INCREASE OF MICROALGAL PRODUCTION THROUGH SYMBIOSIS AND SCALE-UP MICROALGAL CULTIVATION GUO ZHI (B.Eng., Beijing University of Chemical Technology) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION “I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in this thesis This thesis has also not been submitted for any degree in any university previously.” Guo Zhi 10 Jan 2014 a Acknowledgements ACKNOWLEDGEMENTS The completion of my research depends on the encouragement and guidance of many people I take this opportunity to express my sincere gratitude to these people who have been contributory in the successful completion of this thesis First and foremost, I would like to express my deepest and most sincere gratitude to my supervisor, Professor Tong Yen Wah, for giving me the precious opportunities to carry out my research work His passion in scientific research and tireless mentorship are the key motivating factors behind my successful completion for this thesis I am sincerely grateful to his invaluable patience and advice in research He also gives me a lot of helpful suggestions in my life Here, I very appreciate your help and guidance during these challenging but wonderful years Thank you very much! I would like to show my appreciation to Professor Chung, Tai-Shung Neal and Professor He Jianzhong for their generous time and guidance during my research I want to show my thanks to all the past and present members of the Prof Tong’s group, in particular: Koh Shirlaine, Chen Wen Hui, Niranjani i Acknowledgements Sankarakumar, Liang Youyun, Chen Yiren, Anjaneyulu Kodali, Wang Honglei, Xie Wenyuan, Ajitha Sundaresan, He Fang, Wang Bingfang, Sushmitha Sundar, Wang Liang, Ingo Tim Wolf, Zhou Danhua, Lee Jonathan and Liang Yiyun, for their unconditional help, suggestion and support I would like to thank other groups’ members, especially Deny Hartono, Harleen Kaur, Fong Kah Ee, Dai Meng Qiao, Prashant Praveen, Vu-Tran Khanh Linh, Chen Xiyu and Wang Peng, for their valuable advice in my research I am also grateful to my dear friends, Wang Rong, Yu Rui, Zhou Rui, Xu Liqun, Zhang Bin and Han Gang, for their support and encouragement during my hard time In addition, I would like to thank our lab officers, Ms Li Fengmei, Dr Yang Liming, Ms Li Xiang, Mr Evan Stephen Tan, Mr Ang Wee Siong, Mr Lim Hao Hiang, Joey, Mr Qin Zhen, Md Teo Ai Peng, Mr Ng Kim Poi and Mr Chuin Mun Alistair Without their help, I could not finish my research work in time I also want to show my appreciation to the Department of Chemical and Biomolecular Engineering, National University of Singapore for providing the research scholarship and living stipend as well as research opportunity and facilities that make this study possible Last but not least, I would like to thank my parents for their unconditional support, patience, understand and love, which make me successfully overcome all the difficulties and challenges in my life Finally, I would like to thank my dearest girl, Wu Yin, for her selfless support and love ii Table of contents TABLE OF CONTENTS SUMMARY vii LIST OF TABLES x LIST OF FIGURES xii LIST OF ABBREVIATIONS xvii LIST OF SYMBOLS xx CHAPTER 1.1 Background 1.2 Hypothesis 1.3 Research objective CHAPTER 2.1 Global energy problem 2.2 GHG emissions 11 2.3 Biofuels 11 2.4 Biomass 13 2.5 Biorefinery 14 2.6 Algae 15 2.6.1 Cyanobacteria 16 2.6.2 Chlorophyta 17 2.7 Photosynthesis in microalgae 18 2.7.1 Photosynthetic membranes and chloroplast 20 2.7.2 Photosynthetic pigments 20 2.7.3 Photorespiration 21 2.8 Microalgal cultivation 22 2.8.1 Cultivation parameters 23 2.8.2 Cultivation nutrients 28 2.8.3 Cultivation systems 31 2.9 Harvest 34 2.10 Application of microalgae 36 2.10.1 Feedstock for biofuels 36 2.10.2 Food commodities and pharmaceuticals from microalgae 39 2.10.3 Wastes treatment 40 CHAPTER 43 3.1 Microalgae 44 3.2 Culture medium for C vulgaris 44 3.3 C vulgaris culture in the lab 44 3.4 Outdoor cultivation of C vulgaris 46 iii Table of contents 3.5 Analytical methods 