Microbial, Cellular and Molecular Biology

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Browsing Microbial, Cellular and Molecular Biology 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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    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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    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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    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 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
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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
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    Morphological and Molecular Characterization, Diversity and Ethnomycological Studies on Wild Mushrooms of Central and Northwest Ethiopia
    (Addis Ababa Universty, 2017-06) Sitotaw Rediet; Abate Dawit (PhD)
    The diversity and ecology of fungi in general and macrofungi in particular, have been largely neglected in Ethiopia. In this context, this research aimed at providing documentation and analysis of wild mushroom diversity along with the ethnomycological knowledge associated with wild edible and medicinal mushrooms. Mushroom samples were collected from 48 plots (30 m × 30 m) of three different habitat type (forest, grazing land and farming area) over three years (2012 – 2014) from two study sites located in central (Welmera district) and Northwest (Menge district) parts of Ethiopia. Both morphological and molecular (ITS and partial LSU rDNA sequences) data were used for specimen identification. Maximum parsimony (MP), Maximum likelihood (ML) and Bayesian posterior probabilities (PP) were used for phylogenetic analysis. A hierarchical cluster analysis, with statistical program R was used to identify macrofungal community type and synoptic values to indicate dominant species in each community. Shannon-Wiener diversity index and Sorensen's similarity coefficient were used to assess species richness and evenness and to measure similarities among communities. Ethnomycological data were collected using semi-structured interviews, focus group discussions, participant observation and walk-in-the-woods methods. Ethnomycological knowledge held by different informant categories was compared using One-way ANOVA and t-tests. Results in this study showed, a total of 105 species belong to 23 families and 49 genera. About 95% of the species identified in this study are new records for Ethiopia. Family Agaricaceae (49), Lyophyllaceae (12), Tricholomataceae (9) and Psathyrellaceae (6) were represented by more species which together accounted for 74.4% of the total. The observed Shannon-Wiener diversity index (H'=3.76) of WFO showed the highest species diversity and the highest species richness (S= 62). Results of the ethnomycological study revealed 15 wild edible and medicinal mushroom species belonging to 7 genera and 5 families that are used mainly as food in the Menge District. Family Lyophyllaceae with 9 species (45%) was found to be best represented in the area. Ethnomycological knowledge is significantly influenced by gender, age, experience and litracy level parameters. Preference ranking exercise has indicated Termitomyces schimperi was ranked first followed by T. letestui, T. microcarpus and T. eurhizus as the second, third and fourth preferred edible mushrooms respectively. The phylogenetic relationship of 33 Ethiopian Agaricus collection with other members of the genus from tropical/subtropical and temprate region was compared based on morphological and molecular (ITS 1+2 rDNA sequences) characteristics. More than two-third of the Ethiopian Agaricus sequences examined in this study was distributed amongst four of the eight well accepted/temprate sections of the subgenus Agaricus. The remaining six Ethiopian sequences group together with four distinct and exclusively tropical (African and/or Asian) clades with ML/MP/PP branch support 99/100/100, 100/100/100, 83/75/90, 85/75/90 respectively. None of the Ethiopian sequences in the dataset belongs to section Arvenses, Bivelares, Chitonioides and Spissicaules. This study also shown that A. campestris, A. cupreobrunneus, A.bohusii, A. purpurellus, A. subsaharianus and A. heterocystis, which are known to be edible are highly recommended for domestication and cultivation because of their good nutritional and medicinal value. Generally, the present study indicated that country is rich in wild mushroom diversity and associated indigenous knowledge. However, anthropogenic factors together with loss of indiginous knowledge and very poor conservation efforts threaten economically and ecologically important mushrooms survival in the area. Thus, complementary in-situ and ex-situ conservation strategy at national level is highly recommended. Key words: Welmera, Menge, wild mushrooms, diversity, molecular phylogeny, traditional knowledge
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    Optimization of Coffee Wastes for the Cultivation of Pleurotus Ostreatus
    (Addis Ababa Universty, 2011-06) Shimelis, Aster; Abate Dawit (PhD)
