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Addis Ababa University School of Graduate Studies ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES DEPARTMENT OF MICROBIAL, CELLULAR AND MOLECULAR BIOLOGY ISOLATION OF OLEAGINOUS YEASTS AND OPTIMIZATION OF SINGLE CELL OIL FOR BIODIESEL PRODUCTION BY TAMENE MILKESSA JIRU SUPERVISOR DAWIT ABATE (PHD) MAY, 2017 ADDIS ABABA ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES ISOLATION OF OLEAGINOUS YEASTS AND OPTIMIZATION OF SINGLE CELL OIL FOR BIODIESEL PRODUCTION By Tamene Milkessa Jiru A Thesis submitted to School of Graduate Studies of the Addis Ababa University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (PhD) in Biology (Applied Microbiology) Approved by Examining Board Name Signature Dr. Gurja Belay Chairperson ------------------------------- Dr. Dawit Abate Advisor --------------------------------- Prof. James Chukwuma External Examiner -------------------------------- Dr. Amare Gessesse Internal Examiner -------------------------------- Abstract Oleaginous yeasts are known to produce oil with high potential as source of biodiesel. In this study, 340 yeast colonies were isolated from 200 samples that were collected from natural sources in Ethiopia. All the yeast isolates were screened using Sudan III staining for oil production. Among these, 18 were selected as possible oleaginous yeasts. Identification of the 18 isolates was done using morphological and physiological methods as well as sequencing of the internal transcribed spacer regions (ITS; ITS 1, ITS 2 and the intervening 5.8S rRNA gene), and the D1/D2 domain of the 26S rRNA gene. Molecular phylogenetic analyses indicate that isolates PY39, SY89 and SY94 are species of Cryptococcus curvatus, Rhodosporidium kratochvilovae and Rhodotorula dairenensis, respectively, while the rest (SY09, SY18, SY20, PY21, PY23, PY25, SY30, PY32, SY43, PY44, SY52, PY55, PY61, SY75, and PY86) were identified as Rhodotorula mucilaginosa. From these Cryptococcus curvatus PY39, Rhodotorula mucilaginosa SY09, Rhodotorula mucilaginosa SY18 and Rhodosporidium kratochvilovae SY89 were selected for further activities based on their substantial lipid producing capacities. To determine the optimal cultivation conditions for oleaginous yeasts, different carbon and nitrogen sources, carbon to nitrogen ratio, pH and inoculum size were investigated. Moreover, incubation temperature, shaking speed, culture volume (aeration rate) and duration of cultivation were investigated. Wide variations were recorded in the cultivation conditions that lead to maximum lipid production by the yeasts under test. The maximum lipid production was attained within 120-144 h, using 50-70 g/L glucose as a carbon source, 0.50 g/L yeast extract and 0.31-0.85 g/L, (NH4)2SO4 as nitrogen sources, at C/N ratio of 100-140, pH range 5-6, 10% inoculum size as seed culture, 30oC incubation temperature, shaking speed of 200- 225 rpm and 50 mL culture medium. Lipid content was determined by solvent mixture of chloroform and methanol (2:1). Under the optimized cultivation conditions, i Cryptococcus curvatus PY39, Rhodotorula mucilaginosa SY09, Rhodotorula mucilaginosa SY18 and Rhodosporidium kratochvilovae SY89 accumulated lipids up to 7.22±0.26, 5.73±0.62, and 6.47±0.05 and 7.65±0.77 g/L, respectively on dry weight basis. Such values correspond to lipid content of 48.66±0.60, 38.38±3.90, 40.74±0.54 and 51.17±0.72%, respectively. These strains were further grown on media containing peel mixtures of papaya and mango. Under the optimized conditions, Cryptococcus curvatus PY39, Rhodotorula mucilaginosa SY09, Rhodotorula mucilaginosa SY18 and Rhodosporidium kratochvilovae SY89 gave lipid yields and lipid contents of 3.95±0.67 g/L and 35.02±1.63%, 2.66±0.49 g/L and 28.15±1.63%, 3.84±0.19 g/L and 36.76± 0.61%, and 4.31± 0.30 g/L and 35.18±1.40%, respectively. The fatty acids profiles were analyzed using gas chromatography. Data revealed the presence of high amount of oleic acid (47.44±2.14– 54.40±1.15%), palmitic acid (10.69±0.66–24.04±0.39%), linoleic acid (6.34±0.64–21.24± 0.36%) and low amount of other fatty acids in the extracted yeast oils which indicate that the fatty acid profiles fit well with that of conventional vegetable oil. Furthermore, lipid production capacity of Rhodosporidium kratochvilovae SY89 was evaluated using molasses as a substrate in a bioreactor and gave a maximum lipid concentration of 4.82±0.27 g/L which corresponds to 38.25±1.10% of lipid content. The extracted lipid was transesterified into biodiesel and gave a yield of 85.30%. The properties of this biodiesel were determined and found to be comparable to the specifications established by ASTM D6751 and EN14214 related to biodiesel quality. In conclusion, this study revealed the possibility of using the promising yeast isolates in biodiesel production. Keywords/phrases: