalgae as a valuable novel source of molecules with pharmaceutical effects (e.g. antimicrobial, anti-tumoral, anti-inflammatory or antiviral). A big advantage of using less well-investigated organisms, such as microalgae, compared to traditional sources, is an expected much smaller rate of reisolation of known compounds with a higher probability of developing new drugs (Olaizola 2003; Magarvey et al. 2004). A number of studies have demonstrated the ability to produce secondary metabolites, unlike those found in any terrestrial species (Shimizu 1993; Borowitzka 1995; Burja et al. 2001). In terms of their novelty in drug development, already a multitude of compounds with biological activity have been isolated from “free” or symbiotic microalgae, indicating that these mainly phototrophic microorganisms represent an extensive source of new natural substances for medicine (Shimizu 1993; Pietra 1997; Borowitzka 1999; Skulberg 2000; Burja et al. 2001; Simmons et al. 2005; Kalaitzis et al. 2009; Nunnery et al. 2010; Prasanna et al. 2010; Waters et al. 2010; Kehr et al. 2011; Singh et al. 2011; Nagarajan et al. 2012). For a more detailed insight, see Chapter 10.

9.2 Supply of natural compounds from microalgae Despite their huge potential, phototrophic microorganisms are strongly under-represented, and only a few of their compounds have been identified so far or are being developed further. The contradiction between the obvious potential on the one hand and the current status quo in producing natural compounds through microalgae on the other is founded by the amount of biological material needed for structure determination and the clinical development of potential drugs. The use of bioactive compounds from microalgae had not been possible previously, mainly due to problems regarding production, isolation and purification of a sufficient amount of drug candidates in a reproducible manner. Within the range of 0.01–0.5 g (substance) per 100 g (dry weight), naturally occurring yields are generally small (Ishide et al. 1997; Murakami et al. 1997; Okino et al. 1997). The demand for active compounds in the drug-development process is within the range of 0.1 g for drug discovery, 5 g for preclinical phase and 50 g for clinical phases and approval. With regard to drug development, a sustainable and reliable supply of natural compounds as pharmaceutical candidates has to be ensured (Rossi et al. 1997; Zaborsky 1999; Bull et al. 2000; Faulkner 2000; Proksch et al. 2003). Generally, there is a considerable time pressure in many screening investigations. Screening programmes are subjected to cyclic fluctuations and financially scheduled for a certain time. If there is no developable output within a given time frame, even promising compounds will not be pursued further. The reasons are that in the given time and due to unavailable supply, no information about the active compound structure and activity or mode of action can be obtained.

9.3 Sterilizable photobioreactors

147

In general, different sources are conceivable to provide compounds of interest: (1) total synthesis, (2) hemisynthesis, (3) heterologous expression of microalgal gene clusters and fermentation of standard hosts, (4) mass-cultivation methods for microalgae and (5) recollection from nature. Natural stocks are quite limited and are not a viable option for the manufacture of high-value chemicals (Rossi et al. 1997; Burja et al. 2002). In a few cases, especially among small organic molecules, demand can be met by total synthesis of the active metabolite. In many cases of complex compounds, however, this is not a viable option, as synthesis may involve many steps, may be expensive and may produce low yields due to poor selectivity. Great challenges with respect to total synthesis and heterologous expression could be anticipated for complex chemicals from microalgae (see Chapter 10). The supply problem clearly shows the need for mass-cultivation methods for microalgae. The application of cultivation technology and intensification of bioprocesses will become an important strategy to produce complex chemicals that cannot be synthesized chemically or produced by heterologous expression, or to create backbones for hemisynthesis and derivatization from readily available and natural starting material. Furthermore, microalgae should be examined more closely for their ability to provide novel chemical diversity for drug discovery with the ability to manipulate the yields and diversity of metabolites through changes in culture conditions (Harvey 2000).

9.3 Sterilizable photobioreactors A significant advantage of microalgae is its inherent cultivability, for example compared to sponges and other marine invertebrates, which makes its sustainable use for compound screening and production possible. To date, only a very small number of microalgal species have been cultured heterotrophically in conventional bioreactors (Bumbak et al. 2011). Heterotrophic growth in the dark supported by a carbon source replacing the traditional support of light energy is intended to be a specific niche of microalgae cultivation (Perez-Garcia et al. 2011). Additionally, light is the inducer and regulator for the synthesis of some algal metabolites (Lee 2004). Consequently, the most important impediment to the commercial use of algal cultures for production of high-value substances is the availability of suitable photobioreactors (PBRs). Partially, microalgae cultures only show a minor cell density because of the required light supply. As the algae cells naturally feature a very effective light absorption, the total irradiation will be completely absorbed within a couple of millimeters, even if relatively low cell densities are used. Hence, the amount of extractable agents is often very small, with the consequence that a great amount of biomass has to be provided for the use in screens. Cultivation technologies and conditions are important factors for the success of screening. In many cases, substances of interest are secondary metabolites, whose production depends on the environmental factors in a very sensitive way (Skulberg

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9 Screening and development of antiviral compound candidates

2000; Burja et al. 2001; Caicedo et al. 2011). To increase product yields and to ensure consistent product quality, strategies to characterise, optimize and scaleup phototrophic processes are key requirements. The prerequisite for the total exploitation of microalgae is contamination control and, thus, the monoseptic cultivation under defined and therefore reproducible conditions. Most production strategies are carried out at the shake-flask level, offering poor prospects for successful scale-up (Marwick et al. 1999). To date, standardized industrial photobioreactors are not available for monoseptic algae cultivation. The technical difficulty in sterilizing photobioreactors has hindered their application for the production of high-value pharmaceutical products (Lee 2001). The need for controlled conditions, analysis and monitoring of phototrophic growth parameters in screening and scale-up has led to the development of appropriate PBRs. In principle, sterility cannot be achieved in open systems and is often limited in closed reactors due to their construction size and material. Only a few reactors that allow sterile cultivation conditions for microalgae have been described in the literature (Jüttner 1982; Pohl et al. 1988, 1992; Pulz et al. 1995; Gerbsch 1997). Thermal sterilizable stirred-tank reactors for heterotrophic microorganisms are the state of the art of cultivation technology. Therefore, internally illuminated PBRs based on conventional steel vessels with integrated sterilization equipment were developed, where the irradiation is supplied by artificial light sources. The basic reactor concept according to Pohl is the insertion of fluorescent lamps into the fermentation vessel (Pohl et al. 1988, 1992). Another type is a system based on light-diffusing optical fibres (Pulz et al. 1995; Gerbsch 1997). The fibres are arranged at regular intervals of about 4–5 mm throughout the reaction volume. At their upper end, they are bundled and positioned below the observation window (Gerbsch 1997). Light is coupled into the fibres by an external light source such as a xenon flash light or sulfur plasma lamp. As light-diffusing optical fibres are used, the light coupled into the fibres is diffused through the outer surface of the fibres, thereby transmitting the light into the medium. Mori and coworkers also described sunlight-supplied optical fibres (Mori et al. 1987). The performance of these reactors is limited by the small illuminated surfaceto-volume ratio, in the case of the Pohl reactor, and by the light input or light coupling into the fibres in the case of the fibre-optic reactors. Further problems are heat exchange, mixing and the limited scale-up compatibility. Early attempts of sterilizable tubular glass reactors were described by Jüttner (1982). The reactor by Jüttner is a meandering tube reactor with an inner diameter of 40 mm, a length of 79 m and external gas exchange. Fluid velocities within the system were between 12 and 35 m s–1 to prevent sedimentation of biomass in the system. Light was supplied using fluorescent lamps. During cultivations with varying organisms, foam formation was observed, i.e. due to CO2 depletion, and growth on the glass tubes, which could not be overcome. Both effects were caused by the long tube distances and high fluid velocities (Walter et al. 2003).

9.3 Sterilizable photobioreactors

149

Fig. 9.1: Photographic representation of photobioreactor screening modules (version with fluorescent lamps) in operating state (a) and w/o measurement and control devices (b) and Medusa photobioreactor on a 25-litre scale (c).

In order to meet the requirements for a photobioreactor that overcomes the limitations described above, a tubular airlift system of the type “Medusa” following a special simplistic design was constructed (see Figure 9.1), which is suitable for effective thermal in situ sterilization and can be scaled up (Walter and Buchholz 1998; Buchholz et al. 1999; Walter et al. 2003). Thanks to the development of this thermal sterilizable PBR (sPBR), which in principle fulfils Good Manufacturing Practice standards, the high sterile-technical requirements for the development of pharmacologically relevant agents can be achieved (Walter et al. 2003; Rechter et al. 2006). Another requirement for the development and validation of bioprocesses that ensure specific metabolite productivity and that increase yields are small-scale PBRs that provide scalable results. In addition, such systems could improve microalgal exploration and cultivation of new species for their screening, as a large

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number of experimental conditions could be realized in parallel. Simultaneous bioprocessing presupposes the operation of a multitude of bioreactors connected in parallel to study and optimize bioprocesses. Since a proper description of physiological conditions of the phototrophic cultures is required, it is crucial to obtain the relevant measurand. Therefore, photobioreactor screening modules were implemented that mimic a volume element of the Medusa photobioreactor and allow the operation of a multitude of parallel runs (Fig. 9.1). This simple system consists of glass cylinders in 1 litre-scale which are operated as bubble columns and can be sterilized in an autoclave. Light energy can be supplied by fluorescent lamps, light-emitting diodes (LEDs) or even the sun if desired. Additionally, the online measurement of the following parameters is realized: light adsorption via adjustable light control, optical density, chlorophyll fluorescence, pH, partial pressure of O2 as well as O2/CO2 concentration in exhaust gas, and temperature. Furthermore, small-scale systems using high-power LED light sources that illuminate flat-cuvette PBRs and the application of a scale-down approach to represent one volume element of a larger production reactor have been described (Nedbal et al. 2008; Jacobi et al. 2012). Within the scale-down approach, a conventional glass cylinder bioreactor equipped with LED illumination allows for operation at ideal, defined and highly controllable conditions (Jacobi et al. 2012). In a study, conducted at the Institute of Bioprocess Engineering, FriedrichAlexander-Universität Erlangen-Nürnberg, Germany, antiviral compounds in a were screened in a smart screening approach using parallel bioprocessing with screening modules and by scaling the processes to thermally sterilizable Medusa photobioreactors (see Section 9.5).

9.4 Antiviral agents from microalgae Recently, interest in identifying naturally occurring antiviral molecules has increased greatly (Jung et al. 2000; Yang et al. 2001; Beutler et al. 2002; Lee et al. 2004; Abad Martinez et al. 2008). Algae-derived compounds exhibit interesting broad-spectrum antiviral properties with novel modes of action and are thereby a novel source of antiviral drug candidates. An antiviral activity of cell extracts of cyanobacteria, micro- and macroalgae was especially demonstrated towards human immunodeficiency virus type 1 (HIV-1) as well as herpes simplex virus types 1 and 2 (HSV-1, HSV-2) (Schaeffer and Krylov 2000; Serkedjieva 2000). Single studies also document an activity against the human cytomegalovirus (HCMV) (Hayashi et al. 1996a). Gustafson et al. (1989) described for the first time the activity of sulfonic acid-containing glycolipids from the two micro-cultured species Lyngbya lagerheimii and Phormidium tenue against human immunodeficiency virus type 1 (HIV-1). Loya et al. (1998) determined that the sulfonic acid-group and the fatty acid ester of glycolipids exhibit a substantial

9.4 Antiviral agents from microalgae

151

degree of inhibitory effects. The sulfoglycolipids (SGLs) are structural components of thylakoid membranes and thus associated with the photosystem of photosynthetic organisms (Pugh et al. 1995). Several SGLs such as sulfoquinovosyldiacylglycerols, each presenting anti-HCMV activity in a similar range, were discovered in Porphyridium purpureum and further microalgae (Naumann et al. 2007). Cyanovirin-N (CV-N), a novel anti-HIV protein that contains four cysteins that form two intrachain disulfide bonds was isolated from an aqueous extract of cultured cyanobacterium Nostoc ellipsosporum using bioassay-guided discovery (Gustafson et al. 1997). The protein was produced by cultivation of N. ellipsosporum and recombinant by expression of a corresponding DNA sequence in Escherichia coli to further disclose the anti-HIV activity profile (Boyd et al. 1997). Obligate CVN oligomers were created that exhibited an improvement in activity and broad cross-strain HIV neutralization (Keeffe et al. 2011). In different screenings of cyanobacteria strains, about 2–10 % of the extracts tested for inhibition of HIV-1, HSV-2 or respiratory syncytial virus exhibited antiviral activity (Borowitzka 1999). Beside sulfonic acid-containing glycolipids, peptides are a promising biochemically active group of compounds. Chemical investigations to date indicate that cyclic peptides and depsipeptides are common constituents of cyanobacteria (Moore 1996). In a screening programme at Hawaii, extracts of more than 1500 strains, representing some 400 species of cyanobacteria, were tested using cell-based assays. Approximately 10 % of the cultures produced substances that caused a significant reduction in cytopathic effects normally associated with viral infection (Patterson et al. 1994). Furthermore, the natural products of many marine cyanobacteria contain lipopeptides – amino-acid-derived fragments linked to a fatty-acid-derived portion (Burja et al. 2001). For example, analyses of 113 compounds of Lyngbya majuscula isolated to date show that 58 % of the compounds are amino-acid-derived lipopeptides (cyclic or linear). Lipopeptides are an extremely biochemically active group of compounds with approximately 4 % being antiviral active. Moreover, highmolecular-weight compounds with antiviral effects, particularly isolated from cyanobacteria, have been reported: Indolocarbazoles from Nostoc spaericum (Knübel et al. 1990), Ambiguol A from Fischerella ambigua (Falch et al. 1993) and β-Carbolin from Dichothrix baueriana (Larsen et al. 1994). Additionally, sulfated polysaccharides (sPS) from algae were shown to inhibit a variety of human pathogen enveloped viruses, including HSV-1 and HIV-1. The clinical relevance of sPS from micro- and macroalgae as antiviral agents against human pathogen viruses has been well known for many decades (Gerber et al. 1958; Damonte et al. 1996; Witvrouw and De Clercq 1997). A review of antiviral polysaccharides is given by Damonte et al. (2004). The antiviral efficiency is correlated with the molar mass, conformation, charge frequency or charge spreading (Witvrouw and De Clercq 1997; Luscher-Mattli 2000). The generally accepted mechanism of action of negatively charged sulfated polysaccharide-molecules towards HIV and enveloped viruses is an inhibition of

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9 Screening and development of antiviral compound candidates

virus-to-cell-interaction by interacting with positively charged viral membrane glycoproteins (Geresh and Arad 1991; Witvrouw and De Clercq 1997; Damonte et al. 2004). The selectivity index (SI), defined as the relationship between the cytotoxic concentration required to reduce cell viability by 50 % (CC50) and the effective antiviral inhibitory concentration required to reduce susceptibility of cells with virus by 50 % (IC50), is especially high. The documented SI values in the order of 103–104 make sPS very promising antiviral compound candidates (Damonte et al. 2004). In contrast to clinically used antiviral drugs, very slow and poor induction of virus-drug resistance due to sPS treatment has been reported so far (Flexner et al. 1991; Damonte et al. 2004). To date, the clinical application of polyanionic polysaccharides as antiviral agents for systemic virus infections is hindered by their poor absorption from the gut (Naesens et al. 2006). It was reported that sPS isolated from seaweeds are poorly absorbed in vivo with a bioavailability of less than 1 % when given orally (Schaeffer and Krylov 2000, Damonte et al. 2004). This obstacle is mainly due to the polymeric structure and high molecular weight of macroalgae-derived PS. The molecular weight is not an absolute factor for antiviral activity but must be connected with degree and functional distribution of sulfation. Lower-molecular-weight algal sulfated polysaccharides can be prepared by chemical or enzymatic means to obtain oligosaccharides with more diverse bioactivities, whereas chemical degradation methods are easy to perform but lack specificity (Jiao et al. 2011). Dextran sulfate (DS) is used as reference sPS in the plurality of corresponding studies. The intravenous infusion of DS resulted in a significant peak plasma concentration compared to orally administered DS, which was poorly absorbed (Schaeffer and Krylov 2000). The sulfated polymer Carragelose© derived from red seaweed has been approved for marketing in the EU as part of an antiviral nasal spray for treating respiratory diseases (Marinomed 2012). Overall, sulfated polysaccharides have numerous advantages over other classes of antiviral drugs. In addition to the stated characteristics of low cytotoxicity and low induction of viral drug resistance, these are among others: a broad spectrum of antiviral properties, safety, wide acceptability, and novel modes of action (Ghosh et al. 2009). Nevertheless, regarding the potential of sPS as drug candidates, several problematic aspects have to be considered. The production of a standardized commercial product based on algal sulfated polysaccharide constituents will be a challenge (Jiao et al. 2011). Problems may arise from reproducibility of isolation and purification of sufficient amounts of pure bioactive agents (Wagner and Kraus 2000). One major issue associated with complex sPS from macroalgae is the higher size and seasonal variation in their chemical composition (Morya et al. 2012). However, a clear advantage is provided by the fact that PS do not have to be isolated from macroalgae or plants but can be produced by microalgae using the optimized technology of thermally sterilizable photobioreactors (Rechter et al. 2006).

9.5 Antiviral screening

153

9.5 Antiviral screening The antiviral investigations, which are described in the following subsections, were performed in an interdisciplinary approach and conducted at the Institute of Bioprocess Engineering, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany in cooperation with the Institute for Clinical and Molecular Virology, FriedrichAlexander-Universität Erlangen-Nürnberg, and Medical Clinic II, Charité, University Medicine Berlin, Germany.

9.5.1 Primary target of screening The primary targets of the antiviral screening were human pathogenic beta herpesviruses. These viruses are ubiquitously spread and characterized by a high percentage of latently infected persons (60–90 % in adults). After an initial infection, viruses persist in the body, normally without illness symptoms, but in immunocompromised groups, such as transplantation and AIDS patients, these viruses present life-threatening risks. Thereby, the infection and/or reactivation of human cytomegalovirus (HCMV) in risk groups is associated with significant morbidity and mortality (Biron 2006). The interstitial HCMV pneumonia is the most frequent cause of death for AIDS and bone-marrow-transplantation patients. Furthermore, acute HCMV infections can cause gastroenteritis, inflammation of the retina or encephalitis in AIDS and immunocompromised patients (Nguyen et al. 2001, Modrow et al. 2003, Perssons et al. 2003). Human herpesvirus-6 (HHV-6) has been associated with a wide spectrum of neurological syndromes, including: multiple sclerosis, chronic fatigue syndrome and mesial temporal-lobe epilepsy (Naesens et al. 2006). The reactivation of HCMV can be enhanced by HHV-6 and human herpesvirus-7 (HHV-7) (Mendez et al. 2001). While for some of the herpesviruses, there are still no specific therapeutics, drug resistance and undesirable side-effects are the major problems in conventional therapy. Therefore, there remains an urgent need for innovative broad-spectrum antiherpetic drugs that combine high activity and safety with a novel mode of action (Naesens et al. 2006).

9.5.2 Smart screening approach Algae lack an immune system and hence have evolved mechanical, genetic and chemical defences against infection. The mechanisms of chemical defences of microalgae against viral infection are poorly studied, although it is conceivable that sPS, SGL and toxins of cyanobacteria have antiviral effects. Research on extracellular polysaccharides (ePS) possessing anti-HCMV activity indicates an inhibition of virus adsorption and/or penetration. An understanding of the biological function of ePS and other cell constituents can be used to clarify the efficacy of

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9 Screening and development of antiviral compound candidates

these substances as inhibitors of human pathogen viruses. The close degree of relationship of algae viruses (Phycodnaviridae) and herpes viruses (Herpesviridae) is of special importance. This relationship was built up by phylogenetic classification of DNA polymerase genes (Chen and Suttle 1996). Moreover, cellular ePS and surface layer proteins are potential factors blocking attachment and may be a barrier against virus/phage infection. In contrast, it could be that ePS is rather a recognition and attachment site that promotes viral infection (Weinbauer 2004). The strategy of the smart screening approach is a pre-selection of microalgae, producing compounds with known antiviral activities, or a pre-selection of compounds belonging to antiviral compound groups. Those selected compounds are for example sulfated compounds (polysaccharides, lipids). The objective of this “intelligent screening” is to obtain a higher hit rate compared to the high-throughput approach and to identify variants with improved performance. The following descriptions focus on sPS as an example.

9.5.3 Basic process sequence First, a process sequence was established with the model organism Porphyridium purpureum. The scheme of the process includes the selection of axenic strains, cultivation and scale-up, downstream processing and antiviral screening. In the case of a positive hit, product purification, determination of mode of action and structure characterization followed. It is well known that the unicellular red microalga P. purpureum secretes sPS with antiviral effects into the surrounding culture medium. Thereby, the antiviral potential of the sPS towards influenza A virus (Minkova et al. 1996), HSV-1 and HSV-2, varicella zoster virus (VZV) (Huleihel et al. 2001) as well as against a murine leukemia virus (MLV) (Talyshinsky et al. 2002) has been described. Furthermore, the in vivo inhibition of HSV-1 and HSV-2 in rats and rabbits for Porphyridiumpolysaccharides was determined (Huheihel et al. 2002). In order to verify the reproducibility and scalability in the sPBR, the growth and formation of extracelluar polysaccharides (ePS) were analysed under quantitative and qualitative aspects. Optimization of culture parameters was performed in 1-litre photobioreactor screening modules and successfully transferred to the 25-litre scale of the Medusa-type airlift reactor (Fig. 9.2). Further optimization by light adjustment during growth and scale-up of the P. purpureum culture to 100 litres led to final biomass concentrations in the range of 15 g (cell dry weight) l–1 and to ePS concentrations of 3 g l–1 at a minimum. In general, given the reliability and reproducibility of the cultivation, processes were verified with intracellular products such as SGL from P. purpureum and other microalgae as well as for the secondary metabolite Cryptophycin produced by Nostoc (unpublished data). The ePS were isolated from culture supernatant by ultrafiltration plus chromatography and analysed for its biological activity. According to the results of specific

9.5 Antiviral screening

155

Fig. 9.2: Reproducibility (a) and scale-up (b) of microalgal growth and product formation using the example of Porphyridium purpureum and extracellular polysaccharides ePS (adapted from König 2007).

in vitro assays, ePS gathered from P. purpureum exhibit strong inhibition of the herpesviral replication against HCMV and HHV-6A in the absence of cytotoxic effects (König et al. 2006, Rechter et al. 2006, Thulke 2007). The ePS revealed an inhibitory potential of IC50 = 50 μg ml–1 on HCMV replication and IC50 < 75 mg ml–1 towards HHV-6A. Additionally a pronounced in vitro (IC50 = 1.24 μg ml–1) and in ovo activity towards vaccinia virus (VACV), which belongs to the orthopoxviruses,

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9 Screening and development of antiviral compound candidates

was detected (Radonić et al. 2010). VACV is closely related to the variola virus, the causative agent of smallpox. Within the screening programme, the established methods of cultivation and downstream processing were transferred to further phototrophic microorganisms and augmented by further human pathogen viruses. Thereby, a novel extracellular product of the cyanobacterium Arthrospira platensis displayed the most pronounced activity against a number of human viruses (see Section 9.5.5).

