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Saline Microalgae for Biofuels: Outdoor Culture from Small-Scale to Pilot Scale

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Saline Microalgae for Biofuels: Outdoor Culture from Small-Scale to Pilot Scale SALINE MICROALGAE FOR BIOFUELS: OUTDOOR CULTURE FROM SMALL-SCALE TO PILOT SCALE Andreas Isdepsky DIPL.-ING. (BIOTECH) This thesis is presented for the degree of Doctor of Philosophy of Murdoch University, Western Australia 2015 DECLARATION I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. Andreas Isdepsky ii “Do not go where the path may lead, go instead where there is no path and leave a trail.” Ralph Waldo Emerson iii ABSTRACT Three local isolates of the green alga Tetraselmis sp. identified as the most promising microalgae species for outdoor mass cultivation with high potential for biodiesel production due to high amounts of total lipids and high lipid productivity were employed in this study. The aim of the study was to compare three halophilic Tetraselmis strains (Tetraselmis MUR-167, MUR-230 and MUR-233) grown in open raceway ponds over long periods with respect to their specific growth rate and lipid productivity without additional CO2 and with CO2 addition regulated at pH 7.5 by using a pH-stat system. Attention also was given to the overall culture condition including contaminating organisms, biofilm development due to cell adhesion and cell clump formation. All tested Tetraselmis strains in this study were successfully grown outdoors in open raceway ponds in hypersaline fertilised medium at 7 % w/v NaCl over a period of more than two years. A marked effect of CO2 addition on growth and productivities was observed at high solar irradiance and temperatures between 15 – 33 oC. However, differences were identified between the three Tetraselmis strains in biofilm development, specific growth rate and lipid productivity in association with solar irradiance and temperature. Study of Tetraselmis MUR-167 was terminated after two years of outdoor cultivation due to stickiness and biofilm development on the pond walls, with a consequent decline in specific growth and productivities. Tetraselmis MUR-230 and MUR-233 showed quite similar annual specific growth and average productivities of 12 g (ash free dry weight) m-2 d-1 and 5.3±0.4 g m-2 d-1 in semi- continuous culture over a period of 365 days for biomass and lipid respectively; however MUR-233 had a higher specific growth rate and higher lipid productivity at higher temperatures in comparison with MUR-230. Further studies were carried out on Tetraselmis MUR-233 in 2 m2, 20 m2 and 200 m2 raceway ponds at a pilot plant in Karratha, West Australia. Comparison between Perth and Karratha showed that Tetraselmis MUR-233 had up to three times higher biomass productivities in winter and 40 % higher during summer in Karratha. Appreciable differences in specific growth during semicontinuous cultivation were observed in 2 m2 raceway ponds compared to the 20 m2 and 200 m2 ponds accompanied with a decline in biomass productivity of up to 20 % with increasing pond iv size. Highest biomass productivities of 30 g (ash free dry weight) m-2 d-1 and a lipid content of up to 43 % based on ash free dry weight was achieved at an optimum cell 4 -1 density of 60 x 10 cells .mL combined with 0.4 daily dilution and CO2 addition even under hyper saline conditions. These results were obtained at 25 MJ .m-2 solar irradiance and temperatures between 15 – 31 oC. Tetraselmis MUR-230 and MUR-233 with CO2 addition and regulated pH out-competed the major contaminating organisms, the diatoms. Tetraselmis MUR-230 grew successfully with different N-sources such as ammonium chloride, urea and sodium nitrate, the highest cellular lipids obtained with urea and sodium nitrate. Cellular carbohydrates in Tetraselmis MUR-230 increased five times under N-depletion and CO2 addition. In summary, this study demonstrates the feasibility of outdoor large scale cultivation of the halophilic Tetraselmis strains MUR-230 and MUR-233 with a high potential as feedstock for biofuel and carbon dioxide mitigation. The sustainability is underlined by the fact that the Tetraselmis strains were able to grow in hot and dry areas using only seawater as water source. v ACKNOWLEDGEMENTS First and foremost I would like to offer my sincere gratitude to my supervisor, Professor Michael Borowitzka for his dedication, supervision and guidance throughout my PhD candidature. I would like to extend my gratitude to Michael’s wife Dr Lesley Borowitzka and family, for the warm welcome when I first came to Australia. Going back in time, I would also like to thank my previous supervisor Professor Carola Griehl (Anhalt University, Germany), for her faith in me throughout my study in Germany and for given me the opportunities to study abroad twice. Thanks to the