Browsing by Author "Abate Dawit (PhD)"

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    Actinomycetes From Unexplored Environmental Niches in Ethiopia and Their Biotechnological Potentials for Antimicrobial Compound Production
    (Addis Ababa University, 2018-06-04) Kibret Moges; Abate Dawit (PhD); Zotchev Sergey B Professer); Rollinger Judith M. (Professer)
    Actinomycetes have a widely recognized potential for the production of significant bioactive compounds. The major aim of this study was to isolate, screen and evaluate the biotechnological potential of selected actinomycete isolates particularily for antimicrobial compound production using standard bioassays methods, LC-MS, high resolution mass spectrometry (HR-MS) and NMR techniques. It consists of six distinct chapters. In the first chapter, general introduction, statement of the problem and major objectives are presented. The second chapter deals with the review of related literatures. The rest four chapters (3-6) are the experimental sections of the work. Hence, the third chapter concentrated on the isolation, screening, bioactivity detection and phylogenetic analysis of promising actinomycetes capable of producing bioactive secondary metabolites from various unexplored niche habitats in Ethiopia. Among the 416 isolates screened for bioactivities, 101 (24%) isolates were inhibiting the growth of C. albicans, and 88 (21%) isolates were inhibiting both C. albicans (ATCC 62376) and C. neoformans (clinical isolate). Ten isolates having considerable activities were chosen for further investigation and taxonomic identification studies. The polyphasic identification results of these isolates found to be in consistent with the genus Streptomyces described in Bregay’s manual of systematic bacteriology. Identification of the isolates have been verified by the analysis of the 16s rRNA gene sequence. The phylogenetic relationships of the isolates to type strains and best matches based on BLAST search were inferred using the Maximum Likelihood algorithm in MEGA 7 software and confirmed that all the isolates belong to genus Streptomyces. The fourth chapter deals with the cultivation of five promising isolates namely Ac-029, Ac-125, Rv-355, Ac-464 and Go-475 for bioactive secondary metabolite production and subsequently evaluation of SSF process parameters on metabolite yield. Depending on the types of the isolates, variations were observed ii in optimal fermentation process parameters on bioactive secondary metabolite production. It was demonstrated that wheat bran in the presence of supplementary nutrients, an initial moisture content of 65%, a pH value of 7.5, incubation temperature of 30 oC, an inoculum size of 3x107 CFU/mL and incubation period of 12 days were the optimal SSF conditions for most of the isolates studied. The fifth chapter focused on antimicrobial potential of Streptomyces sp. Rv-355 cultivated in submerged culture. In its bioactivity profile, Streptomyces sp. Rv-355 produced antimicrobial compounds with wider spectrum of activities against yeasts, Gram positive and Gram negative bacterial pathogens. It was found that biomass production and bioactivity profiles of Streptomyces sp. Rv- 355 are positively correlated. Bioactivity guided analysis of the crude extract from Streptomyces sp. Rv-355 using TLC, column chromatography, HPLC, LC-MS showed the presence of potential compounds. The partially purified extract showed MIC values of 50μg/mL against Candida albicans and 100μg/mL against Bacillus subtilis. The result is found to be a prelude for further analysis of the crude extract from Rv-355 using HR-MS, and NMR methods. The sixth chapter was targeted on the bioactivity guided identification and structural elucidation of members of benz[a]anthraquinone antibiotics, 8-O-methyltetrangomycin and 8-O-methyltetrangulol from Streptomyces sp. Go-475 extracts using LC-MS, HR-MS/MS and 1H NMR 13C NMR methods. Streptomyces sp. Go-475 displayed potent activity against both yeasts and Gram-positive bacteria with MIC values of the crude extracts 100μg/mL and 50μg/mL against Candida albicans ATCC62376 and Bacillus subtilis ATCC6633 respectively. The analysis revealed that Streptomyces sp. Go-475 is able to produce at least three known secondary metabolites (4-Methoxy-1(3H)-isobenzofuranone, 3-Phenylpropionic acid or 1, 2- Benzenediol and Dehydrocineromycin B) that were not detected in the SmF extract. However, betaine was detected in both SSF and SmF extracts of this isolate. Two important anti-bacterial iii compounds were purified from methanol extract of Streptomyces sp. Go-475 and their structures were elucidated by NMR and HR-MS/MS as 8-O-methyltetrangomycin and 8-Omethyltetrangulol. Besides, many potentially novel metabolites were detected, the majority of which were produced in SSF method. The findings enable us to conclude that Streptomyces sp. Go-475 and other isolates from Ethiopian soil have the capacity to produce potentially new antifungal secondary metabolites and warrant further investigations. The results also proved that SSF as promising economical and best option to produce potential bioactive secondary metabolites from Streptomyces spp. The genome sequence of Streptomyces sp. Go-475 was obtained using a hybrid assembly approach of high quality Illumina short read and low quality Oxford Nanopore long read data. The complete linear chromosome of 8,570,609 bp, featuring a G+C content of 71.96%, contains 7,571 predicted coding sequences, 83 t(m)RNA genes, and six rrn operons. Analysis of the genome for secondary metabolite biosynthesis gene clusters allowed us to connect certain clusters with experimentally confirmed molecules. The findings also verified great potential of Streptomyces sp. Go-475 for the production of chemically diverse secondary metabolites.
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    Alkaline Protease of Alkaliphiles Isolated from Ethiopian Rift Valley Soda- Lakes
    (Addis Ababa University, 2000-05) Hasana Azaga; Abate Dawit (PhD); Gesesse Amare (PhD)
    A prntcolytic alkali phi lie bacteria was selected out of the bacterial isolates obtained from Riftvallcy soda lakes. The isolate was obtained from lake Chitu water sample. The organism was Gram variable aerobic, rod shaped spore forming, motile bacterium. It has been identified to belongs to the genus Bacillus. The growth of the isolate coded as CH-W 1 was observed in the pH and temperature ranges between 7-l l and 25-40°C respectively. Protease production was observed shortly afler inoculation reaching to maximum after 48 h. The crude enzyme had a temperature optimum of 55 °C and a pH optimum of 9. The enzyme was stable in a broad pH range of 8.5- l 0.5 after l hr incubation at 50 ° C . It has a half-life of 30 min. at 60°C . The enzyme was slightly activated by CuSO4 . Where as Ba++ ,Zn ++, Co++ , Hg++ , Mn ++, Fc+r ,.Na', K'and Ca" had very little.or no effect cin the activity of the enzyme. The enzyme was strongly inhibited by I mM PMSF sh o\,,ing that it belongs to the class of serine protease. EDTA at a concentration of IO mM partially inhibits the activity. This shows the requirement of Ca'' for stability. The organism was efficient in degrading Nug meal (GuizotaAbyssinia) and feather when used as a sole carbon source.