    Solid state cultivation (SSC) was carried out to evaluate the feasibility of using coffee waste as substrates for the production of the edible mushroom, Pluerotus ostreatus. Coffee husk and coffee pulp powder media were used as a good culture media for Pleurotus ostreatus. Addition of supplementary substrate such as, Wheat bran (WB), Chicken manure (ChM), Cow dung (CD), Noug meal (NM) and Ash improved yield (production) of the mushroom. Composted coffee waste gave better yield than fresh coffee waste. Highest yield (1361.74 and 1232.18g) was obtained from aerobic composted (for eight (8) days) coffee parchment and husk when it was supplemented with 18% cow dung and 8% chicken manure, respectively. With aerobic composted coffee parchment as a substrate the biological efficiency reached 90.74% with four flushes after 90 days. With coffee husk as a substrate the biological efficiency reached 98.57% in 90 days. In the case of fresh substrate the highest biological efficiency 61.48% and 74.73% was obtained from coffee parchment and coffee husk, respectively. In all cases low biological efficiency was recorded from the substrate supplemented with 18% nug meal and 2% ash. There was significant difference (at P<0.05) observed between fresh and aerobic composted coffee parchment and coffee husk as well as different supplementary substrates on yield and the biological efficiency. In both cases with different supplementary substrate first flash gave high yield. The result of these experiments has showed that the feasibility of using composted coffee husk and coffee parchment as substrates with different supplementary substrate for cultivation of edible mushroom in SSC. Key words: Biological efficiency, coffee husk, coffee parchment, lignocellulosic wastes mushroom cultivation
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    Optimization of Coffee Wastes for the Cultivation of Pleurotus Ostreatus
    (Addis Ababa University, 2011-06) Shimelis Aster; Abate Dawit (PhD)
    Solid state cultivation (SSC) was carried out to evaluate the feasibility of using coffee waste as substrates for the production of the edible mushroom, Pluerotus ostreatus. Coffee husk and coffee pulp powder media were used as a good culture media for Pleurotus ostreatus. Addition of supplementary substrate such as, Wheat bran (WB), Chicken manure (ChM), Cow dung (CD), Noug meal (NM) and Ash improved yield (production) of the mushroom. Composted coffee waste gave better yield than fresh coffee waste. Highest yield (1361.74 and 1232.18g) was obtained from aerobic composted (for eight (8) days) coffee parchment and husk when it was supplemented with 18% cow dung and 8% chicken manure, respectively. With aerobic composted coffee parchment as a substrate the biological efficiency reached 90.74% with four flushes after 90 days. With coffee husk as a substrate the biological efficiency reached 98.57% in 90 days. In the case of fresh substrate the highest biological efficiency 61.48% and 74.73% was obtained from coffee parchment and coffee husk, respectively. In all cases low biological efficiency was recorded from the substrate supplemented with 18% nug meal and 2% ash. There was significant difference (at P<0.05) observed between fresh and aerobic composted coffee parchment and coffee husk as well as different supplementary substrates on yield and the biological efficiency. In both cases with different supplementary substrate first flash gave high yield. The result of these experiments has showed that the feasibility of using composted coffee husk and coffee parchment as substrates with different supplementary substrate for cultivation of edible mushroom in SSC. Key words: Biological efficiency; coffee husk, coffee parchment; lignocellulosic wastes mushroom cultivation
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    Post Harvest Biological Control of Blue Mold on Orange Fruit
    (Addis Ababa Universty, 2014-06) Mequanint Lemlem; Abate Dawit (PhD)
    Orange (Citrus sinensis) is among the most important fruit crop of Ethiopia. Blue mold is the most aggressive economically important wound parasite post harvest disease of orange fruit. The present study was under taken to isolate, characterize and identify indigenous biocontrol microorganisms (yeasts) occur on phyllospher of orange, mango, apple, lemon and avocado plants for their antagonistic activity against orange blue mold development as well as characterization and identification of orange blue mold causing organism has been done. In this study a total of 58 yeast isolates were isolated from fruit surfaces and leaves of orange, lemon, apple, mango and avocado plant. All of them were screened to test their antagonistic effect against orange blue mold development. Among the 58 isolates 9 isolates showed antagonistic activity against the development of orange blue mold. Three isolates namely isolate Y3, Y10 and Y16 were particularly effective in this respect with the antagonistic level of 32%, 36% and 38.7% respectively. All of them achieved significant disease suppression (P<0.005). The identity of the potential antagonists and the pathogen had been studded based on their morphological, biochemical and physiological characteristics. Isolate Y10 and Y16 were identified to the genus Rhodotorula and Y3 was belongs to Trichosporonoides and the pathogen was identified as Penicillium italicum. From this study, it can be concluded that isolate Y16 showed the highest antagonistic activity than the other yeast isolates and Penicillum italicum is one of the responsible fungi for orange blue mold disease in Ethiopia. Keywords: antagonist yeasts, biocontrol, Penicillium italicum, orange, yeast.
