Biotechnology

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An Ecological Study of the Lowland Vegetation of Key Afer-Shala Luqa and Southwest of Lake Chamo, Southern Ethiopia
(Addis Ababa University, 1997-11) Soromessa, Teshome; Derilissew, Sebsebe (PhD); Woldu, Zerihun (PhD)
Studies in the vegetation of Key Afer-Shala Luqa ailG SouthWest of take Chaino Southern Ethiopia were conducted from Nov. 21, 1996 to February 27, 1997. The data on aboveground herbaceous phytomass was collected using 1m2 quadrat from 13 selected sites. To determine the different plant communities, data from 71 quadrats (400 m2) was collected following a 1-9 modified Braun Blanquet scale (van der Maarel, 1979). The environmental data collected consists of soil, altitude, slope and aspect. The average aboveground herbaceous phytomass ranged between 35.9 gm-2 in Shala plain to 1016.8 gm-2 in Southwest of Lake Chamo. Multivariate methods were employed to summarise the vegetation data. Seven community types and an outliers were identified. Statistical test (post-hoc comparison of means) among the community types showed significant variation in altitude indicating that altitude is the most important factor. Analysis of diversity among community types showed that the Hyparrhenia jilipendula- Combretl/Ill moUe dominated community is richer in species than others, while the Commiplwra cyclophylla-Actilliopteris radiata-Sallsevieria ehrenbergii dominated community is the least species rich community. The low aboveground herbaceous phytomass in sites like Shala might be attributed to ecological incompatibility with prevailing stocking rate. To ameliorate the interrelated causes of resource degradation, destocking should be taken into consideration.
Roduction of Specific Antisera Against Selected Mammals and Identification of Blood Meals of Phiembotamine Sandjlies Transmitting Visceral Leishmaniasis (VL) in Ethiopia
(Addis Ababa, Ethiopia, 1999) Mamo, Hassen; Hailu, Asrat
In an auempt to know the feeding preferences of phlebotomine sandflies that transmit visceral leishmaniasis (VL) in Eth iopia, Phlebotomus mani"i and P. orielllaiis. and thus to poilll to the possible reservoir host(s) of the disease in the country, blood meals of the vectors were identified using the counter current immunoelectrophoresis (CCIE) technique. First, genus/species-specific antisera were raised against the immunoglobulin G (lgG) of ten potential hosts of the sand fly vectors: human, sheep, goat, donkey , horse, camel, cattle, squirrel, mongoose and hyrax. The IgG was fractionated from the whole sera of the mammals by the ammonium sulphate precipitation method followed by the ion-exchange chromatography technique. The antisera were developed by prim ing (subcutaneously) rabbits, in triplicates, with water-in-oil emulsion of equal volumes of IgG and Freund's Complete Adjuvant and then boosting with Freund 's Incomplete Adjuvant. The potency of the ant isera was determined by assay ing each lest bleed from each rabbit againstlhe homologous IgG. Cross-reactiv ities were checked by screening each test bleed against heterologous IgGs. Human whole serum and IgG were cross-reacted with anti-cattle antisera and vice versa. All other antisera were specific. The titre of each test bleed was qualitatively determined by visualizing the conspicuousness of the precipitin line. Hyperimmune antisera were used in blood meal analysis. Eight blood meal samples (5 SergelJlomyia spp. and 3 P. manilll) were collected , by sticky and CDC light traps, from Aba-Roba in a total of 42 night catches. One hundred and six blood meal samples (94 P. orielJlalis and 12 P. bergerofl) collected, by CDC, from the Middle Awash va lley and stored for about 4 years were used. Each blood meal sample was tested against cortlmercial anti-dog and anti-rat IgGs vi in addition to the 10 ami-host sera raised in the present study. Of 114 blood meals processed, hosts were identified for the 93 (8 1.6%): 2 P. martilli, 79 P. oriellfalis and all P. bergeroli. None of Sergelllomyia blood meals were identified. 37.6% of the blood meals detected were from single host sources: 20 from cattle, 10 camel, 2 squirrel , I donkey, I human and I from mongoose. The rest 62.4% was from mixed sources. Altogether, 80.6% of the meals identified were from caule origin and 59% from camel, singly or in combination with OIher hosts. Thus. P. oriellfatis appears to be an opportunistic feeder with a preference for cattle and camel at least in the Middle Awash where these hosts occur in large numbers. The CCIE technique is specific and sensi tive enough not only to detect mixed blood meals from close ly related hosts but halfdigested meals. The assay is technically simple and inexpensive in terms of reagents required. Thus, CCIE is a robust technique for identification of blood meals of haemalophagous insect vectors in general and smaller nies such as phlebotomine sandfl ies and of biting midges in particular.