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    Antifungal Metabolites from Submerged Culture of Ganoderna Lucidum (Polypore)
    (Addis Ababa University, 1991-06) Bitew Adane; Abate Dawit (PhD)
    About 60 different basidiomycete cultures were screened for antimicrobial secondary metabolites in submerged culture grown in four different media. Ten (17%) of them produced antimicrobial secondary metabolites. Production medium, duration of growth, and the most susceptible test organisms for each producing strain were established. Among basidiomycetes screened for antimicrobial activity, the culture filtrate extract of the Ethiopian strain of the polypore, Ganodemw lucidum produced the most effective antifungal compounds. The cu/~lral characteristics, growth in submerged culture of the polypore and isolation methods of the two antifungal antibiotics are described. Medium A (Yeast extract malt extract glucose medium) was a better medium for the production of the two antifungal agents. These compounds were released to the culture fluid and the maximum amount of antifungal compounds is obtained after 12 days of submerged growth at 120 revolution per minute (rpm). TIle two antifungal metabolites (20lA and 201B) isolated from culture filtrated were biologically characterized. TIlese metabolites had a wide spectrum of antifungal activity and affect the growth of several saprophytic as well as pathogenic fungi. The minimal inhibitory concentration (MIG) of 20lA against Candida albicans and Candida pseudotropicalis was less than 1 mcg/ml and 1-5 mcg/ml respectively. Inhibition diameter zone of 36 mm was produced when 10 mcg/disc of 20lA was applied on agar medium seeded with AspergHlus flavus. 20lA was also a potent inhibitor of spore gemlination. No spores of Aspergillus niger were germinated at a concentration of 10 mcg/ml of the antibiotic. Bacteria were affected only at high concentration. Antibiotic 20lA was more active than antibiotic 201B. A comparison of antifungal activity against dennatophytes showed that the efficacy of 20lA was comparable to griseofulvin. Treatment of sheep erythrocytes with the two antifungal antibiotics up to 100 mcg/disc did not show any lytic effect on sheep red blood cel/s. Application of 1 mg/1Ocm' of cn/de extract on a shaved rabbit skin showed no demlatotoxic reactions.
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    Biorational Management of Postharvest Anthracnose on Tropical Fruits and Gummy Stem Blight on Cucurbits Biorational Management of Postharvest Anthracnose on Tropical Fruits and Gummy Stem Blight on Cucurbits
    (Addis Ababa Universty, 2014-06) Kefialew Yonas; Abate Dawit (PhD)
    Anthracnose caused by Colletotrichum gloeosporioides and Colletotrichum acutatum and Gummy stem blight caused by Didymella bryoniae (anamorph Phoma cucurbitacearum) are among the most important diseases of fruit and cucurbits. Currently, the control of fruit and vegetable diseases relies mainly on the use of synthetic fungicides. Elucidating non-chemical control methods to reduce postharvest decay is becoming more important. This study investigated biological based approaches to controling these diseases. The first objectives of this study were to analyze morphological, physiological and molecular methods in the differentiation of Colletotrichum isolates obtained from banana, mango and papaya fruits and to evaluate the biocontrol potential of antagonistic bacteria, yeasts and fungal isolates to manage anthracnose disease of banana, mango and papaya during storage. In the first phase, a phenotypic analysis of Colletotrichum isolated from banana, mango and papaya were carried out to identify the species responsible for anthracnose disease on these hosts. A total of 45 isolates from three hosts were used. The overall similarity among different isolates of Colletotrichum was determined using cultural characteristics. According to the results fungal isolates could be divided in to 12 distinct groups based on morphological similarity. Subsequent identification based on ITS sequence lead to the identification of Colletotrichum isolates as C. acutatum and C. gloeosporioides. Isolates from mango were C. gloeosporioides while isolates from banana and papaya was C. acutatum. Further, the study confirmed the cross infection potential of Colletotrichum isolates and absence of host specificity. Effect of different temperature, pH level, culture media, light intensity, carbon and nitrogen sources were tested against the growth of C. acutatum and C. gloeosporioides. Results indicated that the growth of these isolates varied with the different environmental and nutritional conditions tested. The second phase of this study was conducted to isolate, screen and identify indigenous microorganisms found on fruit surfaces in order to find a suitable biocontrol agent against postharvest fruit anthracnose caused by C. gloeosporioides and C. acutatum. Bacteria, yeast and fungal isolates recovered from leaf and fruit surfaces of banana, mango and papaya were tested In vitro and In vivo against C. gloeosporioides and C. acutatum. The microbial antagonists inhibited mycelial growth in the dual culture assay and conidial germination of C. acutatum and C. gloeosporioides isolates in vitro. Studies were carried out to analyze the ability of the antagonists to produce extracellular enzymes on an amended solid media. Fourteen (14) isolates used produced cellulose and chitinase on amended media but only four isolates showed glucanase pectinase and protease activity on solid media. Additional experiments were conducted to extract and determine the nature of antifungal substances produced by antagonists that were inhibitory towards Colletotrichum isolates. Microbial antagonist isolates differ in their preference of culture media. The results of In vivo experiments under artificial infection conditions showed that suspensions containing unwashed cells of antagonists provided the highest levels of inhibition of anthracnose, while the washed cell suspension and autoclaved culture filtrates provided less protection against the disease after 30 d. Nineteen (19) different antagonists were evaluated on their own and in combination with fungicide and hot water for their ability to reduce postharvest fruit anthracnose diseases in vivo under natural infection conditions. Integrating fungicides or hot water with antagonists controlled anthracnose more effectively than fungicide control. Nine antagonists were more effective than other isolates in the control of postharvest anthracnose when fruit were treated under natural infection conditions. After phenotypic and molecular analysis, the bacterial isolates were identified as six Pseudomonas, three Bacillus and a Paenibacillus species. The Pseudomonas and Bacillus strains can neither be sufficiently re-solved by MALDI-TOF MS nor by 16S rRNA gene sequence analysis. The most effective yeast (M-23-L-1) and filamentous fungal (M-30-F-2) isolates were identified as Candida rogousa and Trichoderma longibrachiatum, respectively. This experiment identified six