Browsing by Author "Gessesse, Amare (PhD)"

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    Amylase Production by two Microbial Isolates: Isolation, Enzyme characterization, and optimization of Cultivation condition
    (Addis Ababa Universty, 2017-03) Leykun, Senaite; Gessesse, Amare (PhD)
    Amylases are commercially important enzymes commonly used for the hydrolysis of starch to generate hydrolysates for different food and nonfood applications. Although Ethiopia has a huge potential for the production of starch, to date the starch industry is nonexistent and the country import large quantities of starch hydrolysates from abroad. The aim of this study was production of industrially important microbial amylases through solid state fermentation (SSF), optimize the cultivation condition, and characterize the enzymes. Screening using starch containing media one bacterial isolate producing α-amylase, designated as BI151, and one fungal isolate producing glucoamylase, designated as FI97, were isolated from soil. Both isolates grew well on SSF with wheat bran as a solid substrate. BI151 α-amylase produced 4,636 U/g of amylase after 96 h of incubation at 50% moisture. Isolate FI97 produced a maximum activity of 4,674 U/g after 72 h of incubation under SSF at a moisture content of 67%. For both isolates supplementation with organic or inorganic nitrogen source did not have any impact on enzyme production. This suggests that wheat bran supplemented with mineral salts and water has enough nutrients to support growth of the organisms and make the production process a lot cheaper. Amylase BI151was optimally active in the temperature range of 65-75°C with a peak at 70°C and in the PH range of 4.5 -5.5. On the other hand amylase of FI97 was optimally active at 65°C and at pH 6. These properties showed that both enzymes have properties that make them potentially suitable for starch hydrolysis, especially in the brewery industries. Under lab scale evaluation, both enzymes were able to efficiently hydrolyze high concentration of starch adjuncts (60% of the malt adjunct mixture) indicating their potential for application as malt enzyme supplements. Key word: α-amylase, Glucoamylase, starch hydrolysate, relative activity, residual activity
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    Amylases of Potential Industrial Application from Microbial Sources
    (Addis Ababa Universty, 2006-07) Teka, Muluye; Gessesse, Amare (PhD)
    Glucoamylase and alpha amylase producing fungal and bacterial strains were studied. The fungal isolate previously isolated by Abreham Tesfaye belongs to the genus Aspergillus and was designated as Aspergillus No 43 (Asp 43). The bacterial isolate (designated as Bacillus sp CRC) which was isolated from Hot spring around lake Chitu was Gram positive, rode ,motile catalase positive and central endospor former. Asp 43 glucoamylase was optimally active at pH 4 and temperature of 65 OC. where as Bacillus CRC alpha amylase showed maximum activity at a pH range of 5- 6 and temperature of 80 OC. Addition of 5 mM Ca2+ did not affect the temperature profile of both types of amylases. However the bacterial amylase was Ca dependent for its themostability, and this enzyme retained about 53 % of its original activity after 3 hr incubation at 80 OC. In addition Bacillus CRC was stable over a broad pH range retaining more than 80 % of its original activity in the pH range of 4.5 – 8.0 . Asp.43 grown in SSF medium at 25 OC produced maximum enzyme when the moisture content of the wheat bran used as a sole carbon source was 66.7 %. The optimum pH and temperature for maximum enzyme production by Bacillus CRC in liquid medium was 6.0 and 55 OC respectively. Bacillus CRC produced maximum enzyme when the sole carbon and nitrogen sources were starch and trypton ( at 0.5 and 0.2 % respectively). Enzyme secretion by the fungal and bacterial isolates reached maximum after 96 and 36 hr of incubation respectively.
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    Antibiotic production and optimization of culture condition of Actinomycetes from some Soda Lakes of Ethiopia.
    (Addis Ababa University, 2011-07) Mehabie, Daniel; Gessesse, Amare (PhD)
    The demand for new antibiotics increased due to the misuse and over use of antibiotics in various sectors creating an evolutionary pressure and resulting in multi drug resistant strains which result in increment of morbidity and mortality in the world. The main objective of this study was to explore the potential of alkaliphilic actinomycetes isolated from three Ethiopian soda lakes. About 171 isolates were screened for antibiotic production using cell free culture supernatant taken from the submerged fermentation and ethyl acetate crude extract of the culture, of which 93.6% of them showed antibiotic activity with cell free culture supernatant and 76 % of them with crude ethyl acetate extract against the test microorganisms used. Based on broad spectrum activity, zone of inhibition obtained and their activity with the use of crude ethyl acetate extract, two isolates LCHACT17and LABACT21 were chosen for further characterization. Antibiotic production was favored when phosphate concentrations was 0.2 % for LCHACT 17 and for LABACT 21. Yeast extract and sodium nitrate (for LCHACT 17) and combination of casein and potassium nitrate (for LABACT 21) were suitable nitrogen sources for antibiotic production. From the carbon sources used soluble starch was the best carbon source for antibiotic production by LCHACT17 while sucrose was found to be preferred carbon source by LABACT 21. Similarly it was observed that 1% sodium chloride concentration was favorable for antibiotic production by both isolates and the pH parameter indicated that pH10 (for LABACT 21) and pH11 (for LCHACT17) were the optimum pH for antibiotic production. The cell free culture supernatant of both isolates was stable at alkaline pH and decreases in activity at lower pH (pH 6 and below). The cell free culture supernatant of LCHACT17 was stable up to 60oc while LABACT 21 was stable up to 40 oc. The minimum inhibitory concentration (MIC) of LCHACT17 crude ethyl acetate extract against Streptococcus pneumonia was 12.5 mg/ml. Key words/ phrases: Alkaliphiles, Antibiotics, Disk diffusion.
