AAU-ETD :: Browsing by Author "Muleta, Diriba (PhD)"

Browsing by Author "Muleta, Diriba (PhD)"

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Antimicrobial Properties of Endophytic and Rhizospheric Fungi Associated With Some Medicinal Plants
(Addis Ababa University, 2020-09-09) Shemsedin, Fertuna; Muleta, Diriba (PhD)
Nowadays the development of multidrug resistant human pathogenic microorganisms and the emergence of new diseases are the most challenging problems in public health care on a global scale. To overcome this problem it needs intensive searching for new sources of effective antimicrobial agent producing organisms. Therefore, the main objective of this study was to isolate and identify the antimicrobial properties of entophytic and rhizospheric fungi associated with some medicinal plants. A total of 150 plant parts and 50 soil samples were collected from five medicinal plants around Bale Zone, west Arsi Zone and Chancho Oromia Special Zone, Oromia Regional State of Ethiopia. All collected samples were processed following standard protocols. In vitro antimicrobial activities were tested against common resistant pathogenic organisms (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeroginosa and Candida albicans). A total of 582 (316 endophytes and 266 rhizospheric) fungal isolates were obtained from the collected medicinal plants and soil samples. Accordingly, 78 (19.89%) isolates displayed antimicrobial activities against at least one target microorganism by fungal agar plug method. The ethyl acetate extracts of the crude metabolites of 18 isolates, showed antagonistic activity against at least one tested organisms with higher inhibition zone. Ethyl acetate extracts of isolate 30CRS showed highly significant (p≤0.001) inhibition zone against E. coli (30.33+0.57 mm), E. faecalis (25.33 + 0.28 mm) and S. aureus (19.16+ 0.28 mm) than positive control chloramphenicol whereas fungal isolate 37BRaL showed significantly (p≤0.001) higher inhibition zone against S. aures (19.16+ 0.28 mm) and C. albicans (26.83 + 0.76 mm). The mean MIC, 3.125 - 50mg/ml for gram positive bacteria, 6.25 - 50 mg/ml for gram negative bacteria and 12.5 – 50 mg/ml for yeast test organism. MBC 6.25-50mg/ml and MFC ranged from 12.5-50 mg/ml. The phytochemical screening of the fungal metabolite revealed the presence of flavonoids, alkaloids, glycosides, terpenoids, steroid, saponin, phenol, and tannin. A total of five potential fungi were examined by morphological characterization and Biolog identification, from this, isolates 30CRS and 37BRaL were identified as P. simplicissimum and T. flavus var flavus, also characterized for different Biolog carbon source utilization test using Biolog microbial identification system.
Characterization of Indigenous Wild Yeasts and Evaluation of Their Bread Leavening Capacity
(Addis Ababa University, 2017-08-03) Beyene, Elsa; Muleta, Diriba (PhD); Kiros, Solomon (PhD); Tesfaye, Anteneh (PhD)
Ethiopia is a developing country with a high baker’s yeast demand for bread making and beverage industries. However, there is no baker’s yeast producing plant in the country. Therefore, the objective of this study was to assess bread leavening potential of wild yeasts from teff and wheat dough, shamita, tej, tella, molasses and honey samples, following standard methods. A total of 180 wild yeast isolates were, out of these isolates 23 isolates with high biomass yield (1.7 to 2.6 of 600 nm OD reading) and the efficient gas (CO2) producers in durum tube within 12 to 24 hours were selected for further analysis. Morphological, biochemical and molecular features were used to identify and characterize the yeasts. Isolates identified by biolog were belongs to genera Saccharomyces, Candida humilis and Kluyveromyces and found to utilize glucose, sucrose, raffinose, trehalose and maltose. Genetically, the sequence analysis of the ITS-5.8S rDNA region showed that the isolates belong to 5 species namely, Saccharomyces cerevisiae, Candida humilis (synonyms Candida milleri), Kazachstania bulderi, Pichia fermentans and Pichia kudriavzevii. All the test isolates were found to produce maximum cell mass at a temperature of 30oC, pH of 5.5 and 48 hours of incubation. Isolate AAUTf1 did not produce hydrogen sulfide. The isolates AAUTf5, AAUTj15 and AAUSh17 including commercial yeast produced light level of hydrogen sulfide, while AAUMl20 and AAUWt21 produced heavy level of hydrogen sulfide. The assessment of leavening performance of the yeasts indicated that, isolates AAUTf1 (Candida humilis) and AAUTf5 (Kazachstania bulderi) had higher leavening activity with raising dough by 131 cm3 and 128 cm3 respectively each within 120 min. Similarly, isolates AAUSh17 (Saccharomyces cerevisiae) and AAUTj15 (Saccharomyces cerevisiae), were found rising the volume of the dough by 127 cm3 and 125 cm3, respectively at 60 min compared to the commercial yeast that increased the volume to 117 cm3 within 90 min. The combined bread dough rising effects of AAUTf1 + AAUTf5 + AAUTj15, AAUTf5 + AAUTj15 and AAUTf1 + AAUTj15 + AAUSh17 caused volume increment to 143 cm3 at 90 min, 141 cm3 at 60 min and 140 cm3 at 60 min, in that order. Generally, mixed cultures showed superior leavening potential than that of single cultures and commercial yeast.
