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Tetraselmis Suecica Effects of Stress conditions on Lipid Production By Botryococcus braunii, Tetraselmis suecica, and Coccomyxa By Abdulla Ahmed Al-Temaimi BEng, Chemical Engineering, University of Qatar MSc, Energy & Enviroment Engineering, The University of Sheffield Thesis submitted in part fulfillment of the requirement for the degree of Doctor of Philosophy Department of Molecular Biology and Biotechnology The University of Sheffield, UK March 2018 1 Declaration I hereby declare that this is my own work and effort. Where other sources of information have been used, they have been acknowledged. Signed by: Abdulla Ahmed Al-Temaimi 2 Abstract The effects of stress conditions on neutral lipid accumulation by the microalgae Botryococcus braunii, Tetraselmis suecica and Coccomyxa were investigated to assess their suitability for biodiesel production. The growth media (3N-BBM+V for B. braunii, and Coccomyxa and F/2 for T. suecica) were altered to impose salt stress (0.1 and 0.2 M NaCl for BBM medium) and up to 1.2 M NaCl for F/2 medium. Tetraselmis suecica and Coccomyxa were also cultivated under nitrogen depletion conditions (50%, 25% nitrogen and nitrogen free) to monitor growth and investigate neutral lipid accumulation. It was found that B. braunii grew very slowly despite using the relatively nutrient rich BBM medium. It was difficult to reach sufficient biomass to run experiments for B. braunii, but faster growth was achieved using a 2 litre fermenter. B. braunii had a total lipid content of 29% but only 3.64% were neutral (storage) lipids. This is much less than the 30% neutral lipid normally quoted as the minimum requirement for biodiesel production (Chisti, 2007). The B. braunii strain (CCAP 807/1) used failed to grow in the presence of 0.1 or 0.2 M NaCl. Tetraselmis suecica grew in the higher salinity media, but salt stress did not induce higher lipid accumulation. Nitrogen free medium did induce more neutral lipid accumulation in T. suecica even though the growth rate and final biomass level reached were decreased. Tetraselmis suecica had less than the 30% neutral lipid normally quoted as the minimum requirement for biodiesel production, but it is an excellent candidate for production of biodiesel based on the fatty acids produced which are principally C16:0 and C18:1. Neutral lipid accumulation increased rapidly with increasing nitrogen starvation from 25% nitrogen to nitrogen-free medium as Coccomyxa grew to reach stationary phase over four weeks. Nitrogen free medium induced more neutral lipid accumulation (31%) in Coccomyxa, which was the highest percentage found in the current work and meets the 30% neutral lipid figure suggested by Chisti et al. (2007). 3 Acknowledgment There is no one greater to thank than Allah, our creator, whose blessings, good health, and wellbeing enabled me to complete my research. First of all, I’d like to show my appreciation to my parents who are by far my greatest role models and have shown me great support and believed that I’ll be where I am today. Most importantly, I wish to thank my wife who is always there supporting me in whatever I pursue. I would especially like to thank my mentor Dr. Jim Gilmour, Senior Lecturer and level 3 tutor in Molecular Biology and Biotechnology Department, for his continuous support and great insight that assisted me greatly in the implementation of this project. He taught me more than I could ever give him credit for. He has shown me what a good scientist and person should be. I’d like to thank my sponsors in Qatar; The Ministry of Municipality and Environment and The Ministry of Interior, for their financial, logistical support, and the opportunity to complete my Phd studies in The University of Sheffield. I’d like to thank my fellow lab partners in Gilmour’s lab whom I had the pleasure to work with during this project; Richard Smith, Adel Almutairi, Adnan Almousawi, Hatice Burak, Halima Alhani, Gloria Padmaperuma, Tom Burns, and Fawzia Albluwi. They have been really supportive and were constantly sharing their wisdom and experiences throughout our studies. And to thank them for making my journey filled with memorable moments. Last but not least, I wish to extend my sincere gratitude to Simon Thorpe, faculty of Science, Mass Spectrometry Centre, and Professor Michael Williamson, head of department, for their encouragement, insightful comments, and immense knowledge. I truly admired their spirit of cooperation, beneficial support, and advice. 4 Table of Contents 1.0 Introduction .................................................................................................................................... 12 1.1 Context of Work .......................................................................................................................... 12 1.2 Microalgae as a source for biodiesel ............................................................................................ 13 1.2.1 Carbon Dioxide (CO2)............................................................................................................. 13 1.2.2 Water.................................................................................................................................... 14 1.2.3 Light ...................................................................................................................................... 14 1.2.4 Land ...................................................................................................................................... 15 1.3 Microalgae Strains ....................................................................................................................... 16 1.3.1 Botryococcus braunii ............................................................................................................. 16 1.3.2 Tetraselmis suecica ............................................................................................................... 16 1.3.3 Coccomyxa ............................................................................................................................ 17 1.4 Molecular Identification of Algal Strains and DNA Sequencing...................................................... 19 1.4.1 Polymerase Chain Reaction (PCR) .......................................................................................... 19 1.5 Lipids in Microalgae ..................................................................................................................... 20 1.6 Methods of Lipid Induction .......................................................................................................... 21 1.6.1 Nutrient Starvation ............................................................................................................... 21 1.7 Harvesting and Lipid Extraction of Algal Cells ............................................................................... 23 1.8 Algal Lipid Detection and Quantification ...................................................................................... 24 1.9 Biodiesel Production .................................................................................................................... 25 1.10 Aims and Objectives ................................................................................................................... 26 Chapter Two Materials and Methods ..................................................................................................... 27 2.1 Cleaning and Sterilization Techniques .......................................................................................... 27 2.2 Algae Strains ................................................................................................................................ 27 2.2 Collection of Samples ................................................................................................................... 28 2.3 Medium Preparation .................................................................................................................... 28 2.3.1 3N-BBM+V Medium for Botryococcus braunii and Coccomyxa ............................................... 28 2.3.2 Modified 3N-BBM+V Medium ............................................................................................... 30 2.3.3 F/2 Medium for Tetraselmis suecica ...................................................................................... 31 2.3.4 Modification of F/2 medium .................................................................................................. 32 2.4 Growth of algal strains ................................................................................................................. 36 2.4.1 Botryococcus braunii culture methods................................................................................... 36 5 2.4.2 Botryococcus braunii Fermenter Growth ............................................................................... 36 2.4.3 Tetraselmis seucica culture methods ..................................................................................... 37 2.4.4 Coccomyxa Culture methods ................................................................................................. 37 2.5 Molecular Identification of the Algal Strains ................................................................................. 38 2.5.1 Extraction of DNA .................................................................................................................. 38 2.5.2 Gel Electrophoresis ..............................................................................................................
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