9.5.4 Antiviral activity and immunostimulating effects of Arthrospira platensis In Mexico and many African countries, Arthrospira (formerly Spirulina) is traditionally used as human food. It has gained considerable popularity in the human health food industry, and in many countries of Asia it is used as protein supplement and as a human health food (Pulz and Gross 2004; Habib et al. 2008; see also Chapters 3 and 6). Epidemiological studies suggest that regular consumption of Arthrospira from Lake Chad in Africa might increase the viral dose requirements for HIV infection and might reduce viral replication once infection has taken place (Teas et al. 2004). Arthrospira given as food were shown in animal tests to increase phagocytic activity, increase antibody production, increase accumulation of NK cells into tissue, and mobilize T and B cells into the blood. Arthrospira thus appear to target two different steps, in the viral replication cycle, plus immunostimulating agents, which may support synergistically the antiviral effects (Luscher-Mattli 2003). A clinical study of orally administered Arthrospira to healthy men for three months resulted in an enhanced immune response, including an increased interferon production (Hirahashi et al. 2002). Results of phase I/II trials suggest that Undaria (brown seaweed), Arthrospira and a combination of both were non-toxic. The studies show some evidence that consumption of the algae might have decreased the HIV viral load (Teas and Irhimeh 2012). An aqueous extract of A. platensis biomass has antiviral activities towards HIV-1, HCMV, HSV-1, influenza virus, measles virus and mumps virus (Hayashi et al. 1996a, 1996b, Luscher-Mattli 2003, Khan et al. 2005). An in vitro study of human peripheral blood cells indicated that the aqueous extract of Arthrospira almost completely inhibited HIV-1 absorption and penetration at a concentration of 40 μg ml–1, whereas no cytotoxicity to infected cells was observed (Ayehunie et al. 1998). The activities could be attributed to the sPS calcium spirulan (Ca-SP) (Hayashi et al. 1996a). Ca-SP has a molecular weight of approximately 75 kDa and consists of two types of disaccharide repeating units, O-hexuronosyl-rhamnose and O-rhamnosyl-3-O-methylrhamnose as well as trace amounts of a variety of other saccharidic constituents, i.e. xylose, glucuronic acid and galacturonic acid (Lee et al. 1998, 2000). Even at low concentrations of Ca-SP, no enhancement of HIV-1induced syncytium formation was observed (Hayashi et al. 1996b). Moreover, unde-

9.5 Antiviral screening

157

sirable side-effects of conventional sPS may be overcome by Ca-SP (Hayashi et al. 1996a). Aside from the antiviral effects, sPS, like DS, exhibit several adverse reactions such as anticoagulant activity and heavy, but reversible, thrombocytopenia when applied intravenously (Flexner et al. 1991). These effects depend on compound structure. Studies on structure–activity relationships of sulfated polysaccharides with high antiviral activity demonstrated that the antiviral activity of the sPS varies both quantitatively and qualitatively in relation to their detailed structural features (Ghosh et al. 2009). It should be mentioned that Ca-SP showed a very low anticoagulant activity. Furthermore, as the stability of the substances in the plasma is a crucial point, it should be emphasized that the half-life of Ca-SP in murine blood is much longer than that of other sPS such as DS (Hayashi et al. 1996b). However, in the literature, many indirect modes of measuring antiviral activity of Ca-SP have been performed (Rechter et al. 2006). Beside the choice of strains, the strongest impetus for the development of new natural products is the advancement in modes of measuring antiviral activity and development in bioassay technology (McChesney et al. 2007). In the following study, the characterization of improved spirulan-like compounds with better performance using direct, virusspecific approaches to identify broad-spectrum antiviral activity was executed.

9.5.5 Characterization of novel antiviral spirulan-like compounds In the study, intracellular and extracellular fractions extracted from monoseptic batch cultivations were investigated. To obtain the intracellular polysaccharides as a reference, dry biomass was extracted with water according to Lee et al. (1998). Extracellular fraction TK-V3 was isolated according to König (2007). The supernatant fraction of A. platensis that contains polysaccharide and proteins was purified by bioassay-guided fractionation, analysed for its broad-spectrum antiviral activity and chemically characterized by a gas chromatography– flame ionization detector (GC-FID), gas chromatography–mass spectrometry (GC-MS), elementary analysis and gel-permeation chromatography (GPC) (König 2007). The product TK-V3 was isolated from different cultivations related to the variation in pH and in the harvesting point. Furthermore, TK-V3 was attained from a scale-up approach from the 1-litre to 25-litre scale and subjected to antiviral screening. The active compound in the purified supernatant has been identified to be an anionic exopolysaccharide containing the monomers rhamnose, 3-O-methylrhamnose, xylose, 3-O-methylxylose and 2,3-O-di-methylxylose. The GPC analysis indicated a molecular weight of about 1100 kDa. TK-V3 contains 4.4 % sulfur, most probably as sulfate. Therefore, the identified extracellular compound can be distinguished from known intracellular sulfated polysaccharide Ca-SP particularly because of the absence of uronic acids as well as a somewhat different monomer

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9 Screening and development of antiviral compound candidates

Fig. 9.3: Antiviral activity of extracellular components from P. purpureum (extracellular polysaccharides, ePS) and A. platensis (TK-V3) or reference drug ganciclovir (GCV) against human cytomegalovirus (HCMV) (a) and cytotoxicity in MRC-5 fibroblasts (b) (adapted from Thulke 2007).

composition and a different molecular weight. The cultivation process accompanying analyses demonstrated that TK-V3 is produced and actively secreted into the surrounding medium during the entire cultivation phase up to the end of growth and already can be found in the exponential growth phase (König 2007). This opens up opportunities for beneficial bioprocess strategies. As cost-of-goods is a crucial point, the instance of a simplified downstream processing of an extracellular product is of special importance. Furthermore, routes for continuous or intermittent growth decoupled operation to increase the light conversion efficiency with respect to the bioactive compound can be envisaged.

9.5 Antiviral screening

Arthrospira platensis Intracellular Extracellular Reference drug Ganciclovir

CC50 (μg ml–1)

IC50 (μg ml–1)

Aqueous extract

>5000

39

TK-V3

>5000

4

6000 ± 700

14

+21 –20 + 1.6 – 2 + 5 – 5

159 SI (–)

>128 >1250

430

Abbreviations: CC50: 50 % cytotoxic concentration; IC50: 50 % viral inhibition concentration ± error intervals; SI: selectivity index (SI = CC50/IC50); >: larger concentrations were not tested. Tab. 9.1: Antiviral properties of Arthrospira-derived compounds and reference drug Ganciclovir against HCMV and cytotoxicity in MRC-5 host cells.

Using virus-specific modes of measurement, the novel polysaccharide exhibited eminent in vitro inhibition of human cytomegalovirus (HCMV) and human herpesvirus type 6A (HHV-6A) in the absence of cytotoxic effects (König et al. 2006, Rechter et al. 2006, Thulke 2007). The in vitro activity of the extracellular product against HCMV is even higher than the activity of the therapeutic agent ganciclovir GCV (Fig. 9.3). In comparison, the antiviral activity of the intracellular product from A. platensis is demonstrated, with an inhibitory potential towards HCMV in the same range as ePS from P. purpureum. The substance TK-V3 reduces the susceptibility of human cells (MRC-5 fibroblasts) with HCMV in vitro by 50 % at a concentration of about 4 μg ml–1, and by 90 % at 8.6 μg ml–1, regardless of cultivation variations. The exopolysaccharide is characterized by a complete inhibition of HCMV infection at a concentration not exceeding 20 μg ml–1. In particular, the intracellular fraction reveals one order of magnitude less inhibitory potential against HCMV (IC50 39 μg ml–1) (Tab. 9.1). The mutagenic potential of TK-V3 was assessed using the Ames test, whereas no carcinogenic potential could be detected up to 160 μg ml–1. TK-V3 was additionally studied for its activity against further human pathogen viruses (Walter et al. 2007). As summarized in Table 9.2, in addition to HCMV, TKV3 has been shown to inhibit HHV-6A, HSV-1 and HIV-1 but also VACV, Ebola Zairevirus and SARS Coronavirus (SARS-CoV). All viruses belong to the group of enveloped viruses. Nevertheless, TK-V3 is less or active non-active towards other enveloped viruses such as the Epstein–Barr virus (EBV/HHV-4), which belongs to herpes viruses, and the influenza A virus (Rechter et al. 2006). In order to gain an insight into the mode of action of TK-V3, different approaches were executed. From in vitro infection kinetics, the investigated anionic polymers were shown to reduce DNA- and RNA replication of HCMV and HHV-6A (Thulke 2007). Compared to antiherpal therapeutical agents such as GCV, Aciclovir, Foscarnet and Cidofovir, TK-V3 acts by inhibiting the adsorption of viral particles

5000 Not cytotoxic

CC50 (μg ml–1)

>640

>112

>1250

SI (–)

References

In ovo activity with pronounced decrease in viral load within the CAM Activity comparable to VACV

100 % inhibition at 10–15 μg ml–1

100 % inhibition at 245,000, DNP 2011). Cyanobacterial genomes are extraordinarily large, comprising almost 10 million base pairs in the case of Nostoc punctiforme PCC 73102. A large proportion of the genome seems to be dedicated to genes encoding the biosynthetic machinery for secondary metabolites (Meeks et al. 2001; Kalaitzis et al. 2009). Given that cyanobacteria can be expected to have a biosynthetic capacity at least equal to that of other microorganisms such as myxobacteria (Cragg et al. 2009), the potential of cyanobacteria for future drug-discovery programs becomes clear. Indeed, the rate of rediscovery of already-known compounds when working with cyanobacteria is significantly lower than for other, better-studied organisms (Olaizola 2003; Guyot et al. 2004). The relative disregard for cyanobacteria in natural product research in the past, paired with their high chemical diversity, makes them an attractive source of novel secondary metabolites today. Cyanobacterial secondary products exhibit a high chemical diversity (Guyot et al. 2004). Even though compounds from many chemical classes can be found,

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peptide and polyketide structural elements are predominant among cyanobacterial metabolites (Burja et al. 2001; Welker and von Döhren 2006; Tidgewell et al. 2010). The peptides comprise cyclic, branched and linear structures, depsipeptides, lipopeptides and peptides with uncommon modifications such as N- and O-methylation, sulfation, halogenation, glycosidation, oxidation, dehydration, heterocyclization, prenylation, ketide extensions and others (Jones et al. 2010; Tidgewell et al. 2010). Often, these compounds are synthesized via combined polyketide synthases and non-ribosomal peptide synthetases (PKS/NRPS) (Dittmann et al. 2001; Schwarzer et al. 2003; Challis and Naismith 2004; Finking and Marahiel 2004; Barriosllerena et al. 2007; Jones et al. 2009; Kalaitzis et al. 2009; Marahiel 2009; Rath et al. 2010; Strieker et al. 2010). Although often not recognized at first glance, ribosomally synthesized products play an important role among bioactive metabolites from cyanobacteria as well (Ziemert et al. 2008; Donia and Schmidt 2010; Sivonen et al. 2010). Peptidic structures, especially cyclic peptides, have been postulated as “privileged structures” for bioactivities, because they have a high probability of being able to mimic peptidic substrates or ligands of endogenous proteins such as enzymes or receptors (Hershberger et al. 2007; Driggers et al. 2008). The two morphological sections Oscillatoriales and Nostocales make extensive use of PKS/NRPS for natural product synthesis (Gerwick et al. 2008; Tidgewell et al. 2010), and Lyngbya majuscula especially is known for a very diverse product spectrum from both a chemical and bioactivity point of view – more than 25 % of all secondary products known from cyanobacteria have been isolated from this species (Burja et al. 2001; Shimizu 2003; Tan 2007; Liu and Rein 2010; Tidgewell et al. 2010). Often, several structural variants of one parent compound are found within one strain or related strains, e.g. the compound family of the microcystins comprises more than 80 natural variants, about 20 natural microginins and about 90 variants of the aeruginopeptin/micropeptin/cyanopeptolin/ oscillapeptin/planktopeptin family are known (DNP 2011). This facilitates both the identification of structure–activity relationships at early research stages and the semi-synthetic modification of possible lead structures. The high natural variety within the compound families is due to the variability and flexibility of the various enzymes contained in the aforementioned PKS/NRPS modules as well as transposition and recombination events of biosynthesis genes (“natural combinatorial biosynthesis”) (Kalaitzis et al. 2009; Kehr et al. 2011). The already high diversity of these polyketide/peptides can be enhanced even more by biocombinatorial techniques (Sielaff et al. 2006; Zhang and Tang 2008).

10.1.2.1 Proteinase inhibitors Proteolytic enzymes such as trypsin, plasmin, thrombin, elastase, chymotrypsin and papain control many biological processes and are thus also possibly important drug targets. Proteinase inhibiting activities of cyanobacterial secondary products

10.1 Introduction

Compound a

A90720A

Aeruginosin 102-Ab Anabaenopeptintype compounds Circinamideb Cyanopeptolin 1020a

Lyngbyastatinsa Micropeptin Aa Micropeptin T20a Microginin T1b

175

Source

Enzyme

IC50

Reference

Microchaete loktakensis Microcystis aeruginosa Oscillatoria sp. Anabaena circinalis Microcystis sp.

Trypsin

10 nM

Thrombin

55 nM

Bonjouklian et al. (1996) Matsuda et al. (1996)

Carboxypeptidase U/TAFI

0.2 μM

Bjorquist et al. (2008)

Papain

1 μM

Shin et al. (1997)

Trypsin Factor XIa Kallikrein Plasmin Elastase Plasmin

0.7 nM 4 nM 5 nM 0.5 μM 35 18–57 50–77 15–23

Banarjee et al. (2002) Illman et al. (2000) Chisti (2007) Chisti (2007) Chisti (2007) Chisti (2007) Mata et al. (2010) Chisti (2007) Chisti (2007) Khozin-Goldberg and Cohen (2006) Chisti (2007) Chisti (2007) Khozin-Goldberg and Cohen (2006) Chisti (2007) Khozin-Goldberg et al. (2002) Mata et al. (2010) Chisti (2007) Chisti (2007)

Table 11.1: Lipid contents of several microalgae.

To make microalgae really interesting as a source of biofuels, the cost price for production needs to be reduced, and the scale of production needs to be increased significantly. Technically, this is feasible, but considerable effort is needed to achieve this, because the development of a commercial process will take at least 10 years (Barcley 2009; Wijffels and Barbosa 2010). The next question is whether it is economically feasible to produce biodiesel alone from microalgae if it were possible to reduce the cost price of biomass production to € 0.68/kg (Norsker et al. 2011). Recent studies show that it will not be feasible to produce algae for the production of biodiesel alone (Wijffels and Barbosa 2010; Wijffels et al. 2010), so to refine algal biomass into different products, the total value of the biomass needs to be used (Carioca et al. 2009; Wijffels et al. 2010). The final conclusion that came out of these calculations was that an integrated biorefining concept for microalgae with biodiesel as one of the products as well as proteins and carbohydrates as high valuable and bulk products can lead to a feasible process (Subhadra 2010; Wijffels and Barbosa 2010; Wijffels et al. 2010). Both for proteins/carbohydrates and for biodiesel, the markets are huge, and depending on the composition of the microalgae (Tab. 11.2), proteins can be the main product and biodiesel the by-product, or vice versa. Table 11.2 shows several typical examples of microalgae with the composition of the different compounds; some are rich in lipids, whereas others contain more protein or carbohydrates depending on the microalgae strain. This variation in main compounds could be a very good opportunity to develop biorefining concepts for microalgae yielding different components depending on the strain. It would

11.2 Structural biorefining approach of microalgae

205

Species

Protein (%)

Carbohydrates (%)

Lipids (%)

Nucleic acid (%)

Scenedesmus obliquus Scenedesmus dimorphus Chlorella vulgaris Spirogyra sp. Dunaliella salina Euglena gracilis Prymnesium parvum Porphyridium cruentum Spirulina maxima

50–60 8–18 51–58 6–20 57 39–61 28–45 28–39 60–71

10–17 21–52 12–17 33–64 32 14–18 25–33 40–57 13–16

12–14 16–40 14–22 11–21 6 14–20 22–38 9–14 6–7

3–6 – – – – – 1–2 – 3–4.5

Table 11.2: Typical chemical compositions of microalgae strains (Carioca et al. 2009).

therefore be very interesting to develop a biorefinery plant and fractionate the different products, and depending on the market potential, a strain could be selected to yield more lipids or more proteins, or, eventually, large amounts of carbohydrates. In the following sections, we present a feasible microalgae biorefining continuous production concept integrating cell disruption, extraction and fractionation for the generation of the different components as listed in Table 11.2.

11.2 Structural biorefining approach of microalgae 11.2.1 Approach Unique biorefining concepts for the extraction of lipids, proteins and carbohydrates from complex mixtures are becoming the bottleneck for efficient production of newly developed lipids for the fuel/pharma/food industry and carbohydrates/proteins for the food/feed/pharma/chemical industry. The technologies that need to be developed should be applicable to a variety of feedstocks with variable quality, be easily scalable, maintain the functionality of the compounds, be inexpensive and have a low energy consumption. In our biorefining strategy, we will use coarse fractionation with diluted microalgae streams. Microalgae consist of complex cells containing different organelles, such as mitochondria, lysosomes, endoplasmatic reticulum, Golgi apparatus, etc. (Fig. 11.2), each with a specific composition, some rich in nucleic acids, others in proteins, lipids or carbohydrates. The complexity of the biomass structure provides an opportunity for efficient biorefining. Cells are disrupted in a mild way such that, first, the different components (proteins, lipids, carbohydrates) in the cytoplasm will be released in a functional condition and, second, the larger cell compartments (organelles) also in an intact state. Cell compartments are separated into different fractions, and from these fractions, specific compounds are isolated.

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Fig. 11.2: Cross-section of the microalgae Dunaliella salina (Lamers et al. 2010).

The advantage of such a structure-based approach is that mild- and low-energyinput technologies can be used to isolate components (proteins, lipids, carbohydrates) from the cytoplasm and concentrate the different cell compartments so that more specific (and thus more expensive) isolation techniques can be used for concentrated fractions only. The “broth” from which compounds have to be isolated is less complex because complete cell disruption is omitted as isolation of compounds from a broth with fewer compounds is easier than from a complete cell broth. This approach has never been used for isolation of different biobased products from eukaryotic as well as prokaryotic organisms. As the technologies were not well developed, and focus was directed towards the isolation of one specific product instead of the isolation of multiple products, there was no need to know always the functional state of the product or to isolate multiple products, regarding the cost-effectiveness of the process, and there was no scarcity in products for food/fuel. Complete cell lysis usually triggers the cells to release proteolytic enzymes from different organelles (e.g. lysosomes) leading to irreversible denaturation of the compounds, which should be prevented or inhibited. When developing biorefinery concepts for eukaryotic organisms (e.g. microalgae) it needs to be clear which organelles, present in the cytoplasm, contain also valuable components (proteins, lipids, carbohydrates) so that smart biorefinery strategies can be developed for fractionation of these valuable products from the cytoplasm as well as the organ-

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Organelles

Main components

Nucleus Rough ER Lysosome Smooth ER Golgi apparatus Ribosome Mitochondria Chloroplast Cytoplasm

Proteins, DNA Proteins, carbohydrates, RNA Proteins Proteins, carbohydrates, lipids (e.g. triglycerides) Proteins, carbohydrates Proteins, RNA Phospholipids, proteins, DNA Phospholipids, proteins, DNA, chromophores (carotenoids, chlorophyll) Contains a variety of proteins, carbohydrates and lipids (organism-dependent)

Tab. 11.3: Main components present in important organelles of eukaryotic cells.

elles. Table 11.3 presents a rough overview of the components (proteins, lipids and carbohydrates) present in the different main organelles of eukaryotic cells. The presence of DNA and/or RNA need not to be a problem, as these nucleic acids can be selectively degraded by specific enzymes during the biorefining. A novel integrated biorefining concept with new cell disruption/extraction technologies for the mild recovery and fractionation of high-value components (proteins, lipids, carbohydrates) from cytoplasm and different organelles (e.g. mitochondria, chloroplasts) of microalgae is needed. To make production of these highvalue components economically feasible, it is essential to make use of all biomass components. The greatest benefit will be obtained if we are able to fractionate biomass from microalgae into different components while maintaining their full functionality. The following biorefining steps need to be performed: 1. development of the appropriate mild cell disruption technology (e.g. supersonic flow fluid processing or pulsed electric field) for microalgae: a) localization/mapping of components (proteins, lipids and carbohydrates) in the cell (e.g. cytoplasm or in specific organelles); b) mild breakage of cell walls for fractionation of components from cytoplasm with the supersonic flow fluid processing or pulsed electric field cell disruption techniques at a low energy input; c) increased breakage of cell walls for fractionation of organelles with enriched components with supersonic flow fluid processing or pulsed electric field at an increased energy input; d) final breakage of organelles for fractionation of proteins, lipids and carbohydrates with the mentioned cell-disruption techniques at increased energy levels; 2. extraction and fractionation of valuable components (proteins, carbohydrates, lipids) with detergents and/or the green solutions “ionic liquids” so that the hydrophobic compounds can be separated from the hydrophilic compounds under mild conditions while keeping the different compounds in their fully functional state;

208 3.

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development of an economical feasible continuous biorefining concept for high-value and bulk products by integrating mild cell disruption, extraction and fractionation techniques useful for industrial application.

11.2.2 Cell disruption, fractionation and mild cell disruption of organelles Cell disruption (Foster 1995; Kim and Hong 2001; Darani and Mozafari 2009; Ferrell and Sarisky-Reed 2010; Lee et al. 2010; Mata et al. 2010) is the most crucial technology with which to harvest high-quality biobased products because often the methods used are based on complete disruption of the cells or intended to focus on one specific product using more general techniques such as mechanical (e.g. homogenizers, bead milling, high pressure) or non-mechanical (e.g. ultrasonic, autoclaving, microwaves, osmotic shock, chemicals, enzymatic) cell-disruption methods to isolate specific components. These processes require energy, but also a complex mixture of ingredients is obtained from which the different components are difficult to separate, and in addition, this may lead to denaturation, degradation or irreversible modification of the functional compounds. The most value from the different components should be obtained by performing cell disruption in a subtle and mild way according to new strategies. The first step would be to perform localization studies of the different components in cytoplasm/organelles by labeling studies (e.g. fluorescence). Based upon these localization studies, mild cell-lysis techniques such as supersonic fluid feed or pulsed electric field, which can regulate the energy input, need to be carried out so that the cells can be gradually mild cell disrupted with a stepwise increase in the poresize distribution of the cell walls (Figs 11.3 and 11.4). Supersonic flow fluid processing is an industry-proven, highly sophisticated technology whereby supersonic steam is injected through nanopore channels, initiating a thermofluid interaction with the fermentation feed stream for optimal mass

Fig. 11.3: Cell-disruption techniques.