Pseudomonas and three Bacilli antagonist isolates as novel strains to be used as biological control agents against anthracnose of tropical fruits. The second objectives of this study were to characterize populations of Didymella bryoniae from commercial watermelon and other cucurbit hosts from different parts of the USA on the basis of their biological and molecular diversity and to evaluate the effect of tiadinil and two thymolbased formulations against D. bryoniae and Gummy Stem Blight (GSB) development. In the first phase, morphological characteristics and rDNA Internal Transcribed Spacer (ITS) sequences were analyzed to identify the causal organism of this disease. Thirty five isolates of Didymella spp. and Phoma spp. associated with GSB on watermelon, canary melon, muskmelon and squash from Florida and Georgia were characterized based on morphology on culture media, pathogenicity assays and genetic characterization using ITS sequence analysis. All the isolates were pathogenic on watermelon cv. Melody, but to a varying degree. RAPD and ITS sequence analysis indicated genetic variation between the isolates. The ITS region analysis showed the presence of two isolates, DB-05 and DB-33, which showed a higher similarity to D. bryoniae isolates from China. This is the first description of an isolate with this unique sequence in Florida and Georgia. The present study brings insights into the current genetic profile of D. bryoniae isolates in Florida and Georgia, and its similarity with international isolates. During the second phase, direct antifungal activity of tiadinil and the effect of two-thymol formulations on D. bryoniae were evaluated in vitro. All test materials used in the study affected fungal growth, with tiadinil at 10 ppm and thymol-based formulations at 0.1 ppm significantly (P 0.05) reducing mycelial growth, conidia germination and germ tube elongation. Foliar application of tiadinil (before and after inoculation) on artificially infected watermelon seedlings at 10 ppm significantly reduced the disease severity compared to the untreated controls (P 0.05). The disease severity on seedlings treated with tiadinil at 3000 ppm was statistically comparable to chlorothalonil control (P 0.05). Plants with foliar applications of tiadinil at 1000 ppm (before pathogen inoculation) had significantly lower disease severity than plants with drench application (P 0.05). Foliar application of tiadinil was affected by chemical concentration and frequency of application. Thus, based on this study, it is prudent to say that tiadinil and thymol-based formulations are potential materials for use in watermelon production for effective GSB disease suppression. This study represents a novel report dealing with the biocontrol of anthracnose in banana, mango and papaya fruit by the application of possibly new antagonist bacterial strains and a yeast isolate. It strongly recommends the use of a combination of biological control agents with commercial treatments as a safe and effective disease management option against the postharvest anthracnose of tropical fruits. The study also highlighted the possible utilization of tiadinil and thymol-based formulations against GSB as a management strategy.
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    Characterization of the Causative Agent of Chalkobroood Disease of Honeybee Brood (Apls Melliferal.) in Ethiopia
    (Addis Ababa University, 2006-06) Geremew Teshome; Abate Dawit (PhD)
    Chalkbrood disease is an invasive mycosis of honeybee larvae (Apis mellifera L) caused by Ascosphaera apis (Maassen ex Claussen) Spiltoir and Olive (1955). The causative agent of this disease occurs in most bee keeping countries of the world. This study aims to characterize the causative agent of the disease and compares it with reference strain (MUCL 34668). The mummies were surface disinfected and inoculated onto Sabouards Dextrose Agar for macroscopic and microscopic identification. The local isolates were also compared with the reference strain, assayed for proteolytic ability, toxicity, and virulence on honeybee larvae in the laboratory and in the hive. Microbiological analysis of 45 samples of dead honeybee larvae collected from the regions during 2004-2006 yielded 28 positive cases for chalkbrood disease incidence (62.22%). Spore cyst, spore ball, and ascospores length to width ratios measured with phase contrast inverted computer fitted microscope were 66.15 –97.66 m, 11.00 –19.27 m and 2.00-2.95 m, respectively. The measurements obtained and examination results enabled us to confirm that the local isolates belongs to the genus Ascosphaera and to the species Ascosphaera apis. Key words: Chalkbrood disease, Ascosphaera apis, spore cyst, spore ball, Ascospores, Apis mellifera.
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    Characterization of two Nematodedestroying Fungi from Etillopia
    (1999-06) Bedelu Theodros; Abate Dawit (PhD)
    Under laboratory conditions, ART-l (LDso= 290 conidia/ml) was more efficient in destroying nematodes than ART-2 (LDso= 725 conidia/OIl), Investigation on the growth of MeloidogYlle incognita-infected tomatoes in the greenhouse showed that yield loss was highest in tomatoes treated with only k[, incognita (69.7%). Tomatoes treated with conidia of ART-lor ART·2 had lower losses: 44.5% for ART-l treated & 44% for ART-2 treated tomatoes. ART-2 required 3X the number of conidia of ART-l (180000 conidia/pot) to attain a nematode suppression effect similar to ART-I. Out of 3 solid media compared for their effects on the lateral growths of the isolates at room temperature, oatmeal agar (OA) gave the greatest colony diameters (6.3 cm for ART-l & 7.8 cm for ART-2). Maximal spore yields were also observed on this medium: ART·l, 4960 conidia/ml & ART-2, 31400 conidia/m!. A comparison of lateral growth, on medium A (MA), under different incubation temperatures (room temperature, 28°C, 32°C, & 37°C) showed that greatest colony diameters were attained at 28°C: 5 em & 5.7 cm for ART-l & ART-2 respectively. Broth of MA was found to be suitable for optimal mycelial biomass yield for both isolates (631 mg/IOO ml, ART·l; 755 mg/lOO ml, ART-2). However, ART-I, a slow grower on all solid media tested, gave a higher yit:;ld than ART~::! when grown in c~lej.n ID}ocr:tl :.;:tits·m€rll~~·! (G~S~l) broth (363 mg/lOO ml cr. 273 mg/IOO ml). Optimal mycelial biomass yield in MA broth was found to be in the pH range 4-5 for ART-2 (509·669 mgt 100 ml) & in the range 7·8 for ART-l (487-535 mg/IOO mI). ART·l & ART-2 produced extracellular proteases in CMS-2 broth. The proteases from both fungi had a temperature optimum of 50"C, & were optimally active in the alkaline range (7.5·8, ART-l & 7.5·9, ART- 2). ART-l produced more proteases (19 U/ml/hr) than ART-2 (14 U/ml/hr). ART-l proteases were more efficient in degrading nematodes than those of ART -2 by virtue of being produced in a larger quantity, suggesting that the difference in the nematode-destroying capabilities of ART-l & ART·2 was the result of the difference in the amount of proteases produced.
