Abate Dawit (PhD)Milkessa Tamene2018-07-172023-11-082018-07-172023-11-082017-05http://etd.aau.edu.et/handle/123456789/8830Oleaginous 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,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 oilenBiodieselBiomassCultivation conditionsFatty acidlipid contentlipid concentrationOleaginous yeastSingle cell oilThesis