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Fig. 11.4: Cell disruption of microalgae.

transfer, so as to achieve homogeneous disruption. These controllable forces are achieved fluidically and involve no moving parts, and the energy intensity of the fluid processing can be controlled so that the cells are gradually disrupted, thereby keeping the temperature below the 35 °C to protect the valuable components from denaturation. The second industry-proven technique, pulsed electric field, uses socalled short bursts of electricity to damage the cells and release the products under non-thermal conditions without affecting the valuable components. Also, with this technique, the grade of cell damage can be finely tuned. It should be noted that considerable effort is needed to make both cell-disruption technologies suitable for microalgae, as they are novel techniques. The benefits/advantages of both techniques is that they provide scalable, controlled cell disruption by finely tuned energy release and omitting biomass concentration by centrifugation before cell disruption. A pre-concentration step might be needed, and the best technique for this would be (bio)flocculation (Schenk 2008; Lee et al. 2009; Salim et al. 2011) so that the energy consumption may be kept low. Figure 11.4 shows a schematic impression of the cell-disruption strategy. At first, small holes are pierced in the cells with a low energy so that the different components (proteins, carbohydrates, lipids) from cytoplasm can be easily extracted from the damaged cells (first cycle). Second, the punctured cells are concentrated by filtration and further exposed to increased energy input, generating larger holes in the cells excreting the organelles (second cycle). Finally, depending on the value of the components, the separated organelles and/or cell-wall fragments can be further concentrated and mild cell disrupted by fine-tuning the supersonic flow fluid processing or pulsed electric field technologies to isolate the compounds from these organelles and/or fragments with a higher energy input (third cycle).

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11.2.3 Extraction and fractionation of high-value components After cell lysis, extraction technologies (Raynie 2006; Ferrell and Sarisky-Reed 2010; Lee et al. 2010) are needed to further fractionate the hydrophobic components (lipids) from the more hydrophilic components (proteins, carbohydrates and nucleic acids), to maintain the full functionality and high value, and this can pose quite a challenge. This is because extraction technologies are normally performed using organic solutions (e.g. hexane) to recover the hydrophobic lipids and discard other components that are destroyed in organic solutions. Although the more sophisticated technique of using supercritical CO2, used in recent years for lipid extraction (Krichnavaruk et al. 2008, Macias-Sanchez et al. 2008), is very efficient and can combine lipid extraction with trans-esterification (Pi et al. 2011), it denatures the proteins. A recent review of popular extraction methods for oil recovery from microalgae, together with their respective pros and cons, is provided by Mercer and Armenta (2011). On the other hand, other extraction methods are used to recover the more hydrophilic proteins (e.g. detergents) (Azevedo et al. 2007; Martinez-Aragon et al. 2008) by discarding the more hydrophobic lipids (Fig. 11.5). It would therefore be much more pioneering to extract the hydrophilic and hydrophobic components simultaneously in one step (Fig. 11.6).

Fig. 11.5: Extraction principle.

Fig. 11.6: Extraction of different components.

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The extraction methods to be used need to be able to solubilize both the hydrophobic and hydrophilic compounds as in the use of ionic liquids (Earle and Seddon 2000; Du et al. 2007; Dreyer and Kragl 2008; Martinez-Aragon et al. 2008; Pei et al. 2009; Ge et al. 2010; Louros et al. 2010; Young et al. 2010) or the large variety of new surfactants/polymers (Azevedo et al. 2007) offer alternative approaches for mild extraction of the different components and for keeping the energy costs low, in relation to centrifugation methods. With the use of ionic liquids, the “green chemistry” molecules (Earle and Seddon 2000), hydrophilic components can be extracted from hydrophobic components, and this is very useful as a mild extraction method. A large range of ionic liquids are available depending on their charged state for solubilization of hydrophilic or hydrophobic components. It should be noted, however, that the technique of using ionic liquids is new and still in an exploratory phase, and has not yet been used for large-scale applications. Extraction using surfactants (e.g. polysorbate) and polymers (e.g. polyethylene glycol) for the solubilization of hydrophobic or hydrophilic components is another possibility to keep biobased components in a functional state. Again, also for these extraction methods, the combined approach is novel, so it will require considerable effort to define optimal extraction methods for the selective separation of hydrophilic from hydrophobic components. Finally, the extractants (e.g. ionic liquids, surfactants, polymers) need to be recovered, using ultrafiltration/ diafiltration techniques, for example, so that the valuable components reside in a protective buffer environment for further fractionation in different components or processing in specialized industries (food, feed, chemicals, biomaterials, etc.).

11.2.4 Economically feasible continuous biorefining concept Finally, the cell-disruption and extraction technologies need to be integrated in a continuous biorefining concept together with biomass production as schematically presented (Fig. 11.7). The microalgae biomass production will be fed as a diluted stream through the cell-disruption equipment (e.g. supersonic flow fluid processing or pulsed electric field) without concentrating the cells. By fine-tuning cell breakage, these cell-disruption methods are very attractive, so that the cells can be stepwise cracked and the released components (lipids, carbohydrates, proteins) separated with extraction methods so that hydrophobic (lipids) and hydrophilic (proteins, carbohydrates) components can be isolated and fractionated separately in a sustainable continuous process concept and at low energy costs. The fractionated components from this biorefinery plant, when needed, can be further fractionated to different (non)fuel products in this biorefinery plant or processed at specialized industries (food, feed, chemicals, fuel, etc.). Furthermore, the industrial production process of this microalgae biomass/biorefinery plant should be sustainable, efficient and flexible, with low energy costs, and applicable for a large variety of microalgae feedstocks.

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Fig. 11.7: Biorefinery plant concept.

11.3 Conclusions Biorefining of microalgae for production of proteins, lipids and carbohydrates has been discussed, and it has been shown that although algae are not yet produced on a large scale for high-value products and bulk applications, there are opportunities to develop these processes in a sustainable way. However, it is unlikely that the process will be developed for biodiesel alone, as the other compounds (proteins, lipids, carbohydrates) should be fractionated as well Moreover, a possible integration of algal biofuel-based biorefining with other industries such as livestock, lignocellulosic and aquaculture (Subhadra and Grinson-George 2010) seems to be another opportunity. In order to develop a sustainable and economically feasible process, all biomass components should be used, and so an integrated continuous biorefining technology concept for microalgae is needed. Of course different technological challenges need to be overcome such as the integration of biomass production with cell disruption and extraction technologies, and the simultaneous extraction of hydrophilic and hydrophobic components keeping the components in their full functional state.

References

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Biorefining of microalgae is a new and a promising, albeit immature, area, but and efforts have to be made to develop an economical sector.

References Azevedo, A. M., P. Rosa, I. Ferreira, M. Aires-Barros. 2007. Optimization of aqueous two-phase extraction of human antibodies. Journal of Biotechnol. 132: 209–217. Banarjee, A., R. Sharma, Y. Chisti, U. Banarjee. 2002. Botryococcus braunii: A renewable source of hydrocarbons and other chemicals. Critical Reviews in Biotechnology 22: 245–279. Barcley, B. 2009. Algae oil production. Keynote lecture at the Algal Biomass Organization 2009 summit, San Diego; October 7–9 Carioca, J. O. B., J. Hiluy Filho, M. Leal, F. Macambira. 2009. The hard choice for alternative biofuels to diesel in Brazil. Biotechnol. Adv. 27: 1043–1050. Chisti, Y. 2007. Biodiesel from microalgae. Biotechnol. Adv. 25: 294–306. Clarens, A. F., E. Resurreccion, M. White, L. Colosi. 2010. Environmental life cycle comparison of algae to other bioenergy feedstocks. Env. Sci. Technol. 44: 1813–1819. Darani, K. K., M. Mozafari. 2009. Supercritical fluids technology in bioprocess industries: A review. J. Biochem.Tech. 2: 144–152. Dismukes, G. C. D. Carrieri, N. Bennette, G. Ananyev. 2008. Aquatic autotrophs: efficient alternatives to land-based crops for biofuels. Curr. Op. in Biotechnol. 19: 235–240. Dreyer, S., U. Kragl. 2008. Ionic liquids for aqueous two-phase extraction and stabilization of enzymes. Biotech. Bioeng. 99: 1416–1424. Du, Z., Y. Yu, J. Wang. 2007. Extraction of proteins from biological fluids by use of an Ionic Liquid/Aqueous Two-Phase System. Chem. Eur. J. 13: 2130–2137. Earle, M. J., K. Seddon. 2000. Ionic Liquids. Green solvents for the future. Pure Appl. Chem. 72: 1391–1398. Ferrell, J., V. Sarisky-Reed. 2010. National Algal Biofuels Technology Roadmap. US Department of Energy (Energy Efficiency & Renewable Energy). Foster, D. 1995. Optimizing recombinant product recovery through improvements in cell-disruption technologies. Cur. Op. Biotech. 6: 523–526. Ge, L., X. Wang, S. Tan, H. Tsai. 2010. A novel method of protein extraction from yeast using ionic liquid solution. Talanta 81: 1861–1864. Illman, A. M., A. Scragg, S. Shales. 2000. Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzyme. Microb. Technol. 27: 631–635. Khozin-Goldberg, I., C. Bigogno, P. Shrestha, Z. Cohen. 2002. Nitrogen starvation induces the accumulation of arachidonic acid in the freshwater green alga Parietochloris incise. J. Phycol. 38: 991–994. Khozin-Goldberg, I., Z. Cohen. 2006. The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemistry 67: 696–701. Kim, K. H., J. Hong. 2001. Supercritical CO2 pretreatment of lignocellulose enhances enzymatic cellulose hydrolysis. Bioresource Technology 77: 139–144. Krichnavaruk, S, A. Shotipruk, M. Goto, P. Pavasant. 2008. Supercritical carbon dioxide extraction of astaxanthin from Haematococcus pluvialis with vegetable oils as co-solvent. Bioresource Technol. 99: 5556–5560 Lamers, P. P., C. Van de Laak, P. Kaasenbrood, Lorier. 2010. Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotech. Bioeng. 106: 638–648. Lardon, L., A. Hélias, B. Sialve, J. Steyer. 2009. Life-Cycle assessment of biodiesel production from microalgae. Env. Sci. Technol. 43: 6475–6481.

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Lee, A. K, D. M. Lewis, P. J. Ashman. (2009) Microbial flocculation, a potentially low-cost harvesting technique for marine microalgae for the production of biodiesel. J. Appl. Phycol. 21: 559–567. Lee, J.-Y., C. Yoo, S. Jun, C. Ahn. 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresource Technology 101: S75-S77. Louros, C. L. S., A. Claudio, C. Neves, M. Freire. 2010. Extraction of biomolecules using phosphonium-based ionic liquids + K3PO4 aqueous biphasic systems. Int. J. Mol.Sci. 11: 1777–1791. Macias-Sanchez, M. D., C. Mantell Serrano, M. Rodriquez Rodriques, E. Martinez de la Ossa, L. M. Lubian, O. Montero. 2008. Extraction of carotenoids and chlorophyll from microalgae with supercritical carbon dioxide and ethanol as cosolvent. J. Sep.Sci. 31: 1352–1362. Martinez-Aragon, M., S. Burgoff, E. Goetheer, A. de Haan. 2008. Guidelines for solvent selection for carrier mediated extraction of proteins. Separation and Purification Technology 65: 65– 72. Mata, T. M., A. Martins, N. Caetano. 2010. Microalgae for biodiesel production and other applications: A review. Renewable and Sustainable Energy Reviews, 14: 217–232. Mercer, P., R. E. Armenta. 2011. Developments in oil extraction from microalgae. Eur. J. Lipid. Sci. Technol. 113: 539–547. Norsker, N.-H., M. Barbosa, M. Vermuë, R. Wijffels. 2011. Microalgal production – A close look at the economics. Biotech. Adv. 29: 24–27. Pei, Y., J. Wang, K. Wu, X. Xuan. 2009. Ionic liquid- based aqueous two-phase extraction of selected proteins. Separation and Purification Technology 64: 288–295. Pi, J.-C., T.-B. Du, C. Chuan, L.-P., J. S.-M., J. 2011 SCCO2 Sonication extraction and supercritical methanol transesterification on microalgae. The 13th Asia Pacific Conference of Chemical Engineering Congress, October 5–8, Taipei. Pulz. O., W. Gross. 2004. Valuable products from biotechnology of microalgae. Appl. Microb. Biotechnol. 65: 635–648. Raynie, D. E. 2006. Modern extraction techniques. Anal.Chem. 78: 3997–4004. Salim, S, R. Bosma, M. H. Vermue, R. H. Wijffels. 2011. Harvesting of microalgae by bioflocculation J. Appl. Phycol. 23: 849–855. Schenk, P. M. 2008. Second Generation Biofuels: High-Efficiency Microalgae for Biodiesel Production. Bioenergy Research 1: 20–43. Shi, S., J. Valle-Rodriquez, V. Siewers, J. Nielsen. 2011. Prospects for microbial biodiesel production Biotech. J. 6: 277–285. Subhadra, B., Grinson-George. 2010. Algal biorefinery-based industry: an approach to address fuel and food insecurity for a carbon-smart world. J. Sci. Food. Agric. 91: 2–13. Young, G., F. Nippgen, S. Titterbrandt, M. Cooney. 2010. Lipid extraction from biomass using cosolvent mixtures of ionic liquids and polar covalent molecules. Separation and Purification Technology 72: 118–121. Wijffels, R. H., M. Barbosa. 2010. An outlook on Microalgal Biofuels. Science 329: 796–799. Wijffels, R. H., M. Barbosa, M. Eppink. 2010. Microalgae for the production of bulk chemicals and biofuels. Biofuels, Bioproducts & Biorefining 4: 287–295.

Garry Henderson

12 Development of a microalgal pilot plant: A generic approach 12.1 Understanding the aims of the pilot plant This chapter describes a methodology for the development of a pilot plant design. The methodology is presented from a design-engineer or design-manager perspective and incorporates lessons from many years design experience, including the design of the pilot plant for the Solar Biofuels Research Centre (SBRC; Hankamer, 2012). This chapter does not describe how to conduct a design. It is assumed that the team involved will use the more pertinent information supplied in other chapters to define the specific requirements and sizes of each unit. This chapter is intended to provide a general guide to a team tasked with planning the establishment and execution of a pilot plant project. While it may seem obvious, the first step is to record the aims of the pilot plant. This process will guide all other decisions that are required to be made along the path to establishing the pilot plant. The following points are just some of the questions that should be answered when recording the aims of the pilot plant. These could be a comparison of different reactor designs, for bioenergy or for high-value products, test of shelf-life under realistic conditions, measurement of necessary maintenance energy and manpower. Another important decision is the use of genetically engineered algae or restriction to GRAS strains, and education and training are reasonable goals in these days of emerging technology: – Is the pilot plant to test a single concept, or to compare different concepts? – What do you need to measure and why? – What do you need to control and why? – What will be the size of the plant? – What are you trying to demonstrate or test? – What are the key areas to gather knowledge, and which areas only support that knowledge gathering? You should have the aims recorded and agreed by all participants or stakeholders to the project. This provides a basis to test later decisions. In fact, all considerations are not only targeted to the algae, photobioreactors and infrastructure. Behind the scenes are people, who work there, who see their personal scientific chance conducting experiments on the plant, who have their specific expectations and responsibilities, and last but not least who make mistakes.

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12.2 Pilot plant location and site selection The location of the site for the pilot plant needs to be selected as early as possible. The site constraints will have a major impact on the pilot-plant design. Depending upon the location of the plant, some approval-related items may involve a lengthy process. A list of site-specific conditions that will need to be determined before the detailed design of the pilot plant can be completed is provided below: – development application approval conditions; – environmental approval conditions; – geotechnical conditions at the site; – the existing topography of the site (survey required); a gently sloping terrain is in principle no problem (in contrast to what is written in some LCA studies), but reactor geometry has to consider, for example, orientation of pipes parallel to the slope; – constraints imposed by the surrounding land use, e.g. dust with eutrophication effects from agriculture; – access to the site; technicians and scientist have to work may be only for sampling and go back to the institute; ability to get equipment safely; – orientation of the site, especially if sunlight is required; – other constraints imposed by other institutions (university requirements, land-owner requirements, etc.). While these processes can be undertaken in parallel with the rest of the project development, a late change of site can result in serious delays to the project. Therefore, if a choice of multiple sites exists, either quickly decide on the site to be used, or progress the two most probable sites simultaneously until a decision on the preferred site becomes clear.

12.3 Develop the process flow diagram If your team does not have the expertise to produce the process flow diagram, seriously consider engaging a process engineer to assist. The process flow diagram will allow the team to capture the functionality of the pilot plant as well as help develop plant items such as pumps, compressors, and other items that will be required to make the plant operate. Put as much detail as possible into the diagram from the beginning. This can then be used for the development of the steps that follow. Be aware that the initial diagram will be continually updated throughout the project development until it is expanded to form the piping and instrumentation diagrams.

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12.4 Know what will be required to conduct experiments and measure the data Knowing what is required to conduct experiments and measure results may appear elementary at first, but a pilot plant poses many more challenges than bench scale or laboratory environments. A draft standard operating procedure should be written in the early stages of the project development. The plant lead operator and chief investigator need to imagine themselves on the plant and picture what they are doing to achieve the stated aims of the project. A draft operating procedure is a valuable tool, as it identifies what equipment, methodologies and tools will be required to operate the plant and to gather the required data. Tasks such as making adjustments to test parameters, taking samples, getting materials into and out of reaction vessels, cleaning, and all other required tasks need to be walked through in the mind’s eye at first. The methodology then needs to be recorded. By recording the operating procedures, you are likely to identify items that had previously been overlooked. You should also record the decisions made during this process and the reasons for them. This is particularly helpful when you are later required to make decisions on items to include or exclude if the tender prices are outside the plant budget. The decisions could be to allocate more funds, and this is easier to justify if the reasons for all items are readily justified. Measurement and automation is another issue. On a plant with mainly research tasks, all the reactors will be operated more or less independently. Furthermore, different people with different education want to realize their data-acquisition and process-control ideas. Therefore, a decentralized automation concept has been shown to be adequate, e.g. in large bio-plants for research purposes. Each reactor has a control panel allowing process control from starting a process to selecting sensor signals, up to adjusting control parameters. For this interface, a LabView-based automation system (BioProCon; Posten, 2012) has been employed at the SBRC. An additional central unit is foreseen for the final measurement database and communication with the outer world. During these development steps, you should consider the times required to fill and empty vessels. The time required to clean vessels and pipelines and the time required for any process operations, such as growth cycles, dewatering and inoculation, must all be considered and acceptable time frames agreed.

12.5 Sizing of the units Once the aims and standard operating parameters are defined and agreed, you can size the unit operations, as well as all the ancillary equipment. A list of possible units to be sized is provided below:

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Fig. 12.1: Engineering start progress, all disciplines feedback design requirements.

– – – – – – – – – – – – –

bioreactors; dewatering units; pumps; storage vessels – makeup water, media solutions, waste for treatment, cleaning solutions; blowers/compressors; pipes; valves; motor control panels; electrical distribution panels; control hardware including programmable logic controllers; pad area for locating plant items; supports for all vessels, pipework and cabling; any containment volumes required by occupational health and safety or environmental regulations

These items must be sized in an order that allows the various engineering disciplines to conduct the required sizing. Typically the order of sizing will be as described in Figure 12.1. When the units are sized, you will be able to derive the drive list with drive sizes. The electrical engineer will require this information to size the motor control panel, power distribution board and cabling. At this point, you will need to expand the process flow diagram into logical sections for the completion of the piping and instrumentation diagrams. The piping and instrumentation diagrams will describe to the contractor and control programmer how the control system interacts with the plant. They will allow the contractor and control programmer to define the number and type of instruments required. The details of the piping and instrumentation diagrams need to be incorporated into the general arrangement of the plant to determine power and control cable routes on the plant. The piping and instrumentation diagrams are also

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required to enable the hazard and operability (HAZOP) study on the plant to be conducted. Even the compressed air requirement may appear superficially simple, but there are many things that must be considered. It is important to consider redundancy of the equipment on the plant. Will the operation of the plant be seriously compromised if a piece of equipment is not available? If it is, then you need to check how long it would take to repair or replace that piece of equipment. If the duration is sufficiently quick as to cause minimal impact on the aims of the pilot plant, then a single unit would be sufficient. If, however, it would take a period of time that would cause serious disruption to the ability to achieve the goals of the project, then more than one unit will be required. A cost–benefit analysis is recommended to determine the optimum sizes of the units that allow the achievement of the project goals if one unit fails and remaining within the constraints of the budget. In the case of the Solar Biofuels pilot plant, the air compressors were determined to be vital to the achievement of the project objectives. Therefore, the amount of air required by each unit was determined, and the smallest acceptable compressor size was one that could keep all the vital units operating simultaneously. Items that were deemed to be not vital during a compressor failure incident made up the balance of flow and pressure that must be available when both compressors are available. The extent of design that will be conducted by the design team and the extent that will be left to the contractor to design should be well understood. It is recommended that specialist items that the design team is not specialized in should be left for a competent contractor in the area to design. Leaving design responsibility with a contractor means that they will be taking on the risk for that area, and they will increase their price accordingly. This is typically the cheapest and most robust option for sourcing specialist items.

12.6 Plant layout The layout of each plant will be different due to changing configurations that will vary with the aims of the project, the plant needs to be laid out in such a manner that the process flows logically, and all items that need to be accessed can be safely accessed. Since your pilot plant includes photo-bioreactors, you must also consider how ancillary equipment and tanks will cast shadows and what impact that will have on performance of the photo-bioreactors. If you are in the Northern Hemisphere, you should aim to have tall equipment and tanks on the north side of the plant and vice versa if you are in the Southern Hemisphere. An example of a plant layout that includes access areas required around equipment and consideration of photo-bioreactor location is provided in Figure 12.2. The

12 Development of a microalgal pilot plant: A generic approach

Fig. 12.2: Plant layout of units at SBRC pilot plant.

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Fig. 12.3: Group of several flat plate reactors to perform experiments with different design parameters in a direct comparison.

location of access gateways, security fencing if required and allowances for deliveries of consumables also need to be considered. The layout should also consider the location of pipes and cables. These items need to be protected from damage and should be located, where possible, so that they do not cause a hazard to operators or visitors to the plant. The space you need for the different reactors of course depends on the design, number and research task of the reactors. Figure 12.3 shows a set of flat panel reactors including dummies and control boxes. The reactor set is designed to allow for experiments testing different height of reactors, different surface to footprint ratio and temperature-controlled versus uncontrolled conditions. Simultaneous experiments with different parameters are necessary because changing environmental conditions make evaluation of sequentially performed experiments difficult. The dummies are necessary to mimick the next panel in an anticipated largearea installation. That makes later scale/numbering up investigations more reliable.

