Browsing by Author "Tesfaye, Anteneh (PhD)"
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Item Bioethanol Production Via Fermentation of Waste Substrates Using Yeast Isolates(Addis Ababa University, 2019-05-05) Tsegu, Getu; Tesfaye, Anteneh (PhD)Bioethanol is one biomass derived platform molecules which has a potential to be a sustainable feedstock for a variety of commodity chemicals. The aim of this study was to produce bioethanol from grain waste flour, tella spent, pineapple and papaya peels. Dilute sulfuric acid was used to hydrolyze polysaccharide in the raw materials. Starch content in grain waste flour and reducing sugar concentration in the hydrolysates of each raw material was determined. Indigenous yeast was isolated from areki difdif and tej samples. Indigenous yeast isolates with high fermentation efficiency and high ethanol producing ability were used to ferment hydrolysates in each of raw material to bioethanol. The results indicate that pretreatment of grain waste flour, papaya peels, pineapple peels and tella spent with 2% sulfuric acid at 110oC for 90 minutes yielded 29.7–30.8, 28.2–28.9, 16.2–16.6 and 10.3–10.5% of reducing sugar, respectively. The yeast isolate 1T-10 from tej samples was efficient in converting the D-glucose to ethanol (75.58%) and produced highest yield of ethanol (7.72%) from 20% D-glucose compared to other yeast isolates. Fermentations of grain waste flour with 1T-10 produced the highest yield of ethanol 1.380% (w/v) from pretreatment of substrates with 2% sulfuric acid at 168 hours of fermentation time compared to other combinations or 0% at (72, 120 and 168 hours), 1% at (72, 120 and 168 hours), 1.5% at (72, 120 and 168 hours) and 2% at (72 and 120 hours). Similarly, fermentation of pineapple peels, papaya peels and tella spent produced the highest yield of ethanol; 7.883%, 6.400% and 4.813% (w/v) with pretreatment of 2% sulfuric acid at 120, 72 and 72 hours of fermentation time, respectively compared to other combinations when fermented with the same yeast isolate (1T-10). It was concluded from the present study that it is possible to produce economically viable bioethanol by digesting waste and cheap resources like grain waste flour, tella spent, pineapple and papaya peels using indigenous yeast isolates.Item Characterization of Indigenous Wild Yeasts and Evaluation of Their Bread Leavening Capacity(Addis Ababa University, 2017-08-03) Beyene, Elsa; Muleta, Diriba (PhD); Kiros, Solomon (PhD); Tesfaye, Anteneh (PhD)Ethiopia is a developing country with a high baker’s yeast demand for bread making and beverage industries. However, there is no baker’s yeast producing plant in the country. Therefore, the objective of this study was to assess bread leavening potential of wild yeasts from teff and wheat dough, shamita, tej, tella, molasses and honey samples, following standard methods. A total of 180 wild yeast isolates were, out of these isolates 23 isolates with high biomass yield (1.7 to 2.6 of 600 nm OD reading) and the efficient gas (CO2) producers in durum tube within 12 to 24 hours were selected for further analysis. Morphological, biochemical and molecular features were used to identify and characterize the yeasts. Isolates identified by biolog were belongs to genera Saccharomyces, Candida humilis and Kluyveromyces and found to utilize glucose, sucrose, raffinose, trehalose and maltose. Genetically, the sequence analysis of the ITS-5.8S rDNA region showed that the isolates belong to 5 species namely, Saccharomyces cerevisiae, Candida humilis (synonyms Candida milleri), Kazachstania bulderi, Pichia fermentans and Pichia kudriavzevii. All the test isolates were found to produce maximum cell mass at a temperature of 30oC, pH of 5.5 and 48 hours of incubation. Isolate AAUTf1 did not produce hydrogen sulfide. The isolates AAUTf5, AAUTj15 and AAUSh17 including commercial yeast produced light level of hydrogen sulfide, while AAUMl20 and AAUWt21 produced heavy level of hydrogen sulfide. The assessment of leavening performance of the yeasts indicated that, isolates AAUTf1 (Candida humilis) and AAUTf5 (Kazachstania bulderi) had higher leavening activity with raising dough by 131 cm3 and 128 cm3 respectively each within 120 min. Similarly, isolates AAUSh17 (Saccharomyces cerevisiae) and AAUTj15 (Saccharomyces