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    Cultivation and Yield Performance of Pholiota Nameko on Different Agro Industrial Wastes
    (Addis Ababa University, 2010-04) Gizaw Birhanu; Abate Dawit (PhD)
    Pholiota nameko (T.Ito) S.Ito) is white rot wood inhabiting ligninolytic mushroom species belonged to genus Pholiota, widely distributed through out Far East which has been used as food and medicinal purpose. The research experiment was carried out to investigate the yield and the biological efficiency of Pholiota nameko grown on different agro industrial wastes in Ethiopia. For the cultivation of Pholiota nameko 6 kinds of substrates, namely eucalyptuse shaving (ES), cordia shaving(CAS), coffee husk(CH), Pinus shaving(PS), cotton seed (CS) and teff straw(TS) were used as the main material or substrates. Wheat bran (WB) was used an additive material 100:10 and 100:30 w: w of the main material. Moisture content of the substrate was maintained to50-65 %. Only three substrates, these are eucalyptuse shaving (ES), cotton seed and cordia shaving(CAS) show remakable production of fruiting body. The highest mean yield and biological efficiency were 797.33 g, 53.27% respectively on eucalyptuse shaving supplemented with 30% wheat bran. The higher harvest 732.33g, 48.98% mean yield and biological efficiency respectively obtained from cotton seed supplemented with 30 % wheat bran. While the lowest mean yield and biological efficiency were obtained from Cordia africana shaving supplemented with 10% wheat bran 550.8g, 36.80% respectively. There was no statistical difference observed between substrates supplemented with 10% and 30% wheat bran on yield and the biological efficiency. But substrate supplemented with 30% wheat bran showed a little better quality of fruiting body and cropping time than substrate supplemented with 10% wheat bran. In general the yield of Pholiota nameko mushroom harvested was significantly (P<0.05) greater in eucalyptuses shaving than Cordia africana shaving. The use of eucalyptus shaving as raw material was found better for the production of Pholiota nameko in this study, in fact it is an abundant and chip lignin rich material in Ethiopia. Key Words: Bio conversion effeciency , basdiomycetes, contamination, Lentinus edodes , lignocelluloses, mushrooms, media, mycelium, Pholiota nameko, substrate, shiitake, spawn
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    Cultivation of Medicinal Mushroom: Schizophyllum Commune
    (Addis Ababa University, 2014-06-02) Assefa Woineshet; Abate Dawit (PhD)
    Cultivation of edible and medicinal mushrooms on lignocellulosic wastes represents one of the most economically and cost effective organic recycling processes. Solid-state cultivation (SSC) was carried out to evaluate the suitability of using the logs of Eucalyptus globulus, Cupressus lusitanica, Gravillea robusta, Acacia abyssinica, coffee husk and cotton seed and the logs of Eucalyptus globulus, Cupressus lusitanica, Gravillea robusta and Acacia as substrates for cultivation of S.commune. The number of flushes, yield and biological efficiency of the S.commune grown on Eucalyptus, Cupressus, Gravillea and Acacia spp. were studied. S.commune recorded at least four flushes on all the substrates used and supported fruiting bodies formation on (Eucalyptus, Cupressus, Gravillea and Acacia). Flush 1 gave the highest yield in g/kg while flush 4 gave the lowest yield. Significant differences application.
