Browsing by Author "Milkessa Tamene"

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    Evaluation of Yeast Biomass Production Using Molasses and Supplements
    (Addis Ababa University, 2009-08) Milkessa Tamene; Abate Dawit (PhD)
    Three yeast strains of saccharomyces cerevisiae, namely commercial baker’s yeast (BA), an isolate from teff dough (TE) and an isolate from tella(TL) were cultivated in the laboratory by submerged method to determine biomass yield. The biomass of these yeast strains was compared with respect to molasses concentrations(3% w/v,5% w/v,8% w/v and 10% w/v), pH(3.5,4.0,4.5,5.0 and 5.5),growth temperatures ( 250C,300C and 370C), duration of incubation( 24,48,72 and 96 hrs) and the effect of addition of supplements as treatments; T1-(NH4)2SO4 (0.5 % w/v),T2-(NH4)2SO4 (0.5 % w/v) and KH2PO4 (0.3 % w/v),T3-(NH4)2SO4 (0.5 % w/v), KH2PO4 (0.3 % w/v) and peptone (2% w/v),T4-(NH4)2SO4 (0.5 % w/v), KH2PO4 (0.3 % w/v),yeast extract(1%w/v) ,MgSO4.7H2O (0.05 % w/v ) and CaCL2.2H2O (0. 004 % w/v ),T5-(NH4)2SO4 (0.5 % w/v), KH2PO4 (0.3 % w/v), peptone (2%w/v),yeast extract(1%w/v),MgSO4.7H2O (0.05%w/v) and CaCL2.2H2O (0.004% w/v), biotin(0.005%w/v) and calcium panthetonate (0.0001% w/v). The contents of molasses were analyzed before the cultivation process and it was found that the molasses used for this study contains 43.1 % sugar, 0.25% total nitrogen, 1.56 % crude protein, 17.9 % moisture content, 82.1% dry weight and 11.7 % total ash. With respect to molasses concentration, BA isolate showed maximum biomass yield at 5%, 8% and10% concentrations, whereas TE isolates showed the same trend at 5% and 8% concentrations. TL isolate was found to accumulate the maximum yield at 8% molasses concentrations. In all cases, the isolates showed similarity in high biomass accumulation when they were grown at 8% w/v molasses concentration. Concerning the effect of pH on the growth of yeasts, isolate BA was found to be effective at all pH values except pH 5.5; whereas TE isolate was effective at pH 4.0, pH 4.5 and 4.5. Furthermore, isolate BA and isolate TE were also effective at pH 4.5. At pH 3.5 and 5.5, there was a steady decrease in biomass yield by all the isolates. With respect to incubation temperatures, the different isolates displayed biomass yield ranging from 1.27g/l to 3.25g/l. All isolates showed slow growth at 250C, and 370C with subsequent slow increase as the incubation temperatures increased. The highest biomass was observed at 300C by isolates BA (2.98-3.2g/l in 24-72 hrs), TE (2.91-3.1g/l); whereas isolate TLshowed biomass increase of 2.81g/l .Supplementing molasses media with (NH4)2SO4 (0.5% w/v) (Treatment1) increased the biomass of TL (5.6-5.9g/l), TE (5.6-6.2g/l), and BA (6.1-6.4g/l within 24 and 72 hrs. In all cases the maximum biomass was achieved within 48 hrs. When this compared with biomass accumulation on molasses alone, the inclusion of the supplemental nitrogen source showed 1.5-2 fold increase in yeast dry weight by all isolates. Comparing the growth of the isolates on molasses and ammonium sulphate as control the isolates did not show significant difference in biomass with further treatments (T2-T5). The incorporation of all the necessary supplements resulted in maximum biomass production by BA (8.0 g/l), followed by TE (7.5 g/l) and TL (6.5 g/l). In all the biomass propagation processes, the commercial baker’s yeast strain, BA was superior in giving high biomass yield. Further more the leavening action of the two yeast strains, i.e., an isolate from teff dough (TE) and commercial baker’s yeast (BA) was compared at room temperature and 300C. BA was found to be higher than TE both at room temperature and 300C. Key words/phrases/: Baker’s yeast (Saccharomyces cerevisiae), Biomass, Leavening action, Molasses, Supplements
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    Isolation of Oleaginous Yeasts and Optimization of Single Cell Oil for Biodiesel Production
    (Addis Ababa Universty, 2017-05) Milkessa Tamene; Abate Dawit (PhD)
    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,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