Exploring stress-tolerant plant growth-promoting rhizobacteria from groundnut rhizosphere soil in semi-arid regions of Ethiopia.

The potential of rhizobacteria with plant growth promoting (PGP) traits in alleviating abiotic stresses, especially drought, is significant. However, their exploitation in the semi-arid regions of Ethiopian soils remains largely unexplored. This research aimed to isolate and evaluate the PGP potential of bacterial isolates collected from groundnut cultivation areas in Ethiopia. Multiple traits were assessed, including phosphate solubilization, indole-3-acetic acid (IAA) production, ammonia production, salt and heavy metal tolerance, drought tolerance, enzyme activities, hydrogen cyanide production, antibiotic resistance, and antagonistic activity against fungal pathogens. The identification of potent isolates was carried out using MALDI-TOF MS. Out of the 82 isolates, 63 were gram-negative and 19 were gram-positive. Among them, 19 isolates exhibited phosphate solubilization, with AAURB 34 demonstrating the highest efficiency, followed by AURB 12. Fifty-six isolates produce IAA in varying amounts and all isolates produce ammonia with AAURB12, AAURB19, and AAURB34 displaying strong production. Most isolates demonstrated tolerance to temperatures up to 40°C and salt concentrations up to 3%. Notably, AAURB12 and AAURB34 exhibited remarkable drought tolerance at an osmotic potential of -2.70 Mpa. When subjected to levels above 40%, the tested isolates moderately produced lytic enzymes and hydrogen cyanide. The isolates displayed resistance to antibiotics, except gentamicin, and all isolates demonstrated resistance to zinc, with 81-91% showing resistance to other heavy metals. AAURB34 and AAURB12 exhibited suppression against fungal pathogens, with percent inhibition of 38% and 46%, respectively. Using MALDI-TOF MS, the promising PGP isolates were identified as Bacillus megaterium, Bacillus pumilus, and Enterobacter asburiae. This study provides valuable insights into the potential of rhizobacteria as PGP agents for mitigating abiotic stresses and contribute to the understanding of sustainable agricultural practices in Ethiopia and similar regions facing comparable challenges.

Plant growth-promoting rhizobacterial inoculation to improve growth, yield, and grain nutrient uptake of teff varieties.

Inoculation of plant growth-promoting rhizobacteria (PGPR) improves the growth, yield, and plant nutrient uptake, as well as rhizosphere fertility, without harming the environment and human health. This study aimed to examine the effect of either individual or consortium of PGP bacterial inoculation on the growth, yield, and grain nutrient uptake of teff varieties. Three potential PGPR strains (i.e., Pseudomonas fluorescens biotype G, Enterobacter cloacae ss disolvens, and Serratia marcescens ss marcescens) were used for this study. Field evaluation was carried out in RCBD with 5 treatments. Highly significant (P < 0.001) differences were observed among treatments for plant height (PH), panicle length (PL), number of the total spike (NTS), shoot dry weight (SDW), grain yield (GY), and straw yield (SY). There was also teff variety that significantly (P < 0.01) affects PL, SDW, and SY. However, the interaction effect of the two factors (treatment*variety) did not significantly influence teff agronomic traits and grain nutrient uptake. The highest PH (133.5 cm), PL (53.2), NTS (30.9), SDW (18.1 t/ha), SY (10.7 t/ha), and GY (2.7 t/ha) were observed on Dukem variety (Dz-01-974) inoculated with PGPR consortium. Wherein 2.2 fold increase was observed in grain yield per hectare over the control. Inoculation of PGPR consortium showed better performance in promoting plant growth, yield, and grain nutrient uptake of teff varieties compared with the individual PGP bacterial application, and PGPR consortium could be used as inoculants to enhance teff production and productivity.

Evaluation of inorganic phosphate solubilizing efficiency and multiple plant growth promoting properties of endophytic bacteria isolated from root nodules Erythrina brucei.