Characterization of Indigenous Wild Yeasts and Evaluation of Their Bread Leavening Capacity
(Addis Ababa University, 2017-08-02) Beyene, Elsa; Muleta, Diriba (PhD); Kiros, Solomon (PhD); Tesfaye, Anteneh (PhD)
Ethiopia is a developing country with a high baker’s yeast demand for bread making and beverage industries. However, there is no baker’s yeast producing plant in the country. Therefore, the objective of this study was to assess bread leavening potential of wild yeasts from teff and wheat dough, shamita, tej, tella, molasses and honey samples, following standard methods. A total of 180 wild yeast isolates were, out of these isolates 23 isolates with high biomass yield (1.7 to 2.6 of 600 nm OD reading) and the efficient gas (CO2) producers in durum tube within 12 to 24 hours were selected for further analysis. Morphological, biochemical and molecular features were used to identify and characterize the yeasts. Isolates identified by biolog were belongs to genera Saccharomyces, Candida humilis and Kluyveromyces and found to utilize glucose, sucrose, raffinose, trehalose and maltose. Genetically, the sequence analysis of the ITS-5.8S rDNA region showed that the isolates belong to 5 species namely, Saccharomyces cerevisiae, Candida humilis (synonyms Candida milleri), Kazachstania bulderi, Pichia fermentans and Pichia kudriavzevii. All the test isolates were found to produce maximum cell mass at a temperature of 30oC, pH of 5.5 and 48 hours of incubation. Isolate AAUTf1 did not produce hydrogen sulfide. The isolates AAUTf5, AAUTj15 and AAUSh17 including commercial yeast produced light level of hydrogen sulfide, while AAUMl20 and AAUWt21 produced heavy level of hydrogen sulfide. The assessment of leavening performance of the yeasts indicated that, isolates AAUTf1 (Candida humilis) and AAUTf5 (Kazachstania bulderi) had higher leavening activity with raising dough by 131 cm3 and 128 cm3 respectively each within 120 min. Similarly, isolates AAUSh17 (Saccharomyces cerevisiae) and AAUTj15 (Saccharomyces cerevisiae), were found rising the volume of the dough by 127 cm3 and 125 cm3, respectively at 60 min compared to the commercial yeast that increased the volume to 117 cm3 within 90 min. The combined bread dough rising effects of AAUTf1 + AAUTf5 + AAUTj15, AAUTf5 + AAUTj15 and AAUTf1 + AAUTj15 + AAUSh17 caused volume increment to 143 cm3 at 90 min, 141 cm3 at 60 min and 140 cm3 at 60 min, in that order. Generally, mixed cultures showed superior leavening potential than that of single cultures and commercial yeast.
Characterization of Wild Indigenous Yeasts from Molasses and other Sugary Substrates and their Potential for Bioethanol Production
(Addis Ababa University, 2021-05-09) Degu, Sisay; Muleta, Diriba (PhD); Tesfaye, Anteneh (PhD)
The increasing demand of energy has been supplied through the combustion of petroleum throughout the world. Due to escalating cost of petroleum, its contribution to global warming and non-renewable nature of this oil, there is collectively a need for renewable and ecofriendly energy sources such as bioethanol. Thus, the objective of this study was aimed at isolating, characterizing and evaluating the potent wild yeasts under different stress conditions from locally available resources for bioethanol production to minimize the utility of fossil fuel. In this study, a total of 35 samples of sugary substrates were collected from Metehara Sugar Factory for wild yeasts isolation following the standard protocols. A total of 305 yeast isolates were retrieved and screened using physiological and osmotic stress tolerance tests. Fermentative and potent wild yeasts were identified to species level using morphological and biolog-based biochemical methods. Out of 305 yeast isolates, 20 (6.56%) and 7 (2.29%) of them were found to be tolerant to 18 and 20% of ethanol, respectively. Out of these 20 ethanol tolerant yeast isolates, 17 tolerated the temperature of 45°C for 48 hrs. From 17 ethanol-thermotolerant isolates, 5 (29.41%), 5 (29.41%) and 7 (41.18%) were found producing gas from glucose at 24, 48 and 72 hrs, respectively. Out of the 17 ethanol-thermotolerant yeast isolates, 12 yeast isolates were able to tolerate 35% of glucose. Out of these 12 ethanol-thermo-and-sugar tolerant yeasts, the 7 were found tolerant to pH 2. From the 7 acidic tolerant yeast isolates, 5 yeast isolates were shown tolerant to 7% of NaCl and identified as K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively. During fermentation, K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa were able to produce bioethanol with the values of (% v/v) 12.62, 11.61, 10.58, 10.82 and 10.44, respectively at 48 hrs and 12.56, 12.52, 12.08, 11.11 and 11.48, respectively at 72 hrs. The initial inoculum cell density was increased from (1.83 to 3.08, 0.59 to 1.81, 0.54 to 1.36, 0.48 to 1.69 and 0.45 to 1.56) x108 cells/ml for K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively 0 hr to72 hrs. The fermentation efficiency (%) for K. lodderae, P. guilliemodii B, S. boulardii, T. globosa and Z. rouxii was shown as 98.0, 97.8, 93.1, 90.8 and 88.1, respectively. Thus, on the basis of highest stress tolerant and higher fermentation efficiency features, K. lodderae and P. guilliemodii B wild yeasts were considered to be the best bioethanol producers. Molecular characterization and optimization of fermentation parameters is recommended to utilized these potent yeasts.
