Metagenomic Analysis of Bacterial Community and Isolation of Representative Strains from Vranjska Banja Hot Spring, Serbia.

The hot spring Vranjska Banja is the hottest spring on the Balkan Peninsula with a water temperature of 63-95 °C and a pH value of 7.1, in situ. According to the physicochemical analysis, Vranjska Banja hot spring belongs to the bicarbonated and sulfated hyperthermal waters. The structures of microbial community of this geothermal spring are still largely unexplored. In order to determine and monitor the diversity of microbiota of the Vranjska Banja hot spring, a comprehensive culture-independent metagenomic analysis was conducted in parallel with a culture-dependent approach for the first time. Microbial profiling using amplicon sequencing analysis revealed the presence of phylogenetically novel taxa, ranging from species to phyla. Cultivation-based methods resulted in the isolation of 17 strains belonging to the genera Anoxybacillus, Bacillus, Geobacillus, and Hydrogenophillus. Whole-genome sequencing of five representative strains was then performed. The genomic characterization and OrthoANI analysis revealed that the Vranjska Banja hot spring harbors phylogenetically novel species of the genus Anoxybacillus, proving its uniqueness. Moreover, these isolates contain stress response genes that enable them to survive in the harsh conditions of the hot springs. The results of the in silico analysis show that most of the sequenced strains have the potential to produce thermostable enzymes (proteases, lipases, amylases, phytase, chitinase, and glucanase) and various antimicrobial molecules that can be of great importance for industrial, agricultural, and biotechnological applications. Finally, this study provides a basis for further research and understanding of the metabolic potential of these microorganisms.