BACKGROUND: In soils, phosphorous (P) mostly exists in fixed/insoluble form and unavailable for plants use in soil solution, hence it is in scarcity. P is fixed in the form of aluminium, iron and manganese phosphates in acidic soils and calcium phosphate in alkaline soils. Phosphate solubilizing bacteria, the ecological engineers play a pivotal role in the mobilization of fixed forms of P by using different mechanisms. The objectives of this study were to evaluate inorganic phosphate solubilizing efficiency and other multiple plant growth promoting traits of Erythrina brucei root nodule endophytic bacteria and to investigate effects of the selected endophytic bacteria on the growth of wheat plant under phosphorous deficient sand culture at greenhouse conditions. RESULTS: Among a total of 304 passenger endophytic bacteria, 119 (39%) exhibited tricalcium phosphate (TCP) solubilization; however, none of them were formed clear halos on solid medium supplemented with aluminum phosphate (Al-P) or iron phosphate (Fe-P). Among 119 isolates, 40% exhibited IAA production. The selected nine potential isolates also exhibited potentials of IAA, HCN, NH3 and/or hydrolytic enzymes production. All the selected isolates were potential solubilizers of the three inorganic phosphates (Al-P, Fe-P and TCP) included in liquid medium. The highest values of solubilized TCP were recorded by isolates AU4 and RG6 (A. soli), 108.96 mg L-1 and 107.48 mg L-1, respectively at sampling day3 and 120.36 mg L-1 and 112.82 mg L-1, respectively at day 6. The highest values of solubilized Al-P and Fe-P were recorded by isolate RG6, 102.14 mg L-1 and 96.07 mg L-1, respectively at sampling days 3 and 6, respectively. The highest IAA, 313.61 µg mL-1 was recorded by isolate DM17 (Bacillus thuringiensis). Inoculation of wheat with AU4, RG6 and RG5 (Acinetobacter soli) increased shoot length by 11, 17.4 and 14.6%, respectively compared to the negative control. Similarly, 76.9, 69.2 and 53.8% increment in shoot dry weight is recorded by inoculation with RG6, AU4 and RG5, respectively. These nine potential endophytic isolates are identified to Gluconobacter cerinus (4), Acinetobacter soli (3), Achromobacter xylosoxidans (1) and Bacillus thuringiensis (1). CONCLUSION: AU4, RG6 and RG5 can be potential bio-inoculants candidates as low cost agricultural inputs in acidic and/or alkaline soils for sustainable crop production.

Extracellular enzyme activity of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae and their pathogenicity potential as a bio-control agent against whitefly pests, Bemisia tabaci and Trialeurodes vaporariorum (Hemiptera: Aleyrodidae).

OBJECTIVE: This study was aimed to assess the enzymatic activity and pathogenicity potential of Beauveria bassiana and Metarhizium anisopliae against whiteflies in Ethiopia. RESULTS: The data showed that Beauveria bassiana AAUMB-29, AAUMFB-77, and AAUEB-59 generated the highest chitinase (EI = 3.41), lipase (EI = 4.45), and protease activities (EI = 5.44) respectively. The pathogenicity study of isolates on whitefly nymphs and adults indicated significant variation (P < 0.05) with mortality ranging from 71.67 to 98.33% and 60 to 100% against Bemisia tabaci and Trialeurodes vaporariorum nymphs respectively. The mortality of adults was between 58 and 94.27% against B. tabaci and 59.03 to 95.37% against T.vaporariorum. The result also showed that AAUMB-29, AAUMFB-77, and AAUDM-43 were the most virulent with LC50 values of 2.7 × 104, 5.3 × 104, and 5.4 × 104 conidia/ml against nymphs of B. tabaci, and with LC50 values 6.8 × 104, 8.2 × 104, and 7.2 × 104 conidia/ml against nymphs of T. vaporariorum, respectively. The B. bassiana AAUMB-29, B. bassiana AAUMFB-77, and M. anisopliae AAUDM-43 induced the highest whitefly mortality than other isolates. These isolates can be recommended for further tests under field conditions to fully realize their potential as effective biocontrol agents against whitefly pests in tomato.

Characterization of faba bean (Vicia faba L.) rhizosphere associating rhizobacteria against Botrytis fabae AAUBF-12 and their plant growth-promoting properties.