12.7 HAZOP study A HAZOP study investigates the implications of unplanned occurrences in the plant, how aware the operator will be of them and how safe the plant will be under those circumstances.

Solar

High ambient temperature

High algae temperature

low high high

high

high

Temperature

Compressor failure High aeration

Open drain valve

low

Mixing

Leak

low

Media

Operator error

high

Level

Cause

PBRs

Prompt word

XKTECP-042DW-DR-PID-003

Deviation

Area

Drawing Number

Makeup batch exceeds PBR capacity, wash down to waste tank, dilute and release – covered in SOP Drain, shutdown and repair. Ensure sensors are not dry fpr extended periods – investigate feasibility of providing level sensores and call out alarm; also investigate dry resistant sensors Wash down to waste tank, dilute and release – covered in SOP; remake makeup solution

Comment

Loss of productivity, biological stress affecting product quality

Loss of productivity, biological stress affecting product quality, increased evaporation

Loss of productivity, biological stress affecting product quality, increased evaporation

Provide temp monitoring for each PBR, increased makrup water demand; allow for later implement misting unit for each PBR if required; reactor 1 suitable to be fitted with chiller unit Provide temp monitoring for each PBR, increased makrup water demand; allow for later implement misting unit for each PBR if required; reactor 1 suitable to be fitted with chiller unit Provide temp monitoring for each PBR, increased makrup water demand; allow for later implement misting unit for each PBR if required; reactor 1 suitable to be fitted with chiller unit

Algae setting Establish mixing requirements/design Cell stress, foaming, high DO Ongoing monitoring and adjusting

Release to Bund

Release to bund

Overflow media into bund

Consequence

222 12 Development of a microalgal pilot plant: A generic approach

CO2 underdose

NaOH, KOH, aqueous ammonia overdose

high

high

Low ambient temperature

Table 12.1: Example of a HAZOP study assessment findings.

pH

low

O2 inhibition, loss of productivity (species population shift), biological stress affecting product quality Loss of productivity (species population shift), biological stress affecting product quality

Online pH monitoring with feedback control to CO2 and/or base

Online pH monitoring with feedback control to CO2 and/or base

Loss of productivity (species Provide temp monitoring for each PBR, population shift), biological reactor 1 suitable to be fitted with heater unit; stress affecting product qual- in future geothermal heat ex could be used ity

12.7 HAZOP study

223

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12 Development of a microalgal pilot plant: A generic approach

The aim is to identify risks in operating the plant as it is designed and then to define methods to eliminate or mitigate those risks. The HAZOP study should begin with a workshop led by an experienced facilitator, with the people responsible for the design, operation and budget attending and contributing to the workshop. The decisions recorded in the workshop, or made later based on the results of investigations recommended in the workshop, are to be implemented in the finalization of the plant design. The response of the designers should be recorded in the HAZOP report and recorded as part of the project records. An example of a portion of a HAZOP study is given in Table 12.1. In the table, the action column has been deleted, but an action column is required to allocate any actions and by whom required to address the identified risks. The HAZOP study will help identify omissions or flaws in the initial standard operating procedures and control design. If conducted thoroughly, it has the potential to save effort and budget in the operating phase.

12.8 Multidisciplinary review of the design You should conduct a multidisciplinary review of the design to ensure that the requirements of all the disciplines have been captured and are reflected in the design documentation. The length of the review will vary with the complexity of the plant and the research team’s level of understanding as to how the design will allow the aims to be achieved. If the disciplines disagree on certain elements, the design manager will be required to make a determination based on the technical requirements of each discipline to define the final design.

12.9 Tender for plant construction You should issue the tender documents and contract conditions as defined by the entity responsible for the budget and project management of the plant construction. To provide an accurate price for the construction of the plant, tenderers (contractors) require enough time to understand the design and site constraints, and to determine their methodology for construction, to be able to provide an accurate price for the construction of the plant. If you do not allow them sufficient time, contractors will add sums of money to cover the risk of unknown costs. Inevitably, this will add to the cost of the project. The project manager and a designated team need to review the tender submissions to ensure that they cover all the required costs for the project. It is common

References

225

for contractors to omit items or place exclusions in the tender submission to make their price more competitive. An experienced evaluation team will identify these issues and seek clarification from a tenderer to ensure that all tenders are equal when compared. At the end of this process, you will have identified a preferred tenderer and negotiated a contract.

12.10 Finalize the design To suggest that the design is finalized after the contract is awarded may seem unusual. This is, however, common practice. Every contractor will have its own preferred methods for construction. Until the contractor is known, details that rely on construction method are best not finalized. Typically, changes should be small and only made to accommodate the contractor’s experience and skills. This chapter is intended to provide only general guidelines for a team tasked with planning and executing a pilot plant establishment project. It is not intended as detailed professional advice.

References Hankamer, 2012: http://www.solarbiofuels.org assessed 2012. Posten, 2012: www.bio-ag.de assessed 2012.

Roberto Bassi, Pierre Cardol, Yves Choquet, Thomas de Marchin, Chloe Economou, Fabrice Franck, Michel Goldschmidt-Clermont, Anna Jacobi, Karen Loizeau, Gregory Mathy, Charlotte Plancke, Clemens Posten, Saul Purton, Claire Remacle, Carsten Vejrazka, Lili Wei and Francis-André Wollman

13 Finding the bottleneck: A research strategy for improved biomass production 13.1 Introduction: What do we expect from cell engineering? 13.1.1 The need for domestication of microalgae There is a great diversity of microscopic algae in the natural world, with estimates of over 100,000 different species of algae spanning four of the five major phyla of eukaryotic life (Guiry and Guiry 2012). Only a tiny fraction of these algae have been isolated and characterized, and as a consequence there is a very rich vein that can be mined for novel species with phenotypes suitable for commercial cultivation. Such species might produce copious quantities of hydrocarbons or lipids that can be converted into liquid biofuels, or might be remarkably fast-growing or able to cope with extremes of temperature, light, salinity, etc. However, it is probably the case that no single algal species will be found that has all the attributes required of a commercially successful feedstock – namely, efficient and sustainable lowcost cultivation on an industrial scale, and with a high productivity of the desired product in the photobioreactor conditions. Furthermore, microalgae have naturally evolved to thrive as part of a complex microbial community where symbiotic and competitive interactions between the alga and the rest of the community (comprising bacteria, other algae, fungi, etc.) are necessary for efficient algal growth. The sustained mono-cultivation of a single algal species where it is the only species or is the dominant species in the culture is therefore a rather unnatural situation. Even in the mass algal blooms that can occur in the oceans or large water-bodies, these blooms are accompanied by bacterial growth and inevitably collapse as pathogens and predators invade the blooms. Consequently, there is a need to dramatically reshape the phenotypes of chosen algal species to produce commercial strains better suited to intensive algal farming, in the same way that the last few thousand years has seen the domestication of a handful of weedy plant species to produce today’s robust, high-yielding crop varieties.

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13.1.2 Limitation of traditional approaches to strain improvement While modern agriculture is based on several thousand years of plant breeding that has allowed the selecting and combining of desirable traits, that luxury is not afforded to the algal biologist. First, the pressing need to supplement, and ultimately replace, fossil-derived fuels with fuels from renewable sources within the next 50 years or so means that time is not on our side if we wish to exploit algae as a biofuel feedstock. Second, most microalgal species do not have well-defined and controllable sexual cycles, and therefore the combining of genetic traits by crossing variants of the same species or two closely related species, as is routinely used for flowering plants, is not possible. It might be feasible to create novel algal strains using the technique of somatic hybridization whereby cells of different species are artificially fused with the hope of creating a viable chimeric cell. However, although this technique is well established for closely related plant species, there are only limited reports of success in the algal field. So, we are left with mutagenesis strategies in which large populations of algal cells are treated with a mutagen such as ionizing radiation or a DNA-damaging chemical, and mutant cells are selected that show a desirable phenotype (e.g. a greater tolerance to high light). This new, improved strain is then used as the founder cell line in a second round of mutagenesis for other desirable phenotypes, and so on. Unfortunately, this serial mutagenesis approach is doomed to failure, as each round also introduces many undesirable mutations into the genome, and the cumulative effect is to make the organism less fit, and less able to survive the challenge of mass cultivation. Without sexual recombination and selection to purge the genome of these unwanted mutations, we then fall inevitably into this Darwinian trap. Furthermore, the majority of DNA mutations result in a ‘loss-of-function’ phenotype where the function of a gene product is compromised. While this might yield a trait required of a commercially grown strain (for example, a reduced chlorophyll content allowing greater light penetration in bulk culture), often we are searching for ‘gain-of-function’ mutations that will confer a new facet to the strain, and these mutations are much rarer. Finally, some algal groups (e.g. diatoms and dinoflagellates) have genomes that are diploid or polyploid such that the nucleus possesses two or more copies of each gene. In these algae, mutations in one gene copy often fail to give rise to a novel phenotype, since the other gene copies continue to produce the original gene product. Again, the sexual cycle of diploid plants allows us to overcome this issue by mating the plant with itself (“selfing”) and selecting for offspring in which both copies of the gene carry the mutation, but as discussed above, this approach is not available for most algal species.

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13.2 Algal domestication through chloroplast genetic engineering Genetic manipulation (GM) represents an alternative approach to traditional methods of genetic improvement that could be applied to algae. Although this transgenic technology is still very much in its infancy and currently only applicable to a handful of algal species, it has an advantage over traditional methods in that it is monogenic and predictive (Walker et al. 2005; Radakovits et al. 2010). That is, the genetic manipulation process involves only one (or a few) selected gene(s) that we wish to introduce, to eliminate, or to modify. Furthermore, our prior knowledge of the function of the gene product allows us to predict the effect of the engineered change on the biology of the alga. While most of the current focus has been on developing GM technologies for manipulating the algal nuclear genome, the small circular genome of the chloroplast represents an attractive alternative when it comes to creating designer algae that synthesize novel biofuels or high-value recombinant products (Purton 2007). First, efficient homologous recombination in the chloroplast allows foreign genes to be inserted into precise and predetermined positions in the genome. Second, high expression levels can be obtained without the complications of gene instability and silencing, and the prokaryotic nature of the chloroplast genetic system makes possible the insertion of multiple genes as an operon controlled by a single promoter. Third, the chloroplast compartment is a major storage organelle as well as the site of key biosynthetic pathways including those of carbohydrate, fatty acid, isoprenoid and porphyrin biosynthesis. Consequently, expression of genes for novel metabolic enzymes that tap into these pathways, or those for high-value therapeutic proteins such as vaccines and hormones, should allow accumulation of the recombinant products within the organelle. Unfortunately, significant progress in this field is limited to just one algal species, Chlamydomonas reinhardtii, with only two other isolated reports of success in the 25 years since chloroplast transformation was first achieved. And even with Chlamydomonas, the technology still needs further refinement before it can be fully exploited. Below, we summarize current research efforts in this area.

13.2.1 Chloroplast engineering in Chlamydomonas: progress and challenges Design of a transgene intended for chloroplast transformation requires a careful choice of the flanking cis-acting elements that will drive the expression of the gene of interest. The upstream sequence must be composed of a promoter region, which is responsible for initiating transcription, and a 5′UTR (5′ untranslated region) to govern the stability of the messenger RNA and its translation. Strong constitutive promoters/5′UTRs such as those from the endogenous genes rbcL, atpA, atpB, psaA, psbA and psbD are the most suitable for transgene expression in the Chlamydomonas chloroplast. The choice of an optimal promoter/5′UTR remains somewhat

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empirical and may depend to some extent on the particular open reading frame (ORF) that is placed downstream. To a lesser extent, the 3′UTR can also alter the processing and the stability of the transcript. The 3′UTR of rbcL is frequently used for chloroplast transgene construction in Chlamydomonas because it contains two tandem hairpin loops that act redundantly for the formation of stable 3′ ends. Another factor that needs to be considered is the genomic locus where the integration by homologous recombination will occur after transformation. Indeed, the insertion site must be as neutral as possible to avoid any loss of function due to disruption of an endogenous gene. The selection of rare chloroplast transformation events in which a transgene is delivered into the organelle compartment (usually, via high-velocity bombardment with DNA-coated microparticles of gold or tungsten) and successfully integrated into the genome requires that the transgene is linked to an efficient selectable marker. This selection can be based on different traits such as the ability to perform photosynthesis, on resistance to antibiotics or herbicides, and on other metabolic markers. Efficient photoautotrophic growth is a powerful way to select plastid transformants. This approach involves transforming a host strain mutated for a protein involved in photosynthesis with the corresponding wild-type gene to restore photoautotrophic growth. This approach was used in the pioneering work of Boynton and coworkers (1988) using the atpB gene. It remains a powerful and versatile strategy because adequate host strains with a mutation in any photosynthetic plastid gene can be generated by chloroplast transformation. The prokaryotic origin of the chloroplast is reflected in its translation machinery, which is sensitive to antibiotics targeting ribosomal proteins. Hence, the most frequently used approach is to transform the chloroplast with a bacterial marker gene that confers antibiotic resistance (Day and Goldschmidt-Clermont 2011). Such markers are dominant, so that any host strain can be used for transformation. A selectable cassette containing the bacterial aadA marker gene conferring spectinomycin and streptomycin resistance is widely used. Alternatively, the aphA-6 gene offers resistance to kanamycin and amikacin. Because it is the most efficient marker available to date, a strategy to “recycle” the aadA cassette has been developed. Two direct repeats are placed on each side of the selectable marker. Once a homoplasmic transgenic line has been obtained under antibiotic selection, loss of the cassette by homologous recombination can be induced by growth under non-selective conditions. Finally, integration of the ARG9 gene in the chloroplast genome of the Chlamydomonas nuclear mutant arg9 has been shown to restore arginine prototrophy (Remacle et al. 2009). It should thus be possible to develop the ARG9 gene as a selectable marker for plastid transformation. In addition to the need to select for rare transformation events, there is also a need to ensure that all copies of the polyploid chloroplast genome in a transgenic line carry the trans-gene. Initially, transformants are in a heteroplasmic state where some of the ~80 copies of the genome have acquired the transgenic DNA, but the

13.2 Algal domestication through chloroplast genetic engineering

231

remainder has not. However, by taking these transformant lines through several rounds of single colony isolation on the selective medium homoplasmic lines can be obtained in which all copies of the genome carry the selectable marker and the transgene. Once homoplasmy is achieved, maintenance of the lines on selective media is no longer necessary. The expression of the nuclear and plastid genomes is closely coordinated, and a great number of factors involved in the regulation of plastid gene expression are nucleus-encoded. The most telling example is the trans-splicing of the psaA mRNA, which requires the contribution of no less than 14 nucleus-encoded factors. An approach that deserves to be further developed involves the identification of nuclear mutations that allow an increase in the expression of plastid transgenes. It has been shown with a transgene driven by the psaA 5′UTR that its expression could be enhanced in a nuclear mutant background that prevented trans-splicing of the endogenous psaA gene (Michelet et al. 2011). The absence of PsaA protein alleviated a regulatory negative-feedback loop that limits the expression of the psaA-driven transgene – an example of a regulatory process termed control by epistasy of synthesis. Likewise, deletion of the endogenous psbA genes in the chloroplast leads to enhanced expression of transgenes placed under the control of the psbA promoter/5′UTR (Rasala et al. 2011). A drawback of these current strategies is that the transgenic lines are not photoautotrophic. A better understanding of the regulation of chloroplast gene expression and of the cis- and trans-acting elements that are involved should allow the further development of strategies for improved transgene expression in the future. As some foreign proteins or metabolites can be toxic or detrimental for algal growth and survival, different inducible or repressible systems for transgene expression must be developed. A system based on the anterograde control of chloroplast psbD mRNA stability by the nucleus-encoded NAC2 protein has been demonstrated in Chlamydomonas. The NAC2 gene in the nucleus was placed under a copper-regulated promoter, while the gene of interest in the chloroplast carried the psbD 5′UTR. Expression of the chimeric transgene was induced under copper limitation (Surzycki et al. 2007). An alternative regulatable system was engineered using the lac regulation system from Escherichia coli. Due to its prokaryotic origin, it was possible to develop such an inducible system in Chlamydomonas by inserting a gene encoding the lacI repressor and engineering a lac operator controlling the gene of interest (Kato et al. 2007). Another promising strategy involves the use of riboswitches, which are RNA elements whose structure is altered upon binding of a regulatory molecule, and such structural changes result in an up- or downregulation of expression. In tobacco, a synthetic riboswitch was engineered to control the translation of a foreign gene in the chloroplast (Verhounig et al. 2010). Whether this strategy can be further developed to allow efficient and regulatable expression of transgenes in algal plastids remains to be determined. Finally, plastid proteases play a crucial role in the biogenesis and maintenance of functional chloroplasts. Numerous proteases belonging to several families (such

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13 Finding the bottleneck: A research strategy for improved biomass production

as FtsH, Deg/HtrA or Clp) form a strong proteolytic surveillance system in the plastid (Sakamoto 2006). In several cases where this was examined, accumulation of transgenic proteins in the Chlamydomonas chloroplast was limited by proteolytic degradation. Thus, an important issue to improve the efficiency of algal chloroplasts as bioreactors will be to identify the proteolytic systems responsible for the degradation of foreign recombinant proteins, and to develop host strains mutated for these systems to facilitate the overproduction of proteins of interest.

13.2.2 A synthetic biology approach to chloroplast metabolic engineering As illustrated in Figure 13.1, the assembly of each transformation cassette is a modular process which follows a defined set of rules. Each gene must be correctly flanked by suitable regulatory cis elements, and each cassette must possess left and right flanking elements, and in the correct orientations, to allow integration at the desired locus. If multiple transgenes are to be introduced, the transformation cassette becomes increasingly complex, but the basic rules still remain. As a consequence, the transgenic manipulation of the chloroplast lends itself very much to a synthetic biology approach. Synthetic biology aims to apply the principles of engineering in which DNA “parts” are synthesized and assembled into “devices” (in this case, the transformation cassette) using a standardized assembly process. Furthermore, recent advances in DNA synthesis technology now make possible the synthesis of bespoke DNA parts designed in silico. This allows the redesign of the coding region of transgenes to: (1) adjust the codon preferences to those typically found in Chlamydomonas chloroplast genes where there is a strong preference for AT-rich codons (such codon adaptation can have a profound effect on the efficiency of translation); and (2) alter the protein sequence encoded by the transgene to change the properties of the protein or add an additional sequence such as an epitope tag for protein detection or a polyhistidine tag for purification. Assembly of the DNA parts is best achieved using a synthetic biology method recently developed by Daniel Gibson at the J. Craig Venter Institute (Gibson 2011). This single-step, isothermal technique avoids the use of restriction enzymes, allows all the various parts to be simultaneously joined together in the correct order and orientation, and uses a cocktail of just three enzymes – a T5 exonuclease, a thermostable DNA polymerase and a Taq DNA ligase. Parts are joined together by virtue of an overlap of 20–40 bp shared between two adjacent parts. These overlapping regions are added to the ends of the DNA fragments by PCR using primers with added adapter sequences. The speed of joining multiple pieces of DNA together such as promoter, UTR and terminator sequences (a complete device can be easily assembled within 24 h), in addition to the absence of any restriction site “scar” at the junction of each part, makes the Gibson method an attractive tool for highthroughput chloroplast metabolic engineering. This is particularly useful in testing

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Fig. 13.1: Steps involved in the introduction of a transgene into the chloroplast genome: (a) the coding sequence of the gene of interest is codon-optimized and synthesized; (b) this is linked to suitable endogenous cis elements to mediate efficient transcription and translation; (c) the DNA assemblage is then flanked with left and right arms homologous to the regions flanking the chosen site of insertion on the chloroplast genome; (d) introduction of this “transformation cassette” into the chloroplast of the recipient cells allows integration of the transgene into the genome via homologous recombination events; (e) several rounds of single colony isolation under selective conditions ensure that all copies of the genome in a cell contain the transgene. Note: transformation normally involves a second gene as a selectable marker. This gene could be included at the transformation cassette; could be on a separate cassette introduced as a second locus via cotransformation; or could be an endogenous gene carried on one of the arms.

strategies for expression of multiple transgenes as an operon, since the rules by which ORFs should be assembled into an operon, and how the order of ORFs might affect their efficiency of translation, have yet to be determined. Finally, the synthetic approaches pioneered by Venter and colleagues have made possible the design and assembly of an entire Chlamydomonas chloroplast genome carrying specific gene replacements in which photosystem II genes were replaced with orthologs from the related alga, Scenedesmus obliquus. Construction of this designer genome was achieved by assembling the 204 kb genome as a circular molecule in yeast, amplifying the DNA in E. coli and transforming the Chlamydomonas chloroplast so enabling recombination with the endogenous genome and formation of a hybrid genome (O’Neill et al. 2012). It is envisaged that future work in this area will ultimately allow a complete re-engineering of the genome

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Fig. 13.2: Examples of biofuel molecules that might be produced in the algal chloroplast directly from the CO2 assimilated via photosynthesis. The green text shows organic compounds naturally produced in the chloroplast, and the blue text and structures show possible biofuels that could be produced by the expression of transgenes encoding novel enzymes. The minimum number of enzymes required for each product is illustrated by the blue arrows.

with unwanted repeat elements removed, endogenous genes modified and whole clusters of foreign genes introduced that encode new metabolic pathways for the synthesis of designer biofuels. Figure 13.2 illustrates possible biofuel molecules that could be synthesized in the chloroplast using the carbon fixed through photosynthesis. For example, it should be feasible to channel some of the intermediates of the isoprenoid biosynthetic pathway into isoprene (a C5 hydrocarbon) by the introduction of a gene for isoprene synthase.

13.2.3 Mitigating the risks and concerns of GM algae One of the concerns with GM organisms (GMOs) is their accidental or deliberate release into the open environment where they could propagate and possibly alter the ecology. The GM strains should therefore be free of undesirable markers such as the antibiotic resistance genes which are commonly used for selection after transformation, and which might be transferred to bacterial species by horizontal gene transfer. This issue should be taken into account from the onset in the design

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of the GMO, using one of several alternatives that are available. Markers endogenous to the species can be used for the selection of transformants, such as wildtype genes to rescue the corresponding mutants with defects in photosynthesis, amino-acid biosynthesis or other metabolic pathways. Another possibility is to remove the undesired marker from the genome of the initial transformant in a second step. This involves designing the marker with appropriate flanking sequences that will allow marker excision, either spontaneously driven by the recombination system of the host or directed by a site-specific recombinase (Day and Goldschmidt-Clermont 2011). In addition, two types of containment measures, physical or biological, can be taken to reduce the probability of unwanted release and propagation. The first consists of erecting physical barriers to prevent the release of the GMO. Closed photobioreactors for algal growth offer this type of physical containment. However, with large-scale cultures in open systems such as raceways, it will be difficult and costly to avoid releasing the cultivated algae into the environment. Measures of the second type rely on biological containment. Disabled strains with mutations affecting, for example the cell wall, the motility and/or the autotrophy of the host could be used. Because in algae such as Chlamydomonas, chloroplast DNA is inherited uniparentally from one mating type (mt+), the use of a strain of the opposite mating type as a host (mt–) will strongly reduce the probability that a chloroplast transgene will be transferred to a wild-type population.