46 3.5.1 pH and dissolved oxygen measurement 46 3.5.2 Outdoor cultivation parameters measurement 46 3.5.3 Nitrate and phosphate concentration measurement 47 3.5.4 Extracellular organic carbon measurement 47 3.5.5 Biomass concentration measurement 47 3.5.6 Lipid extraction and measurement 48 3.5.7 Carbohydrate extraction and measurement 49 3.5.8 Protein extraction and measurement 50 3.5.9 Chlorophyll extraction and measurement 51 3.5.10 Photosynthetic efficiency measurement 51 3.5.11 CO2 fixation rate and CO2 to biomass conversion efficiency 52 3.5.12 Fatty acids analysis 53 3.5.13 Elemental analysis 54 3.5.14 Scanning Electron Microscopy 54 3.6 Statistical analysis 55 3.7 Specific experimental section of chapter 55 3.7.1 Isolation of symbiotic bacteria from C vulgaris culture 55 3.7.2 Identification of symbiotic bacteria 56 3.7.3 Purification of C vulgaris culture 56 3.7.4 Co-culture C vulgaris and symbiotic bacteria 57 3.8 Specific experimental section of chapter 58 3.8.1 Adjustment of different CO2 input conditions for outdoor culture of C vulgaris in bubble columns 58 3.9 Specific experimental section of chapter 59 3.9.1 First-stage cultivation in outdoor environment 59 3.9.2 Harvest between the first and second stage 59 3.9.3 Second-stage cultivation in outdoor environment 60 3.9.4 Equations for the determination of biodiesel properties 61 CHAPTER 63 4.1 Introduction 64 4.2 Results 66 4.2.1 Observation of C vulgaris and symbionts 66 4.2.2 Isolation and characterization of symbiotic bacteria 67 4.2.3 Effect of symbiotic bacteria on the growth of C vulgaris 70 4.2.4 Analysis of EOC in culture 74 4.2.5 Analysis of chlorophyll amount 75 4.2.6 Analysis of photosynthetic efficiency 76 4.3 Discussion 77 4.4 Summary 86 CHAPTER 88 5.1 Introduction 89 5.2 Results 93 iv Table of contents 5.2.1 Effects of gas flow rate and different CO2 input conditions on the growth of C vulgaris 93 5.2.2 Effects of different CO2 input conditions on the elemental proportion of C vulgaris 96 5.2.3 Effects of different CO2 input conditions on the CO2 fixation of C vulgaris 97 5.2.4 Effects of different CO2 concentration in aeration on the dissolved CO2 concentration in medium/algal culture 99 5.2.5 Effects of different CO2 input conditions on the yields of FAME, protein and carbohydrate in C vulgaris 100 5.2.6 Effects of different CO2 input conditions on the composition of FAMEs in C vulgaris 102 5.3 Discussion 104 5.4 Summary 111 CHAPTER 113 6.1 Introduction 114 6.2 Results 117 6.2.1 C vulgaris growth and nutrients profiles in the first-stage cultivation 117 6.2.2 C vulgaris lipid profiles and compositions in the first-stage cultivation 119 6.2.3 C vulgaris growth profiles under different stress conditions in the second-stage cultivation 121 6.2.4 C vulgaris lipid profiles and compositions under different stress conditions in the second-stage cultivation 124 6.2.5 The quality determination of C vulgaris FAMEs 129 6.3 Discussion 130 6.4 Summary 136 CHAPTER 143 7.1 Synergistic microalgae-bacterial relationship as an approach to increase microalgal production 145 7.2 Scaling up microalgal culture to increase algal production and optimizing CO2 usage during algal cultivation 147 7.3 Two-stage cultivation strategy to increase algal biomass production and lipid productivity 149 7.4 Suggestions for future work 151 7.5 Preliminary studies for future work: Designing novel PBR for microalgal production 153 BIBLIOGRAPHY 157 APPENDIX A 198 APPENDIX B 200 APPENDIX C 201 v Table of contents vi Summary SUMMARY The global energy crisis has motivated reevaluations of energy-intensive activities and processes in the urban living environment, to identify areas where fossil fuel energy dependence can be reduced through the use of alternative energy Biofuels have triggered an intensification of research into alternative energies, due to their renewable and carbon neutral characters Microalgae are considered the most promising feedstock for the next generation of biofuels because they