Biological and Ecological Studies on Acacia Drepanolobium Harms Ex Sjöstedt in Borana Zone of Oromiya Regional State, Ethiopia
(Addis Ababa University, 2003-06) Maryo, Melesse; Nemomissa, Sileshi (PhD); Kelbessa, Ensermu (PhD)
The biological and ecological studies of A. drepanolobium: the floristic composition in A. drepanolobium wooded grassland, soil properties, seed production, seed dispersal, soil seed bank, percent seed germination at different treatments, capacity of coppicing , and its interactions (symbiosis) with ants, insects and microbes were investigated in four A. drepanolobium wooded grassland sites in Negele Borana , Oromiya Regional State, S. Ethiopia. The results indicated that in A. drepanolobium wooded grassland 114 plant species were identified. Of these 70.2 %, 23.7% and 6.1% were herbs, trees/shrubs and climbers respectively. More over, 33.3 % were forage species whereas 14.4% and 2.6% had socioeconomic and medicinal importance respectively. Asteraceae, Fabaceae and Poaceae have constituted 36 % of the total number of species. The number of species was found to be smaller than previous studies on non- A. drepanolobium wooded grassland of the study area. This may suggest the impact of bush encroachment by A. drepanolobium, which had a mean density of 1798 plants/ hectare with a large number of individuals at the younger stage. The soils studied had higher proportion of clay (> 30 %) with properties that favor the growth of most plant species. An average of 2417 ± 23 (X ± SE) seed production per plant was encountered, and only 1 ± 0.4 (X ± SE) trees bore seeds in average per plot. Seeds are mainly dispersed by wind. 267 seeds (8.3 ± 2.6 seeds /m 2) were found only at the litter layer and none in mineral soil layer. There was statistically significant difference in percent germination among treatments [F (5, 17), P < 0.05]. Fast rate and higher percent germination was achieved by scarification treatments whereas dry heat treatment (90 oC) and moist heat (98 oC for greater than 30 minutes) resulted in almost all mold outgrowths after a week’s period. Tukey’s HSD indicated that moist heat treatments didn’t improve the percentage germination. High percent germination of a control experiment within week’s time may suggest the absence of pronounced seed dormancy in the study species. There is no statistically significant difference among stumping treatments both in number and in height of coppice but the coppice number and height increased down to a tree height (soil surface). Four A. drepanolobium occupant ant species (3 Crematogaster and 1 Tetraponera species) were identified. However, a black cotton soil habitat hosted only 2 Crematogaster species. Although each tree was occupied by a single ant species, Crematogaster mimosae occupied the largest proportion (85%) of seed bearing trees. The mutualistic association of Crematogaster nigriceps is doubtful because this species sterilizes flower buds and new shoots. Two seed feeding bruchid beetles (Callosobruchus maculatus and Acanthoscelides obtectus) were identified and found to reduce the reproductive vigor of the study species by mass predation of its seeds. A. drepanolobium was found to be nodulated by slow growing rhizobia called Bradyrhizobium species. There was no statistically significant difference in nodulation status between two soils (t at 24 df = -1.22 and P=0.268). The mean nodule number and weight were 4.93 ± 0.6 and 0.00381 ± 0.0008 (X ± SE) in clay soils respectively. Reduction in nodulation may be due to 1) richness of clay soil in mineral elements including nitrogen 2) absence of adequate aeration 3) missing of some nutrients elements such as molybdenum and 4) Slowness of the fixer species. From the socio-economic view point, 50% of informants declared the importance of A. drepanolobium, and the rest expressed their hatred for its bush encroachment impacts on their surrounding. However, from the present study , it can be suggested that sterilization of flower buds and young shoots by Crematogaster nigriceps ants, low soil seed bank, seed predation by bruchid beetles and low recruitment being the limiting factors, further expansion of bush encroachment by A. drepanolobium can be managed through integrated bush management systems such as reducing cultivation of dry season grazing areas, encouraging traditional rangeland management systems and applying proper land use policy, reducing excess livestock, stumping late in rainy seasons and periodic burning though complete recovery of the previous range condition is a difficult task. Key words: A. drepanolobium, bush encroachment, seed characteristics, Crematogaster, bruchid beetles.
