Diversity and function of polysaccharide Degrading bacteria from lake magadi, kenya

Abstract

Soda lakes are extreme environments characterized by high alkalinity and salt concentrations and moderate to high temperatures. Despite these conditions, soda lakes support distinctive microbial communities that are a potential source of novel biocatalysts for industrial and biotechnological applications. This study investigated the diversity, structure, and function of bacterial isolates recovered from the soda Lake Magadi, particularly isolates that could utilize polysaccharides. Isolation involved enrichment media supplemented with selected polymers and prepared using sterile lake water. The isolates were identified based on sequencing of the 16S rRNA gene region. A plate radial diffusion assay measured enzyme activity against cellulose, carboxymethylcellulose, xanthan, starch, pectin, and xylan. The bacterial isolates were tested for their ability to grow under various pH, salt, and temperature conditions. The results showed that the isolates were closely related to members of the genus Salipaludibacillus, Halomonas, Alkalibacterium, Alkalihalophilus, Evansella, Shouchella, Halalkalibacterium, Halalkalibacter, Alkalihalobacterium, and Salinicoccus. The highest enzyme activity was recorded among the isolates belonging to Salipaludibacillus, while the least activity was recorded in isolates belonging to Halomonas. High cell densities were recorded at pH 7–9, 40°C, and 5% (w/v) NaCl concentrations. Four isolates (LMS6, LMS18, LMS25, and LMS39) were selected for full genome sequencing. Average nucleotide identity (ANI) analysis revealed that LMS6, LMS18, LMS25, and LMS39 were new species of Shouchella sp., Evansella sp., Salipaludibacillus sp., and Alkalihalobacterium sp., respectively. Analysis of carbohydrate-active enzymes (CAZymes) revealed a higher number of genes for carbohydrate metabolism in the genomes of Salipaludibacillus sp. LMS25 and Alkalihalobacterium sp. LMS39. These genes encode for amylases, cellulase, pectinase, xylanase, and chitinase enzymes, further corroborating the findings of plate screening. Furthermore, the study examined the impact of water chemistry on the composition and structure of microbial communities over time. In this case, the 16S rRNA gene amplicons were sequenced using the Illumina MiSeq platform. Results revealed that the most abundant bacterial phyla were Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Verrumicrobia, Deinococcus-Thermus, Spirochaetes, and Chloroflexi. Euryachaeota, Crenarchaeota, and Thaumarchaeota were representative of archaeal diversity. The dominant bacterial species were: Euhalothece sp. (10.3%), Rhodobaca sp. (9.6%), Idiomarina sp. (5.8%), Rhodothermus sp. (3.0%), Roseinatronobacter sp. (2.4%), Nocardioides sp. (2.3%), Gracilimonas sp. (2.2%), and Halomonas sp. (2%). On the other hand, the dominant archaeal species included Halorubrum sp. (18.3%), Salinarchaeum sp. (5.3%), and Haloterrigena sp. (1.3%). The composition of bacteria was higher than that of archaea, while their richness and diversity varied across the sampling seasons. The alpha diversity indices showed that high diversity was recorded in August, followed by September, June, and July in that order. The findings demonstrated that temperature, pH, P+, K+, NO3-, and total dissolved solids (TDS) contributed significantly to the diversity observed in the microbial community. Multivariate analysis revealed that samples were clustered based on salinity and alkalinity rather than the sampling site or season. Furthermore, this study constructed an artificial metagenome by mixing, in equal amounts, the total chromosomal DNA extracted from 40 bacterial isolates. The aim was to evaluate the total genes encoding for hydrolytic enzymes. To achieve this, Genomic libraries were created and sequenced using paired-end 2 x 250 bp runs on Illumina HiSeq 2500 apparatus. The results revealed a total count of 46,641 putative genes, with a proportion of 10% being those encoding for enzymes involved in the hydrolysis of polysaccharides. Taxonomic assignment showed that more than 99% of the sequences were affiliated with Firmicutes and Proteobacteria. These enzymes included chitinases, cellulases, amylases, and xylanases. In addition, ten metagenome-assembled genomes (MAGs) of medium and high quality were observed. They were affiliated with the genera Salipaludibacillus, Alkalihalophilus, Halalkalibacterium, Evansella, Salinicoccus, Alkalibacterium, and Halomonas. Furthermore, a putative gene encoding for an endoβ-1,4-glucanase (LMP_42667) deduced from Salipaludibacillus was cloned into a pBAD18 vector and heterologously expressed in Top10 E. coli cells. The purified enzyme with 571 amino acids and a theoretical molecular weight of 65.7 kDa demonstrated activity at pH ranges of 4.0 to 8.0 and optimum enzyme activity at 50°C and pH 7.0. The enzyme also demonstrated higher enzyme-specific activity for xylan (9.8 ± 0.1 U/mg). Additionally, enzyme activity was reported against CMC, hydroxyethylcellulose (HEC), microcrystalline cellulose, lichenan, and β-glucan. It depicted stability in the presence of various metallic ions, protein inhibitors, and chemical reagents. Analysis of the structural model showed that the endoglucanase belongs to the glycosyl hydrolase 5 subfamily 4 (GH5_4) group of glycosyl hydrolases. In conclusion, the findings of this study demonstrate that Lake Magadi harbors a rich source of diverse polysaccharide hydrolyzing bacteria with a wide repertoire of genes encoding hydrolytic enzymes with industrial and biotechnological potential. Furthermore, the study reveals the role of salinity and alkalinity in determining the structure of microbial communities in Lake Magadi