13.3 Algal domestication through nuclear genetic engineering 13.3.1 Improving light to biomass conversion by regulation of the pigment optical density of algal cultures According to estimations, algae-based oil production per hectare would be up to 100-fold higher than that of soybean and could meet 50 % of present US transportation demand using less than 3 % of available cropland (Chisti 2008). These promising theoretical estimations assume a solar-to-biomass conversion efficiency in algae of 8–10 %, yielding a maximum productivity of 77 g biomass m–2 day–1 (280 ton ha–1 year–1) (Melis 2009). These estimations, based on lab scale measurements of algal cultures in light-limiting conditions, assume that all available photosynthetically active radiation (PAR) is absorbed and used in photosynthesis. However, real algal biomass productivities achieved so far in larger-scale PBRs do not exceed 73–146 ton dry weight ha–1 year–1 (20–40 g dry weight m–2 day–1) (Lee 1997; Borowitzka 2005; Huesemann et al. 2009) and 3 % of solar-to-biomass conversion efficiency (Melis 2009) in the best cases. These data enlighten an existent problem preventing exploitation of algae to their maximum potential. Currently, algae are mainly cultivated in open ponds, which are easier to construct and to scale up, and less expensive with respect to PBRs. Reasons for this drop in light-use efficiency are

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to be found in the radically different environmental conditions imposed to algae in PBRs, namely high light and high cell density, with respect to the natural environment where algae find the optimal condition for their growth by moving into a water column towards moderate to low light intensities (100–200 μM m–2 s–1). Indeed, in a PBR, a strong light gradient is established due to the high pigment content of cells and the high extinction, thus leading to optimal conditions for photosynthesis in a small fraction of the culture volume, intermediate between a surface layer where light is absorbed in excess and a more inner volume where light is below optimal, if any (Formighieri et al. 2012), thus limiting overall productivity. The stronger factor of energy dissipation is activated in the surface layer of the cell culture where excess light activates non-photochemical quenching (NPQ), a mechanism by which all photosynthetic organisms dissipate the energy absorbed in excess into heat with respect to their maximal capacity for rate of metabolic use. So far, solutions for this problem have been proposed, relying on the mixing of cultures, with a decrease in the photon flux absorbed. In fact, due to random dynamic, each individual cell experiences short periods of high light exposure, alternated with longer periods in low light or in the dark (see Section 13.4). Alternatively, mutations leading to a decrease in the size of photosynthetic antenna systems have been proposed to be useful in order to decrease the number of photons absorbed by each photosynthetic unit and concomitantly decrease optical density (Melis 2009). Nevertheless, rapid changes in light intensity cause photoinhibition and a decrease in light-use efficiency (Leakey et al. 2005) while depletion in lightharvesting antenna proteins impairs photoprotective mechanisms (Havaux et al. 2007; Ballottari et al. 2011) and decreases the time length of positive photosynthetic yield during the day (Formighieri et al. 2012), thus implying that these are not suitable solutions for improving algal productivity. Within the “Sunbiopaths” EEC project, we have followed the alternative strategy of searching for mutants with a reduced pigment per cell content and selecting for small chloroplasts rather than for low antenna size in order to avoid the above limitations and yet obtain a reduced steepness of light gradient within PBRs and yet maintain or increase resistance to photoinhibitory effect in order to counteract the effect of rapid light changes due to mixing. Upon insertion mutagenesis, we have selected three mutants with 50 %, 20 % and 8 % chlorophyll content per cell, respectively, called gun4, As2 and As1 (Bonente et al. 2011a, 2011b), that have been further characterized for their biochemical features (Formighieri et al. 2011, 2012) as well as for their productivity in small-scale PBRs. Experiments on a larger scale are presently ongoing. Results are encouraging, particularly for the As2 mutant which has shown 2× productivity in limiting light conditions and 5× in high light conditions with respect to WT (Bonente, Formighieri and Bassi, unpublished results). The comparative analysis of WT and mutants has underlined the critical importance of controlling NPQ-dependent energy dissipation for light-use efficiency. In particular, C. reinhardtii has been shown to behave hysteretically with respect to light intensity:

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in fact, the amplitude of energy dissipation is determined by the previous growth history of cells, through the level of accumulation of the LhcSR protein, the gene product responsible for NPQ (Peers et al. 2009; Bonente et al. 2012a). However, once LhcSR is accumulated in the thylakoid membrane, a low light intensity is sufficient for triggering NPQ at its maximal amplitude, leading to high levels of thermal dissipation even in moderate light (Bonente et al. 2012b). The above findings suggest that genetic manipulation of the LhcSR protein in order to decrease its reactivity to low pH could be a suitable strategy for decreasing light-energy dissipation into heat in algal cultures. Experiments in this sense are ongoing, and yet the practical interest of these manipulations need to be evaluated in the light of the results with As1 and As2 mutants showing reduced levels of NPQ in these low-pigment phenotypes. We conclude that engineering of pigment cell content is a promising strategy for improvement of light-use efficiency in PBRs and that this strategy is more likely to lead to improved biomass production with respect to the use of truncated antenna mutants.

13.4 Models for predicting growth in photobioreactors 13.4.1 PAM fluorimetry: a keyhole to look into the photosynthetic machinery The quantum efficiency of photosynthesis is the basis of biomass productivity of microalgal mass cultures. As is known from basic studies on the light response of photosynthesis, high photosynthetic quantum yield (around 1 mol of fixed CO2 per 11 mol of absorbed light quanta) only occurs in a limited range of local lightintensity values (usually below 200 μmol.m–2.s–1 PAR for most microalgae). Light saturation of photosynthesis often occurs in outdoor microalgal cultures and limits the efficiency of light usage for the buildup of biomass. Photosynthetic quantum yield is also affected by many factors, such as availability of CO2 and of nutrients, excess dissolved oxygen, presence of toxic substances and various stress factors. It is therefore valuable to monitor the photosynthetic performance of cultured microalgae in order to optimize productivity. PAM fluorimetry is a convenient and sensitive approach to assess how absorbed light quanta are used or dissipated during photosynthesis through measurements of the fluorescence yield of the Chl a associated with photosystem II (photosystem I being very poorly fluorescent at room temperature). Because the different pathways of excited Chl a deactivation (radiative and non-radiative) and reaction (charge separation) compete with each other, this fluorescence yield (F) is controlled primarily by two factors: (1) the quantum yield of electron transport through PSII, which is itself modulated by the redox state of the photosynthetic electron transport chain and (2) the quantum yield of thermal dissipation processes in this photosystem (by antenna or reaction center pigments). Any change in the efficiencies of these processes will be reflected in a corresponding change in F.

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Over the last 20 years, progress in opto-electronics has led to the development of compact “PAM” fluorimeters that are widely used in laboratory research, aquatic sciences and microalgal biotechnology. In PAM fluorimetry, a weak (“analytical’) modulated exciting light is used to monitor the changes in F caused by super-imposed photosynthetically active (“actinic’), continuous lights: an actinic light of tunable intensity (this can be either an in-build light source from the fluorimeter or the ambient light to which the algae are exposed) and a saturating light, given during a short pulse (typically 1 s) to transiently saturate photosynthesis. The use of a saturating light pulse allows for the determination of the reference fluorescence yield corresponding to photochemically inactive photosystems, in either the dark- or light-adapted state. For most applications, three basic parameters can be derived from fluorescence measurements; for measurement protocols and for the relationships between these parameters and fluorescence yield parameters, see Baker (2008): – ФPSII(dark), or ФPSII(max): the maximum PSII photochemical efficiency, measured in the dark-adapted state (usually after a dark-adaptation time in the order of 1 h). – ФPSII(light): the PSII photochemical efficiency, measured in the light-adapted state; this parameter can be evaluated under actinic lights of different intensities, or under the actual light to which microalgae are exposed for growth. Inasmuch as any electron transported through PSII is also transported through PSI in the linear electron transport chain, ФPSII(light) is an estimation of the linear electron transport yield at steady state. – NPQ, or non-photochemical quenching of PSII fluorescence: this parameter is related to the decrease in the fluorescence yield of photochemically inactive PSII (during a saturating light pulse) in the light-adapted state, compared to the dark-adapted state. An important milestone was passed when it was shown that, for higher plants, ФPSII(light) was linearly related to ФCO2, the quantum yield of CO2 fixation, when photorespiration was avoided (Genty et al. 1989). It then became possible to use simple fluorescence measurements to estimate the quantum yield of photosynthesis or at least its variations with experimental conditions. This was found to be true also for the green microalga Scenedesmus obliquus in phototrophic or mixotrophic conditions, in this case by estimating ФO2, the quantum yield of O2 evolution (Heinze et al. 1996). However, the photosynthetic electron transport chain of microalgae is endowed with great flexibility, due to the existence of auxiliary electron transfer pathways (Peltier et al. 2010), such as electron transfer to O2 at PSI (known as the Mehler reaction) or through PTOX (the plastidial oxidase) and cyclic electron transport pathways around PSI. Of these, electron transfer to O2 (most probably Mehler-type) has been found earlier to be very effective in the green microalga Chlamydomonas

13.4 Models for predicting growth in photobioreactors

239

Fig. 13.3: Relationship between ФO2 and ФPSII(light) measured simultaneously under actinic light of different PAR values (from 20 to 500 μmol.m–2.s–1) in C. reinhardtii. Cells were first cultivated in mineral medium in a laboratory photobioreactor under 300 μmol.m–2.s–1 irradiance either with air or with 5 % CO2 as sparging gas. Measurements were performed off-line using a combined PAM fluorimeter–Clark oxygen electrode system, using a Chl concentration of 5 μg.ml–1 in the presence of 20 mM NaHCO3. ФO2 is expressed relative to its maximal value under light-limiting conditions.

reinhardtii, as shown by isotopic measurements of O2 uptake in the light (Sueltemeyer et al. 1986). It follows that electron transport yield estimations, performed fluorimetrically (using ФPSII(light) as a basis), should not necessarily match photosynthetic yield but may include the yield of electron transport to O2 as sink, even in the absence of significant photorespiration (driven by Rubisco-catalyzed O2 uptake). This can be conveniently investigated through measurements of O2 evolution and fluorescence, as shown by the experiment depicted in Figure 13.3. In this experiment, ФPSII(light) and ФO2 were simultaneously measured at various PAR values in phototrophically grown Chlamydomonas reinhardtii using either air or 5 % CO2 as sparging gas during growth. Measurements were performed “off-line”, by transferring microalgal cells from a laboratory photobioreactor to a combined PAM-Clark electrode assembly. Linear relationships between ФPSII(light) and ФO2 were found in both conditions. However, only for cells grown under high CO2 did this relationship pass through the 0–0 origin, indicating a good match between electron transport and photosynthesis. For air-grown cells (“low-CO2” cells) the relationship deviated significantly from the ideal case and indicated lower ФO2 values than expected from ФPSII(light). This can be explained by electron transport to O2, at either PSI or PTOX in low-CO2 cells. These data can be used as a quantitative basis to estimate the fraction of electron transport flux that is directed to O2 in a specific condition. PAM fluorimetry, combined with oxymetry, shows here its potential for analyzing the adaptation of microalgae to limiting CO2 levels.

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13 Finding the bottleneck: A research strategy for improved biomass production

In line with the above example, PAM fluorimetry has become a recognized approach for understanding the regulation of electron transport in microalgae (Ralph et al. 2011). This extends to NPQ analysis, taken as an indicator of thermal dissipation of excess excitation energy. Large NPQ values were found for several algal species in outdoor cultures (Masojídek et al. 2004). It should be pointed out, however, that NPQ can be caused not only by thermal dissipation but also by changes in excitation energy distribution between the two photosystems, a process known as state transitions (Lemeille and Rochaix 2010). State-transition fluorescence quenching can be quite large in C. reinhardtii, compared to what is known for higher plants, and it is part of the photosynthetic response to fluctuations in the cell energy status. There is still much to investigate about the phenomenology of state transitions in microalgae in relation to cell physiology and energetics, which calls for detailed NPQ analysis in microalgal species of biotechnological interest. PAM fluorimetry could in principle be performed “on-line” by applying the branched fibre-optic guide of the fluorimeter against the photobioreactor (Lippemeier et al. 2001). However, for dense cultures such as those generally needed for high volumetric biomass productivity, light attenuation and fast mixing can make correct determination of maximal fluorescence yield difficult. Another promising application of chlorophyll fluorimetry resides in its use as a screening tool for the isolation of photosynthetic mutants. Set-ups are now available for imaging whole fluorescence induction curves from algal clones on Petri dishes during a dark-to-light transition. This approach has recently led to the isolation of Chlamydomonas mutants impaired in cyclic electron flow (Tolleter et al. 2011) in chlororespiration (Houille-Vernes et al. 2011) and NPQ (Bonente et al. 2011). By carefully setting the conditions under which this type of screening is performed, it should be possible in future to isolate new mutants with photosynthetic traits of interest for a better understanding of photosynthetic responses to a variety of conditions as well as for increased performance in biotechnological applications.

13.4.2 Microalgae cultivation in photobioreactors: the fluctuating light effects Productive microalgae cultures are characterized by full light attenuation along the optical path. In other words, light impinging the surface of algae cultures is completely absorbed along the optical path, which results in a light gradient and possibly a dark zone. The length of the dark zone depends on algae concentration, algae pigmentation, length of the optical path and photon flux density (PFD) at the surface. Algae cultures are mixed to ensure a sufficient mass transfer and to keep the algae homogenously suspended. Mixing results in a movement of the algae through the light gradient and the dark zone. The consequence of this movement is an

13.4 Models for predicting growth in photobioreactors

241

Fig. 13.4: Schematic description of light attenuation (PFD versus PBR depth (z)) and its influence on the specific growth rate μ. It is assumed algae respond to local PFDs (PFD(z)) resulting in local photosynthetic rates (PO2(z)). Based on dark respiration (Rd) local specific growth rates μ(z) can be calculated, which result in an average specific growth rate (μavg) for the whole PBR. Photosynthesis (O2 evolution) follows a saturation function with respect to photon flux density (PFD) according to the model of Webb et al. (1974). YC/O2represents the yield of carbon (C, i.e. biomass) on net oxygen evolved.

exposure to fluctuating light (light gradient) and light/dark cycles (alternation between light zone and dark zone). At the surface of a photobioreactor (PBR) or algae production pond, individual algae will be exposed to over-saturating sunlight intensities, whereas the PBR interior can be dark. Darkness is characterized by PFDs below the compensation point of photosynthesis, where gross photosynthesis matches dark respiration. The biological response to these L/D cycles is not fully understood, but general observations will be discussed in the following paragraphs. If it is assumed that algae photosynthesis responds to local PFDs then the local photosynthetic rate (PO2(z)) can be calculated for each layer in the PBR system. The photosynthetic rate depends only on the local PFD, and this dependency can be described by a saturation function (first equation; Fig. 13.4). The local photosynthetic rate can be converted to a local growth rate ( μ(z)), which depends on algae dark respiration rate and a carbon yield (i.e. biomass yield) on produced oxygen (YC/O2 ). The overall growth rate of the whole system ( μavg) can be determined by integrating the local growth rates ( μ(z)) over the whole light path (z) as shown schematically in Fig. 13.4. In this scenario, long exposure to darkness in the bottom layers will result in respiration of internally accumulated storage compounds, and that will lead to reduced PBR productivities. In addition, exposure to over-saturating PFDs at the PBR surface will lead to high PBR productivities but a low light-use efficiency because the light absorption rate is much greater than its maximal utilization rate. The excess absorbed light energy is dissipated as heat. Only a narrow range of PFDs will lead to optimal algae growth and ensure maximal light-use efficiency of the PBR system. PBR productivity in this scenario can be maximized by eliminating the dark zone (no zone with solely dark respiration) and adjusting the biomass concentra-

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13 Finding the bottleneck: A research strategy for improved biomass production

tion such that the last layer of the PBR system receives enough light for net photosynthesis. This response implies that the rate of mixing (and L/D cycling) does not influence the photosynthetic efficiency which can be reached. The optimal biomass concentration and volumetric productivity will decrease with increase in depth, but the areal productivity and overall photosynthetic efficiency of the whole PBR system will remain constant. However, from practice, it is known that a deep and poorly mixed system results in a lower areal productivity than more shallow and well-mixed systems (Norsker et al. 2011, supplement 1). The approach based on local rates is valid for a range of different PBRs with depths in the order of several centimeters (Cornet and Dussap 2009; Takache et al. 2010). In these PBRs, cycle times of cells moving from the PBR interior to the lightexposed surface and back are in the order of seconds. In raceway ponds, cycle times will be in the range of 10–100 s. These conditions lead to a reduction in productivity, which could explain the lower photosynthetic efficiency in raceway ponds in comparison with horizontal tubular PBRs. In addition, lab-scale experiments showed that the photosynthetic efficiency decreases under light/dark cycles in the order of seconds to tens of seconds (Grobbelaar et al. 1996; Janssen et al. 2000; Barbosa et al. 2003). These results could be related to the slow movement of the algae through the light gradient and the fact that they continuously try to acclimate to the local PFD, but acclimation lags behind changing light levels. If algae experience long dark periods, the pigment concentration and optical cross-section will increase, which might lead to photoinhibition due to the over-saturating PFDs at the light-exposed surface and decrease PBR productivity further. In contrast to long cycle time in algae production ponds or PBRs with a light path in the range of centimeters, very short cycle times can be achieved in optically thin PBRs (≤1 cm) if they are intensively mixed. Cycle times can reach tenths of a second, and photosynthetic efficiency is much higher than predicted based on local photosynthetic rates. Under these short cycle times, a phenomenon called “light integration” has been observed (Phillips and Myers 1954; Terry 1986; Richmond 2000). Light integration is based on the assumption that algae do not respond to the fluctuating light levels (PFD(z), Fig. 13.5) but experience a constant PFD equal to the average PFD (PFDavg) along the optical path (or time-averaged PFD based on one L/D cycle). Therefore, the over-saturating PFD impinging the PBR surface is diluted along the optical path to a sub-saturating PFD. A condition for light dilution is fast mixing in combination with a very dense culture (dark zone). As a avg consequence of light dilution, the rate of photosynthesis (PO2 ) and the specific growth rate ( μavg ) are constant over the whole reactor volume and depend solely on the spatial average light intensity (PFDavg; Fig. 13.5). In this case, algae will use light very efficiently because they experience optimal PFDs.

13.4 Models for predicting growth in photobioreactors

243

Fig. 13.5: Schematic description of light attenuation (PFD versus z) and its influence on the specific growth rate μ. It is assumed that algae respond to the spatial-averaged or time-averaged PFD avg (PFDavg ) resulting in an average photosynthetic rate PO2 . Based on dark respiration Rd, the average specific growth rate (μavg ) for the whole PBR can be calculated. Photosynthesis (O2 evolution) follows a saturation function with respect to photon flux density (PFD) according to the model of Webb et al. (1974). YC/O2 represents the yield of carbon (C, i.e. biomass) on net oxygen evolved.

Light integration could be explained by the existence of a temporary storage of electrons and energy released by photosynthesis (Vejrazka et al. 2011). The storage pool is reduced during exposure to over-saturating light at the light-exposed surface of the PBR and oxidized in the darker zones by metabolic reactions (i.e. growth). When the algae return to the light exposed surface, the pool is regenerated, and as a consequence, algae can accept a higher flux of electrons from the photosystems in comparison with continuous exposure to over-saturating light. The nature of such a pool is not clear but it is probably composed of the electron carriers of the photosynthetic electron transport chain and NADPH. Additionally, intermediates of the Calvin–Benson cycle and trans-thylakoid pH gradient could play an important role. Full light integration would be ideal for microalgae mass cultures, but it can probably only be partially reached in well-mixed and optically thin PBRs. Dedicated lab-scale experiments show that full light integration is observed under fast light/dark cycles of 14–24 Hz or more, provided that the dark period is substantially longer than the light period to reach a low time-averaged PFD (Phillips and Myers 1954; Kok 1956; Matthijs et al. 1996; Nedbal et al. 1996; Vejrazka et al. 2011). The highest frequency measured in a PBR was 25 Hz (Perner-Nochta and Posten 2007), but usually frequencies are in the range of 1–10 Hz (Luo et al. 2003, Moberg et al. 2011). Considering the short light flashes, the phenomenon of complete light integration is often referred to as the “flashing light effect”. Partial light integration can occur in optically thin PBRs, but there is still a lack of detailed insight in the dynamic response of microalgae and a need for quantitative and predictive models. For a PBR system with known cycle times, these models will help to determine the optimal size of a dark zone to maximize productivity and efficiency. Furthermore, PBR design can be optimized if the optimal configuration of optimal L/D cycles and mixing frequencies is known.