The rhizobacteria are known to protect plants from different pathogens acting as biocontrol agents and promote growth of plants. This study was conducted to isolate, screen and identify faba bean associating rhizobacteria for their antagonistic properties against Botrytis fabae AAUBF-12 and plant growth-promoting properties under in vitro conditions. In the dual culture assay, the isolates inhibited the mycelia growth of B. fabae AAUBF-12 (6-40 %) upon 3 days of incubation, and the inhibition increased to 9-43 %, 16-50 %, and 24-68 % after five, seven and 9 days of incubation, respectively. The inhibitory activity increased from 6 to 82 % using the culture filtrates of the isolates. Isolate AAUB95 displayed the highest mycelial inhibition (27 %) at 5 % concentration of culture filtrate, followed by AAUB146b that exhibited 21 % inhibition at the same concentration. AAUB146b and AAUB100 effectively inhibited B. fabae AAUBF-12 by 79 % and 80 % at 20 % concentrations of the culture filtrate. The qualitative study demonstrated 75 % of the isolates positive for protease and 60 % for lipase synthesis. Furthermore, the isolates that showed antagonistic activity against B. fabae AAUBF-12, produced IAA and ammonia with 65 % and 60 %, respectively. Moreover, 310-760 µg mL-1 and 200-620 µg mL-1 of tricalcium phosphate (TCP) was released on the 3rd and 6th days of incubation, respectively, due to rhizobacterial solubilization. Nevertheless, the Pearson's correlation analysis between pH and TCP solubilization revealed an inverse relationship (r = -.422∗∗). Based on 16S rRNA sequences analysis, isolate AAUB95, AAUB146b, AAUB100 and AAUB92 were identified as B. subtilis AAUB95, S. nematodiphila AAUB146b, B. tequilensis AAUB100 and B. subtilis AAUB92, respectively. Of the isolates, B. subtilis AAUB95 showed best antagonism of B. fabae AAUBF-12 with multiple plant growth-promoting properties.

Genomic diversity and distribution of Mesorhizobium nodulating chickpea (Cicer arietinum L.) from low pH soils of Ethiopia.

Chickpea is the third most important grain legume worldwide. This is due in part to its high protein content that results from its ability to acquire bioavailable nitrogen when colonized by diverse, nitrogen fixing Mesorhizobium species. However, the diversity and distribution of mesorhizobia communities may depend on their adaptation to soil conditions. Therefore, this study was initiated in order to isolate and investigate the diversity and taxonomic identities of chickpea-nodulating Mesorhizobium species from low pH soils of Ethiopia. A total of 81 rhizobia strains were isolated from chickpea nodules harvested from low pH soils throughout Ethiopia, and their genomes were sequenced and assembled. Considering a representative set of the best-sequenced 81 genomes, the average sequence depth was 30X, with estimated average genome sizes of approximately 7 Mbp. Annotation of the assembled genome predicted an average of 7,453 protein-coding genes. Concatenation of 400 universal PhyloPhlAn conserved genes present in the genomes of all 81 strains allowed detailed phylogenetic analysis, from which eight well-supported species were identified, including M.opportunistum, M.australicum, Mesorhizobium sp. LSJC280BOO, M.wenxiniae, M.amorphae, M.loti and M.plurifarium, as well as a novel species. Phylogenetic reconstructions based on the symbiosis-related (nodC and nifH) genes were different from the core genes and consistent with horizontal transfer of the symbiotic island. The two major genomic groups, M.plurifarium and M.loti, were widely distributed in almost all the sites. The geographic pattern of genomic diversity indicated there was no relationship between geographic and genetic distance (r = 0.01, p > 0.01). In conclusion, low pH soils in Ethiopia harbored a diverse group of Mesorhizobium species, several of which were not previously known to nodulate chickpea.

Molecular phylogeny and pathogenicity of indigenous Beauveria bassiana against the tomato leafminer, Tuta absoluta Meyrick 1917 (Lepidoptera: Gelechiidae), in Ethiopia.