have several major advantages, compared to the feedstock of first(food feedstock) and second- (non-food feedstock) generation biofuels However, one major challenge of microalgal biofuels lies in algal biomass production Therefore, this thesis was designed to increase microalgal production through different approaches The first objective of this project was to investigate potential approach for promoting microalgal growth in the lab Synergistic microalgae-bacterial interaction was proposed to increase microalgal concentration Three symbiotic bacteria were isolated from the long-term operated C vulgaris culture and identified by 16S rDNA analysis One symbiotic bacteria Pseudomonas sp was found to have a growth promoting effect (1.4 times higher algal cell concentration than single algal culture) on C vulgaris when they were co-cultured under photoautotrophic conditions The chlorophyll vii Summary content in C vulgaris cell was higher in co-culture than in single algal culture The mutualistic relationship between microalgae and their symbiotic bacteria could be used as one method to increase microalgal production The second objective was to investigate the feasibility of scaling up microalgal culture to increase the production yield C vulgaris was successfully cultivated in pilot-scale bubble columns (80 L) under tropical outdoor conditions The constant supply of CO2 in microalgal culture was found to be non-essential It was found that when 2% CO2 was intermittently supplied (1 h 2% CO2 enriched air/1 h air) or 2% & 4% CO2 was alternatively aerated (30 4% CO2 enriched air twice and h 2% CO2 enriched air twice), the algal growth was not affected as compared to having a constant supply of 2% CO2 while the amount of CO2 used was reduced by 50% and CO2 to biomass conversion efficiency was doubled Outdoor culture and adjusting proper CO2 input conditions can be suggested in microalgal production so as to save energy and cultivation cost The third objective of this project was to investigate method of increasing the algal biomass and their lipid productivities in outdoor environments A two-stage cultivation strategy was applied, to obtain 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J, Li X, Zhang F, Chen J (2011) Aerobic degradation of methyl tert-butyl ether in a closed symbiotic system containing a mixed culture of Chlorella ellipsoidea and Methylibium petroleiphilum PM1 J Hazard Mater 185:1249-1255 197 Appendix A APPENDIX A LIST OF PUBLICATIONS Journal publications Guo Z, Tong YW (2014) The interactions between Chlorella vulgaris and algal symbiotic bacteria under photoautotrophic and photoheterotrophic conditions J Appl Phycol 26:1483-1492 Guo Z, Phooi WB, Lim ZJ, Tong YW Control of CO2 input conditions on the outdoor culture of Chlorella vulgaris in bubble column photobioreactors Ready to submit Guo Z, Chew CH, Tong YW Algal biomass production, lipids accumulation and fatty acid variation of Chlorella vulgaris under two-stage cultivation strategy in outdoor pilot-scale photobioreactors Ready to submit Conference publications Guo Z, Tong YW (2012) AICHE Annual Meeting, Pittsburgh, USA 198 Appendix A Tong YW, Guo Z (2012) BIOFUEL 2012-Alternative Aviation Fuel in Asia Conference & ASEAN Algae Biofuel Initiative Conference, Singapore 199 Appendix B APPENDIX B (Retrieved from http://www.ccap.ac.uk) 200 Appendix C APPENDIX C Diagram of bubble column PBR 201 ... between microalgae and bacteria could lead to a new potential method to increase microalgal production Secondly, the feasibility of scale- up microalgal cultivation in pilot -scale PBRs in a tropical... in large -scale microalgal production, so as to save energy and cultivation costs (Chapter 5) (3) Two-stage cultivation to increase algal biomass production and accumulate lipids in microalgal. .. microalgal production 145 7.2 Scaling up microalgal culture to increase algal production and optimizing CO2 usage during algal cultivation 147 7.3 Two-stage cultivation strategy to increase