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    Diversity and Efficiency of Cyanide Degrading Alkaliphilic Bacteria from Ethiopian Soda Lakes
    (Addis Ababa University, 2010-06) Mekuaninte, Birhanu; Gessesse, Amare (PhD)
    The aim of this study was to isolate and characterize cyanide degrading alkaliphilic bacteria from Ethiopian Soda Lakes. Seventy two isolates capable of growing on a medium provided with cyanide as a sole nitrogen source has been found. Based on their morphological features, thirty isolates were screened for subsequent identification. ARDRA of the 16S ribosomal genes amplified by PCR was used to screen these isolates. Restriction analysis was done using three endonucleases namely; AluI, HaeIII and RsaI. ARDRA revealed the presence of nine polymorphic groups among the thirty. 16S rDNA amplicons of representative strains were sequenced and compared with sequences from NCBI and RDP databases. This revealed the presence seven bacterial strains from which three belonged to genus Bacillus, three to genus Halomonas, and the other one to uncultured bacterium clone, FJ152630. Based on phylogenetic analysis, the unidentified strain was more related to and clustered with the Halomonas sublineages. Strains CNS10, CNA12 and CNC1 isolated from Lake Shala, Lake Abijata and Lake Chitu respectively were found to be better candidates in that they have utilized up to 99.33% of 200mg/l cyanide and tolerated up to cyanide concentration of 600mg/l in batch mode with pH of 10.22. In addition to cyanide, these strains used other organic and inorganic nitrogen sources, yeast extract being the most utilized. Acetate was being provided as a source of carbon and energy source but most interestingly all the strains have aggressively grown utilizing cyanide when provided with cheap molasses as carbon source. In general, this study indicated the presence of diverse cyanide utilizing bacteria with visible potential for practical application.
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    Dye Biodegradation Using Alkalophillic Consortia in Anaerobic-Aerobic Bioprocess
    (Addis Ababa University, 2009-07) Gessesse, Belay; Gessesse, Amare (PhD)
    The decolorization and Chemical Oxygen Demand (COD) removal efficiency of consortia of alkalophilic microorganisms was evaluated in a contineous anaerobic/aerobic reactor system. The biodegradation products of RR 184 in the continuous anaerobic/aerobic reactor were studied by employing different analytical tools such as Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). When the synthetic dye wastewater was subjected to a two stage reactor, a 96.0 % color removal of RR 184 was attained after the anaerobic treatment. However, after the aerobic treatment, overall decolorization efficiency was 100 % as compared to the anaerobic stage. On the other hand, the removal efficiency of Chemical Oxygen Demand (COD) was greater after the aerobic treatment (66.6%) than the anaerobic treatment (94.26 %). The band of the Thin Layer Chromatography shows fragments of dye decolorization products. On the other hand, the HPLC Chromatograms show that the dye decolorization products that are produced at the anaerobic stage were degraded. From HPLC chromatograms, three new peaks were obtained and one of it was degraded completely and the retention time and peak areas of the other two peaks was decreased. From the consortia, dye decolorizing microorganisms were isolated and tested for their decolorization efficiency in batch test. The isolates from the anaerobic/aerobic reactor were inoculated into a sterilized decolorized media with the addition of glucose as a carbon source to a final concentration of 0.1 % and the isolates were incubated under aerobic condition. After 12 h the isolates growth (OD) was measured and all the isolates tested were able to grow using metabolites of the decolorized media as a sole nitrogen source. Key Words: biodegradation, Reactive Red 184, dye biodegradation products, alkalophilic microorganisms
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    Evaluation of Some Wood Rotting Fungi for Bagasse Pretreatment in Ethanol Production
    (Addis Ababa University, 2011-06) Tewolde, Dawit; Gessesse, Amare (PhD)
    Lignocellulosic biomass is a sustainable feedstock for ethanol production compared to other substrates. In the ethanol generation process, pretreatment makes cellulose more accessible to enzymatic hydrolysis by modification of the lignin barrier. Biological pretreatment employs selective rot fungi that preferentially degrade lignin with minimum loss of polysaccharides. The system is not well developed; but is advantageous over other pretreatments because reactions are enzymatic. These fungi produce ligninolytic enzymes, predominantly lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) in different combinations. Studies on biological pretreatments have shown lignin weight loss and improvement in ethanol yield. However, only few well characterised white rots have been explored. The aim of this study was to evaluate the potential of 15 wood rotting fungi isolated from Ethiopia for pretreatment during 15 days solid state fermentation, using bagasse as lignocellulosic substrate. The production of ligninolytic enzymes by Fomitiporia aethiopica, F. pseudopunctata, Fomitopsis carnea and Vanderbylia vicina were reported for the first time in this study. The white rots, F. aethiopica, Perenniporia tephropora, Inonotus sp. and Pleurotus sajor-caju, were identified as relatively highly selective based on maximum and minimum