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    Ergot Fungus (Claviceps Purpurea), Ergot Alkaloids and Ergotism in the Central Highlands of Arsi, Ethiopia
    (Addis Ababa University, 2017-12-03) Desalegn Asnake; Abate Dawit (PhD)
    Ergot fungi, Claviceps species, parasitize several monocot plants and produce a hardened dark to dark-purple structure called the sclerotia. In Ethiopia, the ergot fungus infects only the wild oat plant (Avena abyssinica) which is endemic to Ethiopia and Yemen. Ergot alkaloids produced in the sclerotia of the ergot fungus, were responsible for mass poisoning in various areas of the world, with the most recent report of mass poisoning in Arsi, Ethiopia. This study was initiated with the objectives of identifying the ergot fungus (Claviceps purpurea) based on morphological and molecular characteristics, ergot alkaloids, and assessing the knowledge of study participants about ergot fungus and ergotism from the previously reported outbreaks areas of Arsi, Ethiopia. Dimensions of the sexual and asexual structures were studied and statistically significant differences (n = 30, P < 0.001) in the length and width of sclerotia collected from the study sites Kechema Murkicha, Bucho Selassie and Shaldo Jigessa were observed. Dimensions of sclerotia collected from all the study sites significantly differed (p < 0.001) from the dimensions of wild oats seeds (n = 30). But, statistically significant differences were not observed (p > 0.05) for the dimensions of conidia of ergot fungi collected from different study sites. Growth (100%) of the sexual stage occurred only on Petri dishes incubated for 21 days at 5 OC followed by incubation at 25 OC. No growth was observed on the Petri dishes incubated under other temperature treatments. The mean length of stromata ranged from 18.5 mm to 19mm and the mean diameter of capitula ranged from 1.8 mm to 2mm. Cylindrical to flask shaped perithecia with mean length and width of 158.8 ± 3.7 μm and 89.2 ± 1.7 μm, and filiform shaped ascospores with mean length and width of 77.1 ± 3.7 μm and 3.3 ± 0.5 μm respectively were observed. Phylogenetic analysis of the β-tubulin intron 3 region using maximum parsimony placed our isolates in a separate cluster with strong bootstrap value of 94. Qualitative studies of the ergot alkaloids using UPLC-QTOF High Definition Mass Spectrometery revealed the presence of ergometrine, ergocryptine, ergocornine, ergosine, ergovaline, lysergyl alanine, lysergyl valine, valine methyl ester, their respective -innine isomers and an ergopeptam (ergocryptam). A crosssectional study conducted to assess the awareness of study participants recruited from Tijo, Digelu and Kechema areas, Arsi, Ethiopia, showed lack of awareness about the fungus and the disease it causes. Among the study participants who were shown the coloured picture of ergot fungus, majority 55 (32.7%) described its name as ‘Sinara Guracha’ which is synonymous with “Black wild ii oat”. A multiple logistic regression model fitted revealed statistically significant association of the study sites with knowledge of ergot (p < 0.05). Finally, morphological and molecular characteristics placed the ergot fungi in the current study under Claviceps purpurea. The presence of the Claviceps purpurea in farmers’ field, detection of additional toxic ergot alkaloids and lack of awareness of the study participants about the ergot fungus, Claviceps purpurea, are the potential risks for the community.
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    Ergot Fungus (Claviceps Purpurea), Ergot Alkaloids and Ergotism in the Central Highlands of Arsi, Ethiopia
    (Addis Ababa University, 12/3/2017) Desalegn Asnake; Abate Dawit (PhD)
    Ergot fungi, Claviceps species, parasitize several monocot plants and produce a hardened dark to dark-purple structure called the sclerotia. In Ethiopia, the ergot fungus infects only the wild oat plant (Avena abyssinica) which is endemic to Ethiopia and Yemen. Ergot alkaloids produced in the sclerotia of the ergot fungus, were responsible for mass poisoning in various areas of the world, with the most recent report of mass poisoning in Arsi, Ethiopia. This study was initiated with the objectives of identifying the ergot fungus (Claviceps purpurea) based on morphological and molecular characteristics, ergot alkaloids, and assessing the knowledge of study participants about ergot fungus and ergotism from the previously reported outbreaks areas of Arsi, Ethiopia. Dimensions of the sexual and asexual structures were studied and statistically significant differences (n = 30, P < 0.001) in the length and width of sclerotia collected from the study sites Kechema Murkicha, Bucho Selassie and Shaldo Jigessa were observed. Dimensions of sclerotia collected from all the study sites significantly differed (p < 0.001) from the dimensions of wild oats seeds (n = 30). But, statistically significant differences were not observed (p > 0.05) for the dimensions of conidia of ergot fungi collected from different study sites. Growth (100%) of the sexual stage occurred only on Petri dishes incubated for 21 days at 5 OC followed by incubation at 25 OC. No growth was observed on the Petri dishes incubated under other temperature treatments. The mean length of stromata ranged from 18.5 mm to 19mm and the mean diameter of capitula ranged from 1.8 mm to 2mm. Cylindrical to flask shaped perithecia with mean length and width of 158.8 ± 3.7 μm and 89.2 ± 1.7 μm, and filiform shaped ascospores with mean length and width of 77.1 ± 3.7 μm and 3.3 ± 0.5 μm respectively were observed. Phylogenetic analysis of the β-tubulin intron 3 region using maximum parsimony placed our isolates in a separate cluster with strong bootstrap value of 94. Qualitative studies of the ergot alkaloids using UPLC-QTOF High Definition Mass Spectrometery revealed the presence of ergometrine, ergocryptine, ergocornine, ergosine, ergovaline, lysergyl alanine, lysergyl valine, valine methyl ester, their respective -innine isomers and an ergopeptam (ergocryptam). A crosssectional study conducted to assess the awareness of study participants recruited from Tijo, Digelu and Kechema areas, Arsi, Ethiopia, showed lack of awareness about the fungus and the disease it causes. Among the study participants who were shown the coloured picture of ergot fungus, majority 55 (32.7%) described its name as ‘Sinara Guracha’ which is synonymous with “Black wild ii oat”. A multiple logistic regression model fitted revealed statistically significant association of the study sites with knowledge of ergot (p < 0.05). Finally, morphological and molecular characteristics placed the ergot fungi in the current study under Claviceps purpurea. The presence of the Claviceps purpurea in farmers’ field, detection of additional toxic ergot alkaloids and lack of awareness of the study participants about the ergot fungus, Claviceps purpurea, are the potential risks for the community.