Characterization of Wild Yeasts Isolated from Selected Fruits for their Bread Leavening Capacity
(Addis Ababa University, 2017-06) Lakew, Eshet; Muleta, Diriba (PhD); Tesfaye, Anteneh (PhD)
Leavening agents are important in raising flour dough. Biological leavening agents are microorganisms that have the ability to produce carbon dioxide from the utilization of Sugar and thereby ferment and raise the dough. The present study was carried out to characterize yeast isolates isolated from selected fruits and to assess their leavening potential of wheat dough under laboratory scale. The collected fruit samples were processed to isolate yeasts using Potato Dextrose Agar (PDA) amended with 0.1 g/L chloramphenicol. Initially, 88 yeasts were isolated from the fruits and were first tested for their carbohydrate fermentation in yeast extract peptone dextrose (YEPD) broth medium. Six yeast isolates with their sugar fermentative abilities were selected and tested for H2S production. Among them, AAUGr5, AAUOr7 and AAUPi3 found not produce undesirable H2S for bread baking quality on both Kligler Iron Agar (KIA) and Bismuth Sulfite Agar (BSA) media. The three yeast isolates were identified as Saccharomyces using colonial, morphological parameters and biochemical tests. The optimum growth pH and temperature values for the three selected yeast isolates were recorded as 5 and 30 oC, respectively, in YEPD medium. In addition, 30% (w/v) D-glucose and 5 %( w/v) NaCl concentrations showed optimum growth of the three selected yeast isolates in yeast extract peptone broth medium. In all the cases, the maximum biomass was achieved at 96 hrs of incubation and there was a rapid decrease in biomass for all the yeast isolates after 96 hrs of incubation. In terms of CO2 and biomass production as well as leavening potential, starter cultures which were formulated from the combination of the three yeast isolates (AAUGr5+AAUOr7+AAUPi3) showed better performance than starter cultures formulated from paired combination of the three isolates or each of the three isolates separately. However, isolate AAUGr5 was found to be satisfactorily potent for leavening action from the single isolates. The present study could therefore be important with respect to screening of wild yeast isolates that possess better bread leavening potential for extending the use of indigenous microbes as starter culture in bakery sector. Keywords/Phrases: biomass; carbon dioxide ;fruits, hydrogen sulphide; laboratory scale leavening; yeast isolation
Ethnomycological Study of Wild Edible Mushrooms of Metu Town, Ilu Abba Bora Zone, Oromia Regional State
(Addis Ababa University, 2020-01-01) Teferi, Alemayehu; Muleta, Diriba (PhD)
This ethnomycological study was conducted to assess interactions of Metu town inhabitants with the wild edible mushrooms, particularly indigenous knowledge and perceptions, diversity of the edible mushrooms and difference among inhabitants in consumption of the WEMs. Cross-sectional study was conducted with the probability and non-probability methods for selection of respondents and also Sem-structured questionnaire with a closed and open-ended questions, deep interviews and observations participants were conducted for data collection methods. In addition, descriptive statistical analysis was conducted to compare indigenous knowledge and perception of inhabitants for WEMs among different categories of informants. All of the respondents revealed that they knew WEMs. Majority (91.4%) of them responded that they had experience to share benefit of the WEMs, but few (8.6%) of them were not beneficial. School, agricultural sector, health sector, NGO and religious office workers were main source of the information of the WEMs. Similarly, majority (91.4%) of them argued that mushrooms were utilized for a food and medicinal purpose, but few (4%),(3%) and (1.5%) of them revealed that they were also utilized for religious case, others and entertainment purposes in respectively. In addition, they uses for ceremonial, hallucinogenic properties and others functions. Among those about 42%, 26.8%, 21.2% and 9.6% of them were experienced to utilize mushrooms for their nutritional values in respectively due to its a good tastes, substitute a meat and fish, delicacy and good smell to get heat and energy, bodybuilding and increase their body resistance. Moreover, among 198 respondents, majority (88.9%) of them confirmed that they were not experienced to utilize mushrooms for medicinal purpose, but few (11.1%) of them revealed that they had experience in using mushrooms for their medicinal value particularly about 28%, 23%, 11.6%, 29% and 4%of them confirmed that they were utilized for the treatment of inflammation, homeostatic, analgesics, diuretic and tumors. in respectively and preferred to use it because of they easily found in environment, too cheap and better to cure than modern medicine. Similarly, majority (73%) of them confirmed that they were involved in collection of WEMs and they differentiate mushrooms based on its smells, colors and tastes during summer, spring and autumn period of seasons particularly from a termite mound area under big tree, on log or wood swampy trees, farmlands, cattle field area and dry lands. About 44.4%, 25.3%, 18.2%, 7.1% and 5.1% of them responded that they have been collected wild edible mushrooms by chance randomly, observing muddy of soil, sound birds, and deliberately. They were collected mainly by men, elders, children and women in respectively. However, they have been not practiced to preserve that might be happened due to lack of an experience share to preserving mushrooms among their parents in the area, lack of well-developed indigenous knowledge of the WEMs. Majority (81.8%) of respondents argued that mushrooms were not practiced to buy/sale in local market but only (18.2%) of them were argued it. In addition, about 78% of them argued to utilize mushrooms in fresh form, but 22% of them confirmed that they were utilized in dried form. Mushrooms include T. schimperi, T. letestui and T. rosbustus and T. microcarpus are the most expensive in area due to its medicinal values, meaty flavor, good smell and delicacy for food consumption and also they are frequently bought by urban inhabitants, rural social group, illiterates and also literates peoples in respectively. Most of time they are transporting by human carrying and domestic animals such as by donkey. About 50.3% of respondents confirmed that mushrooms coverage were decreased due to deforestation, habitat destruction, Overgrazing and populations pressure role. Majority (86.8%) of respondents argued that mushrooms were not possible to adopt/cultivate of WEMs and also about 44.2%, 29.9% of them confirmed that wild mushrooms development was discouraged by both rain and soil properties and heavy thunder rain. In general, anthropogenic activities, negative perception, increasing urbanization, agrochemical, exotic plants and deforestation are main factors cause for the disturbance of WEMs and also they poor conserved efforts of wild edible mushrooms. Therefore, both methods of conservation in-situ and ex-situ mushroom conservation strategy, disseminate information of WEMs to become behavioral change and transformation conception habit of inhabitants to edible mushrooms are the main recommended.
Evaluation of Different Leaf Litters for Oyster Mushroom (Pleurotus Ostreatus) Cultivation in Nekemte City
(Addis Ababa University, 2018-08-08) Gelano, Tadesse; Muleta, Diriba (PhD)
Cultivation of mushrooms using locally available substance is a very cheap and environmentally friendly alternative for producing foods with high nutritional value. The consumption and familiarity of mushrooms to people has been limited in Ethiopia largely due to poor knowledge on the methods of production. This study was conducted to evaluate different locally available leaf litters and their combinations for their productivity and biological efficiency (BE) for cultivation of an edible mushroom strain, oyster (Pleurotus ostreatus); in Nekemte City, east Wollega Zone, Oromia Regional State between January, 2017 and September, 2017. An experiment was conducted to cultivate edible mushroom (Pleurotus ostreatus) using four different leaf litters: namely, Wanza (Cordia africana), Bisana (Croton macrostachyus), Khat (Catha edulis) and Eucalyptus tree (Eucalyptus camaldulensis) and their combinations to determine their biological efficiency for the selected mushroom species. The results showed that, mycelial extension, the highest and lowest mean value 0.521 cm/day and 0.323 cm were measured from Ce (Catha edulis) and CeCmCa (combination of Catha edulis, Croton macrostachyus and Cordia africana), respectively. The results revealed that differences in mean incubation periods and duration of pinning-to-maturation, pileus diameter, and stipe length were insignificant (p>0.05) among substrates. The first pinning took 34.66 days, followed by 9.66 to 10.66 days between different flushes. Mean durations from pinning-to-maturation ranged from 3.66 to 4.33 days. The mean pileus diameter of mushrooms ranged from 4.77 to 5.03 cm, whereas the mean stipe length ranged from 1.98 to 2.16 cm. In this study, the highest biological efficiency for the studied mushroom species was recorded from Croton macrostachyus leaf litter ((94.45%) followed by Eucalpytus camaldulis leaf litters (89.72%). All the substrates that gave over 50% BE could be recommended for oyster mushroom cultivation. The findings generally suggest that the mushrooms species when grown on locally available leaf litters may have the potential to solve the problem of food insecurity in Ethiopia and can also create job opportunity to youngsters, elders and disabled personnel.
