Metagenomics and Culture-Based Diversity Analysis of the Bacterial Community in the Zharkent Geothermal Spring in Kazakhstan

Abstract

Diversity of the microbial community in the Zharkent geothermal hot spring, located in the southeastern region of Kazakhstan, was assessed using both culture-dependent and -independent approaches. Shotgun metagenomic sequencing of DNA extracted from the spring water yielded 11,061,725 high-quality sequence reads, totaling >1,67 Gb of nucleotide sequences. Furthermore, water samples were enriched in nutrient broth at varying high temperatures, and colonies isolated by being streaked onto nutrient agar. Finally, DNA extraction and amplification, as well as sequencing and phylogenetic analysis, were conducted. Bacteria constituted more than 99.97% of the total prokaryotic abundance, with Archaea contributing only an extremely small component; Firmicutes, Proteobacteria, and Actinobacteria dominated the community. At genus level, Firmicutes reads affiliated with Desmospora, Parageobacillus, Paenibacillus, and Brevibacillus, accounting for more than 60% of total prokaryotic abundance. Eight morphologically distinct, aerobic, endospore-forming thermophilic bacteria were recovered; isolates differed significantly in substrate utilization patterns, as well as their production of thermophilic, extracellular, hydrolytic enzymes for degradation of starch, lipids, cellulose, and protein. Five strains could degrade all four macromolecular types at temperatures ranging from 55 to 75 °C. Phylogenetic analyses based on 16S rRNA gene sequences placed all isolates into the genus Geobacillus with some of them possibly representing novel species. The results indicate that this hot spring represents a rich source of novel thermophilic bacteria and potentially useful thermostable enzymes.