Browsing by Author "Gessesse, Amare(PhD)"

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    Alkaline Protease Production by an Alkaliphilic Bacterial Isolate Under Solid State Fermentation
    (Addis Ababa University, 2009-08) Haile, Gizachew; Gessesse, Amare(PhD)
    A total of 240 alkaliphilic microorganisms isolated from samples collected from alkaline soda lakes of Ethiopia were screened for the production of alkaline proteases. Of these, 30% were protease positive indicating the abundance of protease producing microorganisms in these habitats. This again is a reflection of the abundance of protein substrates in the form of bird`s feather and left over from dead cells of spirulina and other microorganisms. Out of the 80 protease positive isolates, 20 (25%) grew well and produce appreciable level of enzyme activity when grown in solid state culture. Of these, one isolate designated as C45 was selected for further study. The protease produced by isolate C45 was characterized to determine its potential industrial application. The enzyme was active in the pH range of 6.5-11.5, with optimum activity at pH 8-9; and stable at alkaline pH. The optimum temperature for activity was 40°C and 50°C in absence and presence of 5mM of Ca+2, respectively. The enzyme displayed appreciable activity and stability at low temperature. These properties suggest that protease C45 could find potential application for dehairing and detergent at moderate temperature. When protease C45 was added to raw hide enabled dehairing, suggesting the potential usefulness of the enzyme in the leather industry. The commercial application of enzymes greatly depends on the cost of the enzyme which again is determined by the production cost of the enzyme. Currently most commercially available enzymes are produced through capital intensive submerged fermentation (SmF). An alternative method for the growth of microorgianims which is currently receiving significant attention is solid state fermentation (SSF). In this study, isolate C45 was grown under solid state fermentation using wheat bran as the growth substrate. Maximum protease secretion was achieved at inoculum size of 20% (v/w), bran to moistening agent ratio of 1:2 when incubated at 30°C for 144 hr. Addition of inorganic nitrogen sources and organic carbon sources as a supplement of SSF medium repressed protease induction. These results indicate that the microbial isolate shows a good potential for production of low cost alkaline protease by using inexpensive substrate such as wheat bran alone and/or low cost complex nitrogen source such as Millettia ferruginea (Berbra) seed flour as supplement in SSF. Key words: Alkaline protease, isolate C45, solid state fermentation (SSF).
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    Biological Degradation of feather Keratin
    (Addis Ababa University, 2006-07) Teka, Zenebe; Gessesse, Amare(PhD)
    A total of 165 bacteria were isolated Ii'om samples collected Ii'om Mojo TannelY and Abijata Lake, and 17% were found to possess feather-degrading capacity. Two best keratinolytic strains were selected Ii'om the 28 feather degrading isolates and tentatively identified as Bacillus lentilS strain Kr-07 and Streptomyces sp. strain Kr-43. The optimal growth period, pH and temperature for the two strains were 96h, 7.5 and 37"C respectively. The proteases of Bacillus lentils strain Kr-07 and Streptomyces sp. strain Kr-43 were inhibited by PMSF and by EDTA indicating that they are serine-type and metallo-type proteases respectively. Protease kr-07 displayed its optimal activity at a temperature of 35°C and at a pH on.5, while protease kr-43 at a temperature of 40°C and at a pH of7.5.In the presence of 5mMCaChprotease Kr-07 showed optimal activity at 45°C at the optimal pH, while protease Kr-43 exhibited optimal activity at 55°C at its optimal pH. Protease Kr-43 displayed 85.3% relative activity at 4°C indicating that it is cold active protease and hence it could have potential application in the detergent industries, especially in cold areas. The strains can utilize feather, fish wastes, and hom wastes. Therefore, they could be potentially important in the development of waste treatment methods in the biotechnological industries involved in the processing ofkeratinacious wastes. Key words: Keratin, Keratinolytic bacteria, Protease, Keratinase, and Degradation.