Taxonomic and Demographic Studies on Three Species Complexes Within the Genus Alole L. (Aloaceae)in Ethiopia
(Addis Ababa University, 2006-06) Dessalegn Fikre; Demissew Sebsebe (Professor); Brysting Anne (Professor)
Taxonomic and demographic studies were conducted on three species complexes within the genus Aloe L. (Aloaceae) in Ethiopia. The studies aimed to contribute new additional knowledge necessary to delineate the Aloe species and their conservation in the Flora of Ethiopia and Eritrea (FEE).In the taxonomic study, the status of 11 species was evaluated using morphological and molecular (AFLP) data. Morphological data were collected from herbarium specimens, fresh materials and literature. Data matrices were constructed for qualitative, quantitative and combined morphological data sets. Molecular (AFLP) data were obtained from leaves of randomly selected germinated seedlings. Total genomic DNA was extracted using a modified 2 x CTAB method described by Doyle and Doyle (1990). AFLP analysis was performed following a protocol modified from Vos et al. (1995). Fragments produced by each primer combination were analyzed and scored for the presence (1) or absence (0) of selected markers/fragments. Binary data matrices were constructed for each of the primer combinations and also for the combined data set. Each of the data matrices constructed for morphological and molecular (AFLP) data sets was subjected to multivariate analyses: cluster analysis (UPGMA) and principal coordinate analysis (PCO).The results from the analyses of both morphological and AFLP data supported the present status of nine out of the eleven species: A. harlana Reynolds, A. monticola Reynolds, A. debrana Christian, A. percrassa Tod., A. yavellana Reynolds, A. megalacantha Baker, A. gilbertii Sebsebe & Brandham, A. calidophila Reynolds and A. sinana Reynolds. The boundary between the two caulescent species from the northern part of the FEE area: A. camperi Schweinf. and A. adigratana Reynolds was not veryclear and particularly the genetic delimitation displayed close relationship. The neighborjoining tree constructed from combined AFLP data revealed thus weak support for A. camperi (51%) and A. adigratana (62%) but kept them together with high bootstrap support (94%).
Assessment of Diversity, Morphological Variation and Description of Grasspea (Lathyrus Sativus) and Other Related Species
(Addis Ababa University, 2007-03) Tsegaye, Martha; Demissew, Sebsebe (Professor); Jorge, Alexandra (PhD)
Lathyrus sativus (grasspea) has been widely cultivated in South Asia and Ethiopia for over 2500 years and is used as a food and feed. It is rich in protein content, around 30 g/100 g edible seeds. Agronomically, the species is able to withstand both severe drought as well as water logging. Although seeds of grasspea are tasty and protein rich, excessive consumption of the seeds causes a motor neuron disease called neurolathyrism which is characterized by the paralysis of the lower limbs. The neurotoxic causal agent of this disease is believed to be a non protein aminoacid called Oxalyl Di aminopropionic Acid (ODAP). Morphological marker analysis and molecular analysis have been used widely to estimate genetic variability of populations. These methods have useful in addressing questions on population genetic structure and genetic conservation. Knowledge of genetic diversity of species is particularly important, since modern breeding practices have narrowed the genetic diversity of cultivated crops. In the case of grasspea, the problem of Lathyrism is leading to the banning of its production which in turn aggravates genetic erosion and loss of diversity of the crop. Fifty one grasspea accessions which were selected from the genebank collection of International Livestock Research Institute (ILRI) were evaluated and characterized for different qualitative and quantitative morphological characters. Cluster analysis was performed to estimate differences between accessions. Randomly Amplified Polymorphic DNA (RAPD) was also used to study the nature of variation. In addition to L. sativus, three other species of the genus (L.cicera, L. clymenum and L. ochrus) and seventeen unidentified populations of Lathyrus were also evaluated for morphological and biochemical characters and characterized accordingly. Cluster analysis of both the morphological and the RAPD data showed that all of the unidentified Lathyrus populations were found to be L. sativus. The result also showed that two of the accessions (5295 and 5296) represented by L. ochrus and one accession (5282) represented by L. cicera were found to be L. sativus. The results would suggest that germplasm evaluation is important for proper characterization of populations. Key words: Lathyrus sativus, grasspea, genetic diversity, morphological characters, RAPD, cluster analysis
