Rock surfaces as reservoirs for airborne halophilic microorganisms in the Bochnia Salt Mine

Airborne halophilic archaea have recently been detected in subterranean salt mines, yet their origin in such dynamic environments remains unclear. We investigated whether exposed salt rock surfaces may serve as biological reservoirs for airborne halophilic microorganisms. Rock surfaces were sampled at three underground sites in the Bochnia Salt Mine (southern Poland), spanning a gradient of distance from the mine entrance and human influence. Surface swabs were collected from rock salt and salty claystone during summer and winter, representing contrasting warm-humid and cool-dry microclimatic conditions, and analyzed using cultivation-based methods combined with 16S rRNA gene sequencing. Across all sites, seasons, and both rock types considered together, halophilic microorganisms dominated rock-associated communities, occurring at approximately 1.2 × 10³ to 4.3 × 10³ CFU/25 cm². In contrast, non-halophilic microorganisms were present at only about 0.5-1.3 × 10² CFU/25 cm². Mean halophilic abundance was approximately 3.8 × 10³ CFU/25 cm² in summer and 1.4 × 10³ CFU/25 cm² in winter, indicating less than two-fold seasonal variation on rock surfaces. In contrast, airborne communities from the same sites previously showed up to 13-fold summer-winter variation. A total of 11 halophilic archaeal species were identified on rock surfaces, dominated by members of the genus Halococcus (Hcc. salifodinae, Hcc. hamelinensis, Hcc. morrhuae and Hcc. dombrowskii), as well as Natrinema versiforme and Halalkalicoccus paucihalophilus. Most taxa detected on rock surfaces overlapped with those previously identified in the mine air. These results indicate that exposed salty rock surfaces constitute favorable habitats and likely persistent reservoirs for halophilic archaea, whereas the mine atmosphere appears to represent a transient, environmentally filtered compartment that receives only a subset of the rock-associated community.