productivity of ligninolytic and polysaccharide degrading enzymes, respectively. The pretreatment by the white rots caused ligninolysis and better cellulose digestibility was obtained with higher lignin loss. Among the selective degraders, P. tephropora caused the highest lignin loss (7.71%) and cellulose digestibility (29.44 %) after enzyme hydrolysis of the pretreated bagasse. This digestibility showed an improvement by 38.74 % in comparison with untreated bagasse. In addition to high MnP productivity (55.87 U/g), P. tephropora also produced high titers of Lac (79.65 U/g) in contrast to the other selective degraders that might have attributed to better lignin loss. The ethanol yield from fermentation of cellulase enzyme hydrolysed P. tephropora pretreated bagasse was 1.87 g/L, which was an improvement by 27.21 % compared with untreated bagasse (1.47 g/L). Therefore, P. tephropora pretreatment enhances ethanol production from bagasse through partial degradation of lignin, which improves the accessibility of cellulose to enzyme hydrolysis. Key words: bagasse, ethanol, ligninolytic enzymes, pretreatment, rot fungi
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    Hydrolysis of Gelatin From Animal Hoof Using Bacterial Gelatinase
    (Addis Ababa University, 2018-01-01) Mekonnen, Miraf; Gessesse, Amare (PhD)
    Gelatinases are enzymes used to hydrolyze gelatin into peptides and amino acids. Enzymatic hydrolysis of gelatin increase it’s functional, textural and nutritional characteristics. The hydrolysates have neutral taste, low bloom and contain high amount of purified protein. In recent years gelatin hydolysates find a number of increasing applications in food and none food products. The main objective of this study was to isolate gelatinase enzyme from soil bacteria and optimize the reaction condition for gelatin hydrolysis. A total of 100 bacterial isolates were isolated from soil samples collected around Addis Ababa University (Arat Kilo Campus) and water samples collected from Lake Afdera. All isolates were screened for the production of gelatinase on gelatin containing media. Based on the properties of the enzyme, one isolate designated as aau17 was selected for further study. The organism can grow under solid-state fermentation and produce gelatinase enzyme with high activity using wheat bran as a carbon source. The enzyme was optimally active at a temperature of 50oC and at pH 8. The optimum enzyme production under solid state fermentation includes an incubation period of 120 h and a moisture content of 66.7%. The isolate produced more enzymes up on addition of peptone as a nitrogen source. The enzyme was used to produce gelatin hydrolysate by hydrolyzing animal bone, animal skin, animal hooves and commercial gelatin. The production process for the gelatin hydrolysis was optimized by varying enzyme concentration and incubation time. The hydrolysis has maximum activity at 4 h incubation period and at high amount of enzyme concentration. This study indicated that by using product of animal waste can be converted to useful gelatin hydrolysates using microbial gelatinases for various applications.
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    Identification and Characterization of Raw Starch Degrading Β-Amylase from Endophytic Bacillus Species
    (Addis Ababa University, 2008-08-06) Gashaw, Metages; Gessesse, Amare (PhD)
    Starch is a major reserve carbohydrate of all higher plants. In some cases it accounts 70% of the wet weight of plant material. It occurs in the form of water insoluble granules. The size and shape of the granules are often characteristic of the plant species from which they are extracted. When heated in water the hydrogen bonds holding the granules together begin to weaken and this permits them to swell and gelatinize. Ultimately they form paste or dispersion, depending on the concentration of polysaccharide (Prasanna, 2005). Starches are produced commercially from the seeds of plants, such as corn, wheat, sorghum or rice; from the tubers and roots of plants such as cassava, potato, arrowroot and the pith of sago palm. The major commercial source of starch is corn from which it is extracted by a wet milling process (Crab, et al., 1997). Starch is a heterogeneous polysaccharide composed of two high molecular weight entities called amylose and amylopectin which are differ in structures and physical properties. The insoluble amylose, composed of linear chains of α-1, 4 linked D-glucose residues can be separated from soluble amylopectin fraction by addition of a polar solvent, e.g. n-butanol. Even if amylose has a degree of polymerization of several thousands of glucose units, because of the molecular shape and structure, it is not stable in aqueous solution and retrogrades (precipitates spontaneously) this is because linear chains align themselves by hydrogen bonding and thus forms aggregates. Due to its molecular shape amylose has also considerable viscosity in alkaline solutions and forms complex with iodine to form intense blue color and this forms the basis of a method for quantitative determination of amylose. Amylopectin accounts for 75 to 85% of most starches. It has molecular weight of 107 – 108 and has a branched structure composed of chains of about 20 – 25 α-1,4 linked D-glucose residues. Amylopectin which is branched by α-1,6 linkages may contain 4 to 5% α-1,6-D-glucosidic bonds. In aqueous solutions, amylopectins are relatively stable due to branched molecules and are not able to form compact aggregates (Fogarty and Kelly, 1990).
