A novel GH3-ß-glucosidase from soda lake metagenomic libraries with desirable properties for biomass degradation.

Beta-glucosidases catalyze the hydrolysis of the glycosidic bonds of cellobiose, producing glucose, which is a rate-limiting step in cellulose biomass degradation. In industrial processes, ß-glucosidases that are tolerant to glucose and stable under harsh industrial reaction conditions are required for efficient cellulose hydrolysis. In this study, we report the molecular cloning, Escherichia coli expression, and functional characterization of a ß-glucosidase from the gene, CelGH3_f17, identified from metagenomics libraries of an Ethiopian soda lake. The CelGH3_f17 gene sequence contains a glycoside hydrolase family 3 catalytic domain (GH3). The heterologous expressed and purified enzyme exhibited optimal activity at 50 °C and pH 8.5. In addition, supplementation of 1 M salt and 300 mM glucose enhanced the ß-glucosidase activity. Most of the metal ions and organic solvents tested did not affect the ß-glucosidase activity. However, Cu2+ and Mn2+ ions, Mercaptoethanol and Triton X-100 reduce the activity of the enzyme. The studied ß-glucosidase enzyme has multiple industrially desirable properties including thermostability, and alkaline, salt, and glucose tolerance.

Production of bioethanol from sweet sorghum [Sorghum bicolor L] juice using yeast isolated from fermented sweet sorghum juice / Producción de bioetanol a partir de jugo de sorgo dulce [Sorghum bicolor L] utilizando levadura aislada de jugo de sorgo dulce fermentado

As a sugar-rich plant with no impact on global warming and food security, sweet sorghum can be exploited as an alternative source of renewable bioenergy. This study aimed to examine the potential of sweet sorghum juice for the generation of bioethanol using yeast isolated from the juice. The °Brix of sweet sorghum juice was measured using a digital refractometer. Additionally, 18 wild yeasts isolated from fermented sweet sorghum juice were subjected to various biochemical tests to describe them to identify potential yeast for ethanol production. The morphological and biochemical analyses of the yeasts revealed that all of the yeast isolates were most likely members of the genus Saccharomyces. The most ethanol-tolerant yeast isolate SJU14 was employed for sweet sorghum juice fermentation. A completely randomized factorial design was used with various fermentation parameters, primarily pH, temperature, and incubation period. Then ethanol content was determined using a potassium dichromate solution. According to the ANOVA, the highest ethanol content (18.765%) was produced at 30/26 °C, pH 4.5, and incubated for 96 h. Sweet sorghum juice was found to be an excellent source of potent yeasts, which have important industrial properties like the capacity to grow at high ethanol and glucose concentrations. Moreover, it can be utilized as a substitute substrate for the manufacturing of bioethanol production to lessen the environmental threat posed by fossil fuels. Further research is, therefore, recommended to develop strategically valuable applications of sweet sorghum for enhancing the food system and mitigating climate change.(AU)

Production of bioethanol from sweet sorghum [Sorghum bicolor L.] juice using yeast isolated from fermented sweet sorghum juice.

As a sugar-rich plant with no impact on global warming and food security, sweet sorghum can be exploited as an alternative source of renewable bioenergy. This study aimed to examine the potential of sweet sorghum juice for the generation of bioethanol using yeast isolated from the juice. The °Brix of sweet sorghum juice was measured using a digital refractometer. Additionally, 18 wild yeasts isolated from fermented sweet sorghum juice were subjected to various biochemical tests to describe them to identify potential yeast for ethanol production. The morphological and biochemical analyses of the yeasts revealed that all of the yeast isolates were most likely members of the genus Saccharomyces. The most ethanol-tolerant yeast isolate SJU14 was employed for sweet sorghum juice fermentation. A completely randomized factorial design was used with various fermentation parameters, primarily pH, temperature, and incubation period. Then ethanol content was determined using a potassium dichromate solution. According to the ANOVA, the highest ethanol content (18.765%) was produced at 30/26 °C, pH 4.5, and incubated for 96 h. Sweet sorghum juice was found to be an excellent source of potent yeasts, which have important industrial properties like the capacity to grow at high ethanol and glucose concentrations. Moreover, it can be utilized as a substitute substrate for the manufacturing of bioethanol production to lessen the environmental threat posed by fossil fuels. Further research is, therefore, recommended to develop strategically valuable applications of sweet sorghum for enhancing the food system and mitigating climate change.