cerevisiae), were found rising the volume of the dough by 127 cm3 and 125 cm3, respectively at 60 min compared to the commercial yeast that increased the volume to 117 cm3 within 90 min. The combined bread dough rising effects of AAUTf1 + AAUTf5 + AAUTj15, AAUTf5 + AAUTj15 and AAUTf1 + AAUTj15 + AAUSh17 caused volume increment to 143 cm3 at 90 min, 141 cm3 at 60 min and 140 cm3 at 60 min, in that order. Generally, mixed cultures showed superior leavening potential than that of single cultures and commercial yeast.Item Characterization of Indigenous Wild Yeasts and Evaluation of Their Bread Leavening Capacity(Addis Ababa University, 2017-08-02) Beyene, Elsa; Muleta, Diriba (PhD); Kiros, Solomon (PhD); Tesfaye, Anteneh (PhD)Ethiopia is a developing country with a high baker’s yeast demand for bread making and beverage industries. However, there is no baker’s yeast producing plant in the country. Therefore, the objective of this study was to assess bread leavening potential of wild yeasts from teff and wheat dough, shamita, tej, tella, molasses and honey samples, following standard methods. A total of 180 wild yeast isolates were, out of these isolates 23 isolates with high biomass yield (1.7 to 2.6 of 600 nm OD reading) and the efficient gas (CO2) producers in durum tube within 12 to 24 hours were selected for further analysis. Morphological, biochemical and molecular features were used to identify and characterize the yeasts. Isolates identified by biolog were belongs to genera Saccharomyces, Candida humilis and Kluyveromyces and found to utilize glucose, sucrose, raffinose, trehalose and maltose. Genetically, the sequence analysis of the ITS-5.8S rDNA region showed that the isolates belong to 5 species namely, Saccharomyces cerevisiae, Candida humilis (synonyms Candida milleri), Kazachstania bulderi, Pichia fermentans and Pichia kudriavzevii. All the test isolates were found to produce maximum cell mass at a temperature of 30oC, pH of 5.5 and 48 hours of incubation. Isolate AAUTf1 did not produce hydrogen sulfide. The isolates AAUTf5, AAUTj15 and AAUSh17 including commercial yeast produced light level of hydrogen sulfide, while AAUMl20 and AAUWt21 produced heavy level of hydrogen sulfide. The assessment of leavening performance of the yeasts indicated that, isolates AAUTf1 (Candida humilis) and AAUTf5 (Kazachstania bulderi) had higher leavening activity with raising dough by 131 cm3 and 128 cm3 respectively each within 120 min. Similarly, isolates AAUSh17 (Saccharomyces cerevisiae) and AAUTj15 (Saccharomyces cerevisiae), were found rising the volume of the dough by 127 cm3 and 125 cm3, respectively at 60 min compared to the commercial yeast that increased the volume to 117 cm3 within 90 min. The combined bread dough rising effects of AAUTf1 + AAUTf5 + AAUTj15, AAUTf5 + AAUTj15 and AAUTf1 + AAUTj15 + AAUSh17 caused volume increment to 143 cm3 at 90 min, 141 cm3 at 60 min and 140 cm3 at 60 min, in that order. Generally, mixed cultures showed superior leavening potential than that of single cultures and commercial yeast.Item Characterization of Wild Indigenous Yeasts from Molasses and other Sugary Substrates and their Potential for Bioethanol Production(Addis Ababa University, 2021-05-09) Degu, Sisay; Muleta, Diriba (PhD); Tesfaye, Anteneh (PhD)The increasing demand of energy has been supplied through the combustion of petroleum throughout the world. Due to escalating cost of petroleum, its contribution to global warming and non-renewable nature of this oil, there is collectively a need for renewable and ecofriendly energy sources such as bioethanol. Thus, the objective of this study was aimed at isolating, characterizing and evaluating the potent wild yeasts under different stress conditions from locally available resources for bioethanol production to minimize the utility of fossil fuel. In this study, a total of 35 samples of sugary substrates were collected from Metehara Sugar Factory for wild yeasts isolation following the standard protocols. A total of 305 yeast isolates were retrieved and screened using physiological and osmotic stress tolerance tests. Fermentative and potent wild yeasts were identified to species level using morphological and biolog-based biochemical methods. Out of 305 yeast isolates, 20 (6.56%) and 7 (2.29%) of them were found to be