16S rRNA gene sequencing reveals bacterial diversity in Khewra Salt Mine walls

The Khewra Salt Mine, also known as the source of Himalayan Pink Salt, is the second largest salt mine in the world and a significant site for salt export. The mine's salt exhibits both amorphous and crystalline textures, with colours ranging from white to deep red, contributing to its distinctive pink hue. While previous research has extensively explored the mine's soil, plants and brine samples, little attention has been given to the salt walls, which are a major tourist attraction. This study aimed to investigate the bacterial diversity within the salt walls from four distinct locations, including an under-construction area sampled for the first time. From these four collection points, 7 samples were gathered, yielding 19 isolates cultured on 2% NaCl nutrient agar. Bacterial isolates from the under-construction area (US1-1, US1-2, US2-1 and US2-2) and the museum walls (MS1-1 and MS1-2) underwent further biochemical, antibiotic sensitivity and salt requirement testing, followed by 16S rRNA sequencing. Biochemical assays identified these isolates as Gram-positive, catalase- and oxidase-positive rods. All isolates were sensitive to chloramphenicol (30 µg ml^-1). Salt tolerance tests classified them as slightly halophilic, thriving at 2-4% NaCl concentrations. Molecular characterization revealed two strains from the under-construction area, US1-1 and US2-2, as uncultured Bacillus species clone 5 and Bacillus sp. PSA3, respectively, both with 94% sequence similarity. The phylogenetic results show monophyletic associations between these two strains, which could be due to their shared salt location and common ancestors. The sequences of US1-2, US2-1 and MS1-1 were 100% identical to Bacillus licheniformis strain P8_B2 chromosome, while MS1-2 matched Staphylococcus saprophyticus strain CJJH1 with 100% similarity. The results of this study point towards a significant diversity of bacteria, slightly halophilic, cocci or bacillus-shaped. These halophilic bacterial strains, possessing salt-tolerant genes, present the need for further identification of bacterial colonies in the mine and their evolutionary patterns, which could also help uncover the role these microbes play in the ecosystem of the salt mine. Additionally, they show promise for industrial enzyme production.