Prokaryotic and eukaryotic microbial diversity from three soda lakes in the East African Rift Valley determined by amplicon sequencing.

Soda lakes are unique poly-extreme environments with high alkalinity and salinity that support diverse microbial communities despite their extreme nature. In this study, prokaryotic and eukaryotic microbial diversity in samples of the three soda lakes, Lake Abijata, Lake Chitu and Lake Shala in the East African Rift Valley, were determined using amplicon sequencing. Culture-independent analysis showed higher diversity of prokaryotic and eukaryotic microbial communities in all three soda lakes than previously reported. A total of 3,603 prokaryotic and 898 eukaryotic operational taxonomic units (OTUs) were found through culture-independent amplicon sequencing, whereas only 134 bacterial OTUs, which correspond to 3%, were obtained by enrichment cultures. This shows that only a fraction of the microorganisms from these habitats can be cultured under laboratory conditions. Of the three soda lakes, samples from Lake Chitu showed the highest prokaryotic diversity, while samples from Lake Shala showed the lowest diversity. Pseudomonadota (Halomonas), Bacillota (Bacillus, Clostridia), Bacteroidota (Bacteroides), Euryarchaeota (Thermoplasmata, Thermococci, Methanomicrobia, Halobacter), and Nanoarchaeota (Woesearchaeia) were the most common prokaryotic microbes in the three soda lakes. A high diversity of eukaryotic organisms were identified, primarily represented by Ascomycota and Basidiomycota. Compared to the other two lakes, a higher number of eukaryotic OTUs were found in Lake Abijata. The present study showed that these unique habitats harbour diverse microbial genetic resources with possible use in biotechnological applications, which should be further investigated by functional metagenomics.

Discovery of novel carbohydrate degrading enzymes from soda lakes through functional metagenomics.

Extremophiles provide a one-of-a-kind source of enzymes with properties that allow them to endure the rigorous industrial conversion of lignocellulose biomass into fermentable sugars. However, the fact that most of these organisms fail to grow under typical culture conditions limits the accessibility to these enzymes. In this study, we employed a functional metagenomics approach to identify carbohydrate-degrading enzymes from Ethiopian soda lakes, which are extreme environments harboring a high microbial diversity. Out of 21,000 clones screened for the five carbohydrate hydrolyzing enzymes, 408 clones were found positive. Cellulase and amylase, gave high hit ratio of 1:75 and 1:280, respectively. A total of 378 genes involved in the degradation of complex carbohydrates were identified by combining high-throughput sequencing of 22 selected clones and bioinformatics analysis using a customized workflow. Around 41% of the annotated genes belonged to the Glycoside Hydrolases (GH). Multiple GHs were identified, indicating the potential to discover novel CAZymes useful for the enzymatic degradation of lignocellulose biomass from the Ethiopian soda Lakes. More than 73% of the annotated GH genes were linked to bacterial origins, with Halomonas as the most likely source. Biochemical characterization of the three enzymes from the selected clones (amylase, cellulase, and pectinase) showed that they are active in elevated temperatures, high pH, and high salt concentrations. These properties strongly indicate that the evaluated enzymes have the potential to be used for applications in various industrial processes, particularly in biorefinery for lignocellulose biomass conversion.

Diversity of Culturable Alkaliphilic Nitrogen-Fixing Bacteria from a Soda Lake in the East African Rift Valley.