BACKGROUND: Tuta absoluta Meyrick 1917 (Lepidoptera: Gelechiidae) is an invasive, pesticide resistant, and a major treat of tomato production in the world. It needs effective management options that naturally infect the insect without causing any identified side effects. Entomopathogenic fungi (EPF) are the most important options. However, geographic origin and climatic condition apparently creates genetic variation among EPF strains that influence on their pathogenicity. Thus, screening of effective EPF strains from the local source is vital to develop environmental friendly pest control tactic for T. absoluta. RESULTS: In this study, 27 indigenous Beauveria were isolated from the various types of soil and 12 of the isolates were screened based on their biological efficiency index (BEI). These isolates scored 65.7-95.7% and 68.3-95% of mortality against second and third instar larvae of T. absoluta at concentration of 1 × 107spores·ml-1 in 7 days post inoculation, respectively. Out of these, five (18.5%) isolates scored above 90% mortality on both instar larvae with LT50 value of 3.33 to 5.33 days at the lowest (104 spores·ml-1) and 1.93 to 3.17 days at highest (108 spores·ml-1) spore concentrations and has LC50 value of 1.5 × 103 to 1.1× 105 spores·ml-1. Moreover, isolates exhibited the promising mortality better (1.5 × 106 to 3.5 × 107 spores·ml-1), sporulated over the larval cadavers, well grown at optimal temperature, and produced chitinolytic enzymes. Molecular analysis showed that isolates have nearly monophyletic characters and grouped under species of Beauveria bassiana. CONCLUSION: Different types of soil in Ethiopia are an important source of B. bassiana, and these isolates showed promising pathogenicity against T. absoluta, which is crucial for ecofriendly biopesticide development. Although isolates were nearly monophyletic in phylogenetic study, five of them were highly effective in the laboratory bioassays against T. absoluta; however, further field evaluation is required for mass production.

Isolation, molecular characterization and pathogenicity of native Bacillus thuringiensis, from Ethiopia, against the tomato leafminer, Tuta absoluta: Detection of a new high lethal phylogenetic group.

Tuta absoluta (tomato leafminer) is one of the devastating agricultural pest that attack mainly tomatoes. The continuous use of chemical pesticides is not affordable and poses a collateral damage to human and environmental health. This requires integrated pest management to reduce chemical pesticides. B. thuringiensis is a cosmopolitan, antagonistic soil bacterium used to control agricultural pests. In this study, effective Bt strains were screened from different sample sources based on their lepidopteran specific cry genes and larvicidal efficacy against tomato leafminer, T. absoluta under laboratory conditions. Of the 182 bacterial isolates, 55 (30 %) of isolates harbored parasporal protein crystals. Out of these, 34 (62 %) isolates possess one or more lepidopteran specific cry genes: 20 % of isolates positive for cry2, 18.2 % for cry9, 3.6 % for cry1, 16.4 % for cry2 + cry9, 1.8 % for cry1 + cry9, and 1.8 % for cry1 + cry2 + cry9. However, 21 (38.2 %) isolates did not show any lepidopteran specific cry genes. Isolates positive for cry genes showed 36.7-75 % and 46.7-98.3 % mortality against second and third instar larvae of the T. absoluta at the concentration of 108 colony forming units (CFUs) ml-1. Cry1 and cry1 plus other cry gene positive isolates were relatively more pathogenic against T. absoluta. However, third instar larvae of the T. absoluta was more susceptible than second instar larvae. Two of the isolates, AAUF6 and AAUMF9 were effective and scored LT50 values of 2.3 and 2.7 days and LC50 values of 3.4 × 103 and 4.15 × 103 CFUs ml-1 against the third instar larvae, respectively. The phylogenetic studies showed some congruence of groups with cry gene profiles and lethality level of isolates and very interestingly, we have detected a putative new phylogenetic group of Bt from Ethiopia.

Phenotypic, molecular, and virulence characterization of entomopathogenic fungi, Beauveria bassiana (Balsam) Vuillemin, and Metarhizium anisopliae (Metschn.) Sorokin from soil samples of Ethiopia for the development of mycoinsecticide.

Fungal entomopathogens are the most effective biocontrol agents against insect pests in the natural ecosystem. This study was conducted for phenotypic, molecular, and virulence characterization of locally isolated entomopathogenic fungi from soil samples of six localities in Ethiopia. Entomopathogenic fungi were isolated from 120 soil samples with the galleria baiting method. A total of 65 (54.2%) entomopathogenic fungal isolates belongs to Beauveria spp and Metarhizium spp were identified based on cultural and morphological features. All isolates were pre-screened based on germination, vegetative growth rate, and spore production as fungal in-vitro virulence determinates. Isolate AAUKB-11 displayed the peak germination of 99.67% and isolate AAUMFB-77 achieved the highest radial growth rate of 3.43 mm day-1 with the highest sporulation 4.60 × 108spores/ml. The phylogenetic analysis of ITS-rDNA confirmed that 7 isolates were identified as B. bassiana and 5 isolates were categorized into M. anisopliae. Selected B. bassiana and M. anisopliae strains were evaluated for their pathogenicity efficiency against G. mellonella larvae and caused 86.67%-100% mortality. The mortality rates of G. mellonella larvae peaked at 100% with 4(33.33%) isolates from B. bassiana and 2(16.67%) isolates from M. anisopliae after 10 days of treatments. The high virulent isolate, B. bassiana AAUMB-29 displayed the least LT50 value of 2.36 days followed by isolate B. bassiana AAUMFB-77 with LT50 of 2.53 days. Future studies should be needed to focus on the evaluation of high virulent isolates against other potential insect pests to assess their vigorous role as favorable biological control agents.