tolerant to 18 and 20% of ethanol, respectively. Out of these 20 ethanol tolerant yeast isolates, 17 tolerated the temperature of 45°C for 48 hrs. From 17 ethanol-thermotolerant isolates, 5 (29.41%), 5 (29.41%) and 7 (41.18%) were found producing gas from glucose at 24, 48 and 72 hrs, respectively. Out of the 17 ethanol-thermotolerant yeast isolates, 12 yeast isolates were able to tolerate 35% of glucose. Out of these 12 ethanol-thermo-and-sugar tolerant yeasts, the 7 were found tolerant to pH 2. From the 7 acidic tolerant yeast isolates, 5 yeast isolates were shown tolerant to 7% of NaCl and identified as K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively. During fermentation, K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa were able to produce bioethanol with the values of (% v/v) 12.62, 11.61, 10.58, 10.82 and 10.44, respectively at 48 hrs and 12.56, 12.52, 12.08, 11.11 and 11.48, respectively at 72 hrs. The initial inoculum cell density was increased from (1.83 to 3.08, 0.59 to 1.81, 0.54 to 1.36, 0.48 to 1.69 and 0.45 to 1.56) x108 cells/ml for K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively 0 hr to72 hrs. The fermentation efficiency (%) for K. lodderae, P. guilliemodii B, S. boulardii, T. globosa and Z. rouxii was shown as 98.0, 97.8, 93.1, 90.8 and 88.1, respectively. Thus, on the basis of highest stress tolerant and higher fermentation efficiency features, K. lodderae and P. guilliemodii B wild yeasts were considered to be the best bioethanol producers. Molecular characterization and optimization of fermentation parameters is recommended to utilized these potent yeasts.Item Characterization of Wild Yeasts Isolated from Selected Fruits for their Bread Leavening Capacity(Addis Ababa University, 2017-06) Lakew, Eshet; Muleta, Diriba (PhD); Tesfaye, Anteneh (PhD)Leavening agents are important in raising flour dough. Biological leavening agents are microorganisms that have the ability to produce carbon dioxide from the utilization of Sugar and thereby ferment and raise the dough. The present study was carried out to characterize yeast isolates isolated from selected fruits and to assess their leavening potential of wheat dough under laboratory scale. The collected fruit samples were processed to isolate yeasts using Potato Dextrose Agar (PDA) amended with 0.1 g/L chloramphenicol. Initially, 88 yeasts were isolated from the fruits and were first tested for their carbohydrate fermentation in yeast extract peptone dextrose (YEPD) broth medium. Six yeast isolates with their sugar fermentative abilities were selected and tested for H2S production. Among them, AAUGr5, AAUOr7 and AAUPi3 found not produce undesirable H2S for bread baking quality on both Kligler Iron Agar (KIA) and Bismuth Sulfite Agar (BSA) media. The three yeast isolates were identified as Saccharomyces using colonial, morphological parameters and biochemical tests. The optimum growth pH and temperature values for the three selected yeast isolates were recorded as 5 and 30 oC, respectively, in YEPD medium. In addition, 30% (w/v) D-glucose and 5 %( w/v) NaCl concentrations showed optimum growth of the three selected yeast isolates in yeast extract peptone broth medium. In all the cases, the maximum biomass was achieved at 96 hrs of incubation and there was a rapid decrease in biomass for all the yeast isolates after 96 hrs of incubation. In terms of CO2 and biomass production as well as leavening potential, starter cultures which were formulated from the combination of the three yeast isolates (AAUGr5+AAUOr7+AAUPi3) showed better performance than starter cultures formulated from paired combination of the three isolates or each of the three isolates separately. However, isolate AAUGr5 was found to be satisfactorily potent for leavening action from the single isolates. The present study could therefore be important with respect to screening of wild yeast isolates that possess better bread leavening potential for extending the use of indigenous microbes as starter culture in bakery sector. Keywords/Phrases: biomass; carbon dioxide ;fruits, hydrogen sulphide; laboratory scale leavening; yeast isolationItem Isolation and Characterization of Potential Bioethanol Producing Indigenous Wild Yeasts from Local Alcoholic Raw Materials (Areke Difdif and Tenses)(Addis Ababa