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    Biological Nitrogen Removal From Tannerywastewater Using Alkaliphilic Sludge
    (Addis Ababa University, 2006-07) Minuta, Tesfaye; Gessesse, Amare(PhD); Leta, Seyoum (PhD)
    Untreated tannery wastewaters contain high levels of organic materials and nitrogen. The principal forms of nitrogen in tannery wastewater are organic nitrogen (mainly proteins) and ammonia obtained from hides and skins. The presence of nitrogen in wastewater discharge can be undesirable because it has ecological impacts and can affect public health. Methemoglobin, eutrophication and depletion of dissolved oxygen in aquatic ecosystems are some of the major problems related to release of nitrogenous wastewater to the environment. Because of these pollution problems, nitrogenous compounds must be eliminated or reduced to the acceptable limits together with the organic carbon during wastewater treatment. The nitrogen in wastewater will be converted to the harmless nitrogen gas by microbial processes mainly through ammonification, nitrification and denitrification. The objective of this study was to evaluate sludge biomass activity obtained from alkaliphilic environment for removal of nitrogen and organic matter from tannery effluent using lab-scale predenitrification/nitrification activated sludge system. This was fed with simulated and actual tannery wastewater. The raw tannery wastewater was obtained from the Modjo Tannery. The system was inoculated with sludge biomass prepared using sediment slurry from alkaline soda lake and artificial wastewater in batch reactor. The potential of sediment sludge to remove nitrogen and organic matter was analyzed using COD, BOD, TN/TKN, NH+ 4-N, NO- 3-N, S2- and SO4 2- concentrations. The sludge activity was also tested for the occurrence of denitrifiaction and nitrification at higher pH using Nitrate and Ammonia Uptake Rates (NUR and AUR). The system was operated at three different organic loading rates (OLR) (10, 5 and 2.5 gm l-1 d-1). The influent had an average concentrations of 8193.33, 2355, 645.67, 686, 222.33 and 564.67 mg l-1 COD, BOD, TKN, NH4 +-N, S-2 and SO4 2-, respectively, at 10 gm l-1 d-1 OLR. Average effluent concentrations of the aforementioned parameters at 5 gm l-1d-1 OLR were 4209.33, 1409.33, 834, 578, 256.67 and 458.67 mg l-1 while at 2.5 gml-1d-1 OLR were 1886.67, 598.33, 672.33, 280.33, 241.67 and 401.33 mg l-1. The sediment sludge biomass was able to achieve 65.3, 89.2 and 97.1 % removal of COD and 59.7, 87.8 and 97.3 % removal of BOD at 10, 5 and 2.5 gm l-1 d-1 of OLRs, respectively. In addition, TKN removal efficiencies of 44.2, 86.7 and 96.6 % and NH4 +-N removal efficiencies of 62.2, 74.5 and 94.8 % were achieved at 10, 5 and 2.5 gm l-1 d-1 of OLRs, respectively. The removal efficiency of sulphide was 38.2, 89.7 and 92.3 % at respective OLRs. Their potential to remove nitrate and ammonia nitrogen during NUR and AUR Test were 69.5 and 82.8 % at range pH values of 10.2 and 10.21 and 9.50 and 9.75, respectively. Maximum removal efficiencies (97.1, 97.3, 96.6, 94.8 and 92.3 %) of COD, BOD, TKN, NH4 +-N and S2- were obtained at 2.5 gm l-1d-1 of OLR. At this OLR, the final COD, BOD, TKN, NH4 +-N, NO3 --N and S-2 were 55.33, 14.67, 22, 14.6, 4.53 and 18.67 mg l-1, respectively. This is in line with the effluent discharge limit values in Ethiopia. Thus, alkaliphiles could be a good alternative to be used as inoculums for nitrogen and organic matter removal from industrial wastewaters. Key words/phrases: Activated sludge, alkaliphiles, ammonification, nitrification, denitrification.