Biodiesel, biomass, cultivation conditions, fatty acid, lipid content, lipid concentration, oleaginous yeast, single cell oil ii Acknowledgements I would like to express my gratitude and appreciation to my supervisor Dr. Dawit Abate for putting long hours of his time in the remarkable supervision, constructive criticisms and guidance throughout this study. I also thank my co-supervisors Professor Carlien Pohl-Albertyn and Dr. Nicholas Kiggundu, who helped me a lot professionally all the way, especially during my stay at the University of Free State, South Africa and Makerere University, Uganda, respectively. I would also like to extend my gratitude to my research colleagues and friends, Moges Kibret, Teshome Geremew, Asmamaw Tesfaw, Alemayehu Amare, Muluye Teka, Ashebir Simiret and Yonas Chekol for their contributions towards my research. I am very grateful to Mr. Fikadu Gadissa for his help in phylogenetic tree construction of the oleaginous yeasts. I am also thankful to Mr. Alemu Beyene for his help in preparing map of sample collection sites. I am grateful to Dr. Marizeth Groenewald from CBS Fungal Biodiversity Center, Utrecht, Netherlands for her help in sequencing my yeast strains. I am also thankful to Zenebech Aytenew, Norma Derby, Prossie Nakayiki, Manda Nangobi, Stella Bykia, Shegz Kuloyo, Aurelia Van Wyk, Luarinda Steyn, and Sarel Marais for their help at various stages during my study. I extend my personal gratitude and respect to Dr. Fassil Assefa and Dr. Gurja Belay for providing me with the necessary administrative and financial assistance in their respective administrative period of the Department of Microbial, Cellular and Molecular Biology as a head. iii I would also like to thank my wife Zinash Deyaso, my father Milkessa Jiru and my mother Abaynesh Begna for their kind support. My daughter Sosina and my son Natnael made my life warmer. I am grateful to Ethiopian Ministry of Science and Technology for their financial support. I would also like to thank my university, University of Gondar for their financial and material overall support. I owe due thanks to the Department of Microbial, Cellular and Molecular Biology, Addis Ababa University for giving me the opportunity to pursue PhD work and for all the cooperations given to me. I am grateful for those who were part of the INTRA ACP-ARISE scholarship; European Commission, University of Cape Town, Addis Ababa University and Makerere University for the opportunity given to me do part of my PhD research at Makerere University, Uganda. I praise Almighty God!!! iv Table of Contents Abstract…………………………………………………………………………………………….i Acknowledgements………………………………………………………………….………...…iii Table of Contents………………………………………………………………………………….v List of Tables……………………………………………………………………………………..xii List of Figures…………………………………………………………………………………...xiii List of Appendices ………………………………………………………………………………xv List of Abbrevations and Acronyms………………………………………..……………………xvi Published and Submitted Papers from the Dissertation........................................................…..xviii Chapter 1: Introduction .......................................................................................................... ….1 1.1 General background………………………………………………………………………...1 1.2 Statement of the problem...................................................................................................... 4 1.3 Research Objectives.............................................................................................................. 5 1.3.1 General objectives.......................................................................................................... 5 1.3.2 Specific objectives ......................................................................................................... 5 Chapter 2:Literature Review....................................................................................................... 6 2.1 Diversity and ecology of oleaginous yeasts.......................................................................... 6 2.2 Isolation and cultivation of yeasts......................................................................................... 8 2.3 Yeast taxonomy and identification …………………………………………………………9 2.3.1 Oleaginous yeast taxonomy ................................................................. ……………….9 2.3.2 Yeast identification ...................................................................................................... 10 2.3.2.1 Classical methods.................................................................................................. 10 2.3.2.2 Kits for yeast identification................................................................................... 13 v 2.3.2.3 Molecular methods of yeast identification...........................................................
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