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    A Study on Ochratoxin a and Toxigenic Fungi on Coffee and Selected Cereal Grains Consumed In Ethiopia
    (Addis Ababa Universty, 2017-02) Geremew Teshome; Abate Dawit (PhD)
    Ochratoxin A (OTA) is toxic fungal secondary metabolite produced by some filamentous fungi belonging to the genus Aspergillus and Penicillium. This mycotoxin is commonly found in coffee and cereal grains and total removal of the toxin by various food processing methods is not possible. The aim of this research is to study ochratoxin A and toxigenic fungi in coffee and cereal grains. This dissertation contains five major chapters in which background information, the rationale for the study and research objectives are presented in the first chapter. The second chapter is dedicated to reviewing the available literature on the properties of OTA, the producing fungi, occurrence of OTA in foods, biosynthesis of OTA, analytical methods, toxicity in human and animals, mechanisms of action and prevention strategy. This section also addresses production and processing of coffee and some selected cereal grains. The remaining three chapters are the main experimental part of this study. The third chapter focuses on the occurrence of toxigenic fungi and OTA in Ethiopian coffee for local consumption. In this chapter, fungal incidences in coffee samples and OTA contamination levels in coffee were evaluated using various mycological techniques and ELISA method for toxin analysis. The identities of fungal isolates were revealed using species-specific primers and DNA sequencing techniques. The result indicated that total fungal incidence mounted up to 87%. The predominant fungal genera were Aspergillus (79 %), Fusarium (8 %) and Penicillium (5 %). Ochratosin A producing species of A. westerdijkiae, A. ochraceus, A. melleus, and A. steynii were identified for the first time using molecular techniques from locally sold coffee in Ethiopia. A. westerdijkiae and A. ochraceus were found to be the predominant OTA producers. The median OTA level in the locally sold Ethiopian coffee was 1.53 μ g/kg. Although samples from wet processing resulted in low median levels of OTA, they were not significantly different from other processing types (p < 0.306). Significant differences in fungal incidences were observed between the different coffee processing types (p < 0.001), coffee sample types (p < 0.005), and storage materials (p < 0.03). An in vitro assessment of OTA and Aflatoxins (AFs) production potential of all Aspergillus isolates reviled that A. westerdijkiae isolates were clearly the most potent producers of OTA while AFs were only produced by two A. flavus isolates. This chapter is published in Food Control 69 (2016) 65-73. The fourth experimental chapter deals with the association of coffee bean defects with Ochratoxin A contamination in the beans: A possible implication for visual judgment of OTA contaminated coffee beans. In this chapter coffee bean defects were investigated based on Ethiopian coffee quality and liquoring manual and Specialty Coffee Association of American (SCAA). The predominant coffee bean defects encountered in this study in descending order were black beans (47.5 %), cherry (13.5 %), immature bean (8.1 %), sour bens (7.9%), insect infested (7.9 %), moldy beans (4.4 %), and broken beans (3.7%). Ochratoxin A contamination levels in coffee beans were positively correlated with black beans (r = 0.33, p < 0.03), visible mold overgrowth (r = 0.80, p < 0.00), beans damaged by insects (r = 0.71, p < 0.00), and presence of husk (r = 0.67, p < 0.00). These types of defects could be used as a possible visual marker for OTA presence in locally sold coffee. The fifth chapter deals with High Performance Liquid Chromatography Variable Wavelength Detector (HPLC-VWD) method of detection and quantification of Ochratoxin A in teff (Eragrostis teff) and wheat (Triticum spp.) flour samples intended for local consumption. In this section, HPLC method was developed and validated to detect and quantify OTA in teff and wheat flour samples. Based on this validated method teff and wheat four samples were analyzed for OTA contamination using HPLC-VWD apparatus. The results indicate that 20% of the teff and 50 % of the wheat flour samples were contaminated with median OTA level of 0.99 μg/kg (LOD = 0.78) and 6.76 μg/kg (LOD = 0.58 μg/kg) respectively. This validated method for OTA detection and quantification in teff and wheat using HPLC-VWD detector generates meaningful data that satisfy performance criteria set by European Commission, EC 401, 2006 and Eurachem Guideline validation requirements. Moreover, an insight into the occurrence of OTA in teff is very valuable because the cereal might provide a low OTA risk alternative for wheat in Ethiopia. Keywords: Aspergillus spp., Aflatoxins, toxigenic fungi, coffee, defect count, mycotoxin, ochratoxin, ELISA, HPLC-VWD, teff, wheat