Lake Chitu is a highly productive soda lake found in the East African Rift Valley, where Arthrospira fusiformis (Spirulina platensis) is the main primary producer. High biomass accumulation requires an adequate supply of nitrogen. However, Lake Chitu is a closed system without any external nutrient input. A recent study has also demonstrated the presence of a diverse group of denitrifying bacteria, indicating a possible loss of nitrate released from the oxidation of organic matter. The aim of this study was to isolate culturable nitrogen-fixing alkaliphiles and evaluate their potential contribution in the nitrogen economy of the soda lake. A total of 118 alkaliphiles belonging to nine different operational taxonomic units (OTUs) were isolated using a nitrogen-free medium. Nineteen isolates were tested for the presence of the nifH gene, and 11 were positive. The ability to fix nitrogen was tested by co-culturing with a non-nitrogen-fixing alkaliphile, Alkalibacterium sp. 3.5*R1. When inoculated alone, Alkalibacterium sp. 3.5*R1 failed to grow on a nitrogen-free medium, but grew very well when co-cultured with the nitrogen-fixing alkaliphile NF10m6 isolated in this study, indicating the availability of nitrogen. These results show that nitrogen fixation by alkaliphiles may have an important contribution as a source of nitrogen in soda lakes.

Plant growth-promoting characteristics of halotolerant endophytic bacteria isolated from Sporobolus specatus (Vahr) Kunth and Cyperus laevigatus L. of Ethiopian rift valley lakes.

Understanding plant microbes' intimate relationship and search for beneficial microbes is a sustainable alternative to improve plant growth and yield under a wide range of biotic and abiotic stress conditions. More than 20% of the total global agricultural land is affected by salinity. High salinity challenges crop plants by affecting several metabolic pathways and decreasing plant growth and yield. Unlike chemical fertilizers and pesticides, endophytic microbes offer an eco-friendly approach to increasing crop yield via various metabolites during salinity stress. The objective of this study was to isolate and characterize endophytic halotolerant bacterial isolates from haloalkaliphytes, investigate their plant growth-promoting (PGP) properties and tolerance for various stress conditions. Sporobolus specatus (Vahr) Kunth and Cyperus laevigatus L. grass samples were collected from the shores of two Ethiopian soda lakes (Lakes Abijata, and Chitu, respectively). A total of 167 halotolerant endophytic bacterial isolates, that clustered into 21 ARDRA (Amplified ribosomal DNA restriction analysis) groups, affiliated to members of 11 bacterial genera, namely Halomonas, Agrobacterium, Exiguobacterium, Jonesia, Stenotrophomonas, Pseudomonas, Alishewanella, Kosakonia, Bacillus, Paracoccus and Pannonibacter, were identified based on 16S rRNA sequencing. Most of the strains were able to produce IAA (indole-3-acetic acid) and hydrogen cyanide, grow on a nitrogen-free medium and solubilize phosphate. In vitro tolerance tests reveal that isolates were tolerant to: 5.0-15% NaCl, up to 40% PEG 6000, temperatures up to 50 °C, and pH 5-11. These characteristics of the isolates indicate their potential PGP application under various plant stress conditions.

Microbial community shifts association with physicochemical parameters: Visualizing enset bacterial wilt from different states of enset health.

