Isolation Of Oleaginous Yeasts And Optimization Of Single Cell Oil For Biodiesel Production

Oleaginous yeasts are known to produce oil with high potential as source of biodiesel. In this study,rn340 yeast colonies were isolated from 200 samples that were collected from natural sources inrnEthiopia. All the yeast isolates were screened using Sudan III staining for oil production. Amongrnthese, 18 were selected as possible oleaginous yeasts. Identification of the 18 isolates was donernusing morphological and physiological methods as well as sequencing of the internal transcribedrnspacer regions (ITS; ITS 1, ITS 2 and the intervening 5.8S rRNA gene), and the D1/D2 domain ofrnthe 26S rRNA gene.Molecular phylogenetic analyses indicate that isolates PY39, SY89 and SY94rnare species of Cryptococcus curvatus, Rhodosporidium kratochvilovae and Rhodotorularndairenensis, respectively, while the rest (SY09, SY18, SY20, PY21, PY23, PY25, SY30, PY32,rnSY43, PY44, SY52, PY55, PY61, SY75, and PY86) were identified as Rhodotorula mucilaginosa.rnFrom these Cryptococcus curvatus PY39, Rhodotorula mucilaginosa SY09, Rhodotorularnmucilaginosa SY18 and Rhodosporidium kratochvilovae SY89 were selected for further activitiesrnbased on their substantial lipid producing capacities. To determine the optimal cultivationrnconditions for oleaginous yeasts, different carbon and nitrogen sources, carbon to nitrogen ratio,rnpH and inoculum size were investigated. Moreover, incubation temperature, shaking speed, culturernvolume (aeration rate) and duration of cultivation were investigated. Wide variations werernrecorded in the cultivation conditions that lead to maximum lipid production by the yeasts underrntest. The maximum lipid production was attained within 120-144 h, using 50-70 g/L glucose as arncarbon source, 0.50 g/L yeast extract and 0.31-0.85 g/L, (NH4)2SO4 as nitrogen sources, at C/Nrnratio of 100-140, pH range 5-6, 10% inoculum size as seed culture, 30oC incubation temperature,rnshaking speed of 200- 225 rpm and 50 mL culture medium. Lipid content was determined byrnsolvent mixture of chloroform and methanol (2:1). Under the optimized cultivation conditions,Cryptococcus curvatus PY39, Rhodotorula mucilaginosa SY09, Rhodotorula mucilaginosa SY18rnand Rhodosporidium kratochvilovae SY89 accumulated lipids up to 7.22±0.26, 5.73±0.62, andrn6.47±0.05 and 7.65±0.77 g/L, respectively on dry weight basis. Such values correspond to lipidrncontent of 48.66±0.60, 38.38±3.90, 40.74±0.54 and 51.17±0.72%, respectively. These strains werernfurther grown on media containing peel mixtures of papaya and mango. Under the optimizedrnconditions, Cryptococcus curvatus PY39, Rhodotorula mucilaginosa SY09, Rhodotorularnmucilaginosa SY18 and Rhodosporidium kratochvilovae SY89 gave lipid yields and lipid contentsrnof 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±rn0.61%, and 4.31± 0.30 g/L and 35.18±1.40%, respectively. The fatty acids profiles were analyzedrnusing gas chromatography. Data revealed the presence of high amount of oleic acid (47.44±2.14–rn54.40±1.15%), palmitic acid (10.69±0.66–24.04±0.39%), linoleic acid (6.34±0.64–21.24± 0.36%)rnand low amount of other fatty acids in the extracted yeast oils which indicate that the fatty acidrnprofiles fit well with that of conventional vegetable oil. Furthermore, lipid production capacity ofrnRhodosporidium kratochvilovae SY89 was evaluated using molasses as a substrate in a bioreactorrnand gave a maximum lipid concentration of 4.82±0.27 g/L which corresponds to 38.25±1.10% ofrnlipid content. The extracted lipid was transesterified into biodiesel and gave a yield of 85.30%.rnThe properties of this biodiesel were determined and found to be comparable to the specificationsrnestablished by ASTM D6751 and EN14214 related to biodiesel quality. In conclusion, this studyrnrevealed the possibility of using the promising yeast isolates in biodiesel production.rnKeywords/phrases: Biodiesel, biomass, cultivation conditions, fatty acid, lipid content,rnlipid concentration, oleaginous yeast, single cell oil

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