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13 Finding the bottleneck: A research strategy for improved biomass production

13.4.3 Standard model for growth under an exponential light gradient This model can be used to determine light gradients in photobioreactors in a particular plate reactor illuminated form one side. Especially the effect of different antenna-reduced microalgal mutants can be investigated in this system and modeled to compare the light penetration into deeper parts of the reactor with the characteristics of the corresponding wild type. Light attenuation is described by a correlation based on the Lambert–Beer Law. This is normally only applicable for absorption (in a cuvette), but similar behavior can be observed in a plate reactor with an infinite planar illuminated area where all the scattered light is absorbed finally: ε =

dI (l )

% I (l) = I0 ⋅ e

I (l)

−ε⋅l

Relevant for the kinetics of light absorption are the two following parameters: kI, the irradiation transition point between two linear parts of the kinetics; rX,max, the maximum specific growth rate. Then, the specific growth rate can be calculated according to: I⋅rX,max for I < kI kI rX (I) = rX,max for I ≥ kI

rX (I) =

The transition point from light saturation to limitation on the light path, lI, is calculated using I0 ⋅ e

−ε⋅lI

( )

k 1 = kI % lI = − ε ⋅ ln I I0

The calculation of rX as a function of the light path l results in rX (l) = rX,max for l < lI rX,max rX (l) = I0 ⋅ e −ε⋅l ⋅ for l ≥ lI kI Finally, the apparent specific growth rate as a volumetrically weighted average over rX is calculated according to: dcx d dt 1 ∫ rx(l) = ln e ⋅ dl ⋅ cx d 0

(

)

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13.4 Models for predicting growth in photobioreactors

This integral can be solved and results in five relevant solutions: rX,app = rX,max for l < lI For l ≥ lI the simplification of two terms of the solution (being positive and/or negative) (details not shown) leads to the following solutions:

( ( ((

rX,app = − ln ε − ln d + ln − Ei 1, −

rX,app = − ln ε − ln d + ln Ei 1, − −e

rX,max

(

)

rX,max⋅I0⋅e rX,max⋅I0 + Ei 1, − kI kI

rX,max⋅I0⋅e kI

))

−ε⋅d

) − Ei (1, − r

−ε⋅d

X,max )

)

⋅ ln (kI) + e rX,max ⋅ ln (I0)

(

(

rX,app = − ln ε − ln d + ln e rX,max ⋅ ε ⋅ d + Ei (1, − rX,max ) − Ei 1, −

)

rX,max⋅I0 kI

)

+ e rX,max ⋅ ln (kI) − e rX,max ⋅ ln (I0)

(

)

rX,app = − ln ε − ln d + ln e rX,max ⋅ ε ⋅ d

Ei is the exponential integral with the following definition: ∞

−k⋅z −a ⋅ k ⋅ dk Ei (a, z) = ∫ e 1

Exemplarily the results for calculation at I0 = 200, ε = 0.5 / 0.75 / 1, kI = 100, rX,max = 1 are shown in Figure 13.6 a, b and c. Figure 13.6 d illustrates the dependence of apparent growth rate in the whole reactor volume from the absorption characteristics of the suspension for a given reactor geometry (light path 2/5/10 cm, single side illumination). It is obvious that for a given reactor geometry (light path length), the light gradient becomes more pronounced for increasing values of ε (Fig. 13.6 a). This could be the case for example when using a wild-type microalgae stain compared to an antenna-reduced mutant. These appear less optically thick at equal cell densities due to a reduced absorption coefficient. For measured data of absorption (normalized to applied irradiation) for different mutant strains compared to the wild type, see Fig. 13.7. Based on the growth kinetics (Fig. 13.6 b) of the microalgae, the growth rate depending on the position on the light path can be calculated (Fig. 13.6 c). As expected, the same behavior is observed for variations of ε. Strains with lower absorption coefficients show maximum growth rate into deeper layers of the reactor from the illuminated surface.

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13 Finding the bottleneck: A research strategy for improved biomass production

Fig. 13.6: Calculation example for I0 = 200, ε = 0.5 / 0.75 / 1, kI = 100, rX,max = 1; (a) shows the decline of light intensity [μE/m²/s] depending on the light path [cm], (b) displays the growth kinetic (growth rate depending on light intensity), and (c) visualizes the growth rate depending on the light path. Finally, (d) illustrates the dependence of apparent growth rate in the whole reactor volume from the absorption characteristics of the suspension for a given reactor geometry (light path 2/5/10 cm, single side illumination).

Figure 13.6 d finally shows the calculated apparent growth rate in the whole reactor volume for a given reactor geometry (in this case, with light path 2/5/10 cm, single side illumination) depending on the absorption coefficient of the suspension. Using these data, it is possible to select the light path of the reactor for maximizing efficiency (= maximum apparent growth rate) for a microalgal strain with specific absorption characteristics (vertical lines in diagram). The graph also enables a comparison of the performance of a given reactor geometry for cultivation of algae stains with different absorption characteristics, e.g. in a plate reactor with a 2 cm layer thickness, the apparent growth rate will reach values of 0.8 for ε = 0.8 compared to 0.9 for ε = 0.6.

13.5 Cells’ response to changing environments: the example of nitrogen limitation

247

Fig. 13.7: Absorption characteristics (normalized to the applied irradiation) for a Chlamydomonas wild type and three different antenna-reduced mutants depending on dry biomass.

13.5 Cells’ response to changing environments: the example of nitrogen limitation With the growing interest in microalgae as a source for renewable biofuel production (Wijffels and Barbosa 2010), macronutrient-limited growth conditions are becoming commonly used to boost storage of reduced carbon in starch granules or lipid droplets. Indeed, any stress treatment that compromises cell division without altering the intracellular production of reduced carbon ends up in carbon storage in either of these two macromolecular bodies. Most of the oil storage involves neutral lipids, namely triacylglycerol (TAG) (Hu et al. 2008), whose accumulation in nitrogen-starved cells of Chlamydomonas was reported to be further increased upon a block in starch synthesis (Wang et al. 2009; Work et al. 2010), although the view that the two pathways compete for storage of reduced carbon has been challenged recently (Siaut et al. 2011). Transcript profiling studies demonstrated that an extensive remodeling in gene expression underlies these metabolic changes in N-starved Chlamydomonas (Miller et al. 2010; Merchant et al. 2011). These geneexpression changes reflect the gametic differentiation program and the metabolic diversion previously established from genetic and cell-biology studies. Quite surprisingly, the bioenergetics of nitrogen-starved cells has received less attention, although a decrease in the photosynthetic ability of Chlamydomonas to reduce carbon when starved in nitrogen sources should have critical consequences for its use as a biofuel generator. In an earlier work (Bulté and Wollman 1992), we had shown that photosynthetic competence was lost upon nitrogen starvation when

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13 Finding the bottleneck: A research strategy for improved biomass production

using a common laboratory strain of Chlamydomonas, strain 137c, that requires ammonium for growth, being unable to use nitrate/nitrite as nitrogen sources because of a mutation in nit1/nit2 genes. We then showed that, after transfer to an ammonium-free medium containing acetate for about 30 h, the Chlamydomonas fluorescence pattern converted to a typical cytochrome b6 f mutant pattern: when dark-adapted cells were subjected to a continuous illumination, their fluorescence yield increased steadily to a maximum level close to the value attained when using DCMU, an inhibitor of photosynthetic electron flow. However, the kinetics of this rise matched those observed upon a block in electron transfer at the level of cytochrome b6 f complexes. Probing the protein content of the thylakoid membranes from N-starved cells confirmed the specific loss of all subunits of the cytochrome b6 f complexes, due to an active degradation process (Majeran et al. 2000), whereas the content in PSI, PSII and ATP synthase remained unaltered. We subsequently identified a number of auxiliary proteins that are specifically required for the biogenesis of the cytochrome b6 f complex. These include transacting factors of nuclear origin that are required for post-transcriptional expression of chloroplast genes encoding subunits of the cytochrome b6 f complex (Wostrikoff et al. 2001; Boulouis et al. 2011) as well as proteins required for heme attachment to either of its two cytochrome subunits (Xie et al. 1998; Kuras et al. 2007). Recently we examined the fate of these proteins upon nitrogen starvation and concluded that most of them were degraded along with the cytochrome b6 f subunits. The same proteolytic systems, combining the stromal protease Clp and the transmembrane protease FtsH, control the degradation of these various subsets of proteins. Moreover, their loss is subjected to a common regulation that is sensitive to the intracellular carbon flux: when starved in nitrogen sources but placed in phototrophic (no acetate added) or anaerobic conditions (no consumption of reduced carbon by mitochondrial respiration), Chlamydomonas cells retained their photosynthetic competence, and the level of all cytochrome b6 f related proteins remained unaltered. Although these studies aimed at a better understanding of protein remodeling in a changing environment – an issue seemingly unrelated to the issue of biofuel production – they provide useful knowledge for devising a robust TGA generator. As described in previous studies (Merchant et al. 2011; Msanne et al. 2012), both photoheterotrophically and phototrophically grown cells placed in nitrogen-free conditions produce TAG. However, it is apparent from our studies on the degradation of cytochrome b6 f-related proteins induced by nitrogen starvation that mixotrophic conditions should be less suitable for a robust TAG production, because of the reduced carbon fixation capability owing to the degradation of cytochrome b6 f. Still, engineering a robust TAG generator from nitrogen-starved cells of Chlamydomonas should require a better control of another degradation process that develops in the chloroplast stroma during nitrogen deprivation, that of the RuBisCO enzyme itself!

References

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Acknowledgments Research presented in this chapter is funded by a FP7 grant, Sunbiopath, KBBE 2009-3 (GA245070).

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Gibson, D. G. 2011. Enzymatic assembly of overlapping DNA fragments. Methods Enzymol. 498: 349–61. Guiry, M. D. and G. M. Guiry. 2012. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org. Grobbelaar, J. U., L. Nedbal and V. Tichy. 1996. Influence of high frequency light/dark fluctuations on photosynthetic characteristics of microalgae photoacclimated to different light intensities and implications for mass algal cultivation. J. Appl. Phycol. 8: 335–343. Havaux, M., L. Dall’Osto and R. Bassi. 2007. Zeaxanthin has Enhanced Antioxidant Capacity with Respect to All Other Xanthophylls in Arabidopsis Leaves and functions independent of binding to PSII antennae. Plant Physiol. 145: 1506–1520. Heinze, I., H. Dau and H. Senger. 1996. The relationship between the photochemical yield and variable fluorescence of photosystem II in the green alga Scenedesmus obliquus. J. Photochem. Photobiol. B. Biol. 32: 89–95. Houille-Vernes, L., F. Rappaport, F.-A. Wollman, J. Alric and X. Johnson. 2011. Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas. Proc. Natl. Acad. Sci. USA. 108: 20820–20825. Hu, Q., M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert and A. Darzins. 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54(4): 621–39. Huesemann, M. H., T. S. Hausmann, R. Bartha, M. Aksoy, J. C. Weissman and J. R. Benemann. 2009. Biomass Productivities in Wild Type and Pigment Mutant of Cyclotella sp (Diatom). Appl. Biochem. Biotechnol. 157: 507–526. Janssen, M., M. Janssen, M. de Winter, J. Tramper, L. R. Mur, J. Snel and R. H. Wijffels. 2000. Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/ dark cycles. J. Biotechnol. 78: 123–137. Kato, K., T. Marui, S. Kasai and A. Shinmyo. 2007. Artificial control of transgene expression in Chlamydomonas reinhardtii chloroplast using the lac regulation system from Escherichia coli. J. Biosci. Bioeng. 104: 207–213. Kok, B. 1956. Photosynthesis in flashing light. Biochim. Biophys. Acta 21: 245–258. Kuras, R., D. Saint-Marcoux, F.-A. Wollman and C. de Vitry. 2007. A specific c-type cytochrome maturation system is required for oxygenic photosynthesis. Proc Natl Acad Sci USA. 104: 9906–9910 Leakey, A. D., J. D. Scholes and M. C. Press. 2005. Physiological and ecological significance of sunflecks for dipterocarp seedlings. J. Exp. Bot. 56: 469–82. Lee,Y.K. 1997. Commercial production of microalgae in the Asia-Pacific rim. J. Appl. Phycol. 9: 403–411. Lemeille, S. and J.-D. Rochaix. 2010. State transitions at the crossroad of signaling pathways. Photosynth. Res. 106: 33–46 Lippemeier, S., R. Hintze, K. H. Vanselow, P. Hartig and F. Colijn. 2001. In-line recording of PAM fluorescence of phytoplankton cultures as a new tool for studying effects of fluctuating nutrient supply on photosynthesis. Eur. J. Phycol. 36: 89–100 Luo, H. P., A. Kemoun, M. H. Al-Dahhan, J. M. F. Sevilla, J. L. G. Sanchez, F. G. Camacho and E. M. Grima. 2003. Analysis of photobioreactors for culturing high-value microalgae and cyanobacteria via an advanced diagnostic technique: CARPT. Chem. Engineering Science 58: 2519–2527. Majeran, W., F. -A. Wollman and O. Vallon. 2000. Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex. Plant Cell 12: 137–150. Masojídek, J., J. Kopecký, M. Koblížek and G. Torzillo. 2004. The xanthophyll cycle in green algae (Chlorophyta): its role in the photosynthetic apparatus. Plant Biol. 6: 342–349

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Vítor Verdelho, Ana P. Carvalho, Diana Fonseca and João Navalho

14 Trends driving microalgae-based fuels into economical production 14.1 Introduction From an economic point of view, microalgae can be seen as microorganisms with the ability to convert solar energy into valuable products, at the expense of (theoretically) low-cost resources: with an input of ca. 3,000 kg CO2, 400–500 kg of nitrates and 40–50 kg of phosphates, microalgae are able to deliver outputs of 1,000 kg of biomass and 2,000 kg of O2. This biomass contains 17–71 % proteins, 4–40 % lipids and 5–57 % carbohydrates (Cohen 1999; Richmond 2004; Andersen 2005). The idea of large-scale production of microalgae first occurred during World War II in an attempt to derive inexpensive sources of proteins so as to replace those of animal origin that were difficult to obtain; subsequently, the changes brought about by the global economy, gave rise to other targets for microalgae production from wastewater treatment to the production of fine chemicals, among others (Carvalho et al. 2006). By 2010/2011, the global production of microalgae biomass was ca. 17,000 tons/year, of which ca. 15,000 tons was sold as dried biomass, mainly for health food and cosmetic industries, and the remaining ca. 2,000 tons commercialized as refrigerated paste or frozen for aquaculture (Global Market 2010, unpublished data from Necton, S.A.). Until recently, microalgae species with commercial value were limited to Spirulina, Chlorella, Dunaliella and Haematococcus strains, used essentially in feed, food health and cosmetic industries. This positioning is about to change. In recent years, due to increasing constraints associated with the first generation of biofuel sources, microalgae biotechnologies have been identified as a prime opportunity for future bioenergy solutions, and therefore different species and algal utilizations are expected to enter the commercial markets (Tab. 14.1). To become economically profitable, microalgal production must achieve maximum productivity with minimum operation costs. Therefore, one major challenge of industrial microalgae production is the devising and development of technical equipment, cultivation procedures and algal strains susceptible of undergoing substantial increases with an efficient usage of solar energy and carbon dioxide. Despite several research efforts developed so far, there is no such thing as “the best system conditions”. In fact, the choice of “the most suitable system” is situationdependent, as the species of algae available and the intended final purpose both play a role.

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Microalga

Average price/dry biomass (€/kg)

Annual production volume (tons)

Spirulina sp. Chlorella sp. Dunaliella sp. Haematococcus sp. Other species Total

5–50 10–60 60–100 150–340

5,900–7,000 2,700–3,000 1,000–1,600 280–350 2,500–3,000 12,500–15,000

Tab. 14.1: Microalgal species with commercial significance, average price and production volume (dry weight).

Based on our experience and knowledge in the field of microalgae biotechnologies during the last 20 years, we give here an insight of several emerging strategies able to support the economic production of microalgae-based fuels, comprehensively addressed and discussed, presenting commercial applications as examples. These comprise (1) microalgae biorefinery for food, feed, fertilizer and energy production; (2) biofuel production from low-cost microalgae grown in wastewater; (3) biogas upgrading with microalgae culture for production of electricity; (4) hydrocarbon milking of modified Botryococcus microalgae strains; (5) hydrogen production combining direct and indirect microalgae biophotolysis; and (6) direct ethanol production from autotrophic cyanobacteria. Furthermore, existing production platforms (oceans, lakes, raceways/ponds, photobioreactors (PBRs) and fermenters) are also addressed, as not every platform can be used for all of the emerging technologies.

14.2 Leading trends Today, the main biofuels used on a worldwide scale include bioethanol, biodiesel and biogas. The usage of microalgae to produce such resources will be addressed here. From a wide range of possibilities, six technologies are emerging as the main drivers for the microalgal biotechnology sector. Their key characteristics, necessary innovations and examples are summarized in Table 14.2 and detailed in the following text.

14.2.1 Microalgae biorefinery for food, feed, fertilizer and energy production Microalgae, as one of the most productive and sustainable sources of raw materials, can be transformed into a variety of products. A broad list of applications of microalgae cultures has been widely described and discussed in the literature. Those that have commercial significance include the areas of health foods, food additives, pigments, diets for aquaculture, growth-regulating agents, secondary metabolites and wastewater treatments. The production of several bioactive com-

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pounds such as hydrocarbons, isotopes, polysaccharides and antifungal, antitumor, antibacterial and antiviral substances is currently under study; uses of microalgae for CO2 fixation, removal of nitric oxide from flue gas, fuel production, recovery of heavy metals from effluents and outer-space technologies are also on the agenda. The majority of the mature industrial microalgae producers are focused on a single product application for the biomass, which has a high market value. For products with a lower market value, such as biofuels, protein or animal feed, the economic viability of the process is achieved only within the logic of a biorefinery, meaning that all its secondary products or biomass residues are leveraged and sold to the market. The production of biofuel, apart from responding to the growing demand of its usage as a replacement of traditional petroleum-based fuels, puts to use its residual biomass by-products from algae oil processing as an alternative high-grade protein source for aquaculture and livestock feed. On top of it, algae can also be cultivated to serve many additional commercial and industrial uses, including the production of health and nutrition products, as well as bio-based materials. Economical constrains of biofuel production can be significantly reduced by using a biorefinery-based production strategy, where the biomass produced and its components can be used as raw material of other useful products. For example, in addition to biofuel production, other valuable products such as eicosapentaenoic acid (EPA) and carotenoids can also be obtained. Furthermore, such an approach also combines the maximization of economic and environmental benefits, thus decreasing waste and pollution. A possible example of this concept is Cellana, a company using marine microalgae to produce feedstock for biofuels, skin and personal care products, aquaculture and livestock feeds, nutritional oils and renewable chemicals, while simultaneously reducing industrial emissions of CO2 (Cellana, Inc. 2012).

14.2.2 Biofuel production from low-cost microalgae grown in wastewater Microalgae appear to be an interesting source of biofuel feedstock because, among other parameters, they can grow in conditions that require a minimal freshwater input and utilize non-productive land to crop plants, therefore avoiding competition for resources. In fact, they can grow in salty, brackish or even wastewaters derived from municipal, agricultural and industrial activities (Pittman et al. 2011). These wastes are generally characterized by high levels of N and P, which promote eutrophication of rivers and lakes if discharged without treatment. Microalgae have been shown to be particularly tolerant to many wastewater conditions, removing large amounts of N and P, as they require high amounts of these nutrients for protein, nucleic acid and phospholipid synthesis (Pittman et al. 2011; Rawat et al. 2011). The necessary CO2 may be provided from nearby industries, at low cost.

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The usage of microalgae for treatment of municipal wastewater has been under study for more than 40 years, although it has been used together with physical and chemical removal methods. By simultaneously supplying an effective and cheap growth medium for microalgal biomass production, as well as providing sewage treatment in a low-cost and environmentally friendly procedure, the use of wastewater resources may thus offer a viable means to increase the sustainability of algal biofuel production. The biomass and lipid productivities described in many of the reviewed studies suggest that there is a strong potential for the utilization of this nutrient resource, and therefore this dual cultivation method offers a real potential as a viable approach for the production of sustainable and renewable energy. Furthermore, because much of the infrastructure is already in place, the synergy of wastewater treatment and biofuel production from microalgae may already be experienced. One example of wastewater treatment with microalgae is the Algaewheel system (OneWater Inc. 2012), which provides conventional biological wastewater treatment as well as advanced nutrient removal utilizing algae. In this technology, the wheel is designed to be significantly buoyant in water, requiring no mechanical drive mechanism and rotated using a constant airflow. Functionally, the wheel offers a suitable environment where bacteria and algae work in a symbiotic fashion to synthesize living organic mass efficiently from the nutrients in a variety of wastewaters. The algae supply the oxygen required by the bacteria, and likewise the bacteria supply the carbon dioxide required by the algae. Thus, an ecological balance is established, making the system very stable and resistant to the fluctuations normally experienced with bacteria-based wastewater treatment systems. Furthermore, oxygen produced by algae through photosynthesis replaces the need for the costly mechanical oxidation of wastewater. In addition to ecological balance, this system has several other advantages, such as elimination of greenhouse-gas emissions, and nitrogen and phosphorus removal, all at a low cost. Another example is wastewater treatment, CO2 scrubbing and biofuel production with microalgae, using enhanced ponds. This was the world’s first large-scale demonstration of both wastewater treatment in high-rate algal ponds (HRAP) with CO2 addition and super critical water reactor (SCWR) algal bio-oil conversion. The project was intended to demonstrate the large-scale construction, operation and performance of the technologies mentioned and provides an in-depth life-cycle economic analysis of the complete process pathway (NIWA 2012). However, several challenges still need to be solved, such as lowering the cost of harvesting, and harvesting in a way that allows for the production of bio-products, i.e. harvesting without using chemical coagulants. One possible solution may involve harvesting with pH-change flocculation-sedimentation or through specific electrical modulations (OriginOil, Inc. 2012). Another solution involves eliminating the harvesting step, which may be pursued by using biphasic systems (Hejazi et al. 2002).

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14.2.3 Biogas upgrading with microalgae production for production of electricity Due to resource depletion, lipid-based algal biofuels have been indicated as an interesting alternative, mainly because of the high productivity of algae per hectare and per year, as well as their ability to recycle CO2 from flue gas. However, as has been recently shown in life-cycle assessment (LCA) or energy analyses, the need for high amounts of N and P (fertilizers), the harvesting of large volumes of diluted cultures and the oil-extraction processes represent a high cost, which might surpass overall interest in algal biofuel (Collet et al. 2011). In fact, harvesting costs may account for 20–30 % of the total production cost, exceeding 50 % when combined with oil extraction (Molina Grima et al. 2003). It is therefore worth investigating other transformation processes that do not include the extraction step; a possible solution would be to carry out direct anaerobic digestion (AD) of raw harvested algae, to produce biogas (a mixture of methane and CO2): the biomethane produced can be burned to generate electricity or heat, whereas CO2 may be reintroduced into microalgal autotrophic cultures. The remaining biomass can be used for fiber or fertilizer, or re-introduced in anaerobic digestion fermentation for biogas. The anaerobic digestion process is a well-known technology, widely used in the treatment of concentrated polluted streams as distillery or piggery effluents. As it bypasses the harvesting/concentration and oil-extraction steps, it could significantly reduce costs and the total energy debt. Usually, the biogas produced by anaerobic digesters and landfills is used locally, via co-generators or boilers. However, it can also be enriched to become a fuel used by internal combustion engines (e.g. the buses in Lille, France or Linköping, Sweden) (Börjesson and Mattiasson 2006). Schmack Biogas AG developed studies on a new process to purify biogas, using microalgae. The process was presented as an economic and sustainable one and involves feeding raw biogas to algae, which scrub out the CO2 and other impurities during their photosynthetic activity and growth. The company carried out three successful tests using three different algae species in photobioreactors (FNR 2012).