University, 2020-12-12) Admas, Alene; Muleta, Diriba (PhD); Tesfaye, Anteneh (PhD)Shortage of fossil fuel supplies has increased a great deal of interest in worldwide production of renewable energy. Bioethanol produced from renewable biomass like molasses has received considerable attention as an alternative biofuel in recent years. Thus, the main purpose of this research work was to isolate, screen and characterize ethanol tolerant wild indigenous yeasts with high bioethanol yield from areke difdif and tinses samples collected from different areke producing sites. The wild yeast isolates were retrieved from the alcoholic beverages following standard methods. From a total of 270 isolates, ten (3.7%) of the yeast isolates tolerated 22% and four (1.5%) isolates from the same 10 isolates tolerated 23% ethanol concentration, and were selected for further characterization. Based on morphological appearance of vegetative cells under microscope, colony characters and molecular analysis, the isolates were identified as Saccharomyces cervicae and Kluyvermyces marixianus. Ten (Mda, Mdb, Mdd, D5f, D5b, D5d, D5e, Dt1e, Dt1c and Dtlj) isolates were found thermotolerant (45oC and Mda able to tolerate 47 oC) and also all ten isolates grow at low pH (>=2.5). Ethanol production from sugar cane molasses by the yeast isolates was determined by using Hall method and was crosschecked with Ebulliometer. From 10 isolates, two isolates (Mda and Dt1e) showed the highest bioethanol production capacity of 14.3% v/v and 13.2% v/v, respectively at pH 4.39 and temperature 35oC in 30 degree brix molasses concentration at 150 rpm shaking condition. During this study potentially useful and excellent ethanol producing indigenous yeasts with high ethanol tolerant features from areki difdif and tenses were isolated, identified and characterized. This study exhibited the potential of local fermentation processes and products (areke difdif and tenses) as possible sources of industrially useful microbial isolates.Item Lactic Acid Production from Agro-Industrial Wastes Using Lactic Acid Bacterial Isolates(Addis Ababa University, 2020-07-07) Adane, Agurash; Tesfaye, Anteneh (PhD)Lactic acid is a chemical compound that plays an important role in food, pharmaceutical, cosmetics, chemical and polymer industries. It can be produced either through fermentation or chemical synthesis methods. Hence, the objective of this study was production of lactic acid from the fermentation of spent grain of beer and banana peels using indigenous lactic acid bacteria isolated, screened and selected during this study. The effects of temperature, pH, carbon sources, nitrogen sources, and incubation period and inoculum size on lactic acid production during the fermentation processes were also studied. From a total of 90 lactic acid bacteria isolated from ergo and siljo, 66 (84.4%) and 14 (15.6%) LAB isolates were found producing 7.30 to 15.91 and 16-25.2 g/l LA at 48 hrs from 5 ml of MRS broth, respectively. Out of the 14 isolates AS1S that produced the highest amount LA (23.35g/l and 25.20 g/l at 24 and 48 hrs, respectively) from 5 ml MRS broth was selected and subjected to further analysis of LA from produced spent grain of beer (SGB) and banana peel (BNP) during this study. The moisture, ash, protein, fat, fiber and carbohydrate contents SGB and BNP were shown as 7.38% and 5.08%, 3.85% and 5.09%, 4.70% and 6.65%, 3.11% and 1.70%, 4.41% and 12.65%, and 76.56% and 66.59% respectively. The proximate analysis result exhibited that the substrates were nutritionally rich enough to be used as medium to grow lactic acid bacteria to produce LA. During this study the optimum conditions for production of the maximum amount of LA (27.73 and 25.98 g/l from SGB and BNP, respectively), and were obtained at a temperature, pH, nitrogen content, carbon source, and inoculum size of 37 ᵒC, 5.5, 15 g/l NH4)2SO4, 50 g/l glucose and 5% at 72 hrs in that order. The results of this study displayed the potential use of indigenous microbial resources for producing high value biotech products like lactic acid from locally available agro-industrial wastes.Item Microbial Diversity and Probiotic Potential of Lactic Acid Bacteria Isolated from Traditionally Fermented Food Products and Their Applicability for Prevention of Selected Foodborne Pathogens(Addis