Phosphate solubilization and multiple plant growth promoting properties of Mesorhizobium species nodulating chickpea from acidic soils of Ethiopia.

The main purpose of this study was to screen and select strains from seven Mesorhizobium spp. for efficient phosphate solubilizing and other plant growth-promoting traits. Mesorhizobium species were tested for their ability to dissolve inorganic phosphate sources and multiple plant growth-promoting attributes. From a total of 62 Mesorhizobium strains, 47(76%) strains formed clear zones with an average PSI of 1.9-2.7 on Pikovskaya's agar plate. The selected strains also released soluble phosphorus [125-150 P (µgml-1)] from tri-calcium phosphate and low level of phosphorous i.e., 15.4 µg/ml and 14.5 µg/ml from inorganic ferrous and aluminum phosphates, respectively, in a liquid medium after 4 days of incubation. The release of soluble P was significantly (P < 0.01) correlated with a drop in pH of the medium. Moreover, screening for multiple plant growth-promoting attributes showed that 40, 28, 26, 21, and 38% of the strains were capable of producing indole-3-acetic acid, hydrogen cyanide, siderophores, ACC deaminase, and antagonism against Fusarium oxysporum f.sp. ciceris under in vitro conditions. The Mesorhizobium strains were endowed with the presence of ACC deaminase which was rarely reported elsewhere. All taken together, the acidic soils harbor numerous and more diverse phosphate solubilizing and plant growth-promoting Mesorhizobium spp. However, greenhouse and field conditions can be further studied within the context of improving chickpea production in Ethiopia.

Optimization of ethanol production using newly isolated ethanologenic yeasts.

Yeasts are important microorganisms used for ethanol production; however, they are not equally efficient in the amount of ethanol production under different environmental conditions. It is, therefore, necessary to screen for elite strains to utilize them for commercial production of these commodities. In this study, yeasts were isolated from different Ethiopian traditional fermented alcoholic beverages (teji, tella, shamiata and areqe tinisis), milk and ergo, teff and maize dough, soil and compost, flowers, and fruits to evaluate their potential use for ethanol fermentation process. Isolates were screened for efficient ethanol production and the selected ones were identified using phenotypic and genetic characters using D1/D2 region of LSU rDNA sequence analysis. The yeast isolates were evaluated based on their growth and fermentation of different carbon sources. Response surface methodology (RSM) was applied to optimize temperature, pH and incubation time using central composite design (CCD) in Design-Expert 7.0.0. A total of 211 yeasts colonies were isolated of which 60% were ethanologenic yeasts (ethanol producers) and 40% were non-ethanol producers. The yeast population detected from various sources was in the range of 10 5 CFU from traditional foods and beverages to that of 10 3 CFU from fruits and soil samples. The data also showed that the number of colony types (diversity) did not correlate with population density. The highly fermentative isolates were taxonomically characterized into four genera, of which 65% of the isolates (ETP37, ETP50; ETP53, ETP89, ETP94) were categorized under Saccharomyces cerevisiae, and the remaining were Pichia fermentans ETP22, Kluyveromyces marxianus ETP87, and Candida humilis ETP122. The S. cerevisiae isolates produced ethanol (7.6-9.0 g/L) similar with K. marxianus ETP87 producing 7.97 g/L; comparable to the ethanol produced from commercial baker's yeast (8.43 g/L) from 20 g/L dextrose; whereas C. humilis ETP122 and P. fermentans ETP22 produced 5.37 g/L and 6.43 g/L ethanol, respectively. S. cerevisiae ETP53, K. marxianus ETP87, P. fermentans ETP22 and C. humilis ETP122 tolerated 10% extraneous ethanol but the percentage of ethanol tolerance considerably decreased upon 15%. S. cerevisiae ETP53 produced ethanol optimally at pH 5.0, 60 h, and 34 o C. pH 4.8, temperature 36 o C, and 65 h of time were optimal growth conditions of ethanol fermentation by K. marxianus ETP87. The ethanol fermentation conditions of P. fermentans ETP22 was similar to S. cerevisiae ETP53 though the ethanol titer of S. cerevisiae ETP53 was higher than P. fermentans ETP22. Therefore, S. cerevisiae ETP53, K. marxianus and P. fermentans ETP22 are good candidates for ethanol production.