Evaluation of Ground Water Resources Potential of the Teji River Cathment South West Shoa Zone, Oromia Region
(Addis Ababa University, 2008-01) Eshetu, Andualem; Kebede, Seifu(PhD)
The groundwater potential assessment of Teji river catchment is important to the currently increasing demand of water resources for different developmental activities and its quantification is vital for the efficient and sustainable groundwater resource management. The most important parameter contributing to recharge in the area are rainfall, evapotranspiration rate, and soil types. The area has an annual rainfall of 1104mm. Quantification of potential and actual evapotranspiration of the area is made using different techniques. The results obtained from are compared and representative one is determined to the area and qualitatively described. Water balance studies of the catchment indicate that 325.39mm of water is recharged annually and the months October, November, December, January, February, March, April, and May have soil moisture deficit and the remaining months are soil moisture surplus. The main aquifer identified in the catchment is alluvial sediment, weathered and fractured ignimbrite, and weathered and fractured basalt. Graphical presentation using Piper, Shoeller, Stiff and Wilcox diagram have been used for the effective analyses and interpretation of data. The groundwater in the catchment is characterized by Ca-Mg-Hco3, Ca-Na-Hco3, Ca-Hco3 and Na-Hco3-cl type water. TDS, EC and PH values range 184mg/l-566mg/l, 354μs/cm-838μs/cm and 6.6-7.68 respectively. The water sample analyses made in the catchment indicate the water is suitable for domestic and agricultural purpose. Almost all the water samples analyzed are with in the limits of WHO water quality standards except bore holes drilled at Urogotade PA’s (fluoride = 1.68mg/l) and Asgori town (fluoride = 2.01mg/l & iron=0.39mg/l).
Effects of Feed Quantity on Growth Performance and Water Quality in Cage Culture System for Production of Nile Tilapia (Oreochromis Niloticus, L., 1758) in Lake Babogaya, Ethiopia
(Addis Ababa University, 2008-07) Hailu, Solomon; Getahun, Abebe(PhD)
This study was conducted in Lake Babogaya, one of Bishoftu’s crater lakes in Bishoftu town, to determine the appropriate feeding level for tilapia, Oreochromis niloticus and the impacts of supplementary feed on water quality. A 16.9% crude protein diet, local made feed (Mill sweeping, oilseed cake and chicken dug cake) was fed to duplicate groups of O. niloticus fingerlings (29.70 ± 0.45 – 30.43 ± 0.70 gm) and (120.00 ± 0.56 -122.97 ± 0.54 mm) in cages (1 m x 1 m x 1 m). Fingerlings obtained from Lake Babogaya and stocked at a density of 100 fish/cage. They were feed at 1, 2, 3, 4, and 5% of body weight daily. Water quality parameters, pH, temperature and dissolved oxygen (DO) were measured every month during the culture periods. The results of the experiment revealed that the final weight (224.63 ± 1.23, 223.07 ± 1.23, and 223.40 ± 1.23) and specific growth rate (1.22 ± 0.08, 1.20 ± 0.07, and 1.21 ± 0.06) of the fishes fed at 3, 4 and 5% body weights were similar (p>0.05) but differed significantly (p <0.05) from the final weight (118.30 ± 0.43 and 152.87 ± 0.40) and SGR (0.81 ± 0.08 and 0.99 ± 0.07)of the group of fishes fed at 1 and 2% body weight respectively. Moreover, there were significant differences in FCR of fishes fed at the various body weights (1.76 – 6.33). Survival rate was not affected by feeding level. In addition, pH, DO and temperature of the culture water were not affected by the treatments. The study established the optimum feeding rate for O. niloticus at 3% body weight daily reared in cages. Keywords: Cage, Ethiopia, Feeding Levels, Lake Babogaya, Oreochromis niloticus
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
Phenotypic and Symbiotic Characterization of Grass Pea (Lathyrus Sativus) Rhizobial Isolates from Some Major Growing Areas Of South Wollo and West Shoa , Ethiopia
(Addis Ababa University, 2009-07) Adal Musa; Assefa Fassil (PhD)