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    Ergot Fungus (Claviceps Purpurea), Ergot Alkaloids and Ergotism in the Central Highlands of Arsi, Ethiopia
    (Addis Ababa University, 2017-12-03) Desalegn Asnake; Abate Dawit (PhD)
    Ergot fungi, Claviceps species, parasitize several monocot plants and produce a hardened dark to dark-purple structure called the sclerotia. In Ethiopia, the ergot fungus infects only the wild oat plant (Avena abyssinica) which is endemic to Ethiopia and Yemen. Ergot alkaloids produced in the sclerotia of the ergot fungus, were responsible for mass poisoning in various areas of the world, with the most recent report of mass poisoning in Arsi, Ethiopia. This study was initiated with the objectives of identifying the ergot fungus (Claviceps purpurea) based on morphological and molecular characteristics, ergot alkaloids, and assessing the knowledge of study participants about ergot fungus and ergotism from the previously reported outbreaks areas of Arsi, Ethiopia. Dimensions of the sexual and asexual structures were studied and statistically significant differences (n = 30, P < 0.001) in the length and width of sclerotia collected from the study sites Kechema Murkicha, Bucho Selassie and Shaldo Jigessa were observed. Dimensions of sclerotia collected from all the study sites significantly differed (p < 0.001) from the dimensions of wild oats seeds (n = 30). But, statistically significant differences were not observed (p > 0.05) for the dimensions of conidia of ergot fungi collected from different study sites. Growth (100%) of the sexual stage occurred only on Petri dishes incubated for 21 days at 5 OC followed by incubation at 25 OC. No growth was observed on the Petri dishes incubated under other temperature treatments. The mean length of stromata ranged from 18.5 mm to 19mm and the mean diameter of capitula ranged from 1.8 mm to 2mm. Cylindrical to flask shaped perithecia with mean length and width of 158.8 ± 3.7 μm and 89.2 ± 1.7 μm, and filiform shaped ascospores with mean length and width of 77.1 ± 3.7 μm and 3.3 ± 0.5 μm respectively were observed. Phylogenetic analysis of the β-tubulin intron 3 region using maximum parsimony placed our isolates in a separate cluster with strong bootstrap value of 94. Qualitative studies of the ergot alkaloids using UPLC-QTOF High Definition Mass Spectrometery revealed the presence of ergometrine, ergocryptine, ergocornine, ergosine, ergovaline, lysergyl alanine, lysergyl valine, valine methyl ester, their respective -innine isomers and an ergopeptam (ergocryptam). A crosssectional study conducted to assess the awareness of study participants recruited from Tijo, Digelu and Kechema areas, Arsi, Ethiopia, showed lack of awareness about the fungus and the disease it causes. Among the study participants who were shown the coloured picture of ergot fungus, majority 55 (32.7%) described its name as ‘Sinara Guracha’ which is synonymous with “Black wild ii oat”. A multiple logistic regression model fitted revealed statistically significant association of the study sites with knowledge of ergot (p < 0.05). Finally, morphological and molecular characteristics placed the ergot fungi in the current study under Claviceps purpurea. The presence of the Claviceps purpurea in farmers’ field, detection of additional toxic ergot alkaloids and lack of awareness of the study participants about the ergot fungus, Claviceps purpurea, are the potential risks for the community.
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    Ergot Fungus (Claviceps Purpurea), Ergot Alkaloids and Ergotism In the Central Highlands of Arsi, Ethiopia
    (Addis Ababa University, 2017-12-03) Desalegn Asnake; Abate Dawit (PhD)
    Ergot fungi, Claviceps species, parasitize several monocot plants and produce a hardened dark to dark-purple structure called the sclerotia. In Ethiopia, the ergot fungus infects only the wild oat plant (Avena abyssinica) which is endemic to Ethiopia and Yemen. Ergot alkaloids produced in the sclerotia of the ergot fungus, were responsible for mass poisoning in various areas of the world, with the most recent report of mass poisoning in Arsi, Ethiopia. This study was initiated with the objectives of identifying the ergot fungus (Claviceps purpurea) based on morphological and molecular characteristics, ergot alkaloids, and assessing the knowledge of study participants about ergot fungus and ergotism from the previously reported outbreaks areas of Arsi, Ethiopia. Dimensions of the sexual and asexual structures were studied and statistically significant differences (n = 30, P < 0.001) in the length and width of sclerotia collected from the study sites Kechema Murkicha, Bucho Selassie and Shaldo Jigessa were observed. Dimensions of sclerotia collected from all the study sites significantly differed (p < 0.001) from the dimensions of wild oats seeds (n = 30). But, statistically significant differences were not observed (p > 0.05) for the dimensions of conidia of ergot fungi collected from different study sites. Growth (100%) of the sexual stage occurred only on Petri dishes incubated for 21 days at 5 OC followed by incubation at 25 OC. No growth was observed on the Petri dishes incubated under other temperature treatments. The mean length of stromata ranged from 18.5 mm to 19mm and the mean diameter of capitula ranged from 1.8 mm to 2mm. Cylindrical to flask shaped perithecia with mean length and width of 158.8 ± 3.7 μm and 89.2 ± 1.7 μm, and filiform shaped ascospores with mean length and width of 77.1 ± 3.7 μm and 3.3 ± 0.5 μm respectively were observed. Phylogenetic analysis of the β-tubulin intron 3 region using maximum parsimony placed our isolates in a separate cluster with strong bootstrap value of 94. Qualitative studies of the ergot alkaloids using UPLC-QTOF High Definition Mass Spectrometery revealed the presence of ergometrine, ergocryptine, ergocornine, ergosine, ergovaline, lysergyl alanine, lysergyl valine, valine methyl ester, their respective -innine isomers and an ergopeptam (ergocryptam). A crosssectional study conducted to assess the awareness of study participants recruited from Tijo, Digelu and Kechema areas, Arsi, Ethiopia, showed lack of awareness about the fungus and the disease it causes. Among the study participants who were shown the coloured picture of ergot fungus, majority 55 (32.7%) described its name as ‘Sinara Guracha’ which is synonymous with “Black wild ii oat”. A multiple logistic regression model fitted revealed statistically significant association of the study sites with knowledge of ergot (p < 0.05). Finally, morphological and molecular characteristics placed the ergot fungi in the current study under Claviceps purpurea. The presence of the Claviceps purpurea in farmers’ field, detection of additional toxic ergot alkaloids and lack of awareness of the study participants about the ergot fungus, Claviceps purpurea, are the potential risks for the community.
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    Evaluation of Ethiopian Isolates of Entomogenous Fungi as Potential Biological Control Agent of the Desert Locust, Schistocerca Gregaria.