Genetic and metabolic diversities of rhizobacteria isolated from degraded soil of Ethiopia.

Genetic and metabolic diversities of rhizobacteria are the fundamental sources for their adaptation to cope with abiotic and biotic stresses in order to enhance growth and health of plants in the soil. Thus, this study was initiated to assess the genetic and metabolic diversities of rhizobacteria isolated from plants grown in degraded soil through BOX-PCR and partial sequencing of 16S rRNA genes. A total of eighty isolates were recovered and subjected to phenotypic profiling of carbohydrate and amino acid utilization, BOX PCR and 16S rRNA profiling. The phenotypic profiling showed remarkable metabolic versatility with Ochrobactrum spp, Pseudomonas spp and Klebsiella spp, and BOX-PCR showed greater discriminatory power for fingerprinting of rhizobacterial isolates with high degree of polymorphism. Bacillus spp showed the highest Simpson's diversity Index. The 16S rRNA genes sequence assigned the rhizobacteria to phyla Proteobacteria with Gammaproteobacteria and Alphaproteobacteria classes and Firmicutes with Bacilli class. The data also showed that the most dominant species were Pseudomonas and Ochrobactrum. Genetic and metabolic diversities of the rhizobacterial isolates reveal the potential of these microbes for plant growth improvement under water deficient soil after testing other inoculant traits.

Plant Growth-Promoting Rhizobacteria Isolated from Degraded Habitat Enhance Drought Tolerance of Acacia (Acacia abyssinica Hochst. ex Benth.) Seedlings.

Drought stress (DS) is the most impacting global phenomenon affecting the ecological balance of a particular habitat. The search for potential plant growth-promoting rhizobacteria (PGPR) capable of enhancing plant tolerance to drought stress is needed. Thus, this study was initiated to evaluate the effect of inoculating Acacia abyssinica seedlings with PGPR isolated from rhizosphere soil of Ethiopia to enhance DS tolerance. The strains were selected based on in vitro assays associated with tolerance to drought and other beneficial traits such as salinity, acidity, temperature, heavy metal tolerances, biofilm formation, and exopolysaccharide (EPS) production. The strains with the best DS tolerance ability were selected for the greenhouse trials with acacia plants. The results indicate that out of 73 strains, 10 (14%) were completely tolerant to 40% polyethylene glycol. Moreover, 37% of the strains were strong biofilm producers, while 66 (90.41%) were EPS producers with a better production in the medium containing sucrose at 28 ± 2°C and pH 7 ± 0.2. Strains PS-16 and RS-79 showed tolerance to 11% NaCl. All the strains were able to grow in wider ranges of pH (4-10) and temperature (15-45°C) and had high tolerance to heavy metals. The inoculated bacterial strains significantly (p ≤ 0.05) increased root and shoot length and dry biomass of acacia plants. One of the strains identified as P. fluorescens strain FB-49 was outstanding in enhancing DS tolerance compared to the single inoculants and comparable to consortia. Stress-tolerant PGPR could be used to enhance acacia DS tolerance after testing other phytobeneficial traits.

Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation.