Grass pea (Lathyrus sativus) is the most widely distributed and the third most important pulse crop in Ethiopia. It grows in a wide range of altitudes and survives in scarce moist conditions and fixes nitrogen with rhizobia and performs well in less fertility soils. However, the effectiveness in nitrogen fixation varies depending upon the host varieties, type of strain and different environmental characters. It is, therefore, necessary to evaluate the efficiency of different isolates from different sampling areas. Thus, 68 grass pea rhizobial isolates (47 from South Wollo and 21 from West Shoa) were isolated and characterized on the basis of different characters. These isolates were reinoculated on pouch culture using “Wasse” variety of grass pea to authenticate nodulation and determine their symbiotic effectiveness. All but 5 isolates were authenticated as root nodule bacteria with their capacity to infect and nodulate their parent host. Most of the isolates displayed fast doubling time (1.2-4hrs) and large colonies (2-6.0 mm) and changed the BTB-YEMA media to yellow which showed that they are fast growing rhizobia and then categorized into R. leguminosarum bv. viceae. The preliminary screening of the isolates for symbiotic effectiveness on pouch culture showed that isolates AAUGR (31, 37, 41 and 47) of South Wollo and AAUGR (48, 49, 52, 59, 60, 61, 66 and 67) of West Shoa that comprised of 19% of the isolates were highly effective whereas, 43% were found to be effective. Three highly effective isolates from South Wollo (AAUGR 31, AAUGR 37, and AAUGR 41) and four highly effective isolates from West Shoa (AAUGR 48, AAUGR 59, AAUGR 61, and AAUGR 67) were reinoculated into soil culture to evaluate their performance in the natural environment (soil). All the selected isolates were found to accumulate 112-149 % of SDM that was higher than the controls. Isolate AAUGR 37 (SW) accumulated the highest SDM of all the inoculated treatments and the controls. All the selected isolates were found to perform better on pot culture than on pouch culture. Almost all of the isolates were able to grow at extremely low and high pH, low and high salt, low and high temperature, and utilized a wide range of carbohydrate and nitrogen sources and displayed high resistance to several antibiotics indicating that the isolates can compete in their natural environment. The result of the numerical analysis also showed the existence of diversity among the tested isolates.
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).
Studies on Anopheles Mosquitoes Host Preference and Malaria Transmission Intensity Using Immunological Diagnostic Methods in Gilgel-Gibe Dam Area, Southwestern Ethiopia
(Addis Ababa University, 2010-06) Kelel, Musin; Getu, Emana (PhD); Yewhalaw, Delenasaw (PhD)
The Host feeding pattern and sporozoite rate of Anopheline mosquitoes were determined for mosquitoes collected from September to November 2010 in Gilgel-Gibe hydroelectric dam area, southwestern Ethiopia. Mosquitoes were collected from 16 villages surrounding the dam by hand-capture and light trap catches. Mosquitoes resting outdoors were also collected from potential natural resting sites and artificial pit shelters. Overall, 520 anopheline mosquitoes were collected. Anopheles arabiensis was the most predominant (88.65%) species followed by An. coustani s.l (7.12%) and An. demelloni (4.23%). All the 520 mosquito specimens were tested for circumsporozoite protein (CSP) and host feeding pattern using ELISAs. Of those tested for CSP, 10 specimens were found positive for Plasmodium vivax infection. None were found positive for P. falciparum. The CSP rate was 1.92 and entomological inoculation rate (EIR) was 9 infectios bites/person/month. Moreover, a single specimen of An. coustani s.l was screened positive for Plasmodium CSP. Results of blood meal ELISA showed that An. arabiensis have a wider host range and more opportunistic feeder than the other two anopheline species. The human blood index (HBI) of An. arabiensis was 4.12%, but no human blood was found in the other two species. The host preference index and forage ratio were higher for Equine for all tested species, indicating that equine was the most preferred host by the three mosquito species. An. arabiensis fed more on equine, bovine and ovine than human though the number of humans was significantly higher in the study area. Thus, zoo prophylaxis can be employed as possible vector control strategy. Multiple blood feeding was observed in all the 3 mosquito species and this multiple blood feeding behavior especially, in An. arabiensis population might have resulted in elevated EIR in contrast to the low HBI. Keywords: Anopheles, Blood meal, ELISA, An. arabiensis , sporozoite
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.