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    Diversity of Culturable Alkaliphilic Denitrifying Bacteria in Four Soda Lakes of Ethiopia
    (Addis Ababa University, 2010-12) Tilahun, Lulit; Gessesse, Amare(PhD)
    Denitrifying bacteria (95 in number) were isolated from the four Soda Lakes of Ethiopia namely, Lake Abijata, Lake Arenguade, Lake Chitu and Lake Shalla. Similar species of denitrifying bacteria were identified from the four lakes. The sequence and phylogeny relation of the isolates show that Halomonas campisalis, Halomonas salina, Halomonas nitritophilus, Bacillus cohnii as well as Bacillus pseudofirmus exhibited high similarities with the isolates studied. In addition, 9 isolates from the four lakes show similarity with the novel bacterial group, Nitricola lacisaponesis. Fast denitrifyers were among the isolates that are capable of producing N2 gas only in 2 hours after inoculation. Molecular, morphological and some biochemical studies conducted on the DN-C18 isolate from L. Chitu, showed high similarity on all accounts with previously obtained isolate BACC180 from L. Chitu. These two isolates showed high denitrification activity and tolerance to high pH and salt concentration. Both isolates were found to be closely related the Halomonas sp. Key words: denitrification; alkaliphilic denitrifiers; soda lakes; Lake Abijata; Lake Arenguade; Lake Chitu; Lake Shalla; Halomonas; Bacillus; Nitricola lacisaponesis
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    Production, Characterization, and Potential Application of A Keratinolytic Alkaline Protease Produced by Alkaliphilic Vibrio Sp.
    (Addis Ababa University, 2011-05) Seid, Muhammed; Gessesse, Amare(PhD)
    At present alkaline proteases are widely used in leather processing, detergent formulations, silver recovery process, and in the production of protein hydrolysates. All alkaline proteases are dervied from microbial sources that grow on expensive growth substrate. Many studies showed that nearly 40% of the production cost of alkaline proteases is accounted for by the growth substrate. To reduce the production cost it is important to search for microorganisms capable of growing and producing suffiecient amount of the enzyme using cheap substartes. In this regard keratinacious wastes released by poultry and leather tanning industries has an enourmous potential to serve as growth substartes for protease production. In this study, a protease producing Vibrio sp. capable of growing on bovine and sheep hair was isolated from Lake Arenguade. The organism produced appreciable level of keratinolytic protease using hair as the sole source of carbon and nitrogen. The enzyme was active in the pH range of 7.0- 11.5 and 40-70°C with an optimum pH of 11.0 and 50oC. The enzyme showed good stability in the presence of oxidizing agents and detergents. Application of the enzyme at the inner side sheep skin at a dosage of 58U/ml, pH 10.0 brought about complete removal of hair within 24 h at room temperature and 12 h at 37oC. Protease R-11 was also tested for the recovery of silver from used x-ray films. At enzyme dose of 11.6 U/ml and at 55oC complete removal of the gelatin layer of used x-ray films was achieved within 3 min at pH 10.00. These results indicate the potential of protease R-11 for multipurpose industrial application. Because the organism produces the enzyme using cheap substrates, hair, its production cost is expected to be very low. Key words: Dehairing; Gelatin hydrolysis. Hair degrading; Keratinolytic protease; Keratinous wastes; Used x-ray film; Vibrio sp. strain R11
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    Xylanase and Cellulase production by a termite associated Xyiaria species
    (Addis Ababa University, 2006-08) Degefu, Tulu; Gessesse, Amare(PhD)
    Xyalaria sp. are known to be associated with termites. However, the benefit the termites get from this association is not yet known. Termites collect wood pieces from the surrounding and collect it in the mound. The wood is then converted to soft spongy mass, called comb. The comb is normally invaded with fungal mycelia. It is well known that termites use cellulose as energy source after degradation to glucose with the help of microbial cellulases in the gut. But lignified celluloses can not be digested by cellulases. We hypothesize that the fungus probably helps to delignify cellulose fiber either directly through lignin degradation or through removal of the hemicellulose that cement the lignin to the cellulose fiber. To test this hypothesis we collected termite comb from Zuway and extracted proteins. The extract showed high xylanase activity (24U/g comb) and no detectable cellulase activity. This indicates that the role of the fungus is probably to remove lignin from the cellulose fiber. The fact that there was no detectable cellulase in the comb indicates that the fungus and the termite are not competing for cellulose. The fungus was isolated from the comb in pure culture. It was then grown m culture usmg submerged fermentation (SmF) and solid·state fermentation (SSF). However, enzyme production in SSF was much higher than in SmF. Maximum enzyme production in SSF using wheat bran was obtained at a substrate to moisture level ratio of 1:0.5 to 1:2. Addition of different sugars to the SSF substrate didn't affect enzyme production, indicating that enzyme production is probably constitutive. The xylanase was optimally active in the pH range of 4 to 6 and at temperature of 40°c. These properties make Xylaria xylanase potentially attractive as animal feed supplements. Key words: Xylaria, Xylanase, Cellulase, Termites