    (Addis Ababa University, 1998-05) Aysheshim, Seneshaw; Abate Dawit (PhD); Seyoum Emiru (PhD)
    Ten native fungal isolates were collected from the different regions of Ethiopia. Eight of the isolates were identified at International Institute of Tropical Agriculture, UTA, Benin. Five of the isolates were identified as Beallveria, two were Paecilomyces and one belonged to Metarhizilllll species. Eight of the tested fungal isolates showed pathogenecity to fifth instar Schistocerca gregaria. The Beallveria spp. isolate FF and MetarhiziulII spp. EE were more virulent compared to the rest. Thus two isolates were further assayed in peanut (Arachis hypogaea) and noug (GlIizotia abyssinica) oil formulations, and compared with a known entomopathogen, M anisopliae ICIPE 30. The results indicated that there were strong doseresponse patterns. Peanut oil formulation enhanced infectivity of both FF and EE. Infectivity of EE was significantly (P<0.05) reduced in noug oil formulation. Comparison of virulence revealed that isolates FF and EE were more pathogenic than ICIPE 30. FF in peanut oil formulation had the lowest LCso value of2.02xlOs conidia/ml followed by EE, 2.93x10s and ICIPE 30 with 4.98x10s conidia/m!. The highest mortality was achieved by FF at Ixl08 conidia/ml, resulting in 50% and 100% mOliality in 4.2 days and seven days, respectively. The median lethal time taken to fOUlih, fifth and adult FF treated insects were 3.47, 4.14 and 4.83 days; respectively that indicated fourth instars are more susceptible. Insects killed as a result of FF infection developed red pigmentation and produced white powdery mass of spores on the external surface. The Metarhizilllll isolates EE and ICIPE 30 produced green sporulation on the integument when the cadavers were kept at high humidity. FF and EE grew at a temperature range of 24°C and 37°C, with peaks at 24°C and 28°C for FF and 28°C for EE. Malt extract agar favoured growth and sporulation of FF, whereas EE grew and sporulated faster on sabouraud dextrose agar. Germination of both isolates was significantly (P<0.05) high in peanut oil formulation than in noug oi!.
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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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    Fusarium and Fusarium Toxins in Maize in Some Regions of Ethiopia.
    (Addis Ababa University, 1997-06) Wubet, Tesfaye; Abate Dawit (PhD)
    Maize Grain Samples Were Collected from stores and markets in Shashemene and Alemaya regions. Samples were rated as damaged, Hornlal and malted. Nannal and damaged samples were rated based on percentage of kernel discolouration, wrinkledness, floating ofke~le1s in water and attack by insects. Mycofloral study of the three sets of samples showed that the three tox.jgenic fungal genera: Fusarium, Aspergillus and Penicillium were COllunon in maize in Ethiopia. Among these toxigenic fungi Fusarium was the most common genus, comprising 17.5 % of the total fungi isolated and was recovered from 80.5 % of the samples investigated, Species of fusarium were most prevalent in damaged and malted samples than in nonnal samples. TIle ftmgus was represented in 91.7 % and 28 % of the total fungal isolates in damaged and malted samples respectively. In the normal samples, however, Aspergillus and Penicillium spp were more prevalent and they were represented in all the samples examined. Preliminary test result of screening toxigenic fusarium isolates using the brine shrimp (Artemia salina) bioassay showed that 93 % of them were tmdgenic. The frequently occurring toxigenic fusarium were identified to be Fusarium prolijeratum, Fusarium gramil1earum, Fusarium subglutillGns and Fusarium alllhophilum. Isolates of these frequent toxigenic species were grown on sterile shredded maize grain and their extracts were assayed for toxicity on the brine shrimp larvae. The results indicate production of methanol and/or chloroform soluble toxic metabolites. Bioassay directed isolation of toxic principles from chloroform extract of F. graminearum isolates showed production of zearalenonc and trichothecene compounds (TA and TB). Based on their physico· chemical data four compOlUlds that belong to the fumonisins were isolated from methanol extracts of F. proliferatum isolates. TIle results of the study on screening of toxigenic fusarium and bioassay directed isolatfon and purification of toxic principles indicated the importance of brine sluimp bioassay in screeuing toxigenic fusarium and detection of fusarium toxins. Toxicity assay results from extracts of selected grain samples of the three sets of samples indicated that 93 % of the tested sample extracts were toxic. In the natural occurrence study of the toxins, zearatenone and the trichothecenes (TA and TB) were detected in 50, 50, and 75 % of the damaged samples examined respectively. The estimated concentration ranges from 0-0.42 J.tglg for zearalenone, 0-1.5 J.tglg for TA and 0·2.3 J.tglg for TB. The fumonisins, on the other hand, were detected in all the samples examined with the highest concentrations in the malted and damaged samples. The results of this study showed that toxigenic fusarium are associated with maize samples and their toxins are fOlmd on the grain. TIle biological effect and thermostability of some of the toxins indicates the health hazard of consuming mould infected grains, particularly damaged and malted grains. This necessitates the quality control of maize grain destined for human consumption.
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    Growth Inhibition of Grain Spoilage Fungi by Some Herb and Spice Essential Oils Grown in Ethiopia
    (Addis Ababa University, 2009-06-05) Chewaka Diriba; Abate Dawit (PhD)
    Microbial food contamination is an on-going limiting factor in crop production that can determine the shelf life of processed and unprocessed foods. Spice plants and herbs are commonly used as food flavoring and seasoning agents. Their antimicrobial properties as food preservatives are also well documented. In this study, essential oils of seven spice plants were tested for their antimicrobial properties against Aspergillus flavus and A. niger, two of the most important food and feed spoilage organisms. Agar disk diffusion assay was used for screening of the most effective essential oils, agar dilution assay was used to determine Minimum Inhibitory Concentration (MIC) of the essential oils and broth dilution assay was employed to the spore germination inhibition assay. Tests were also conducted to examine the effects of the essential oils for sorghum kernel protection against the tested fungi, and the optimal protective dosages on the sorghum grains were also determined. From the preliminary tests, essential oils of Cinnamomum zeylanicum (Cinnamon) and Thymus schimperi (thymus) were found to be the most effective. However piper nigrum (black pepper) had no effect on the test organisms. In MIC, spore germination inhibition and grain protection assay, cinnamon essential oil was found to be superior where its MIC on the isolates was found to be 0.0156% and its optimum protective dosage on the grain was 5%. It inhibited spore germination at a concentration of 3 _L/ml. The effect of thymus oil was also very much comparable to these results (no significant difference at P < 0.05). Finally, it could be concluded that some plant essential oils can be a useful source of antifungal agents for protection of grain spoilage by fungi.