Molecular Characterization of the Bovine CSN3 and LGB Milk Protein Genes Using Sequencing and PCR-RFLP Markers in Ethiopian Indigenous Cattle Populations
(Addis Ababa University, 2010-07) Getachew, Robel; Zegeye, Abiy (PhD); Dessie, Tadelle (PhD)
In the last few decades, genetic polymorphism of bovine milk protein genes such as caseins and whey proteins has achieved considerable research interest mainly due to their significant associations with fat and protein contents as well as the manufacturing properties of milk. In this study, genetic polymorphisms of two bovine milk protein genes namely CSN3 and LGB were analyzed in five indigenous and one crossbred cattle breeds using PCR-RFLP markers and sequencing. Moreover, the magnitude of genetic variability within and among these cattle populations at these two loci was estimated hence their population structure and phylogenetic relationships characterized. For this purpose, genomic DNA was extracted from 83 animals belonging to Abigar, Boran, Guraghe, Horro, Sheko and Holestin-Barka crossbred cattle populations. Both of the markers used to analyze polymorphisms in the CSN3 and LGB genes were informative and enabled us to detect the widely reported CSN3-A and B and LGB-A and B variants. Furthermore, sequencing revealed additional genotypes among which were the zebuspecific CSN3-AI and H haplotypes observed in 50% of the sequences from the indigenous cattle and CSN3-E in one sequence derived from a Holstein-Barka crossbred cow. These CSN3 haplotypes were deduced analyzing a total of 7 point mutations found in the 633bp region sequenced of which 5 were base transitions and 2 transversions. Two of these sites were silent substitutions while the rest were non-synonymous. At LGB locus, 12 SNPs were detected in the amplified 529 bp region where only 3 resided in the coding region corresponding to 2 silent and one non-synonymous substitution. Analysis of these mutations yielded two new haplotypes (LGBB 1 and B*) that where results of silent mutations. The PCR-RFLP assay revealed that CSN3-A and LGB-B were more prevalent than CSN3-B and LGB-A variants in the indigenous cattle population investigated. The frequency of allele CSN3-A (0.687) was more than twice that of B (0.313) where as LGB-B had a very high frequency of 0.861 compared to LGB-A (0.139). All of the breeds met HWE at both loci with the exception of Sheko that deviated significantly (P<0.05) at the LGB locus. The total population revealed a rather moderate level of genetic variability (0.34). The highest variability was observed in Sheko (0.414) while the lowest was in Guraghe (0.273). When the two loci were considered separately, CSN3 locus presented higher (43.04%) level of gene diversity compared with LGB locus (24.42%) which was rather more homogenous. A higher level of inbreeding was detected at LGB locus (FIS=0.173) compared with CSN3 which instead showed excess heterozygosity with FIS of -0.0282. When considering both loci, the mean FIS and FIT estimates were 0.044 and 0.07 respectively. The overall FST value (0.028) revealed little and insignificant (P>0.05) genetic differentiation among the populations studied. Most of the total genetic variation was attributed to the within population (97.23%) whereas little and insignificant level of variation was witnessed among the different cattle breeds (2.77%). Pairwise genetic distances revealed very small distances among the breeds studied. However, Sheko had larger distances when paired with each breed where the maximum of which was with Abigar and the minimum that involving Guraghe indicating its relative departure from the rest. Similarly, the dendrogram generated clearly depicted Sheko forming a separate branch where the rest shared a second main branch. Key words: PCR-RFLP, Sequencing, diversity, Ethiopian cattle, SNPs, CSN3, LGB, HWE
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
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.
