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Investigation of Neutral Lipid Production in Desmodesmus armatus and Synechocystis sp PCC6803 By ADNAN ABDULAH HAMAD AL-MOUSAWI BSc, in Biology, University of Basrah, Iraq, 2001 MSc, in Biotechnology, University of Baghdad, Iraq, 2004 Thesis submitted in part fulfilment of the requirement For the degree of Doctor of Philosophy Department of Molecular Biology and Biotechnology The University of Sheffield, UK SeptemBer 2017 Declaration I hereBy declare that this thesis is my own work and effort. Where other sources of information have Been used, they have Been acknowledged. Signed: By Adnan Al-Mousawi I Dedication To My beloved country (IRAQ) & MY Father (Dr. ABDULLAH AL-MOUSAWI) My Cousins: Naofal (Abo Sharar) &Mujtaba (May Allah have mercy them) II Abstract Microalgae has been introduced in the last decades to be an optimum source for biofuel production that can be utilised efficiently to replace the conventional fossil fuels as a source of energy. In our study, a local strain of green microalgae was isolated from the Weston Park pond (Sheffield, UK) and identified, using different molecular markers including 18S rDNA, ITS1, ITS2, and 5.8S rDNA, as Desmosdesmus armatus. The neutral lipid content (as the basis of renewable biodiesel production) of both D. armatus and the model cyanobacterium Synechocystis sp. was measured using the lipophilic fluorescent dye Nile Red. The two strains were grown in normal BG11 medium and under stress conditions including high salinity (0.2, 0.4 M NaCl), different concentrations of sodium nitrate in the BG11 media (10% NaNO3, N-free), and different sources of nitrogen (NH4Cl, urea). The results showed that N-free BG11 medium was the best stress conditions in both strains for inducing a significant (p<0.05) increase in neutral lipid content. Further work concentrated on D. armatus and the Fatty Acid Methyl Esters (FAME) conversion yield was examined using a direct transesterification method and the composition of fatty acids was investigated using GC-MS. Desmodesmus armatus grown in N-free BG11 medium showed the highest yield and the contents of C16 and C18 fatty acids (useful for biodiesel production) increased significantly under this stress condition. Further analysis of D. armatus lipid content was conducted using 1H NMR and the results confirmed that the fatty acid content is much higher in the N free grown cells. The final set of experiments focused on random mutation of D. armatus cells using Ultra Violet light (254 nm) to generate new strains with high neutral lipid content. The high lipid containing cells were isolated using Nile Red staining and automated fluorescence assisted cell sorting (FACS) flow cytometry technique. A mutant strain was isolated with 5 times greater yield of neutral lipid than the wild type strain based on the median of the Nile Red fluorescence of the wild type and the mutant cells. This significant increase in the lipid yield using UV-induced random mutation, Nile Red staining and FACS has III paved the way for further investigation of the molecular and genetic approaches to identify the key genes that control neutral lipid biosynthesis in microalgae. IV Acknowledgement The supreme thanks belong to Allah, the Almighty, the creator, without his care I could not imagine who to finish my hard four years work. I am massively grateful my gorgeous supervisor Dr. James Gilmour for his excessive support, infinite patience, calm resolve, and huge experience that help me to overcome all the obstacles that I have suffered during my project. I would like to thank my sponsor The Higher Education Committee of Education development in Iraq -Iraqi Prime minister office (HCED) who has offered me an opportunity to finish my PhD in the United Kingdom and for their financial and emotional support for me during my project. Also, I would like to thank the Ministry of Higher Education and Scientific Research-Republic of Iraq, University of Basrah, College of Science-Department of Biology, who provide me all the official documents that help me to achieve the scholarship. My great thank to the University of Sheffield, and the Department of Molecular Biology and Biotechnology, for their marvellous efforts to facilitate all the requirements that I require to finish my study. I could not find an appropriate word to express my immensely grateful to my close friends Murtakab Al-Hejjaj, Ehssan Al-Bermany, Naer AlKabbawi, Yasir Alabdali, and all my Iraqi colleagues in Sheffield for their support, help, and love that help me to continue my work. A huge thanks to all my previous and current members of Gilmour¢s lab Richard Smith, Adel Almutairi, Abdullah AlTemaimi, Hatice Burak, Halima Alhani, Gloria Padmaperuma Tom Burns, and Fawzia for their scientific and emotional support during my study. Also I would to thank staff members in the university of Sheffield which have helped me in many experiment, including Simon Thorpe (Chemistry) for GC-MASS assistance, Professor Mike Williamson for processing NMR samples, Flow cytometry group V (Medical school) for flow cytometry running. And my colleague Ibrahim Yaseen(MBB) for analysing the flow cytometry results. Finally, I have a deep grateful and thank to my beloved wife, my children Reema, Abdulah, and Deema for their endless patience and emotional support and love that have offered to me during these four years. Also, I would like to thank my beloved mum, brother, sisters and my relatives especially my uncle Fakhir Al-Mousawi and his family for their hugely support, prayer, to help me finish this project. VI Table of Contents Abstract .................................................................................................................. III Acknowledgement ................................................................................................... V List of Figures ........................................................................................................... X List of Tables ......................................................................................................... XIII AbBreviation ......................................................................................................... XIV 1 Chapter One Literature Review ........................................................................ 15 1.1 Introduction ........................................................................................................ 16 1.1.1 Global warming and fossil fuel .......................................................................... 16 1.1.2 Renewable energy ............................................................................................. 18 1.1.3 Biofuels .............................................................................................................. 20 1.1.4 Biodiesel ............................................................................................................ 27 1.1.5 Microalgae ......................................................................................................... 28 1.1.6 Microalgal Lipid ................................................................................................. 29 1.1.7 Algal lipid biosynthesis ...................................................................................... 30 1.1.8 Microalgal cultivation ........................................................................................ 32 1.1.9 Strategies to induce lipid synthesis in microalgae ............................................. 36 1.2 Aims of the project .............................................................................................. 42 2 Chapter Two Materials and Methods .............................................................. 43 2.1 Cleaning and Sterile Techniques .......................................................................... 44 2.2 BG11 Medium ..................................................................................................... 44 2.2.1 Modified BG11 Media ....................................................................................... 45 2.3 Collection of Samples .......................................................................................... 46 2.4 Cell Density Evaluation ........................................................................................ 48 2.4.1 Spectrophotometer ........................................................................................... 48 2.4.2 Cell Counting and Growth Curve Determination ............................................... 48 2.5 Microscopic Examination of the Pond Samples .................................................... 49 2.6 Assessing Bacterial Contamination and Purification Methods .............................. 49 2.6.1 PercollTM Treatment .......................................................................................... 50 2.6.2 Centrifuge Washing Technique (Purification of Algal Samples) ........................ 50 2.7 Molecular Identification of the Strains ................................................................ 51 2.7.1 Extraction of DNA .............................................................................................. 51 2.7.2 Gel Electrophoresis ............................................................................................ 54 2.7.3 Polymerase Chain Reaction amplification ......................................................... 54 2.7.4 PCR Purification ................................................................................................. 58 2.7.5 DNA Quantification