Commonwealth of Australia for funding the current research project as part of the Asia-Pacific Partnership (APP) on Clean Development and Climate. The success of a major research project like this is due to the expertise, dedication and hard work of many different people including in my case, Murdoch University, Adelaide University and various industry partners. I would like to thank all contributors from Rio Tinto for the assistance provided at all levels during planning, building and operating the pilot plant in Karratha. I am grateful to Muradel Pty Ltd, for giving me the opportunity to continue my research work on Tetraselmis via the development of the demonstration plant in Whyalla, South Australia following my work at the pilot plant in Karratha, Western Australia. There are numerous people to thank at Murdoch University for their help and support during my PhD candidature: Karen Olkowski, Anne Roberts, Dr. Carolyn Jones, Ian Gell and Anushia Suppiah. My sincere thanks also goes to my colleagues at the Algae R&D centre at Murdoch University, including Nathan Good, Edin Omerhodzic, Chia Lee and the many others for their assistance, including social aspects. In particular, I thank Dr Navid Moheimani for his assistance and support during the early stage of my PhD candidature; Eric Raes, for the collaborative work on the growth comparison of Tetraselmis MUR-233 in open raceway ponds and the Biocoil and Frances Suckale for the collaborative work on fatty acid composition for Tetraselmis MUR-233 in Chapter 4. Thanks also to Bruno Pais for his friendship and hospitality in Perth and Karratha. I am also grateful to Tracy Marshall for her assistance and collaborative work during the transition period from Perth to Karratha. vi A very special acknowledgment to Dr Sophie Fon Sing her support and friendship has been invaluable at both an academic and a personal level, for which I am extremely grateful. The time and effort we both put into assisting each other during our PhD candidature was priceless, particularly the set-up of the Algae R&D Centre, several cultivation trials and improvements of various analytic methods to make it through. Thank you very much for being part of the adventure. The writing of this dissertation has been one of the most significant academic challenges I have ever had to face. Without the patience, guidance and support in editing this dissertation of the following people, this study would not have been completed. Thanks to Professor Michael Borowitzka, Dr Lisa Lines, Dr Bailey Green and Dr Sophie Fon Sing. It is to them that I owe my deepest gratitude. Finally, I thank my family, especially my parents for encouraging and supporting me throughout all my studies around the globe. Words cannot express my gratitude for everything you have done. vii TABLE OF CONTENTS DECLARATION ii ABSTRACT iv ACKNOWLEDGEMENTS vi TABLE OF CONTENTS viii PUBLICATIONS xi List of Abbreviations xii CHAPTER 1 Introduction 1 1.1 Applications of microalgae .............................................................................. 3 1.2 Mass culture of microalgae .............................................................................. 5 1.2.1 Open-culture systems .................................................................................. 7 1.2.2 Enclosed-culture systems .......................................................................... 11 1.3 Limits to growth in open raceway ponds ....................................................... 13 1.3.1 Light .......................................................................................................... 13 1.3.2 Photosynthesis and photon utilisation efficiency ...................................... 14 1.3.3 Temperature .............................................................................................. 16 1.3.4 CO2, pH and O2 ......................................................................................... 18 1.3.5 Nutrients .................................................................................................... 22 1.3.6 Salinity ...................................................................................................... 25 1.3.7 Mixing ....................................................................................................... 27 1.3.8 Contamination and other limitations / inhibitions ..................................... 28 1.3.9 Scaling up of microalgal culture – constraints and requirements ............. 30 1.4 Microalgae as a source of renewable biofuel ................................................. 31 1.5 Lipids and Lipid synthesis .............................................................................. 37 1.6 Selection of microalgae species and strains ................................................... 45 1.6.1 Chosen
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