Evaluation of Phyto Beneficial Traits of Indigenous Phosphate Solubilizing Bacteria and Fungi as Microbial Inoculants for Enhancing Growth and Production of Coffee (Coffea Arabica) Under Greenhouse and Field Conditions in Jimma South West of Ethiopia
(Addis Ababa University, 2021-12-17) Abafita, Reshid; Assefa, Fassil (PhD); Muleta, Diriba (PhD)
Exploitation of phosphate solubilizing bacteria (PSB) and fungi as microbial inoculants is known to promote plant growth through the supply of plant nutrients and supression of pathogens. In view of this, the present investigation was planned to assess the phytobeneficial traits of phosphate solublizing bacterial and fungal isolates recovered from coffee (Coffee arabica) and vermicompost to determine their potential in growth promotion of coffee seedlings under low input agriculture.. The microbes were isolated and purified following standard methods. The selected isolates were investigated for their plant growth promoting properties, eco-physiological tolerance under laboratory conditions, and further tested under greenhouse and nursery experiments. The greenhouse and nursery experiments were conducted with completely randomized design (CRD) in three (3) replications per treatments. Thus, a total of 154 bacteria and 72 fungi isolates were recovered from which twelve potent bacterial and nine fungal isolates were selected and investigated for their plant growth promoting properties. Among the twelve bacterial isolates, three of them were tentatively identified to the genera of Pseudomonas (RCHVCB1) and Bacillus (RScB1.19 and RMaB2.11), and showed significant potential to solubilize Ca3 (PO4)2 and posessed several phytobeneficial traits, viz, indole acetic acid, NH3, HCN productions and N-fixing ability. They also exhibited remarkable tolerance to ecophysiological factors such as heavy metal, acidity and salinity, and inherent antibiotic resistance (IAR). Similarly, three fungal isolates with superior phosphate solubilizeation ability were characterized and identified as genera of Penicillium (RSCF1.19) and Aspergillus (RCHVCF2 and RLVCF2). During co-culture, RSCF1.19 (Penicillium sp.) slightly inhibited the test pathogen, Fusarium xyloriodes. The bacterial (RCHVCB1, RScB1.19, RMaB2.11) and fungal isolates (RSCF1.19, RCHVCF2, RLVCF2) enhanced rate of coffee seed germination under laboratory conditions and promoted coffee seedlings growth under glasshouse conditions. The results of inoculated seeds showed significant (p≤0.05) differences in germination rate and vigor index compared to the control. Isolates RScB1.19, RMaB2.11+RSCF1.19 and RMaB2.11 + RLVCF2 showed high germination rate (20.59%) over the control (13.33%). Moreover, a single inoculation of RLVCF2, RSCF1.19 and co-inoculation of RMaB2.11 with RLVCF2 also showed significant (p≤0.05) mean root length (1.31 cm) and mean shoot length (1.48 cm) over the control. Under greenhouse conditions, single inoculation of RSCF1.19+phosphate and dual inoculation of RSCF1.19 and RCHVCB1 in the presence of inorganic phosphate fertilizer led to significantly higher plant height, root length, stem girth, leaf number, leaf area, fresh and dry weights. Due to high pH value of the potting medium (vermicompost alkaline pH-pH>7.5), all the treatments combined with vermicompost showed suppressive effect and no any seedlings were emerged at all. Under nursery conditions, co-inoculation of RSCF1.19 with three bacterial isolates (RCHVCB1, RScB1.19, and RMaB2.11) in combination with inorganic phosphate led to significantly increase the tested growth parameters. Similar increase in growth attributes was observed in both single and dual inoculations due to vermicompost used compared with both positive and negative controls. Higher NPK-uptake was observed in a combination of bioinoculants and inorganic phosphate fertilizer compared to the positive and negative control. In general, inoculation of RSCF1.19 and RLVCF2 isolates to coffee 74110 variety combined with inorganic phosphate fertilizer resulted in good vigor and healthier coffee seedlings (RSCF1.19, 34.42%) and (RLVCF2, 37.09%) when compared to control (28.49%). Therefore, both RSCF1.19 and RLVCF2 fungal isolates could be used as bioinoculants after field trials in coffee 74110 variety productions.