Bacterial wilt of enset caused by Xanthomonas campestris is a devastating disease in Ethiopia, where enset is domesticated and served as a staple food for about 20 million people in the country. While enset is infected by bacteria, it shows different wilting stages. However, the microbial community shifts at the different stages of enset infection and associated physicochemical parameter changes remain poorly understood. This study was aimed to visualize the proportion of enset wilt bacterium from other microbial community and its association with physicochemical parameter at different states of enset health. Soil and enset (zero, first, second and third stages) samples were collected from three districts in Gamo Highlands for physicochemical and biological (culture dependent and16S rRNA gene sequence) analysis. The results of culture dependent analysis which has been complemented by 16S rRNA gene sequence confirmed that increasing trends were observed for Xanthomonadaceae, Pseudomonadaceae, Lactobacillaceae and Flavobacteriaceae, while Bacillaceae and Enterobacteriaceae showed progressive decrease from zero to the third stage. Particularly, the 16S rRNA data showed that Xanthomonadaceae increased significantly from zero to different (2.5 × 102 times at the onset of disease and 1.0-2.0 × 104 times at the second and third) stages of enset infection. Most physicochemical results showed that a decreasing trends from zero to third stage, while few parameters are showing an increasing trend. Moisture content (R2 ≥ 0.951, P ≤ 0.049) of the soil and plant samples positively influenced Xanthomonas abundance, while this bacterium showed a strongly negative significant correlation with pH (R2 ≥ -0.962, P ≤ 0.038), temperature (R2 ≥ -0.958, P ≤ 0.042), OM (R2 ≥ -0.952, P ≤ 0.048), and TN (R2 ≥ -0.951, P ≤ 0.049). A strongly negative significant correlation (R2 ≥ -0.948, P ≤ 0.050) was also observed between Xanthomonas and nutrients (K, Mg, Ca, and Cu). Overall, this study implies that different environmental factors found a key driving force of Xanthomonas proportional increment from low abundance at zero stage to higher abundance at the last stage of enset infection suggesting that considering these factors help to design an effective enset disease management strategy, for which further studies will be needed.

Prediction of Genes That Function in Methanogenesis and CO2 Pathways in Extremophiles.

Gaet'ale (GAL) and Mud'ara (MUP) are two hypersaline ponds located in the Danakil Depression recharged by underground water from the surrounding highlands. These two ponds have different pH, salinity, and show variation in the concentration of many ionic components. Metagenomic analysis concludes that GAL is dominated by bacteria as in the case of the other hypersaline and acidic ponds in the Danakil Depression. However, Archaea dominated the ponds of MUP. In the current study, the application of SEED and KEGG helped to map the ordered steps of specific enzyme catalyzed reaction in converting CO2 into cell products. We predict that highly efficient and light-independent carbon fixation involving phosphoenolpyruvate carboxylase takes place in MUP. On the contrary, genes encoding enzymes involved in hydrogenotrophic and acetoclastic methanogenesis appeared solely in ponds of GAL, implying the biological source of the hazardous methane gas in that environment. Based on the investigation of the sources of the genes of interest, it is clear that cooperative interactions between members of the two communities and syntrophic metabolism is the main strategy adapted to utilize inorganic carbon as a carbon source in both MUP and GAL. This insight can be used to design biotechnological applications of microbial communities in production of methane biogas or to minimize CO2 emissions.

In silico determination of nitrogen metabolism in microbes from extreme conditions using metagenomics.

The acid ponds of the Danakil Depression in northern Ethiopia are polyextreme environments that exceed the normal physicochemical limits of pH, salinity, ion content, and temperature. We tested for the occurrence of DNA-based life in this environment using Metagenomic Shotgun DNA sequencing approaches. The obtained sequences were examined by the bioinformatic tools MetaSpades, DIAMOND and MEGAN 6-CE, and we were able to bin more than 90% of the metagenomics contigs of Dallol and Black Water to the Bacteria domain, and to the Proteobacteria phylum. Predictions of gene function based on SEED disclosed the presence of different nutrient cycles in the acid ponds. For this study, we focused on partial or completely sequenced genes involved in nitrogen metabolism. The KEGG nitrogen metabolism pathway mapping results for both acid ponds showed that all the predicted genes are involved directly or indirectly in the assimilation of ammonia and no dissimilation or nitrification process was identified. Furthermore, the deduced nitrogen fixation in the two acid ponds based on SEED classification indicated the presence of different sets of nitrogen fixing (nif) genes for biosynthesis and maturation of nitrogenase. Based on the in silico analysis, the predicted proteins involved in nitrogen fixation, especially the cysteine desulfurase and [4Fe-4S] ferredoxin, from both acid ponds are unique with less than 80% sequence similarity to the next closest protein sequence. Considering the extremity of the environmental conditions of the two acid ponds in the Danakil depression, this metagenomics dataset can add to the study of unique gene functions in nitrogen metabolism that enable thriving biocommunities in hypersaline and highly acidic conditions.