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    Isolation and Characterization of Alkalophilic Cyanide- Degrading Bacteria from Ethiopian Alkalophilic Soda Lakes (Lake Chitu)
    (Addis Ababa University, 2007-07-03) Lako, Wajana; Gessesse, Amare (PhD)
    Currently, alkalophillic microbial cyanide degradation has gained greatest attention due to its vital advantages over the acidophilic and /or neutral and conventional cyanide removal methods from industrial wastes. The objectives of this study were to determine how readily alkalophillic cyanotrophic microorganisms could be isolated and screened from enrichment cultures supplied with cyanide as sole source of nitrogen. A total of 63 potential microorganisms were isolated from Lake Chitu mud sediments. Two relatively more active isolates (designated as AUCN-54 & AUCN-60) in resisting and utilizing cyanide were selected for morphological and physiological characterizations. Biomass (dry weight) and protein measurements were the bases for selection. The optimum cyanide concentrations (for maximum growth yield- dry weight and protein) were found to be 10.2mM and 17.34mM for AUCN-54 and AUCN-60, respectively. Thus, AUCN-60 was found to be better in utilizing and resisting cyanide. Both isolates were capable of managing to grow and utilize sodium cyanide a t a salt concentration of 0.25-10 % ( w/v), which was consistent with the salinity of the sample site (microbial original habitat). Concerning the pH, temperature and antibiotics resistance study, clear variations were observed between the two isolates. Interestingly, both of the bacterial isolates were good in utilizing sugarcane molasses as carbon source during cyanide degradation, indicating the need for further study in large scale applications. In addition, both of the cyanotrophic isolates were capable of utilizing potassium thiocyanate as a sole source of nitrogen. On contrary, they were poor in utilizing K2CU(CN)4 as a sole source of nitrogen. This may be attributed to the stability of the cyanide complex due to strong attraction of CNgroup to the central atom in the ligand.The bacterial isolates screened and characterized in this particular study and in their original habitat (sample site) could be of great importance due to their good cyanide utilization capacity and high salt and pH tolerance, which are commonly encountered influential factors in cyanide contaminated waste treatments.
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    Isolation and Characterization of Alkalophilic Denitrifying Bacteria From Lake Chitu
    (Addis Ababa University, 2008-07-04) Abubeker, Rajiha; Gessesse, Amare (PhD); Leta, Seyoum (PhD)
    The objectives of the study were to isolate and characterize alkaliphilic denitrifying organisms from Ethiopia soda lake, Lake Chitu. Eighty seven potential denitrifying organisms were isolated. Two isolates were selected (designated as BACC280 and BACC118) as efficient denitrifiers for further characterization. These were Gram-negative, oxidase and catalase positive cocci, mesophilic and facultative anaerobe. Isolate BACC118 can optimally grow from 0 to 5% NaCl and 0.25 to 1% Na2CO3 concentration but optimum salt concentration for BACC280 is relatively higher. Salt requirement for denitrification activity of the isolates were found to be similar with growth concentration of NaCl but Na2CO3 optimum was at 0.25% to 2% for BACC280 and 0.5 to 3% for BACC118. The pH optimum of both buffered and un buffered were 12 for BACC280 and BACC118. Both isolates were able to denitrify aerobically and anoxically. These isolates may play great role in recycling of nitrogen compounds in to gaseous end product in their natural habitat. They have also potential to treat wastes that contain high amount of nitrogen, high salt concentration and high pH values.