Genetic and Physiological Diversities of Bacteria from the Rhizosphere of Woody Plants Grown on Soil from Girar Jarso District and their Effect on the Growth and Establishment of Tree Seedlings in North Shewa Zone Ethiopia
(Addis Ababa University, 2020-04-19) Getahun, Alemayehu; Muleta, Diriba (PhD); Assefa, Fassil (Professor)
Land degradation (LD) is one of the major problems the planet earth has been facing. It has severely affected 1.9 billion hectares and decreased ecosystem services by 60%. In Eastern Africa, particularly in the northern highlands of Ethiopia, 1 billion tons of topsoil are lost annually. Thus, the land becomes bare, nutrient-depleted, water-stressed and abandoned by the community. Hence, it needs urgent rehabilitation with the application of eco-friendly microbes and organic amendments (OAs) through the exploitation of different types of trees/shrubs. Therefore, the overall objective of the study was to rehabilitate degraded habitat through the application of OAs and phytobeneficial bacteria for the establishment of multipurpose trees. Soil samples were collected from nine random corners at depth of 30 cm for soil physicochemical analysis before and after OAs. Six different treatments (biochar, compost, manure, mixed, bacterial inoculation (BI), and control) were considered at a 1:1 ratio of OAs per pot. Application BI was done for field trials during transplanting and quarterly for a year. Six plots measuring 41 m x 4 m were established in completely randomized block design and assigned at the random block for the field trial. Following OAs, microbial counts were done for one year every month. Bacteria were isolated from the rhizosphere of Acacia and Juniperus. The primary selection of isolates was based on drought stress (DS) tolerance and phosphate solubilization, other stress, and plant growth-promoting traits. The potential isolates were subjected to carbohydrate and amino acid utilization tests, BOX PCR, and 16S rRNA profiling. DS tolerant, phosphate solubilizer strains with multiple plant growth-promoting traits were chosen for Acacia seeds germination and field application alone and in combination with OAs. Plant growth parameters and their survival rate at each amended plot were assessed. There is a significant increase in soil pH (5.69-8.13), CEC (43.78-49.98 cmolc/kg), OM (2.43-3.91%), total nitrogen (0.13-0.76%), and available P (18.9-26.31 ppm) following OAs compared the control. Combined treatment had the largest effect on cover crops biomass with 3.43 g, 4.54 g, 0.7 g, 2.07g in alfalfa, grass pea, and control respectively p ≤ 0.05. The C and N utilization revealed metabolic versatility of the strains (14.29 to 100%). Ochrobactrum, Pseudomonas, and Klebsiella spp expressed remarkable metabolic versatilities. BOX-PCR showed greater genetic diversity and confirmed by Simpson’s Index of Diversity (0.883) took the leading position with Bacillus species. The 16S rRNA genes sequence showed 21.92% Firmicutes and 78.08% Proteobacteria with Pseudomonas 23% and Ochrobactrum 21% dominant species. Out of 73 isolates, 10 (14%) were highly tolerant of 40% polyethylene glycol. All the isolates can grow in wider ranges of pH (5-9), temperature (15-45°C). The inoculated bacterial strains significantly p ≤ 0.05 increased root, shoot length, and dry biomass of acacia. According to solubilization index (SI) 45% isolates were classified as high and medium phosphate solubilizers with 195 to 373 μg/mL. The maximum P and IAA were produced by Pseudomonas FB-49 (373 and 659.07μg/mL), respectively. The highest (100%) seed germination caused by Pseudomonas BS-26 and Pseudomonas FB-49. There is a significant difference in microbial counts following OAs compared to the control. The greatest counts in bacteria, actinomycetes, and fungal (21.66, 2.29, 0.82 x 105 CFU g-1) of soil, respectively in the combined amended plot. There was a significant increase in stem height, girths, and branch numbers in amended plots relative to the control. The survival rate was observed in apple (80%) followed by acacia (66%) and prunus (51%). The survival is in the order of BI x OAs > BI > biochar > compost > manure > control amended soil. This study concluded that degraded land could be rehabilitated with cheap OAs, potential bacterial strains, and bring multipurpose tree establishment with greater survival rate and best performance.
Isolation and Characterization of Potential Bioethanol Producing Indigenous Wild Yeasts from Local Alcoholic Raw Materials (Areke Difdif and Tenses)
(Addis Ababa University, 2020-12-12) Admas, Alene; Muleta, Diriba (PhD); Tesfaye, Anteneh (PhD)
Shortage of fossil fuel supplies has increased a great deal of interest in worldwide production of renewable energy. Bioethanol produced from renewable biomass like molasses has received considerable attention as an alternative biofuel in recent years. Thus, the main purpose of this research work was to isolate, screen and characterize ethanol tolerant wild indigenous yeasts with high bioethanol yield from areke difdif and tinses samples collected from different areke producing sites. The wild yeast isolates were retrieved from the alcoholic beverages following standard methods. From a total of 270 isolates, ten (3.7%) of the yeast isolates tolerated 22% and four (1.5%) isolates from the same 10 isolates tolerated 23% ethanol concentration, and were selected for further characterization. Based on morphological appearance of vegetative cells under microscope, colony characters and molecular analysis, the isolates were identified as Saccharomyces cervicae and Kluyvermyces marixianus. Ten (Mda, Mdb, Mdd, D5f, D5b, D5d, D5e, Dt1e, Dt1c and Dtlj) isolates were found thermotolerant (45oC and Mda able to tolerate 47 oC) and also all ten isolates grow at low pH (>=2.5). Ethanol production from sugar cane molasses by the yeast isolates was determined by using Hall method and was crosschecked with Ebulliometer. From 10 isolates, two isolates (Mda and Dt1e) showed the highest bioethanol production capacity of 14.3% v/v and 13.2% v/v, respectively at pH 4.39 and temperature 35oC in 30 degree brix molasses concentration at 150 rpm shaking condition. During this study potentially useful and excellent ethanol producing indigenous yeasts with high ethanol tolerant features from areki difdif and tenses were isolated, identified and characterized. This study exhibited the potential of local fermentation processes and products (areke difdif and tenses) as possible sources of industrially useful microbial isolates.
Isolation and in-Vitro Characterization of Extracellular Phytase Producing Bacterial Isolates for Potential Application in Poultry Feed
(Addis Ababa University, 2020-07-07) Amede, Lubaba; Muleta, Diriba (PhD); Tafesse, Mesfine (PhD)
Phytase is an enzyme that can be breaking down the complex organic forms of phosphorous such as phytate into simpler forms by sequential hydrolysis of phosphate ester bonds and liberate the usable form of inorganic phosphates. Supplementation of feeds with microbial phytase enhances the bioavailability of phosphorus and micronutrients. Hence, the aim of this study was to isolate and characterize phytase producing bacteria from Rhizospheresoil, fresh poultry excreta, Cattle shed in order to evaluate their potential in improving poultry feeds. Phytase producing bacterial were isolated using Wheat Bran Extract Medium. A total of 169 bacterial isolates were purified and screened for phytase activity. Out of which36 were found positive for the desired purpose. The bacterial isolates were identified by cultural, morphological, and biochemical features. Finally, the isolates were identified to species level by 16S RNA genes sequencing using universal primers. The isolate (RS1, RS8, RS10 and RS15) correlated with shared the highest similarity indices, Enterobacter asburiae, Klebsiella pneumonia, Pseudomonas aeruginosae and Escherichia coli respectively. all isolates alone possible to increase the phytase production in cultures medium (wheat bran extract medium) by supplementing with 1% galactose and ammonium sulphate, temperature (50oC), pH (6), incubation time (72 hr) and size of inoculum (at 800μl). Proximate composition analysis after supplementation of phytase released the bound phytate phosphorus and improved bioavailability of phosphorus, calcium, potassium and sodium in poultry feed. Overall, this study is to improve the nutritional value of poultry feed, reducing the cost of diets and supplementation inorganic phosphate. Lastly, it will be maximizing poultry products by supplementation of phytase with poultry feed.