The Role of Private Health Sector for Tuberculosis Control in Debre Markos Town, Northwest Ethiopia.

BACKGROUND: Tuberculosis has been declared to be a global epidemic. Despite all the effort, only less than half the annual estimated cases are reported by health authorities to the WHO. This could be due to poor reporting from the private sector. In Ethiopia, tuberculosis has also been a major public health problem. The aim of this study was to assess the role of the private health sector in tuberculosis control in Debre Markos. METHODS: An institution based cross-sectional descriptive study was carried out in private health facilities. A total of 260 tuberculosis suspects attending the private clinics were interviewed. Focus group discussion, checklist, and structured questionnaire were used. RESULTS: Majority of the private clinics were less equipped, poorly regulated, and owned by health workers who were self-employed on a part-time basis. Provider delay of 4 and more months was significantly associated higher likelihood of turning to a private provider (OR = 2.70, 95% CI = (1.20, 6.08)). CONCLUSIONS AND RECOMMENDATIONS: There is significant delay among tuberculosis patients. Moreover, there is poor regulation of the private health sector by public health authorities. The involvement of the private sector in tuberculosis control should be limited to identification and refer to tuberculosis cases and suspects.

Prokaryotic Community Diversity Along an Increasing Salt Gradient in a Soda Ash Concentration Pond.

The effect of salinity on prokaryotic community diversity in Abijata-Shalla Soda Ash Concentration Pond system was investigated by using high-throughput 16S rRNA gene 454 pyrosequencing. Surface water and brine samples from five sites spanning a salinity range of 3.4 % (Lake Abijata) to 32 % (SP230F, crystallizer pond) were analyzed. Overall, 33 prokaryotic phyla were detected, and the dominant prokaryotic phyla accounted for more than 95 % of the reads consisting of Planctomycetes, Bacteroidetes, candidate division TM7, Deinococcus-Thermus, Firmicutes, Actinobacteria, Proteobacteria, and Euryarchaeota. Diversity indices indicated that operational taxonomic unit (OTU) richness decreases drastically with increasing salinity in the pond system. A total of 471 OTUs were found at 3.4 % salinity whereas 49 OTUs were detected in pond SP211 (25 % salinity), and only 19 OTUs in the crystallization pond at 32 % salinity (SP230F). Along the salinity gradient, archaeal community gradually replaced bacterial community. Thus, archaeal community accounted for 0.4 % in Lake Abijata while 99.0 % in pond SP230F. This study demonstrates that salinity appears to be the key environmental parameter in structuring the prokaryotic communities of haloalkaline environments. Further, it confirmed that the prokaryotic diversity in Lake Abijata is high and it harbors taxa with low or no phylogenetic similarities to existing prokaryotic taxa and thus represents novel microorganisms.

Surprising prokaryotic and eukaryotic diversity, community structure and biogeography of Ethiopian soda lakes.

Soda lakes are intriguing ecosystems harboring extremely productive microbial communities in spite of their extreme environmental conditions. This makes them valuable model systems for studying the connection between community structure and abiotic parameters such as pH and salinity. For the first time, we apply high-throughput sequencing to accurately estimate phylogenetic richness and composition in five soda lakes, located in the Ethiopian Rift Valley. The lakes were selected for their contrasting pH, salinities and stratification and several depths or spatial positions were covered in each lake. DNA was extracted and analyzed from all lakes at various depths and RNA extracted from two of the lakes, analyzed using both amplicon- and shotgun sequencing. We reveal a surprisingly high biodiversity in all of the studied lakes, similar to that of freshwater lakes. Interestingly, diversity appeared uncorrelated or positively correlated to pH and salinity, with the most "extreme" lakes showing the highest richness. Together, pH, dissolved oxygen, sodium- and potassium concentration explained approximately 30% of the compositional variation between samples. A diversity of prokaryotic and eukaryotic taxa could be identified, including several putatively involved in carbon-, sulfur- or nitrogen cycling. Key processes like methane oxidation, ammonia oxidation and 'nitrifier denitrification' were also confirmed by mRNA transcript analyses.