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    Isolation and Evaluation of Azo Dye Decolorizing Microorganisms from Ethiopian Alkaline Soda Lakes
    (Addis Ababa University, 2008-07-04) Guadie, Awoke; Gessesse, Amare (PhD)
    Textile dye decolorizing microorganisms were isolated from Ethiopian alkaliphilic Soda lakes. A total of 121 morphological distnict colonies were isolated from mineral salts medium-agar plate. When these isolates were subjected to confirmed decolorizer and non-decolorizer using mineral salts media containing 10 mg/l of Reactive Red 184, 95 isolates were found to be decolorizer while the rest were non-decolorizer. By selecting 25 isolates decolorizing the dye rapidly, further screening were carried out using 25 mg/l of Reactive Red 184. Among the 25 isolates, 8 isolates which decolorized the dye > 80% in the first 24 h were selected and subjected to further screening using various concentration(50-150 mg/l) of Reactive Red 184. A55 which decolorized > 82 % of the tested dye concentration in one day was selected as an efficient isolate for further studies. The effect of nutrient composition (carbon and nitrogen), culturing condition (anaerobic, anoxic, shaker, and aerobic), dye concentration and dye type on decolorization of Reactive red 184 was evaluated using isolate A55. Maximum extent of decolorization at the end of the fourth day was observed when carbon (98-100%) and organic nitrogen (100%) sources were used in the medium. The presence of sodium nitrate inhibited decolorization activity ranging from 1-29 % through four consecutive days of incubation. Decolorization was efficient (100%) in anaerobic and anoxic cultures. However, A55 showed good growth in aerobic and agitated culture, color removal was strongly inhibited. The decolorizing activity was found to decrease with increasing dye concentration from 50 - 250 mg/l in the first day, but at the end of the fourth day almost equal percentage of decolorization was observed. The isolate was found to decolorize a total of seven dyes ranging from 51-100 % at the fourth day of incubation.
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    Optimization, Characterization, and Potential Industrial Applications of Alkaline Protease isolated from Alkaliphilic Actinobacteria BACC-15
    (Addis Ababa University, 2014-07) Worabo, Birhanu; Gessesse, Amare (PhD)
    Microbial alkaline proteases are stimulating tremendous interests in the research and enzyme market since there is a great demand for developing biotechnological alternatives for many industrial processes. Hence, this study was conducted with the aim of screening for the best alkaline protease producing alkaliphilic Actinobacteria, optimizing enzyme production in SSF; characterizing and investigation of the different industrial applications. One best producer isolate (Actinobacteria BACC-15) was selected based on different parameters. Alkaline protease production by BACC-15 in SSF was maximum at 37 (591.67±12.99 U/g) and 1:1.8 initial moisture content (595.75±9.60 U/g). The enzyme was active and stable in the pH ranges of 6.0-11.5, with optimum activity at pH 10.0 and stability at pH 7.5-9.5. It was active in temperature range of 35-75 , with optimum activity at 60 and 65 in presence of 5mM Ca2+ and highly stable at 30-50 . The enzyme was active and stable in presence of NaCl, and 8.62% enhancement was observed at 3M. Presence of high concentrations of H2O2, NaBO3.H2O, SDS, Tween-80, Triton X-100, sodium citrate and saponins had no effect on enzyme stability. The protease (84.21U/g) alone or 25% protease + 75% endod preparation in combination completely removed proteinaceous stains. Using 1% w/v E/S ratio, BACC-15 alkaline protease hydrolyzed SBPI (DH, 77.09%) and MSPI (DH, 66.19%). SBPH and MSPH were suitably used for growing pathogenic bacteria with PR of 1 compared to commercial peptone. Therefore, BACC-15 alkaline protease is potential candidate in many industrial applications as alternative to environmentally unfriendly hemicals and production of valuable products. Key words: Alkaline protease; biodetergent formulation; isolates BACC-15; protein hydrolysates; SSF;
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    Production and Characterization of Alkaline Chitinase and Protease From an Alkaliphilic Actinomycete
    (Addis Ababa University, 2007-07-04) Kassa, Firew; Gessesse, Amare (PhD)
    Chitinase and protease are enzymes produced by a number of organisms and have wide industrial applications. This study was aimed at production and characterization of alkaline chitinase and protease from alkaliphilic microorganisms and evaluation of their possible applications. Alkaliphilic actinomycetes isolated from alkaline soda lakes were screened for the production of chitinase and protease. Out of 122 isolates tested 54 % produced appreciable chitinase activity indicating the presence of diverse chitinolytic microorganisms in these habitats. Based on the level of chitinase and protease production, isolate designated as A1 was selected for further study. When grown in colloidal chitin, the isolate A1 showed peak production of chitinase after 72 h while it was 120 h for protease production. The maximum amount of chitinase obtained at the peak production time was 22.6 U/ml for and 110.4 U/ml for protease. Both of the enzymes showed wide temperature and pH optima where the peak pH for chitinase activity was 8.5 while it was 10.5 for protease. The optimum temperatures for chitinase and protease activity were 45 °C and 55 °C respectively. Chitinase was stable over the wide range of pH and maintained more than 80% of its original activity after incubation in different buffers of pH 6.5-11 for one hour at room temperature. Protease A1 was stable in a wide range of pH and temperature. It has kept over 75% of its original activity in the pH range of 9-11 after one hour at 50°C. Protease does not require CaCl2 for its stability. It showed good stability in the presence and absence of CaCl2 where its stability was maintained after 60 min of incubation at 45°C. Both protease and chitinase can be used in wide applications of biotechnology and various industries
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    Production of Alkaline Amylase from A Facultatively Anaerobic Alkaliphilic Bacteria.