Microbial Diversity and Probiotic Potential of Lactic Acid Bacteria Isolated from Traditionally Fermented Food Products and Their Applicability for Prevention of Selected Foodborne Pathogens
(Addis Ababa University, 2019-11-11) Mulaw, Guesh; Tesfaye, Anteneh (PhD); Muleta, Diriba (PhD); Sisay, Tesfaye (PhD)
Probiotics are live microorganisms which when administered in adequate amounts promote the health of the consumer. The major purpose of this study was to determine bacterial diversity of traditionally fermented food products (teff dough, Ergo and Kocho) and to assess their probiotic potential of lactic acid bacteria in prevention of foodborne pathogens. Culture-independent metagenomic methods, particularly 16S rRNA amplicon sequencing were applied to study the bacterial diversity of some fermented foods. The sequencing of the amplicons resulted in an average of 76,594.33 ± 2,876.25 high-quality reads per sample. The bacterial communities of the three traditional fermented foods were dominated by the phylum Firmicutes, followed by Proteobacteria and Actinobacteria. At the genus level, the prevalent microorganisms were Lactobacillus, Zymomonas, Streptococcus, Leuconostoc, Bacillus and Weissella. The genus Lactobacillus was the most abundant genus in the three studied food products, whereas Zymomonas was the most dominant genus in fermented ergo. In order to assess the probiotic potential of strains isolated from these fermented products, a total of 450 LAB were isolated from the fermented products, and their ability to survive the gastrointestinal conditions was tested. Among them, 34 (7.56%) isolates survived at pH 2 for 3 and 6 h, showing a survival rate of 45.35- 96.33% and 33.33- 91.75%, respectively. All the 34 acid-tolerant isolates survived in the presence of 0.3% bile salts for 24 h with 82.58 to 99.44% rate of survival. The acid and bile salt-tolerant LAB isolates shown inhibiting some bacterial foodborne pathogen into varying degrees. Among the 34 acid and bile salt-tolerant isolates, 9 (26.47%) isolates produced bacteriocins to varying degrees. All ii the 34 acid and bile salt-tolerant isolates displayed sensitivity to ampicillin (10 μg/ml), tetracycline (30 μg/ml) and erythromycin (15 μg/ml), but showed resistance to kanamycin (25 μg/ml), while 17/34 of the isolates were found sensitive/resistant to streptomycin (10 μg/ml). The in vitro adherence to stainless steel plates of the 34 screened probiotic LAB isolates showed adhesion rates that ranged from 29.21 to 41.94%. The identification of 34 screened potential probiotic LAB strains and their genetic relatedness was performed using 16S rRNA gene sequencing and whole-genome (WGS) sequence comparisons. Hence, four isolates were identified as Lactobacillus species including Lb. plantarum, Lb. paracasei subsp. tolerans, Lb. paracasei and Lb. plantarum. The remaining 30 potential probiotic LAB strains were identified by using WGS comparisons. Thus, these isolates belonged to Lactobacillus and Lactococcus species including 22 Lb. plantarum, 1 Lb. paracasei, 1 Lb. brevis and 6 Lc. lactis. Among the 30 potential probiotic LAB strains, BAGEL predicted 1 class III bacteriocin, 7 class II bacterioicins and 3 class I bacteriocins in the genome of 9 strains. The compatibility test among the selected potential probiotic strains (Lb. plantarum, Lb. paracasei and Lc. lactis) showed the absence of antagonism among them. The in vitro antagonism testing of the potential probiotic LAB as separate pure or mixed culture against Salmonella showed a remarkable anti-Salmonella activity with 96.50 to 100% growth inhibition. The combination of strains that showed the highest growth inhibition rates against Salmonella Typhimurium, were used to test their in vivo effect on the colonization of mice by Salmonella Typhimurium. The survival rate of mice on day 21 after the oral challenge with Salmonella Typhimurium was significantly higher (p<0.05) in the experimental pro-typ, treated group (100% survival) when compared with the positive control group (20% iii survival). The counts (colony-forming unit/ml) of Salmonella in feces were significantly lower (p<0.05) for the pro-typ group compared to the positive control group (typ). In general, this study indicates a high level of diversity in the bacterial community structure in traditional fermented foods and the LAB of the traditional fermented food samples was found to be good candidates for food industries as prospective probiotic cultures with health benefits.