Genetic Diversity Study of Five Ethiopian Indigenous Bovine Breeds Using Microsatellite Markers
(Addis Ababa University, 2011-05) Diriba, Solomon; Tesfaye, Kassahun (PhD); Dessie, Tadelle (PhD)
The current study describes the genetic variability within and among five Ethiopian indigenous bovine breeds viz; Abigar, Boran, Horro, Guraghe and Sheko based on nine microsatellite markers (TGLA227, ETH3, ETH10, ETH152, ETH225, BM2113, BM1824, ILSTS049 and TGLA53). The Abigar breed represents the Sanga (cross between Bos taurus and Zebu breed) cattle breed, the Boran and Guraghe highland are grouped under Zebu breed, the Horro breed fall under Zenga (cross between Sanga and Zebu breed) cattle and the Sheko cattle belongs to humpless shorthorn (Bos taurus). Blood samples were taken randomly from10 unrelated animals from each cattle breed and DNA was isolated for PCR amplification. The PCR products were resolved on eight or 10% nondenaturing polyacrylamide gels, based on the size of the products, and visualized by GelRed staining. Allelic frequencies, hetrozygosity values, polymorphic information content (PIC), genetic differentiation and genetic distance were estimated. The allelic frequencies were also tested for deviation from Hardy-Weinberg equilibrium (HWE). The result of the current study indicated that the total numbers of alleles observed overall loci in Abigar, Boran, Guraghe, Horro and Sheko were 67, 65, 54, 58 and 59, respectively. The breeds showed deviation from HWE at least at one of the loci examined, except the Boran breed which was found to be at HWE for all loci studied. The overall observed and expected hetrozygosity values and PIC were 0.782±0.079, 0.847±0.050 and 0.828±0.057, respectively. The mean observed hetrozygosity value in Abigra, Boran, Guraghe, Horro and Sheko were 0.800±0.071, 0.803±0.157, 0.761±0.109, 0.781±0.133 and 0.763±0.084, respectivey, The Nei’s expected hetrozygosity values in these breeds were found to be 0.820±0.036, 0.798±0.065, 0.788±0.070, 0.780±0.063 and 0.783±0.049, respectively. Generally, the genetic differentiations between the breeds were low. Moreover, non-significant (P>0.05) differentiation were noticed in the study (between Boran and Guraghe, between Guraghe and Horro and between Boran and Horro).The partitioning of genetic variability based on overall FST values indicated 2.2 % of variation was due to between the breeds studied whereas the remaining (97.8%) was due to variation among individuals. The inbreeding coefficients within and among were small, 0.070±0.031and 0.090±0.27, respectively. Genetic distance based on Nei’s corrected distance was found to be least (0.027) between Boran and Guraghe and highest (0.229) between Boran and Sheko. Generally, a dendrogram following UPGMA clustering generated based on Nei’s genetic distance matrix agreed with the evolutionary history of the breeds studied.
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
Factors in Fluencing Micropropagation and Somatic Embryogenesis of two, Kello and Qulle, Cassava Varieties
(Addis Ababa University, 2011-06) Berhanu, Roza; Feyissa, Tileye (PhD)
Cassava (Manihot esculenta Crantz) is a perennial shrub of the Euphorbiaceae family grown in the tropics, African and Asian continents for its starchy tuberous roots. Its cultivation is constrained by the lack of high quality seed, low productivity and profit, virus and insect pests, high heterozygosity, low natural fertility, poor seed set and seed germination. These problems are difficult to be dealt with traditional breeding system. Therefore, other biotechnological methods are required to solve the above mentioned problems. The objective of this study was to overcome the above mentioned tribulations and come up with the best way to get a maximum number of shoots and morphologically fit in vitro materials through micropropagation and along with that to study the different factors that are associated with the micropropagation of the two cassava varieties; ‘Kello’ and ‘Qulle’. The study also includes a method of somatic embryogenesis that is an ideal source of in vitro materials for large-scale propagation. In this experiment a Solid MS medium with different salt strengths, sucrose concentrations, TDZ, a two-step procedure involving pre-soak of nodal explants into a liquid MS medium prior to culture on a solid MS medium, with different pH values, repeated subcultures and somatic embryo induction on MS medium supplemented with 2,4-D alone and in combination with 2 μM CuSO4 were used. Maximum mean number of shoots per explant as well as a better morphological property of in vitro material was obtained at 0.2 mg/L TDZ for both ‘Kello’ and ‘Qulle’. In both varieties, the two-step culture system resulted in a maximum mean number of shoots/explant at a TDZ concentration of 0.2 mg/L. A medium salt strength of quarter produced maximum mean number of shoots per explants in case of ‘Kello’ while in ‘Qulle’ at a full strength MS medium. The highest mean number of shoots per explant for ‘Kello’ and ‘Qulle’ was obtained at pH 5.6 and 6.6, respectively. For all parameters maximum mean numbers for both ‘Kello’ and ‘Qulle’ were obtained at 0.15 % sucrose. During somatic embryo induction the treatments with different 2,4-D concentrations induced FEC and somatic embryos but the somatic embryos failed to develop beyond the globular stage. The micropropagation property of the two varieties through successive subculturing indicated that repeated subculturing results in loss of multiplication property of the in vitro materials. Key words/phrases: Manihot esculenta; Salt strength; Shoot multiplication; Sucrose; FEC; 2,4-D;TDZ
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
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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