Ababa University, 2019-11-11) Mulaw, Guesh; Tesfaye, Anteneh (PhD); Muleta, Diriba (PhD); Sisay, Tesfaye (PhD)Probiotics are live microorganisms which when administered in adequate amounts promote the health of the consumer. The major purpose of this study was to determine bacterial diversity of traditionally fermented food products (teff dough, Ergo and Kocho) and to assess their probiotic potential of lactic acid bacteria in prevention of foodborne pathogens. Culture-independent metagenomic methods, particularly 16S rRNA amplicon sequencing were applied to study the bacterial diversity of some fermented foods. The sequencing of the amplicons resulted in an average of 76,594.33 ± 2,876.25 high-quality reads per sample. The bacterial communities of the three traditional fermented foods were dominated by the phylum Firmicutes, followed by Proteobacteria and Actinobacteria. At the genus level, the prevalent microorganisms were Lactobacillus, Zymomonas, Streptococcus, Leuconostoc, Bacillus and Weissella. The genus Lactobacillus was the most abundant genus in the three studied food products, whereas Zymomonas was the most dominant genus in fermented ergo. In order to assess the probiotic potential of strains isolated from these fermented products, a total of 450 LAB were isolated from the fermented products, and their ability to survive the gastrointestinal conditions was tested. Among them, 34 (7.56%) isolates survived at pH 2 for 3 and 6 h, showing a survival rate of 45.35- 96.33% and 33.33- 91.75%, respectively. All the 34 acid-tolerant isolates survived in the presence of 0.3% bile salts for 24 h with 82.58 to 99.44% rate of survival. The acid and bile salt-tolerant LAB isolates shown inhibiting some bacterial foodborne pathogen into varying degrees. Among the 34 acid and bile salt-tolerant isolates, 9 (26.47%) isolates produced bacteriocins to varying degrees. All ii the 34 acid and bile salt-tolerant isolates displayed sensitivity to ampicillin (10 μg/ml), tetracycline (30 μg/ml) and erythromycin (15 μg/ml), but showed resistance to kanamycin (25 μg/ml), while 17/34 of the isolates were found sensitive/resistant to streptomycin (10 μg/ml). The in vitro adherence to stainless steel plates of the 34 screened probiotic LAB isolates showed adhesion rates that ranged from 29.21 to 41.94%. The identification of 34 screened potential probiotic LAB strains and their genetic relatedness was performed using 16S rRNA gene sequencing and whole-genome (WGS) sequence comparisons. Hence, four isolates were identified as Lactobacillus species including Lb. plantarum, Lb. paracasei subsp. tolerans, Lb. paracasei and Lb. plantarum. The remaining 30 potential probiotic LAB strains were identified by using WGS comparisons. Thus, these isolates belonged to Lactobacillus and Lactococcus species including 22 Lb. plantarum, 1 Lb. paracasei, 1 Lb. brevis and 6 Lc. lactis. Among the 30 potential probiotic LAB strains, BAGEL predicted 1 class III bacteriocin, 7 class II bacterioicins and 3 class I bacteriocins in the genome of 9 strains. The compatibility test among the selected potential probiotic strains (Lb. plantarum, Lb. paracasei and Lc. lactis) showed the absence of antagonism among them. The in vitro antagonism testing of the potential probiotic LAB as separate pure or mixed culture against Salmonella showed a remarkable anti-Salmonella activity with 96.50 to 100% growth inhibition. The combination of strains that showed the highest growth inhibition rates against Salmonella Typhimurium, were used to test their in vivo effect on the colonization of mice by Salmonella Typhimurium. The survival rate of mice on day 21 after the oral challenge with Salmonella Typhimurium was significantly higher (p<0.05) in the experimental pro-typ, treated group (100% survival) when compared with the positive control group (20% iii survival). The counts (colony-forming unit/ml) of Salmonella in feces were significantly lower (p<0.05) for the pro-typ group compared to the positive control group (typ). In general, this study indicates a high level of diversity in the bacterial community structure in traditional fermented foods and the LAB of the traditional fermented food samples was found to be good candidates for food industries as prospective probiotic cultures with health benefits.