This study reports the optimization of milk-clotting protease production from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) in both one-variable-at-a-time and response surface methodology (RSM). The production and optimization of milk-clotting protease obtained from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) using different agro-industrial wastes as solid substrates were studied. The agro-industrial wastes used included wheat bran, rice bran, pea bran, and grass pea bran. The chemical composition of the best solid substrate was tested using standard methods. Others cultivation parameters were studied, and the results showed that the optimum fermentation medium composed of wheat bran, casein (1% w/w), and glucose (0.5% w/w) and the conditions for maximum milk-clotting protease production were at the moisture content of 55.0%, inoculum of 0.5*106 spores/mL, incubation temperature of 30 °C, pH of 6.0, and fermentation time of 5 days. The highest milk-clotting activity was obtained from the crude enzyme extracted using 0.1 M NaCl and partial purification of the crude enzyme using chilled acetone, and 80% (NH4)2SO4 increased the ratio of MCA/PA from 0.56 to 1.30 and 0.65, respectively. Moreover, the highest MCA (137.58 U/mL) was obtained at a casein concentration of 0.5%, pH 4.0, and 25 °C, using RSM. Thus, results from the present study showed that the optimization of milk-clotting protease production from A. oryzae DRDFS 13 under SSF by both one-variable-at-a-time and RSM significantly increased the milk-clotting activity. This is the first report from a fungus in the Ethiopian setting and a modest contribution to highlight the potential of harnessing microbial protease enzymes for industrial applications.

Global-level population genomics reveals differential effects of geography and phylogeny on horizontal gene transfer in soil bacteria.

Although microorganisms are known to dominate Earth's biospheres and drive biogeochemical cycling, little is known about the geographic distributions of microbial populations or the environmental factors that pattern those distributions. We used a global-level hierarchical sampling scheme to comprehensively characterize the evolutionary relationships and distributional limitations of the nitrogen-fixing bacterial symbionts of the crop chickpea, generating 1,027 draft whole-genome sequences at the level of bacterial populations, including 14 high-quality PacBio genomes from a phylogenetically representative subset. We find that diverse Mesorhizobium taxa perform symbiosis with chickpea and have largely overlapping global distributions. However, sampled locations cluster based on the phylogenetic diversity of Mesorhizobium populations, and diversity clusters correspond to edaphic and environmental factors, primarily soil type and latitude. Despite long-standing evolutionary divergence and geographic isolation, the diverse taxa observed to nodulate chickpea share a set of integrative conjugative elements (ICEs) that encode the major functions of the symbiosis. This symbiosis ICE takes 2 forms in the bacterial chromosome-tripartite and monopartite-with tripartite ICEs confined to a broadly distributed superspecies clade. The pairwise evolutionary relatedness of these elements is controlled as much by geographic distance as by the evolutionary relatedness of the background genome. In contrast, diversity in the broader gene content of Mesorhizobium genomes follows a tight linear relationship with core genome phylogenetic distance, with little detectable effect of geography. These results illustrate how geography and demography can operate differentially on the evolution of bacterial genomes and offer useful insights for the development of improved technologies for sustainable agriculture.

Assessment of Bacteriological and Physicochemical Quality of Drinking Water from Source to Household Tap Connection in Nekemte, Oromia, Ethiopia.

In Ethiopia, access to improved water supply and sanitation has been very low and hence majority of the communicable diseases are associated with unsafe and inadequate water supply. Thus, the aim of this study was to assess physicochemical and bacteriological characteristics of water from sources to household connection in Nekemte town. A cross-sectional study was conducted from November 2015 to March 2016. Water samples were collected in triplicates from selected 30 sampling points from source, disinfection point, main distribution system tank, and household taps. All samples were analysed for bacteriological, chemical, and physical quality parameters using standard procedures. The results showed that temperature, pH, turbidity, total dissolved substances, and electrical conductivity of the water samples were varied between 16.9 and 22°C, 6.8-7.0, nil-12 NTU, 50-70 mg/l, and 40-46 µS/cm, respectively. Phosphate and nitrate concentrations of the water samples also ranged between 0.65 and 1 mg/l and 2.2-6.5 mg/l, respectively. Free residual chlorine concentration in the majority of the water samples was less than 0.5 mg/l. All samples were positive for total coliform with counts ranging from 12 to 120 CFU/100 ml, whereas faecal coliforms were detected in only 37% of tap water samples. In general, the majority of the tested parameters were within the permissible range of both the WHO and Ethiopian drinking water standards. However, Fe, Mn, faecal coliforms, total coliforms, and temperature did not conform to both WHO and Ethiopian drinking water standards. Based on the results, we can conclude that water quality deterioration was both at the sources and in the supply networks. Hence, proper drainage, sewage disposal systems, and sufficient disinfection of water with chlorine are of prime importance to deliver safe drinking water to the residents of Nekemte town.