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    Investigation on the Genus Bacillus in the Control of Mosquitoes
    (Addis Ababa University, 1993-10) Wahab Abdel; Abate Dawit (PhD); Gemetchu Teferi (PhD)
    Screening programmer to isolate strains of the genus Bacillus for their potential use in the control of mosquitoes was done. Over 90 spore-forming bacillus strains were examined. Six isolates AA-2, AA-6, AA-10, AA-l1, AA-71, and AA-82) have sho\m killing ability of which four (AA-2, AA-11, AA-71, and AA-82) were found to be effective. Biochemical and morphological characterizations have revealed similarity between these isolates and the Well Known mosquito pathogens (B. thuringiensis ancl B. sphaericus). The effect of temperature and aeralion supply on biomass production was studied. Isolateswere found to dliffen:nlly responcl to temperJIUle. Isol"Ias fnunu to qro\•: h'l'll at 30')C. A,;-ll 11ds been found to huve a widerange of temperature (23- 40)AA-71 has Sl10WIl io\'erse relation \,ith temperature, h'hereas isolate "\A-82 demonstrated critical decrease in growth with increase in temperature and 30~ was found to be optimal. Dry biomass was founcl to increase with the increase in aeration levels. The efficacy of the isolates was tested on different larval stages of mosquito. LC IO that ranged from 43-170 llg/ml \,as obtained. ~o siqnificant difference has been observed in sensitivity of different larval stages of mosquito to different bacteria at a significance level of 1% except isolate AA-11. Efficacy of the isolates under natural conditions was also tested. Some strains (eg. AA-82) have shown good results.Further confirmatory characterization is needed as well as investigation for optimal conditions to increase toxin yield.
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    Isolation and Characterization of Rennin Enzyme from Mucor Species and Utilization for Cheese Making
    (Addis Ababa University, 2006-01-31) Getu Abebe; Abate Dawit (PhD)
    Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Rennet, a microbial coagulant, can be obtained from Mucor species isolated from cow dung. The aim of this work was to extract milk-clotting enzyme from Mucor species by submerged fermentation and to produce cheese. Among the physico-chemical parameters tested, the best results were obtained in a medium having initial pH of 7.0 and incubated at 25oC for 5 days, using glucose and peptone as carbon and nitrogen sources respectively. A partial purified extract of the enzyme was obtained by fractional precipitation with (NH4)2SO4 and its maximum activity were obtained at pH 5.5 and 550C. The clotting activity of the purified enzyme was stimulated with increasing CaCl2 concentration up to 0.05%.The enzyme was completely inactivated by heating for 5 min at 70oC. This enzyme was stable at pH 4.5–6 and below 45 °C, and this was convenient for storage and transportation. The result showed that the enzyme from Mucor was a promising microorganism for industrial milk-clotting enzyme production in Ethiopia.
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    Isolation and Characterization of the Dominant Yeast in the Traditional Beverages of Ethiopia; Tella and Tej
    (Addis Ababa Universty, 2011-01) Abebe Haimanot; Abate Dawit (PhD)
    To date the source and type of the dominant yeast in tej are not known. In this study yeasts were isolated from gesho powder and honey. Both the ingredients contained the dominant yeast, S.cerevisiae, of tej at its consumption stage. However, gesho contained 102 to 103 cfu/g while honey contained 0 to 102 cfu/g. Thus, gesho was considered the major source of the dominant yeast in tej. The source and type of the dominant yeast in tella are not known also. Yeasts were isolated from gesho and bikil powder. S.cerevisiae, the dominant yeast at consumption stage of tella was found in both the ingredients. Bikil contained 103 to 105 cfu/g yet gesho contained 102 to 103 cfu/g. Thus, bikil was considered the major source of the dominant yeast in tella. In this study, the source and type of the dominant yeast in the beverages at consumption stage was identified. The findings of this study help to define and standardize the fermentation of the traditional beverages of Ethiopia, tella and tej. Key words: tella, tej, bikil, gesho, honey
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    Isolation and Selection of Ethanol Tolerant Yeasts for the Production of Ethanol
    (Addis Ababa University, 2010) Tolessa Dechassa; Abate Dawit (PhD)
    Twenty yeasts colonies were isolated from Tela, Teji, Honey and teff dough selection for ethanol tolerance on yeast extract peptone dextrose agar medium (YEPDA). All the isolates were first tested for carbohydrate fermentation using Durham tube fermentation method in yeast extract peptone dextrose broth using common fermentative carbohydrates. Ten isolates which were relatively high fermentative in Durham tube fermentation method were selected for testing of isolates for ethanol tolerance. Ethanol tolerance was tested using different concentration made from 96 %( v/v) of absolute ethanol in yeast extract peptone dextrose broth and their growth was determined by measuring optical density of the cells in broth using spectrophotometer at 615nm. Two strains of isolates showed measurable growth in medium containing above 14% (v/v) of ethanol were selected for further study. Strain TBY1 tolerates15.5% (v/v) ethanol and strain TGY2 tolerates 16% (v/v) of ethanol. Both isolates were classified under genus saccharomyces based morphological appearance of vegetative cell under microscope, ascospore production, ascospore character, colony character and physiological testing of carbohydrates using baker yeast as reference. They were found to be equally sugar tolerant having good growth in medium containing 24-28 % (w/v) sucrose. Fermentation of sucrose was optimized with respect to temperature, pH and sugar concentration. Results revealed that pH 5 and 28% sucrose concentration as optimum for fermentation for the three yeasts. The optimum temperature for selected strains and reference yeast was 300C. The maximum ethanol produced by TBY1and TGY2 from 28% of sucrose at pH 5 and temperature of 30 0C were 11.3 and 11.5% (v/v) of ethanol respectively, and 9.6%(v/v)ethanol by the standard baker’s yeast. The amount of ethanol produced in the fermentation broth was measured using ebulliometer. The biomass was determined after the end of fermentation and the result showed it the biomass of more ethanol tolerant strain is greater than the others. Keywords/phrases: Baker yeast, biomass, Durham tube, ethanol, ethanol tolerance, optical density Spectrophotometer, sugar tolerance