    (Addis Ababa University, 2011-07) Damte, Ayalew; Gessesse, Amare (PhD)
    Bacillus sp. LCBC245, which grew at a temperature of 30 0C, pH of 10.3 and under anaerobic condition, produced an alkaline amylase. The growth of the organism and amylase production reached a maximum of 0.46 and 74.09U/ml, respectively, after 36 h of incubation. The organism secreted a maximum of 69.12 and 76.96 (U/ml) of amylase when grown in liquid media containing (w/v) 1% maize and 0.5% peptone as the sole carbon and nitrogen source, respectively. On the other hand, the level of enzyme production reached a peak value of 59.52 U/ml at 0.1M NaCl and 34.23U/ml at 1% of raw starch and thereafter progressively declined in both cases. The enzyme performed optimally in the range 55-65 0C, with a peak value of 100%, and 50-600C, with a peak value of 77.59%, in the presence and absence of 5mM CaCl2, respectively. LCBC245 amylase lost more than 70 % of its activity for NaCl concentrations exceeding 1M at incubation temperature of 50 0C for 15min and in the presence of 5mM CaCl2. After the enzyme was incubated at 55 0C for 1 h it retained 66.91% and 61.42% of its original activity corresponding to the presence and absence of 5mM CaCl2 in the reaction mixture. The optimum pH of the enzyme was 10.5 and it was stable in broad pH ranges (8-11). It had a relative residual activity of 86.33 after it was incubated for 1 h at pH 11 in 50 mM Glycine- NaOH buffer. As far as its reducing power is concerned LCBC245 amylase was able to liberate 504.25μmol/ml of reducing sugar equivalents by hydrolyzing 36.31% of a previously gelatinized starch [25 % (w/v)] after 10 h of incubation at 60 0C and pH of 10. Thus, the amylase from LCBC245 is a liquefying type. Therefore, the amylase from Bacillus sp., LCBC245 was stable at high alkaline pH ranges and required Ca+2 for enhancing its thermoactivity and thermostability. The organism produced amylase maximally when grown in media containing undefined carbon sources, 0M NaCl and organic nitrogen sources, and incubated under anaerobic condition. Key words/phrases: Amylase ; alkaline ;liquefying ; Ca-dependent.
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    Production of Bacterial Amylase and Evaluation For Starch Hydrolysis
    (Addis Ababa University, 2018-05-03) Belay, Eleni; Gessesse, Amare (PhD)
    In starch Degrading industry, the first step to be preformed is gelatinization of starch granules at elevated temperature. This process tends to form a viscose suspension, thus making mixing and pumping a huge challenge. Therefore, to lower viscosity of starch paste thermo stable enzymes are added for breaking down the polysaccharides into oligosaccharides by a process known as liquefaction. Therefore, availability of these enzymes can be considered a prerequisite to start a successful starch liquefaction and saccharification industry. The main objective of this study was, therefore, to produce efficient microbial amylolytic enzymes for starch hydrolysis. One bacterial isolate designated as AAU 2107 was isolated from locally collected samples. The enzyme has an optimum activity at pH of 5.0 and 70°C. Addition of calcium in the reaction mixture was shown to stabilize the enzyme. The isolate AAU 2107 was able to grow under solid state fermentation (SSF) using wheat bran as a solid substrate. Optimum enzyme production was recorded at 32°C, and at 120 h of incubation and at a moisture content of 75%. Addition of peptone to the SSF medium as an organic nitrogen supplement enhanced enzyme production. The enzyme was used to hydrolyze a 25 % (w/v) of starch at 70°C and pH 5.0, and the resulting hydrolysate was analyzed on TLC plates. After 4 h hydrolysis, the TLC chromatogram showed release of glucose, maltose, maltotriose, and other oligosaccharides, indicating that the enzyme is endo acting alpha amylase. The results of this study show that amylase from AAU 2107 has a good potential to be used in starch liquefaction and saccharification processes. The ability of the isolate to grow and produce the enzyme under SSF using cheap agro industrial wastes could help to greatly reduce the production cost of the enzyme.