Microbial Safety and Quality of Fresh Beef Supplied to Gullele Sub-City Market, Addis Ababa
(Addis Ababa University, 2017-02) Zerabruk, Kibrom; Muleta, Diriba (PhD); Reta, Nigusse (PhD)
Food-borne pathogens are the leading cause of illness and death in developing countries. The widespread habit of raw beef consumption is a potential cause of food-borne illnesses in Ethiopia. Therefore, this study was initiated to evaluate the microbiological quality and safety of beef samples supplied to Gullele sub-city Addis Ababa and also to assess the associated risk factors in handling the product. A questionnaire survey was used to answer some questions concerning the current status of food hygiene and sanitation practiced in abattoir and butchery shops. Fresh beef samples from different sources and swab samples from contact surface were collected for microbial analysis, following standard methods. This study revealed that the mean microbial counts of total aerobic mesophilic, staphylococci, Enterobacteriaceae, total coliform, fecal coliform, aerobic spore and yeasts and molds of the abattoir were 4.788, 3.287, 4.882, 4.763, 4.072, 1.476, 4.024 log cfu/100cm2, respectively. Likewise, the mean microbial counts of total aerobic mesophilic, Staphylococci, Enterobacteriaceae, total coliform, fecal coliform, aerobic spore and yeasts and molds of the butcher shops were as great as 6.4, 4.8, 5.98, 5.9, 5.5, 1.84 and 5.3log cfu/g, respectively. Microbial load on meat was found to be low in the carcass sample compared to the morning and afternoon samples from butcher shops. The mean microbial count of total aerobic mesophilic, staphylococci, Enterobacteriaceae, total coliform, fecal coliform, aerobic spore and yeasts and molds of the contact surface samples were > 6.3, 3.7, 4.8, 5.3, 4.4, 1.8 and 4.9 log cfu/cm2, respectively. The aerobic mesophilic flora was dominated by Enterobacteriaceae (29.5%) followed by Staphylococci ssp (26.67%) and Bacillus spp (17.4%). The prevalence of E. coli, Salmonella and S. aureus in meat samples were 29.55%, 3.4% and 28.41%, respectively. The isolated food-borne pathogens were resistant mostly to Penicillin (>60%), Amoxicillin (>40%) and Ampicillin (>40%). The overall facilities, slaughtering techniques, cold chain management, handling of slaughter cattle in the abattoir and personal hygiene, environmental sanitation, knowledge on food handling and educational status of the butcher shops ‘workers were among the predominant factors of contamination of the beef. Therefore, implementations of GMP and HACCP principles as strategy should be adapted to control pathogenic microbes which pose public health risks. Keywords: beef samples; dominant microflora; microbial counts; microbial contamination
Physicochemical Properties, Microbial Qualities and Antimicrobial Activities of Honeys from Kellem and West Wollega Zones, Western Ethiopia
(Addis Ababa University, 2018-01-04) Tesfaye, Ofijan; Muleta, Diriba (PhD); Dessalegn, Asnake (PhD)
Honey is a natural substance synthesized by honey bees. Its physicochemical properties and biological composition differs among honey types. The aim of this study is, therefore, to determine the physicochemical and antioxidant properties, antimicrobial activities, microbial qualities and floral source of honeybee and stingless bee honeys collected from Kellem and West wollega zones of western Ethiopia. The physicochemical, antioxidant and pollen grain analysis of honey were determined using standard methods. Agar disc and well diffusion were used for antimicrobial susceptibility test while agar dilution was used to determine minimum inhibitory concentration (MIC). A microbial quality was carried out by pour plate technique. Honey from Meliponulla beccarii L. (MBH) was found to be more acidic (pH 3.00 ±0.11) with high level of free acidity (113.1±18.43 meq/kg) and moisture (33.55±2.82%) content, but with lower concentration of reducing sugar (37.84±4.04%) compared to honey from Apis mellifera L. samples. There was no statistically significant (p > 0.05) difference in the total phenol, flavonoid and antioxidant content of honey obtained from Meliponulla beccarii L (MBH), Apis mellifera market honey (AMMH) and Apis mellifera L fresh honey (AMFH). The overall average and the highest diameter of inhibition zone (mm) against clinical and standard isolates of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhi was recorded for MBH (15.71±2.78 mm) followed by AMFH (14.83±2.45 mm) and AMMH (10.51± 2.63 mm), while 25.16±2.50 mm for the positive control (chloramphenicol) by well diffusion method. On the other hand, the overall average inhibition zone (mm) recorded by disc diffusion from MBH, AMFH and AMMH were 10±1.21 mm, 9.04±1.67 mm and 7.62±1.32 mm, respectively against the tested bacterial pathogens. However, none of the honey samples inhibited Candida albicans. The overall mean of minimum inhibitory concentration (%) and minimum bactericidal concentration (%) against all the tested bacteria were 9.92 and 53.57 by MBH, 11.00 and 56.24 by AMFH, and 15.40 and 64.28 by AMMH, respectively. The highest Staphylococci (2.1 x 103cfu/g) and least spore forming bacteria (1.17 x 102 cfu/g) were counted from AMMH sample. MBH sample had the highest mean of 3.16 x 102 cfu/g by yeast and lowest mean of 1.0 x 102 cfu/g by mould, while AMFH sample became free of any microbial contamination. All types of honey samples were safe from Enterobacteriaceae and coliforms. Coffee arabica L., Plantago lanceolate L. and Guizotia scabra L. were the major floral sources of the tested honeys. Thus, Honey from the study area had good antioxidant, antibacterial and nutritional values.
Public’s Awareness and Practice of One Health Approach in Preventing Zoonotic Diseases in Sibu-Sire District, East Wollega Zone,West Ethiopia
(Addis Ababa University, 2018-10-03) Asfaw, Ephrem; Muleta, Diriba (PhD)
This study was conducted with the objective of assessing public‘s conception and practice of One Health Approach in preventing zoonotic diseases in Sibu- Sire district, Oromia regional state, Southwestern Ethiopia using questionnaires and face-to-face interview technique. The total number of participants consisted of 428. Males (n=316) accounted for 73.8 % and female (n=112) 26.2%. The respondents were stratified into four groups namely, teachers (n=13), students (n=79), farmers (n=299), and nurses (n=37). Among the study groups 83.4% were rural residents and the rest 16.6% urban dwellers. Around 73% of them were married. A total of 251 (58.6%) of the study subjects were illiterate, whereas 41 of them (9.6%) had basic education .The rest 32% (n=137) of the respondents attended from primary school to university education. Many of them (72.6%) of farmers and 63.3% of the students did not know about One Health Approach. Public‘s practice of One Health Approach to prevent zoonotic disease in the area is very low, 66.1% (n=283) of the respondents said that zoonosis prevention through One Health Approach was little. Rabies as a zoonotic disease was known to majority of the respondents (99%), With regard to rabies as a zoonotic disease, there was no significantly (P>0.05) different level of awareness among the different respondent groups which can imply that rabies is a well known disease in the area whereas brucellosis was known by too few respondents (0.7%). There was no statistically (p>0.05) significant association between knowledge about brucellosis and educational status of the participants in this study. A large number (71.1%) of the respondents consumed raw meat regardless of their knowledge about transmission of zoonotic diseases. Majority (95.7%) of farmers do not know the occurrences of zoonosis through inhalation. Over 56% of them do not know direct contact with animals could cause zoonosis. According to this study 74.2% of these farmers do not know handling animals with cut and 62.9% of them sharing the same room with animals could cause disease to human. Similarly 86.6% of the farmers do not know whether human could be infected by zoonotic diseases through vectors. In general, the present study revealed a very low level of awareness by the public about One Health Approach and major zoonotic diseases, signifying the need for public health promotion through education and inter-disciplinary One Health Approach with close collaboration among health extension workers, agricultural extension workers, wild life experts, environmentalist and kebele leaders.