Microbial community structure and diversity in an integrated system of anaerobic-aerobic reactors and a constructed wetland for the treatment of tannery wastewater in Modjo, Ethiopia.

A culture-independent approach was used to elucidate the microbial diversity and structure in the anaerobic-aerobic reactors integrated with a constructed wetland for the treatment of tannery wastewater in Modjo town, Ethiopia. The system has been running with removal efficiencies ranging from 94%-96% for COD, 91%-100% for SO4(2-) and S(2-), 92%-94% for BOD, 56%-82% for total Nitrogen and 2%-90% for NH3-N. 16S rRNA gene clone libraries were constructed and microbial community assemblies were determined by analysis of a total of 801 unique clone sequences from all the sites. Operational Taxonomic Unit (OTU)--based analysis of the sequences revealed highly diverse communities in each of the reactors and the constructed wetland. A total of 32 phylotypes were identified with the dominant members affiliated to Clostridia (33%), Betaproteobacteria (10%), Bacteroidia (10%), Deltaproteobacteria (9%) and Gammaproteobacteria (6%). Sequences affiliated to the class Clostridia were the most abundant across all sites. The 801 sequences were assigned to 255 OTUs, of which 3 OTUs were shared among the clone libraries from all sites. The shared OTUs comprised 80 sequences belonging to Clostridiales Family XIII Incertae Sedis, Bacteroidetes and unclassified bacterial group. Significantly different communities were harbored by the anaerobic, aerobic and rhizosphere sites of the constructed wetland. Numerous representative genera of the dominant bacterial classes obtained from the different sample sites of the integrated system have been implicated in the removal of various carbon- containing pollutants of natural and synthetic origins. To our knowledge, this is the first report of microbial community structure in tannery wastewater treatment plant from Ethiopia.

Current Trends in Bioethanol Production by Saccharomyces cerevisiae: Substrate, Inhibitor Reduction, Growth Variables, Coculture, and Immobilization.

Bioethanol is one of the most commonly used biofuels in transportation sector to reduce greenhouse gases. S. cerevisiae is the most employed yeast for ethanol production at industrial level though ethanol is produced by an array of other yeasts, bacteria, and fungi. This paper reviews the current and nonmolecular trends in ethanol production using S. cerevisiae. Ethanol has been produced from wide range of substrates such as molasses, starch based substrate, sweet sorghum cane extract, lignocellulose, and other wastes. The inhibitors in lignocellulosic hydrolysates can be reduced by repeated sequential fermentation, treatment with reducing agents and activated charcoal, overliming, anion exchanger, evaporation, enzymatic treatment with peroxidase and laccase, in situ detoxification by fermenting microbes, and different extraction methods. Coculturing S. cerevisiae with other yeasts or microbes is targeted to optimize ethanol production, shorten fermentation time, and reduce process cost. Immobilization of yeast cells has been considered as potential alternative for enhancing ethanol productivity, because immobilizing yeasts reduce risk of contamination, make the separation of cell mass from the bulk liquid easy, retain stability of cell activities, minimize production costs, enable biocatalyst recycling, reduce fermentation time, and protect the cells from inhibitors. The effects of growth variables of the yeast and supplementation of external nitrogen sources on ethanol optimization are also reviewed.

Coffee husk composting: an investigation of the process using molecular and non-molecular tools.

Various parameters were measured during a 90-day composting process of coffee husk with cow dung (Pile 1), with fruit/vegetable wastes (Pile 2) and coffee husk alone (Pile 3). Samples were collected on days 0, 32 and 90 for chemical and microbiological analyses. C/N ratios of Piles 1 and 2 decreased significantly over the 90 days. The highest bacterial counts at the start of the process and highest actinobacterial counts at the end of the process (Piles 1 and 2) indicated microbial succession with concomitant production of compost relevant enzymes. Denaturing gradient gel electrophoresis of rDNA and COMPOCHIP microarray analysis indicated distinctive community shifts during the composting process, with day 0 samples clustering separately from the 32 and 90-day samples. This study, using a multi-parameter approach, has revealed differences in quality and species diversity of the three composts.