14.2.4 Hydrocarbon milking of modified Botryococcus microalgae strains Among the costly downstream processing steps, it is agreed that harvesting/dewatering and the following extraction of fuel precursors from the biomass are the most energy-demanding steps (Radakovits et al. 2010). In order to decrease their cost, the possibility of integrating the steps of harvesting and extraction seems to be a potential solution. In this integrated process, algal cells can be cultured in biphasic bioreactors, consisting of (1) an aqueous phase, in which algae grow, and (2) an organic phase, immiscible with the former, which continuously extracts the lipids. This type of in situ extraction process, also named “milking”, has been

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14 Trends driving microalgae-based fuels into economical production

found to be successful in the pigment production from algae (Leon et al. 2001; Hejazi and Wijffels 2004; Mojaat et al. 2007). Botryococcus braunii is one of the most promising algal species for lipid milking due to its high lipid content (25–75 % dry weight biomass) (Mata et al. 2010). Algal lipid of Botryococcus braunii could be produced continuously and extracted in situ in an aqueous-organic bioreactor. In a study by Zhang et al. (2011), the cell ultra-structure and cell membrane permeability of B. braunii FACHB 357 were investigated in order to understand the mechanism of lipid extraction within the biphasic system. Results showed that the biocompatible solvent tetradecane could induce algal lipid accumulation and increase cell membrane activity. On August 2011, IHI Corporation (IHI), Gene and Gene Technology Y. K. (GGT) and Neo-Morgan Laboratory Incorporated (NML) have announced a joint venture company to research and develop the technology for biofuel production using Botryococcus braunii (IHI Corp. 2012).

14.2.5 Hydrogen production combining direct and indirect microalgae biophotolysis Hydrogen gas is considered as a future energy carrier because it is renewable, does not evolve CO2 in combustion and provides large amounts of energy per unit weight in combustion. Hydrogen may be produced by several processes, such as electrolysis of water, thermocatalytic reformation of hydrogen-rich organic compounds and biological processes. Currently, hydrogen is produced almost exclusively by electrolysis of water or steam reformation of methane. The technologies to produce biological hydrogen provide a wide range of approaches, including direct biophotolysis, indirect biophotolysis, photo-fermentations, and dark fermentation. Biological hydrogen production has several advantages over hydrogen production by photoelectrochemical or thermochemical processes, e.g. biological hydrogen production by photosynthetic microorganisms requires the use of a simple solar reactor such as a transparent closed box, with low energy requirements, whereas electrochemical hydrogen production via solar battery-based water splitting requires the use of solar batteries with high energy requirements (Levin et al. 2004). Photobiological production of hydrogen by photosynthetic microorganisms is of interest due to the promise of generating clean carbon-free renewable energy from abundant natural resources, such as sunlight and water. Photobiological hydrogen production has advanced significantly in recent years, and nowadays a variety of photosynthetic and non-photosynthetic microorganisms, including unicellular green algae, cyanobacteria, anoxygenic photosynthetic bacteria, obligate anaerobic and nitrogen-fixing bacteria, are endowed with genes and proteins for H2 production (Eroglu and Melis 2011). The possibility of using cyanobacteria for the production of molecular hydrogen has been the subject of several reviews (Levin et al. 2004; Tamagnini et al.

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259

2007). These organisms can use two natural pathways for H2 production: (1) H2 production as a by-product during nitrogen fixation by nitrogenases; and (2) H2 production directly by bidirectional hydrogenase (Angermayr et al. 2009). Integration of hydrogen production among these organisms and enzymatic systems is a recent concept and a rather interesting development, as it may minimize feedstock utilization and lower the associated costs while improving yields of hydrogen production. PBR development and genetic manipulation of the hydrogenproducing microorganisms are also key parameters to improvement in the yield of the overall process. OriginOil Inc. (2012) developed its algae extraction concept with its Hydrogen Harvester™, a continuous, passive extraction system for removing hydrogen gas from algae culture. The process uses viable, high-growth, high-oil-content algae strains in a photosynthetic technology platform. In contrast to previously reported developments in the area, the new Hydrogen Harvester uses little to no external energy inputs, requires no sulfur deprivation or other “stressing” of the algae, and requires no genetic modification.

14.2.6 Direct ethanol production from autotrophic cyanobacteria Liquid fuels derived from biomass represent one sustainable option for alternative sources of energy, and among these, corn-based ethanol has led the way in providing a source of first-generation biofuel. However, in recent years, corn-based ethanol has received criticism for its reliance on fossil fuel. Cyanobacteria and algae are able to produce glucose and sucrose, which in turn can be converted to ethanol by anaerobic fermentation under dark conditions. Advances in the development of genetic tools to increase energy production in microalgae and cyanobacteria have recently been achieved, and are being used to manipulate central carbon metabolism in these organisms. Manipulation of metabolite pathways can redirect cellular functions towards the synthesis of preferred products. It is likely that many of these advances can be extended to industrially relevant organisms, in order to improve cyanobacteria/microalgae as a biofuel platform for the production of bioenergy. Photosynthetic cyanobacteria can be redesigned for highly efficient ethanol production by the combination of gene transformation, strain/process development and metabolic modeling/profiling analysis. Dexter and Fu (2009) have transformed pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) genes from Zymomonas mobilis into Synechocystis sp. PCC 6803. This strain can convert CO2 phototrophically to ethanol. In another example, Lan and co-workers were able to modify the metabolic engineering of cyanobacteria (Synechococcus elongatus PCC 7942) for 1-butanol production from CO2 (Lan and Liao 2011). Advances in the genetic manipulation of metabolic networks will generate a platform of knowledge able to be applied in the production of numerous valuable compounds

Hydrocarbon milking of modified Botryococcus microalgae strains

Key characteristics

System integration and microalgae inoculation procedures

System integration and scale-up

System integration and separation of O2 from the culture

Biomass can be used for fiber, fertilizer or re-introduced in AD fermentation for biogas

PBRs used for inoculation of ponds

location must be close to large industrial units as power-plants or oil refineries

GMO strains and culture media optimization in advanced photobioreactors

Hydrogen production combining direct and indirect microalgae biophotolysis

GMO efficient and fast growth strains

System integration and optimization

Ethanol membrane separation with pervaporation technologies

GMO strains and culture media optimization in advanced photobioreactors

Direct ethanol production from autotrophic cyanobacteria

GMO efficient strains and H2 separation process

GMO efficient strains and cost-effective separation of ethanol

System integration and System integration and optimization optimization

Botryocene separation and Membrane separation biomass harvesting technol- technologies for O2 ogies and H2

Use of biogas from landfills GMO strains and culture and other AD waste media optimization in fermentation advanced photobioreactors

Biomethane is burned to produce electricity or heat and CO2 reintroduced into microalgal culture

Use of existing infrastructures, sources of water and nutrients

High-quality biomass is fractionated into proteins, lipids, carbohydrates and other products

Biogas upgrading with microalgae for (carbon neutral low cost) production of electricity

5–10 % photobiore- CO2 piped from actors and 90– nearby industries 95 % open systems as raceways or cascade systems

Biofuel production from low-cost microalgae grown in wastewater

Microalgae biorefinery for food, feed, fertilizer and energy production

260 14 Trends driving microalgae-based fuels into economical production

Key innovations

Industrial examples

http://onewater works.com/ index.html www.niwa.co.nz

http://www.fnr-server.de/ ftp/pdf/berichte/ 22017105.pdf http://www.genifuel.com

http://www.ihi.co.jp/en/ press/2011/2011-8-01/ index.html http://www.originoil.com/ technology/overview.html

Tab. 14.2: Key characteristics, necessary innovations and examples for each trend.

http://cellana.com http://ispt.eu/ news_and_press/ news/ ISPTandAlgaePARC partnersstartnew projectonAlgae biorefinery

http://www. originoil.com http://sgth2.com/

http://www. algenolbiofuels.com http:// www.biofields.com/ index.php?lang=en& lang2=es

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(Rosenberg et al. 2008). The development of a number of transgenic strains with recombinant protein expression, engineered photosynthesis and enhanced metabolism, strengthens the prospects of using modified cyanobacteria for biofuel generation in the near future. Founded in 2006, Algenol Biofuels Inc. (2012) uses strains of algae or cyanobacteria capable of enhanced production of ethanol (Christenson and Sims 2011).

14.3 Production platforms Production platforms are of utmost importance in microalgal production, because their shape and operational conditions dictate the final productivity rates achieved. The choice of the most suitable platform is case-dependent, as well as the production mode. In general, available platforms can be divided between (1) those operating in heterotrophic growth conditions – fermenters and photobioreactors (when operated mixotrophically), always closed, and (2) those used for autotrophic growth, which can have either open (lakes, raceways/ponds) or closed (PBRs and offshore membrane enclosures) configurations. Mixed growth modes are also used. Their ranking in terms of size and (general) operating complexity is depicted in Figure 14.1.

Fig. 14.1: Relationships between dimension and (general) complexity of existing production platforms.

Commercial (or experimental) examples of each of the existing configurations are presented below:

14.3.1 Ocean Offshore Membrane Enclosure for Growing Algae (OMEGA) is a concept project under development by NASA’s Aeronautics Research Mission Directorate and California Energy Commission, aiming to demonstrate the viability and scalability of producing large amounts of algae for carbon-neutral biofuels, foods, fertilizers and

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other valuable products, on the one hand, and treat wastewaters and sequester CO2 on the other, without competing with traditional farming by land, freshwater or fertilizers. Large amounts of algae-producing units, composed of floating bags in the sea, would sequester CO2 and use seawater to grow (NASA 2012).

14.3.2 Lakes The technology to cultivate microalgae using natural lakes was first developed in the 1970s and is still in use. One such example can be found in Cognis, which produces natural β-carotene and other carotenoids in 400 ha of salt lakes in Australia (BASF/Cognis GmbH).

14.3.3 Raceways In 1976, the parent company of Earthrise, Proteus Corporation, was founded to develop Spirulina blue-green algae as a world food resource. Proteus began cultivation in the hot desert area in the southeastern part of California in the late 1970s. Earthrise® developed a partnership with a Japanese company, Dainippon Ink, and Chemicals, Inc. (DIC), a global, diversified chemical company with a commitment to developing microalgae for food, biochemicals and pharmaceuticals (Earthrise Nutritionals, LLC 2012).

14.3.4 Photobioreactors Algatech was founded in 1999 to develop and commercialize astaxanthin and other microalgae-derived products for the nutraceutical and cosmeceutical industry. Astaxanthin is an important naturally occurring molecule and the most abundant carotenoid in the marine world (Algatechnologies,Inc. 2012).

14.3.5 Fermenters Martek Biosciences Corporation, now acquired by DSM, developed and patented two fermentable strains of microalgae, which produce oils rich in docosahexaenoic acid (DHA) in fermenters that range in size from 80,000 to 260,000 liters (DSM/ Martek Biosciences Corp. 2012).

14.4 Conclusions At present, the available options for microalgal cultivation may be classified according to (1) the production platform used (oceans, lakes, ponds/raceways,

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Fig. 14.2: Available options for microalgal cultivation and possible combinations.

PBRs and fermenters), (2) the nutrition mode (autotrophic, heterotrophic and mixotrophic) and (3) the types of organisms (wild, from those prevailing in the region; selected, from microalgal collections; and genetically modified). Within these three main options (summarized as where to grow, how to grow and what to grow), there are many possibilities and combinations (Fig. 14.2). There is no optimal unique solution for microalgal-based biofuel production. However, several approaches have been developed by researchers, by using alternative cheaper nutrients (e.g. wastewaters), developing the production of other fuels than ethanol or biodiesel (e.g. biogas or biohydrogen), improving technologies in order to bypass the costly harvesting and extraction steps (by directly “milking” the lipids from algae or directly producing ethanol) and operating in a general overview concept of biorefinery. Integrated systems, with several technological options combined in the same operation unit, may provide increases in productivity and reduction in costs. Examples are the utilization of photobioreactors for inoculation (5–10 % of total production volume) and open systems as raceways, ponds or cascade systems (90–95 % of total production volume). These system configurations must always be adapted to the microalgae species cultivated, final market features and production unit location.

References Algatechnologies, Ltd. 2012. www.algatech.com, accessed on 2012.04.20. Algenol Biofuels Inc. 2012. www.algenolbiofuels.com, accessed on 2012.04.20. Andersen, R. A. 2005. Algal culturing techniques. 1st edition. PSA/ Elsevier Academic Press, USA Angermayr, S. A., K. J. Hellingwer, P. Lindblad and M. J. Teixeira de Mattos. 2009. Energy biotechnology with cyanobacteria. Curr. Opin. Biotechnol. 20: 257–263.

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BASF Personal Care and Nutrition GmbH / Cognis GmbH. 2012. www.cognis.com (http://www.basf.com/group/corporate/de), accessed on 2012.01.19. Börjesson, P., B. Mattiasson. 2006. Biogas as a resource-efficient vehicle fuel. Trends Biotechnol. 26: 7–13. Carvalho, A. P., L. A. Meireles and F. X. Malcata. 2006 Microalgal reactors: a review of enclosed system designs and performances. Biotechnol. Prog. 22: 1490–1506. Cellana, Inc. 2012. http://cellana.com, accessed on April 2012. Christenson, L. and R. Sims. 2011. Porduction and harvesting of microalgae for wastewater treatment, biofuels and bioproducts. Biotechnol. Adv. 29: 686–702. Cohen, Z. 1999. Chemicals from microalgae. 1st edition. Taylor & Francis, London Collet, P., A. Helias, L. Lardon, M. Ras, R. A. Goy and J. P. Steyer. 2011. Life-cycle assessment of microalgae culture coupled to biogas production. Bior. Technol. 102: 207–214. Dexter, J. and P. Fu. 2009. Metabolic engineering of cyanobacteria for ethanol production. Energy Environ. Sci. 2: 857–864. DSM Nutritional Products Ltd. / Martek Biosciences Corp. 2012. http://investors.martek.com/ releases.cfm, accessed on 2012.04.01. Earthrise Nutritionals, LLC. 2012. www.earthrise.com/about.html, accessed on 2012.04.20. Eroglu, E. and A. Melis. 2011. Photobiological hydrogen production: Recent advances and state of the art. Biores. Technol. 102: 8403–8413. FNR Fachagentur Nachwachsende Rohstoffe e.V. 2012 www.fnr-server.de/ftp/pdf/berichte/ 22017105.pdf, accessed on 2012.04.20. Global Market 2010: Natural Products Insider. http://www.naturalproductsinsider.com/. “Betacarotene leads carotenoid sales”, accessed in 05-10-2011 Hejazi, M. A., C. Lamarliere, J. Rocha, M. Vermuë and J. Tramper. 2002. Selective Extraction of Carotenoids from the Microalgae Dunaliella salina with Retention of Viability. Biotechnol Bioeng. 79: 29–36. Hejazi, A. M. and R. H. Wijffels. 2004. Milking of microalgae. Trends Biotechnol. 4:189–194. IHI Corp. 2012. www.ihi.co.jp/en/press/2011/2011-8-01/index.html, accessed on 2012.04.20. Lan, E. I. and J. C. Liao. 2011. Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide. Metabolic Eng. 13: 353–363. Leon R., I. Garbayo, R. Hernandez, J. Vigara and C. Vilchez. 2001. Organic solvent toxicity in photoautotrophic unicellular microorganisms. Enz. Microb. Technol. 29: 173–180. Levin, D. B., L. Pitt and M. Love. 2004. Biohydrogen production: prospects and limitations to practical application. Int. J. Hydrogen Energy 29: 173–185. Mata, T. M., A. Martins and N. Caetano. 2010. Microalgae for biodiesel production and other applications: A review. Renewable Sustainable Energy Reviews, 14: 217–232. Mojaat, M., A. Foucault, J. Pruvost and J. Legrand. 2007. Optimal selection of organic solvents for biocompatible extraction of -carotene from Dunaliella salina. J. Biotechnol. 133: 433–441. Molina Grima, E., E. H. Belarbi, F. G. Acien Fernandez, A. Robles Medina and Y. Chisti. 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 20: 491–515. NASA. 2012. http://inhabitat.com/nasas-omega-project-creates-carbon-neutral-food-and-fuel/, accessed on 2012.04.20. NIWA. 2012. www.niwa.co.nz, accessed on 2012.04.20. OneWater Inc. 2012. http://onewaterworks.com/index.html, accessed on 2012.04.20. OriginOil, Inc. 2012. www.originoil.com, accessed on 2012.04.20. Pittman, J. K., A. P. Dean and O. Osundeko. 2011. The potential of sustainable algal biofuel production using wastewater resources. Biores. Technol. 102: 17–25. Radakovits, R., R. E. Jinkerson, A. Darzins and M. C. Posewitz. 2010. Genetic engineering of algae for enhanced biofuel production. Eukaryot.Cell 9: 486–501.

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Rawat, I., R. R. Kumar and T. M. Bux. 2011. Dual role of microalgae: phycoremediation of domestic wastewater and biomass production for sustainable biofuels production. Applied Energy 88: 3411–3424. Richmond, A. 2004. Handbook of microalgal culture. 1st edition. Blachwell Publishing, Oxford Rosenberg, J. N., G. A. Oyler, L. Wilkinson and M. J. Betenbaugh. 2008. A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Curr. Opin. Biotechnol. 19: 430–436. Solazyme Roquette Nutritionals, LLC. 2012. www.srnutritionals.com, accessed on 2012.04.20. Tamagnini, P., E. Leitao, P. Oliveira, D. Ferreira, F. Pinto, D. J. Harris, T. Heidorn and P. Lindblad. 2007. Cyanobacterial hydrogenases: diversity, regulation and applications. FEMS Microbiol. Rev. 31: 692–720. Zhang, F., L. H. Cheng, W. L. Gao, X. H. Xu, L. Zhang and H. L. Chen. 2011. Mechanism of lipid extraction from Botryococcus braunii FACHB 357 in a biphasic bioreactor. J. Biotechnol. 154: 281–284.

Evan Stephens, Liam Wagner, Ian L. Ross and Ben Hankamer

15 Microalgal production systems: Global impact of industry scale-up 15.1 Microalgal biotechnology Microalgal biotechnology has been commercially viable for several decades, but only for a restricted range of applications (Benemann et al. 1987). Owing to the relatively high capital cost of microalgal production systems, successful applications have generally focussed either upon niche areas in which both modern agriculture and microbial fermentation systems lack a competitive advantage or upon unique microalgal products, for which no competition exists. Although the largest existing algae farms are still for health food production (e.g. Spirulina production in China) and natural products (e.g. Dunaliella in Australia for β-carotene), those undergoing the most rapid expansion are currently aimed at biofuel production and associated R&D. The microalgal industry is growing rapidly, and while microalgal biofuel technologies generally remain in the basic and applied R&D stage (IEA 2011a), commercial-scale facilities are starting to come online. For photoautotrophic production, these include Sapphire Energy’s 120ha (300ac) Integrated Algal Biorefinery (IABR) facility currently under construction in New Mexico, USA (Sapphire 2011; US D.O.E. Energy Efficiency & Renewable Energy 2011), while Solazyme’s factories have focused on heterotrophic conversion of sugars to oils and other products (Solazyme 2011; Dillon 2011). Interest in microalgal biofuels (Stephens et al. 2010a; Wijffels and Barbosa 2010) has increased rapidly in recent years, but arguably the real transition is one from a narrow range of high-value products (HVPs) to a broad range of lower-value biocommodities and bioremediation applications – which necessitates improvements in microalgal productivity and reductions in the cost of microalgal production. Microalgal production systems are now being promoted as a solution to global issues, including fuel and food security and the reduction in carbon emissions to minimize climate change effects. However, to achieve real global impact requires a massive scale of deployment – which ultimately raises the prospects of production efficiency, resource and space constraints. Here, we examine microalgal mass cultivation at scales of 1 %, 10 % and 100 % of global food and fuel demand, to define what levels of production could reasonably be achieved and to identify critical limiting factors. The importance of this is that the aim to develop a sustainable industry with a beneficial global impact will only be achieved if rigorous analysis of feasibility is conducted early in the scale-up process.

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15.2 Global challenges, production and demand To evaluate the effects of scaling up microalgal biotechnologies, the first step is to define the limits of fuel and food required globally both now and towards 2050. The reader is advised that the projections towards 2050 provided below can only be taken as an estimate, and unavoidably, uncertainties are large. Nevertheless, the 2050 scenario has been included to provide a broad scope that can, in turn, contribute to early phase planning and to evaluating approximate outcomes. It should also be noted that a unified model for food and fuel, based on energy content (J) has been examined. Microalgal biotechnologies drive the conversion of solar energy to chemical energy (e.g. food and fuel) and so the use of a unified energy term (J) greatly assists in the calculation of preliminary area and resource requirements as will be shown later in this chapter.

15.2.1 Global fuel production and demand Total global primary energy use in 2010 was reported to be 12,002.4 million tonnes of oil equivalent (Mtoe) which at a standardised energy density of 41.868 GJ T–1, is equal to ~0.5 ZJ and was provided by oil, gas, coal, nuclear, hydroelectric and other renewables (BP 2011). This primary energy is required for power generation, heat, transportation and industrial processes (e.g. coking coal for steel production). Total global electricity generated is 21,325 TWh (~0.08 ZJ) (BP 2011) which is consistent with reports of ~15 % (BP 2011) to ~17 % (IEA 2009) of global primary energy in Mtoe. Some electricity is generated directly from nuclear, hydroelectric and other renewable sources (~6,896 TWh or ~0.02 ZJ), while a sizeable proportion of fuels are combusted to generate the majority of electricity (~14,429 TWh or ~0.05 ZJ). At BP’s assumed 38 % conversion efficiency (BP 2011), equivalent to 9.47 GJ MWh–1, it can be calculated that ~27.2 % of primary energy as fuel is combusted to generate ~10.3 % of primary energy as electricity (in addition to 4.9 % of primary energy as direct electricity from nuclear, hydro, and other renewables). This suggests that from the ~87 % of primary energy represented by oil, gas, and coal fuels – after electricity generation the remaining volume of fuels corresponds to ~60 % of total primary energy in Mtoe’s. This corresponds closely in size to the combined oil and gas market (~57 %). We have therefore specifically chosen to model global fuel use on the size of the oil and gas market. This has the added advantage that they are both considered to be potential products of microalgal biotechnologies which are currently being developed for future large scale production. The total oil (4,028.1 MT or ~0.17 ZJ) and gas (2,858.1 Mtoe or ~0.12 ZJ) consumption in 2010 was 6,886.2 Mtoe (~0.29 ZJ), which was equivalent to ~57 % of total energy consumption that year (BP 2011). To put this in perspective, the global average oil field size is 22 M barrel oil equivalents (boe) (~0.14 EJ), and while the average deep water oil discovery in the Gulf of Mexico is reported to be approxi-

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mately four times larger at ~83 M boe (~0.51 EJ) (Tippee et al. 2010), this is still less than the world’s current daily consumption of ~86.7 M bbl d–1 (~12.8 Mtoe d–1) in 2010 (IEA 2011a). This explains the industry’s focus on drilling approximately 4,000 wells yr–1 in the Gulf of Mexico alone (Tippee et al. 2010) and the associated costs that this will incur. Currently, oil and gas supply ~33 % and ~24 % of the global energy market, respectively (BP 2011). It is unknown if oil will be able to maintain this share of the energy market given the decline in reported reserves (Meng and Bentley 2008). For the purposes of this chapter, we have therefore assumed that if the oil industry contracts, gas production will likely expand to meet this energy demand and that collectively their share can reasonably be expected to remain around the current level of 57 % unless major policy shifts are implemented. The US Energy Information Administration furthest projections of global fuel supply are to 2035 and predict a rise to 0.721–0.852 ZJ by 2035. Assuming a simple extrapolation of global energy demand towards 2050, it could conceivably rise towards 1.02 ZJ (~24,300 Mtoe) and indeed beyond, if annual energy efficiency gains are less than the combined effects on energy demand of continued population growth and per capita economic growth. In summary, given the uncertainties, for the purposes of our analysis we have therefore assumed a current fuel market size of 0.29 ZJ (~6,900 Mtoe) and the 2050 scenario to be 0.58 ZJ (~13,800 Mtoe – 57 % of 1.02 ZJ), providing the fuel input data for Table 15.1. The 10 % and 1 % scenarios used in Table 15.1 are calculated from these values.