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    Production of Ethanol Fromcane Molasses by Using Awild Yeast And Brewery Yeast
    (Addis Ababa University, 2008-07-04) Mama, Ebrahim; Gessesse, Amare (PhD)
    A fermenting isolate TAYI 4-2 and brewer yeast were utilized for alcoholic fermentation using sugarcane molasses. The fermentation of molasses was optimized with respect to temperature, pH and sugar concentration. Results revealed a pH 4.5 and 11% sugar concentration as optimum for fermentation for both microorganisms studied. The optimum temperature for TAYI 4-2 was 350C while for brewer yeast 30 0C. Under optimized conditions, TAYI 4-2 and brewer yeast produce 50.50 and 48.76 g/l of ethanol, respectively. The time required to produce a maximum alcohol by TAYI4-2 was around 54h and productivity was limited to 1.04 g/l/h. In order to improve the productivity, the immobilization of TAYI 4-2 was simply performed by the enriched cells cultured media harvested at exponential growth phase. Immobilization of yeast cells was carried out by entrapment in 2% calcium alginate and tested for ethanol production. In a batch culture of immobilized cells, the ethanol productivity was improved to 2.12 g/l/h. To further optimize productivity, the immobilized TAYI 4-2 was loaded continuous immobilized cell reactor (ICR). The performance of continuous fermentation system using immobilized TAYI 4-2 in ICR was evaluated in terms of ethanol productivity and fermentation efficiency with varying sugar concentration in the medium, at different initial pH and dilution rate. Ethanol productivity was higher at 11% initial sugar concentration similar to batch fermentation but the productivity was improved to 14.34 g/l/h. High ethanol productivity was achieved with a medium containing 11%( w/v ) total sugar concentration at a dilution rate of 0.15 h.1. And it was found that an increase in dilution rate from 0.15 h.1 to 0.25 h.1 resulted in lowering ethanol concentration in the fermented broth. The dilution rate increased the amount of sugar un-utilized found to increase. The high speed through the continuous system which cause a short residence time in the fermenter which may leads sugars in the medium to pass un-fermented and cause declining in fermentation efficiency. On the basis of the results obtained ,the potential yeast isolate TAYI 4-2 screened from .tella. showed the highest fermentation efficiency and productivity when immobilized in continuous system of fermentation within short period of time. This clearly indicates potential ethnologic microbe from .tella. was highly effective when it is immobilised and run in continuous system of fermentation at its optimum physico-chemical conditions.
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    Production, Characterization and Evaluation of Bacterial Protease as a Potential Additive to Enhance Detergency of Endod
    (Addis Ababa University, 2018-01-03) Tilahun, Rahel; Gessesse, Amare (PhD)
    At present alkaline proteases are widely used in the detergent, leather tanning, pharmaceutical, food, and feed processing industries. Although proteases are found in all living organisms (plant, animal, and microorganisms), the bulk of commercially important enzymes are from microorganisms. The aim of this study was to isolate protease producing bacteria, characterize the enzyme, and evaluate potential application as detergent additive to enhance the washing performance of endod berries. Different bacterial strains were isolated from soil and screened using an alkaline casein agar media. One isolate designated as aau 2106 was selected for further study because of its high proteoliytic activity, level of enzyme production and, ability to grow under solid state fermentation (SSF). Cultivation condition for the production of the enzyme under SSF, such as, moisture content, nitrogen content, and incubation period were optimized. The enzyme was active in the pH range of 6 to 10.5, temperature of 40 to 75OC. And optimum in; 96h incubation period, 1:1 ratio of moisture content and, casein in the production of enzyme under SSF and the enzyme was stable in the presence of endod and commercial detergents. The potential of the enzyme as a detergent additive was tested by adding the protease (57 U/g) in detergent formulations and used to clean pieces of cloth stained with blood and egg. Addition of the enzyme improved the cleaning efficiency of both endod and commercial detergents. This indicates that enzyme supplementation of endod could result in the development of an ecofriendly detergent.
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    Tannery Wastewater Treatment using Alkaliphilic Sediment Inoculum in Anoxic-Oxic Bioprocess
    (Addis Ababa University, 2011-04) Admassu, Tesfaye; Gessesse, Amare (PhD)
    Tanning is one of the oldest industries known by its voluminous wastewater and the use of huge type and amount of chemicals. In Ethiopia there are over twenty tanning industries. Most of which release their wastes without pretreatment. TWW is known particularly by its high organic load, nitrogenous, sulfide and chromium contents and by its salinity and alkalinity. In the presence of these toxic substances and saline and alkaline conditions, TWW treatment by biological methods using conventional neutrophilic organisms is hardly efficient. The objective of this study was to treat TWW by naturally alkaliphilic sludge obtained from Soda Lake Chitu water and sediment using lab-scale anoxic-oxic activated sludge treatment system and to evaluate the adaptability of the inoculated sludge to the toxic tannery wastes. The efficacy of the alkaline sludge and performance of the reactors was evaluated by analyzing COD, TN, NH4 +, S2-, SO4 2-, NO3 -, Crtot, MLVSS, MLSS and pH of the raw and treated TWW. The system was operated on continuous basis, but physicochemical data was obtained at specific OLR of 1.92gCOD/L/d and 1.64gCOD/L/d at feed 1 and feed 2 respectively. The system was able to remove 99% S2-, 93%TN and 89% NO3 - at feed 1 and 96% NH4 +, 93% COD and 92% Crtot at feed 2 from raw TWW. The result showed that at relatively high OLR there was slightly better removal of sulfides, TN and nitrate but higher removal of COD, ammonia and chromium was observed at comparatively lower OLR. In addition, the treatment process was effective in removing the color and notorious odor of TWW. Among the key factors of the activated sludge in treating the nitrogenous TWW are denitrifying bacteria. Adaptability of inoculated microorganisms to the TWW was evaluated by isolating denitrifying bacteria from the inoculant lake sediment and from the steady state treatment system. Comparative characterizations of these isolates showed that there was little difference in their pH optima (9.5-10.5) and range, chromium tolerance (250-2500mg/l) and preferences to energy sources. Also the differences in their cell wall chemistry (<3%), colony morphology, starch hydrolyzing ability and catalase reaction were minimal. But remarkable difference was evident in nitrate reducing ability and salt tolerance. Denitrifying isolates obtained from the treatment system were found to be complete nitrate reducers, incomplete nitrate reducers, exclusively nitrite reducers and both nitrite and nitrate reducers, but isolates from the inoculum were only complete and incomplete nitrate reducers. Presence of exclusively nitrite reducers, both nitrite and nitrate reducers and tolerance to high salinity by the denitrifying isolates obtained from the treatment system (DS) showed a shift in denitrification function by the inoculated microbial community. The shift in denitrification function and tolerance to high salinity conditions by the DS isolates was supposed to be associated to the adverse nature of TWW and strict anoxic condition to which the bacteria adapt the new environment than resisting it. Key words: Tannery wastewater; Lake Chitu, alkaliphiles; denitrifying bacteria; lab scale activated sludge.