Synergetic effects of Mandillo [Crassocephalum macropappum (Sch. Bip. ex. A. Rich.) S. Moore] during Enset fermentation: chemical nature and efficiency
(Addis Ababa University, 2016-04) Gonfa, Alemu; Megersa, Negussie (PhD); Mehari, Tetemke (PhD); Muleta, Diriba (PhD); Young-Mog, Kim (Professor)
Enset (Ensete ventricosum (Welw.) Cheesman) is an ornamental plant in Asia, widely distributed in Africa and cultivated only in some regions of Ethiopia. Enset plant withstands prolonged period of drought and known for its high yielding potential. Fermentation of Enset produces a starchy food named Kocho, however, it is not practiced extensively. The principal reasons for low popularity of Enset are its uncommon sensory attributes among non-Enset consuming people, short shelf-life, nutrient losses, long fermentation period and lack of awareness. In Ethiopia, the Shekacho society adds Mandillo (Crassocephalum macropappum (Sch. Bip. ex. A. Rich.) S. Moore) stem during traditional Enset fermentation without knowing its scientific significance to the fermentation processes. Thus, the present study was initiated with the main purposes of investigating the indigenous knowledge of Shekacho society in Enset cultivation, Kocho consumptions, bioactive components of Mandillo parts and to determine its effects in Enset fermentation processes. The study was conducted using standard methods. To evaluate the indigenous knowledge of Shekacho society, analytical data were collected using semi-closed questionnaire, field observations, interviews and discussion with key informants with local annotations review. Enset plant physical structures were assessed. Phytochemical composition of Mandillo was evaluated. Its DNA damage protective and antioxidant activities were analyzed. Antimicrobial activity of Mandillo was tested against standard bacterial and yeast strains. Cytototoxicity of Mandillo was also evaluated. Fermentation process was carried out from 0-90 days. The pH value, titratable acidity, lactic acid and acetic acid contents were measured. Microbial counts were made on their respective agar media. Lactic acid bacteria (LAB) and yeast isolates were identified using morphological, biochemical and molecular techniques. Proximate composition, mineral contents, phytic acid, tannin, water holding capacity, and sensory attributes iii of Kocho were evaluated and the main findings are described as follows. The descriptive analysis of the data and the corresponding results indicated the existence of a high degree of consistency among the respondents with regard to Enset histories, fermentation processes, productivity, shelf-life of Kocho and food preparation. About 115 Enset cultivars were identified. Phytochemical analysis of Mandillo showed the presence of saponins, tannins, anthraquinones, steroids, terpenoids and flavonoids. Total phenolic and flavonoid contents of the aerial part calculated as 101.48 mg GAE (Gallic acid equivalent)/g and 293.25 mg QE (Quercetin equivalent)/g, respectively. Mandillo showed strong 2, 2-diphenyl-1-picryl hydrazyl (DPPH) scavenging potential with IC50 ≤ 50 μg/mL. The stem of Mandillo showed promising protective activity against oxidative DNA damage. The minimum inhibitory concentration of stem against E. coli KCTC 1682 and Candida utilizes KCCM 11355 was ≥ 15.62 μg/mL. The final pH of Kocho fermented with starter culture but no Mandillo (Koki) and with Mandillo (Kom) decreased to 4.49 and 4.10; respectively, which are significantly different and didn’t allow the growth of spoilers in Kom. Titratable acidity, lactic acid and acetic acid contents of Kom increased by 64.34%, 103.90% and 40.04%, respectively when compared to Koki. Koki and Kom samples had LAB counts of 1.16×107 and 2.83×108 CFU/g, respectively, on the day 37. Treatments with Mandillo stem significantly (p < 0.05) lowered counts of aerobic mesophilic bacteria, aerobic spore formers and coliforms. Staphylococci were below detectable level. The 16S rRNA gene sequences analysis and phylogenetic tree reconstruction identified LAB species as Lactobacillus plantarum (69.37%), Leuconostoc mesenteroides (9.91%), Lac. paracasei (9.01%), Lac. brevis (3.60%) and other rare species. Lac. paracasei and Lac. brevis were dominantly found in Kom during the final days of fermentation. An 18S rRNA gene sequences evaluation identified yeast isolates as Candida boidinii (30%), Wickerhamomyces sp. ES22S03 (16%), Pichia fermentans (16%), Williopsis sp. (10%) as dominate species and others minor strains. Kocho composition analysis showed significant (p < 0.05) differences between Koki and Kom in moisture contents (8.34 to 8.80%) and (9.14 to 7.95%), fiber (7.92 to 5.56%) and (6.25 to 3.96%), protein (3.24 to 3.68%) and (3.59 to 3.95), phytic acid (5.83 to 4.11 mg/g) and (4.95 to 4.33 mg/g), water holding capacity (8.55% and 6.37%); respectively. Mineral contents of Koki and Kom also calculated as Ca (9.84 mg/100 g and 10.08 mg/100 g), Fe (2.12 mg/100 g and 2.08 mg/100 g), Zn (1.99 mg/100 g and 1.77 mg/100 g) and Cu (0.12 mg/100 g and 0.16 mg/100 g); respectively. Sensory evaluation revealed overall acceptability values of 73.27% (Koki) and iv 97.20% (Kom). Cytotoxicity evaluation of Mandillo stem against Raw Cells 264.7 showed no toxicity at ≤ 4 μg/mL. From the results of the descriptive analysis of the data and the corresponding results suggested by the respondents, it is understood that Enset is very important for food security and environmental protection. Phytochemical contents and activities of Mandillo revealed consistency with the starter culture role played by the addition of the stem part of the plant. The effect of adding Mandillo during Enset fermentation showed significant differences in all of the measured Kocho quality parameters, food composition, microbial dynamics and diversities and sensory attributes of experimental Kocho product compared to the control sample. In general, improvements of the basic components of Kocho dough are responsible for the enhancement of its shelf-life, safety and sensory attributes of the food as a result of good fermentation processes. In the light of the current finding, extensive investigation are required to qualify Mandillo as food preservative and shelf-life enhancer for its application in food sectors.

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