15.2.2 Global food production and demand Based on an average calorie intake per person between the age of 18 and 60 of 10,200 kJ (~2,430 cal) (FAO 2001) and a current population of ~7 billion people, the total energy requirement (assuming no losses in production and distribution) would be 10,200 kJ person–1 day–1 × 365 days × 7B people = ~2.61 × 1019 J = ~0.0261 ZJ. Recent data on food production indicate that in 2010, approximately 7.6 BT of food products were produced (FAO 2011). This consisted of approximately 2.43 BT of cereals, 1.69 BT sugar cane, 1.69 BT of vegetables/tubers, 0.72 BT of pulses, 0.61 BT of fruit, 0.29 BT of livestock meat, 0.07 BT of eggs, 0.01 BT of nuts, and additionally some other minor food classes. If simplified but reasonable calorific values are assumed for these production categories, i.e. cereals (15 MJ kg–1 based on wheat), sugar cane (3.9 MJ kg–1), pulses (14.7 MJ kg–1 based on lentils), vegetables/ tubers (2.0 MJ kg–1 based on potatoes, carrots, turnips, green beans, corn and soybean mean), fruit (2.4 MJ kg–1 based on apples, bananas and citrus mean), livestock meat (8 MJ kg–1), eggs (5 MJ kg–1), and nuts (25 MJ kg–1 based on almonds), this estimate of global food production in 2010 can be roughly calculated as equivalent to 0.063 ZJ. The discrepancy between total production of food

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products (0.063 ZJ) and the total calorific demand of the human population (0.026 ZJ) is in part explained by the following. First, global livestock populations are large (~3.5 B cattle, sheep and pigs) and some crop outputs are utilized as feedstocks for animal production. Second, there are also processing losses such as for specialized and refined food products (e.g. sugar, oil and flour) as well as wastage. Third, an increasing proportion of particular crops (e.g. corn and soybean) are being utilized for first-generation biofuel production. Based on the BP statistical review 2011, biofuel production yielded ~0.002 ZJ in energy (BP 2011). In summary, for the purposes of this chapter, the 100 % value for energy embodied in food has been attributed a current value of 0.061 ZJ (i.e. 0.063–0.002 ZJ). Reports from the FAO’s 2009 forum “How to Feed the World 2050” concluded that food demand is expected to increase ~70 % by 2050 (FAO 2009). Based on the above levels, this would be up to ~12.9 BT of food yr–1, corresponding to a similarly calculated food energy equivalent value of 0.104 ZJ in 2050.

15.2.3 Solar irradiance and areal requirement Having established the total amount of food and fuel required currently in ZJ equivalent units, the next stage of analysing the effects of scale up is to calculate the available solar energy and the theoretical area required to supply 100 % of global food and fuel demand. Over the course of a year, the Earth receives ~5,500 ZJ yr–1 of solar energy. Approximately 26 % is reflected back into space, while the atmosphere and clouds absorb a further 19 % of the total incident energy (Smil 2005), leaving ~3,020 ZJ of radiant energy available for absorption by terrestrial and marine systems. Of this, approximately 43 % (~1,300 ZJ yr–1) is photosynthetically active radiation (PAR) which can be used for the production of biomass-derived food and fuel. It is worth noting that this annual level of irradiance (1,300 ZJ yr–1) dwarfs the total of all reported oil, coal, gas and uranium reserves (82.7 ZJ) and current global energy demand (~0.5 ZJ) (BP 2011). From the photosynthetically active radiation (1,300 ZJ yr–1), it can be calculated that to produce 0.56 ZJ yr–1 of food and energy (in captured chemical energy as microalgal biomass) at a 2 % photosynthetic conversion efficiency (PCE), which is already achievable using conventional microalgal production systems, the area required would be 0.56 ZJ yr–1 × 2 %/1,300 ZJ yr–1 = 1.8 % of the Earth’s surface at an average solar irradiance. Annual, time-averaged local solar irradiance values generally fall between 57 W m–2 (1.8 GJ m–2 yr–1) and 285 W m–2 (9.1 GJ m–2 yr–1), depending on the geographical location and local weather conditions. This is equivalent to annual average values of ~5–25 MJ m–2 d–1. Theoretical irradiance levels may be higher but can also be attenuated by weather conditions. As highlighted in the recent study by Weyer et al. (2010), analysis of the Kuala Lumpur site in the Malaysian

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tropics had a theoretical irradiance level of ~11,400 MJ m–2 yr–1 (~31 MJ m–2 d–1) but had an actual irradiance value of only ~5,600 MJ m–2 yr–1 (~15 MJ m–2 d–1) while at the much drier site Phoenix, Arizona, USA, the actual irradiance level of ~7,400 MJ m–2 yr–1 (~20 MJ m–2 d–1) matched the theoretical irradiance (9,800 MJ m–2 yr–1, or ~26 MJ m–2 d–1) more closely. For high solar irradiance regions of 15–25 MJ m–2 d–1, it can therefore be calculated that at 2 % efficiency, the total food and energy production for 2010 (0.56 ZJ) would require 3.4–5.7 M km2 (2.3–3.8 % of total global land area). At a PCE of 5 %, this area could be reduced to 1.4–2.3 M km2 (0.9–1.5 % of total global land area). To match the potential of world production in 2050 (up to ~1.1 ZJ) the areas required for higher solar irradiance regions of 15–25 MJ m–2 d–1 at 2 % and 5 % efficiencies can be calculated to be 6.8–11.3 M km2 (4.6–7.6 % of total global land area) and 2.7–4.5 M km2 (1.8– 3.0 % of total global land area) respectively. Even at the maximum calculated land area of 11.3 M km2, this is still less than the current area of cropped arable land in 2010 of ~13.8 M km2 (FAO 2010) (see Section 15.3.3) to produce both food and fuels. In summary, based on available solar energy, microalgal production systems could theoretically match total world production levels for food and fuel, using 0.9–3.8 % of global land area currently, and an area of 1.8–7.6 % of global land by 2050 (for captured energy in biomass, subject to processing losses). This exercise demonstrates that solar energy and total land area are not limiting for this technology. Of course, microalgal production systems will not be expected to match the total world production, even if it were possible, and in subsequent calculations we examine large-scale production of food and fuels (based on oil and gas) only. While this significantly reduces the total areal requirement, areas of suitable land are more constrained (see Section 15.3.3) and so may increase demand for high efficiency systems with a smaller footprint.

15.2.4 Global challenges The global population is currently undergoing unprecedented growth from ~7 to 9 billion people by 2050, which corresponds to a ~28 % increase (PRB 2009). Although the rate of population growth is in fact decelerating, this partial benefit is overshadowed by the increasing demand for energy and food production by the rapid advancement of developing nations (most notably China and India). Clearly all nations should have an equal right to development; however, the sheer magnitude of collective human expansion is inevitably increasing pressure on future sustainability, and an urgent response is required to address three primary challenges of our time: protecting against climate change, an increasing need for energy security, and putting in place strategies to support sustainable populations and economic development. Energy consumption and economic growth are intimately linked. Despite the economic downturn of the global financial crisis in 2008, energy consumption and

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related CO2 emissions rebounded rapidly in 2009 (mainly in developing nations), and in 2010 reached a historic high (IEA 2011b; EIA 2011). This illustrates the strong linear correlation between economic growth and energy demand. Gross domestic product (GDP) is in fact strongly correlated with global energy consumption (Brown et al. 2011). It therefore follows that to sustain positive economic growth into the future as the global population increases, more energy and more energetically efficient processes for the production of food and fuel will be critical to increase or indeed even maintain living standards. In summary, failure to secure future energy supplies will negatively affect economic growth and world production. The international community recognizes the need for sustainable energy solutions despite the complex challenges posed by the deployment of these technologies. The EU Renewable Energy Directive for 10 % renewable fuels by 2020 (IEA 2010) and the EU carbon trading scheme, which is to include aviation from 2012 (Ellerman and Buchner 2007; IEA 2010), the carbon tax legislation recently passed in Australia at AU$ 23 T–1 C (Sydney Morning Herald 2011), as well as the Renewable Fuel Standard (RFS) in the USA (IEA 2010; Olmstead and Stavins 2012), are testimony to this, although the international unification of policy frameworks is arguably as complex as the technological development itself (Metcalf and Weisbach 2012). Unfortunately, the required speed of political change required to achieve this transition is hampered by misinformation due to vested interests and a resultant negative public perception. This is however increasingly being countered by energy security concerns, which suggest that fuel security is an issue not for the distant future but for this century (Heinberg and Fridley 2010; IEA 2010). Energy-security concerns generally focus upon gaseous and liquid fuels rather than electricity, and also vary geographically. For example, currently natural gas dominates energysecurity concerns in Europe, while petroleum is the central focus of the US, whereas Australia (as an example of the Asia Pacific region), though rich in coal and gas, is limited in oil supply and is geographically more sensitive to the cost of fuel importation. Increasingly it is also recognized that energy security and climate security are coming into alignment, and a broad diversity of fuel technologies are now in development. Consequently, from a climate change, energy security and economic perspective, the scale-up of renewable fuel technologies will be increasingly important over the coming decades.

15.3 Potential production and limitations 15.3.1 Solar energy and geographic location The initial consideration for any solar technology is clearly the available amount of incident solar energy (dependent on geographic location) and the efficiency of its utilization. As has been demonstrated above, at a global level, solar energy is

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not limiting for light-dependent technologies (1,300 ZJ yr–1 PAR versus global food and fuel demand ~0.35 ZJ yr–1 – comprising ~0.29 ZJ fuel and ~0.06 ZJ food). However, the distribution of solar energy is not uniform across the globe, and some regions are better suited to solar technologies than others, for example regions having solar irradiance levels above 15 MJ m–2 d–1 (~55,000 GJ ha–1 yr–1). For microalgal production systems, this can be readily demonstrated through productivity modelling at different geographic locations (Weyer et al. 2010; Franz et al. 2012). A recent analysis for Germany with a reported light energy input of 1,000 kWh m–2 yr–1 (~14.4 MJ m–2 d–1) over 250 operational days to account for productivity losses during winter concluded that the system could yield 40 T ha–1 yr–1, which is equivalent to a productivity level of 16 g m–2 d–1 (~2.4 % PCE assuming a biomass energy density of 22 MJ kg–1) (Wilhelm and Jakob 2011). A similar calculation for more tropical climes with a light intensity of 25 MJ m–2 d–1 and 330 operational days per year (allowing one month of downtime) would yield 89 T ha–1 yr–1 at 2.4 % PCE. The corresponding production levels assuming a 5 % PCE would therefore rise to 83 T ha–1 yr–1 (at 14.4 MJ m–2 d–1) and 185 T ha–1 yr–1 (at 25 MJ m–2 d–1). In summary, current yields are estimated to range between ~40 and 90 T ha–1 yr–1, and these might sensibly be expected to rise towards ~80–185 T ha–1 yr–1, based on these data. Theoretically, further improvement is possible in the time frame towards 2050. Despite the differences in the estimated productivity of different geographical regions, there have been initiatives to grow microalgae in higher-latitude locations having lower solar irradiation levels of 5–15 MJ m–2 d–1, by incorporating methods such as waste-heat utilization to warm microalgal cultivations to productive operating temperatures. Because available light levels and temperatures are lower, and the variation between summer and winter irradiance is much greater, process optimization can be more challenging than in tropical locations where such variations are less severe. Despite this, ongoing algae R&D suggests that there could be tailored solutions for different markets and geographical locations. Seasonal variations can be at least partially countered through strain selection (i.e. winter strains and summer strains), while markets may be extended by increasing the efficiency of systems and reducing capital costs. In the final analysis, it is expected that whereas global capacity may be affected markedly by efficiency (photons per carbon fixed), market acceptance will be based primarily on economics ($ per carbon fixed). Efficiency is related to optimal light dilution. In some system configurations, lower light levels can be seen as an advantage as they reduce photoinhibition, effectively due to a natural form of light dilution. Furthermore, while heating of cultures may present an obstacle in cooler climates, overheating is a concern in warmer climates. Both regions therefore have specific challenges and opportunities. However, while it is technically feasible to produce algae at higher latitudes (5–15 MJ m–2 d–1), the extra cost of production and lower productivities will likely

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mean that these systems are used predominantly in the production of HVPs unless provided with support via external market mechanisms (e.g. rising food and fuel prices, carbon tax or trading schemes, or renewable fuel tax benefits). Consequently, it is anticipated that an emerging microalgal industry that aims to be costcompetitive with conventional food and fuel production is more likely to be initially deployed in regions with higher insolation (e.g. >20 MJ m–2 d–1) before expanding into the 15–20 MJ m–2 d–1 zones (and less likely below) as food and fuel prices rise. Even for high annual mean levels of 20 MJ m–2 d–1 (~73,000 GJ ha–1 yr–1), desirable for the most productive systems with the smallest footprint, a high 5 % PCE would lead to the storage of 1 MJ m–2 d–1 (~3,650 GJ ha–1 yr–1) of chemical energy in the form of biomass, which is subject to further parasitic/processing losses prior to real energy output. This clearly illustrates the restrictions on energy and economic inputs if the technology is to be adopted and deployed up to a globally significant scale. In summary, we anticipate that the deployment of microalgal production systems will be favoured at higher solar intensity regions and may expand from there in a demand led fashion, unless external market drivers assist a broaderbased development.

15.3.2 Potential productivity Overly optimistic and unrealistic predictions accompanied the reascendance of microalgal biofuels in the last decade (Waltz 2009), and wild claims of productivity yields from 400 T biomass ha–1 yr–1 to 200,000 gal oil ac–1 yr–1 (~2 M L oil ha–1 yr–1), which are above the theoretical maximum, were reported. Some of these extravagant claims still persist today, but realistic, conventionally designed commercial systems generally benchmark at up to ~70 T ha–1 yr–1 (Richmond et al. 1990). The first step to evaluating the capacity of this technology set for global impact is to define the maximal productivities and the factors that impact upon them such as available solar irradiation. Even in the scientific literature, a wide range of microalgal productivities have been predicted (Huntley and Redalje 2007; Chisti 2008; Campbell et al. 2009; Lardon et al. 2009; Pienkos and Darzins 2009; Rodolfi et al. 2009; Batan et al. 2010; Wijffels and Barbosa 2010). Waltz (2009) described the resultant “algae fervor” in the microalgal biofuels sector, and reported that reasonable productivity claims for oil production were ~20,000 L oil ha–1 yr–1 (~2,000 gal oil ac–1 yr–1) at present, and a reasonable estimated productivity of future commercial systems might be ~60,000 L oil ha–1 yr–1 (~6,000 gal oil ac–1 yr–1). These estimates were again supported at the 5th Algae Biomass Summit (2011) (Algae Biomass Summit Plenary Panel 2011) by a panel representing some of the largest microalgal biofuel start-up companies. Potential yields of up to ~100,000 L ha–1 yr–1 are theoretically possible at high irradiances (approximately 5 % PCE at 25 MJ m–2 d–1 irradiance yields, 50 g biomass dry weight m–2 d–1 at 50 % oil content, or equivalent) but this will be difficult to attain in practice.

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Recent studies have aimed to address commercial over-promotion of microalgal productivity and to highlight limitations to photosynthesis and reasonable assumptions for photosynthetic output (Zhu et al. 2008; Larkum 2010; Stephens et al. 2010b; Weyer et al. 2010; Blankenship et al. 2011; Wilhelm and Jakob 2011). It has been argued that metabolic generation of biomass cannot theoretically achieve PCEs of much over ~8 % (Zhu et al. 2008) even with genetic improvement. Other reports suggest that higher efficiencies are possible for fuels such as H2 (Melis 2009), the production of which is closely coupled to the photosynthetic electron transport chain and avoids the inefficiencies associated for example with oil biosynthesis. Furthermore, mixed cultures of green microalgae, cyanobacteria and purple bacteria collectively have a broader absorption range, which in theory could yield a higher photon-to-chemical-energy-conversion efficiency. Extending the breadth of the absorption range could also be achieved by using strains with chlorophyll D (Chen and Blankenship 2011), or with genetically engineered strains capable of utilizing a broader range of the incident solar irradiation. If individual strains, mixed community systems or engineering solutions can be developed to exploit the UV or near-infrared wavelengths of light then theoretical boundaries of productivity could potentially be lifted. Some modelling by us and others has therefore explored production at PCE rates up to 10 % for photosynthesis to biomass (Stephens et al. 2010b; Williams and Laurens 2010), but we support the view that these are unlikely to be achievable for individual native species. The reader should also be aware that this subject is confused by some publications that report efficiency as a percentage of photosynthetically active radiation rather than a full spectrum. For transparency, we advocate that percentage efficiencies are calculated on the basis of the full solar spectrum. This has the added advantage that it allows comparison with other solar technologies that are benchmarked against the AM1.5 spectrum. In summary, based on the full solar spectrum, cultivations with extended wavelength absorption range, exceeding 8 % PCE could theoretically be achieved (the precise percentage depends on many factors including the absorption range and end product), though for single species ~8 % is the approximate upper theoretical limit, and a range of 2–5 % is a sensible estimate for the time frame between now and 2050, although further improvement cannot be excluded. For the purposes of our calculations, we have used a 2–5 % range. Currently, for microalgal biomass production, levels of ~2 % PCE are achievable in conventional high rate pond (HRP) systems, and higher levels of ~4.5 % are reportedly achievable in photo bio-reactors (PBRs) (Tredici 2010). In production terms, levels of 100–200 T biomass ha–1 yr–1 (~27–55 g m–2 d–1 as a 365-day average) represent a reasonable target range for advanced systems in development. As a comparative example, corn or soybean typically yields a harvest of up to ~10 T ha–1 yr–1 and ~3.5 T ha–1 yr–1 respectively (FAO 2011). A key reason for the increased level of productivity in microalgal production systems relative to agricultural systems is that light, CO2, nutrients, temperature and water management can be opti-

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mized. It should be noted, however, that there are inconsistencies between dryweight determinations of crops and algae. For example, microalgal biomass is generally reported as “dry weight” or “ash free dry weight” (primarily for saline strains in laboratory studies), while corn and soybean yields are based on product that retains up to 15 % moisture. Furthermore, corn/soybean totals are for harvested yield, not total plant growth. Also, the production of algae requires energy inputs in terms of mixing and gas supply. The production of corn and soy bean also requires energy inputs, but the two differ substantially, and for a proper comparison, a life-cycle analysis would have to be conducted (see below). In the case of crops, usable biomass production could effectively be higher if the rest of the plant were used as biomass energy, but soil condition and recycling of nutrients must also be considered – mulching of residuals is often practised to retain soil carbon and nutrients. Consequently, despite the considerable costs of installing microalgal production systems, they have significant potential advantages over conventional agriculture. In summary, because of the many uncertainties above, we simply conclude that microalgal production systems can compare favourably with crops in terms of yield and so it is sensible to consider them for use as both food and fuel.

15.3.3 Land resources One of the most widely presented advantages of microalgal production systems is their capacity to be sited on non-arable land, in contrast to most conventional agricultural systems, which depend on soil fertility and freshwater. Of the Earth’s 510 M km2 surface area, ~148.9 M km2 is reportedly land (CIA 2012). This can generally be divided into arable, non-arable and marginal land. Arable land is land that is presently used for growing non-permanent crops or is temporarily fallow, and excludes land that is not cultivated. Therefore, it does not represent the amount of land that could potentially be cultivated (FAO 2010). A single negative factor (e.g. poor soil fertility, low freshwater availability, excessive salt levels or poor climatic conditions) can result in land being classified as non-arable or marginal. According to the FAO definition, “agricultural land” includes all arable land and additionally includes land under permanent crops as well as permanent pastures. According to the FAO, in 2008, the world’s total arable land amounted to 13,805,153 km² (10.6 % of total land area), with 1,462,421 km2 (1.1 % of total land) of permanent crop land (e.g. fruit orchards and plantations) and 33,569,402 km2 as pastures (25.8 % of total land). This results in 48,836,976 km² (37.6 % of total land) being classified as “agricultural land” (FAO 2010). As outlined above, these numbers can be mistakenly interpreted to suggest that there is a massive amount of non-agricultural land available, but in fact, most of this consists of the polar and high-latitude regions, deserts, and mountain ranges, and only a fraction is available for any type of production system including microalgal systems. At an average

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of 20 MJ m–2 d–1 and 2–5 % PCE, to match 2010 production of food (~7.6 BT yr–1) and fuels (~6,900 Mtoe yr–1) would require ~2.0–5.2 M km2, and ~3.8–9.6 M km2 would be required to match 2050 levels of food (~12.9 BT yr–1) and fuels (~13,800 Mtoe yr–1) production (see Table 15.1). If production streams could be integrated, this could be further reduced (see Section 15.4.4). The classification of “marginal” lands is more problematic. Recent assessment of marginal lands identified only ~4 M km2 of lands in APEC countries suitable for marginal biomass production (Milbrandt and Overend 2009), while marginal land globally has been estimated at ~11 M km2 (Cai et al. 2011). As previously mentioned, given that total food and fuel production in 2050 could require up to ~3.8–9.6 M km2, this demonstrates how suitable land is more limiting than total land, leading to questions of land-use efficiency. Unfortunately, the term “marginal lands” is poorly defined, as highlighted by Milbrandt and Overend (2009). Even their attempts at clarification fall short of providing a clear delineation. For example, they propose that acidic soils below pH 5.5 (~0.6 M km2) be classified as marginal, which overlaps with classifications of arable land given that many farming regions have a pH range of 4.5–5.5 (e.g. many farming areas in Australia). Soil amendment requirements (e.g. aglime (CaCO3), or hydrated lime (Ca(OH)2)) to return highly acidic soils (especially high Cation Exchange Capacity (CEC) soils) to optimal fertility and productivity, can be well beyond the financial capacity of many farming enterprises, but the lands continue to be economically viable at suboptimal productivity. Marginal lands have also been defined as including glacial, desert and mountainous areas, which are non-arable lands. Although a yield classification of