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    Thermastable and Alkaline Xylanase from an Alkaliphilic Actinomycete
    (Addis Ababa University, 2006-07) Siraw, Amare; Gessesse, Amare (PhD)
    A xylanase producing alkaliphilic actinomycet~ strain designated As-19 was isolated from Lake Abjata, an alkaline Soda Lake in Ethiopia. The strain prodnce xylanase in solid state fermentation (SSF) using different agricultural residues (wheat bran, sugarcane bag ass, and saw dust) as substrates without enrichment of the medium .The highest level of enzyme was produced in wheat bran (165 U/g). Maximum production of xylanase was observed at wheat bran-tomoisturing agent ratio of I: 1. 5 (w/v) at 37°C for 72 h. Birch wood xylan enhances enzyme production with 110 % while xylose, aribinose, glucose, lactose and flUctose strongly repressed the enzyme production. In the presence of metal ions such as HgCIz, and, ZnS04 the activity of the enzyme was strongly inhibited. Birch wood xylan was hydrolyzed more rapidly than oat spelt xylan. The optimum pH of the enzyme was 8.5-10 and stable at alkaline pH (8-10). The optimum temperature for enzyme activity was 70-80 °c at pH 9. The enzyme after 4 h incubation retained 77.7 and 54 % of its original activity at 75 and 80°C respectively, at pH 9. The enzyme is also stable over wide temperature range (50-80°C) for I h at pH 9. Thus, the result showed that the enzyme is both alkaline and thermostable. The enzymatic products of xylan hydrolysis were a series of shOtt-chain xylooligsaccharides, indicating the enzyme was an endoxylanase. These are some characteristics that make this enzyme potentially very attractive for industrial applications. Key words: Actinomycete; xylem; xylanase; alkaliphile.
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    Utilization of Starch from Selected Crops as a Partial Substitute for Barley Malt in Brewing Technology
    (Addis Ababa University, 2009-06) Admassu, Habtamu; Zegeye, Adamu (Mr); Gessesse, Amare (PhD)
    Brewing process generally involves the steps of malting, mashing and fermentation. The main purpose of malting is the development of amylolytic enzymes in the grain with simultaneous degradation of high molecular substances in the cell walls enabling the achievement of a distinctive character. Barley malt is the principal ingredient in the manufacturing of beer and has traditionally been the grain of choice in the brewing industry. However, it is not always economically feasible to brew with 100% malted barley, and today's breweries are forced to minimize their costs without changing the quality or the character of their beer. Therefore, the present study was initiated to utilize Maize, Potato and Enset starch as a partial substitute for barley malt and to evaluate some physico-chemical quality attributes of the beer. All the experiments were conducted at Addis Ababa University’s laboratories(Science Faculty) and Meta Abo Brewery. The quality parameters of the starch (composition and degradability) were tested. The beer underwent four series of experiments in triplicate involving the starch from the three crops (10%, 20% and 30% starch substitute from each) with full barley malt serving as a control. The major attributes of the beer (alcohol content & flavor) were evaluated for each of the 10%, 20% and 30% substitutes from the three crops with reference to the control beer. Accordingly, the collected data were subjected to statistical analysis using SPSS software with emphasis on alcohol content and sensory attributes (flavor).The results showed that 30% substitution of barley malt with Maize and Enset starch is promising in the beer production. Beer produced using these two crops showed no statistically significant difference from the control barley malt beer (p≤0.05).The present study indicated that it is possible to partially substitute full barley malt up to 30% as it is feasible in many ways. Based on the findings, a production technology involving maize starch as a partial substitute for barley-malt has been suggested. Key words: Barley; Beer; Partial Substitution; Starch