Skip to main content
NCBI home page
Frontiers in Microbiology logoLink to Frontiers in Microbiology
. 2026 Apr 22;17:1813537. doi: 10.3389/fmicb.2026.1813537

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

Magdalena Kowalewicz-Kulbat 1, Gabriela Arciszewska 1, Maciej Manecki 2, Dominika Drzewiecka 3, Michalina Rachubik 1, Karolina Stępniak 1, Luciana Albuquerque 4,5, Conceição Egas 4,5,6, Krzysztof Krawczyk 1, Camille Locht 1,7, Aleksandra Puławska 2,*
PMCID: PMC13144014  PMID: 42100702

Abstract

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.

Keywords: halite, halophilic archaea, hypersaline environments, microbial aerosolisation, rock-air interface, salt mine microbiology

1. Introduction

Salt rocks host diverse microbial communities, including halophilic microorganisms, that play an important role in geomicrobiological processes, long-term microbial survival and mineral–microbe interactions in hypersaline environments (Lowenstein et al., 2011; Jaakkola et al., 2016). Such communities are relevant not only for understanding the limits of life in evaporitic systems but also for applied contexts in which salt rocks constitute stable geological matrices, including underground resource storage and the preservation of biological signatures over geological timescales (Onstott et al., 2019; Dopffel et al., 2024). Beyond their geological and geomicrobiological relevance, subterranean salt mines are also widely used for therapeutic purposes based on inhalation of salt-saturated mine air, which has further motivated research into the composition, dynamics and origin of airborne microbial communities in these environments (Puławska et al., 2025).

Subterranean salt mines represent tightly coupled systems in which halite-rich rock surfaces, saline aerosols and mine air interact through ventilation and microclimatic gradients (Puławska et al., 2021a). Recent studies in the Bochnia Salt Mine demonstrated the presence of viable, cultivable halophilic archaea in mine air and revealed that their abundance and diversity are strongly modulated by distance from the mine entrance, relative humidity and seasonal changes (Puławska et al., 2025; Puławska et al., 2026). In contrast to non-halophilic microorganisms, whose occurrence in mine air can largely be attributed to external inputs or human activity, airborne halophilic archaea are evenly distributed across underground sites and largely absent at the mine entrance, suggesting an indigenous subterranean source (Puławska et al., 2025; Puławska et al., 2026).

The origin of these airborne halophilic archaea, however, has remained unresolved. Among potential reservoirs, salty rocks represent one of the most plausible persistent sources, as they provide long-term stability, high salinity, and protection from rapid environmental fluctuations (McGenity et al., 2001; Jaakkola et al., 2016). Seasonal changes in relative humidity are likely to influence the potential transfer of microorganisms from salt rock surfaces into the mine air via halite deliquescence and aerosol formation, whereas the persistence of rock-associated communities appears less sensitive to short-term atmospheric variability (Robinson et al., 2015; Puławska et al., 2026).

In this study, we tested the hypothesis that salt rock surfaces act as reservoirs supplying airborne halophilic microorganisms in the Bochnia Salt Mine. We compared the abundance and taxonomic composition of cultivable halophilic and non-halophilic microorganisms inhabiting the surfaces of two dominant rock types—rock salt (RS) and salty claystone (SC)—at three underground sites differing in distance from the mine entrance and human activity. Samples were collected during both summer and winter seasons, representing contrasting microclimatic regimes characterized by warm, humid summers and cool, relatively dry winters, and coinciding with previous airborne sampling campaigns. This design enabled direct comparison between rock-associated and airborne microbial communities under seasonally distinct environmental conditions. As in our previous studies (Puławska et al., 2025; Puławska et al., 2026) we focused here only on culturable microorganisms, as they may be valuable sources of novel compounds for biotechnological applications, including antimicrobials (Moopantakath et al., 2023) and anticancer compounds (Kowalewicz-Kulbat et al., 2023), which warrant further investigation.

2. Materials and methods

2.1. Sampling

Rock surfaces were sampled in June 2022 (summer season) and February 2023 (winter season) at three locations within the Bochnia Salt Mine, designated BL-2, BL-3, and BL-4 during the summer campaign and BZ-2, BZ-3, and BZ-4 during the winter campaign. Sites BL-3/BZ-3 (the Ważyn Chamber) are accessible to visitors, whereas sites BL-2/BZ-2 and BL-4/BZ-4 are not open to public access. Detailed descriptions of the mine layout, ventilation regime, microclimatic conditions and the exact spatial distribution of sampling sites are provided in our previous studies (Puławska et al., 2025; Puławska et al., 2026).

At each site, samples were collected from two lithologically distinct rock types based on surface texture and mineralogical composition: smooth RS surfaces and rough SC surfaces. RS surfaces consisted predominantly of halite (NaCl) and formed compact, crystalline walls with smooth to slightly polished surfaces resulting from mining activity and prolonged exposure to mine air. These evaporitic rocks formed from the evaporation of Miocene seawater deposits, which formed approximately 13.6 million years ago (de Leeuw et al., 2010). In contrast, SC surfaces were rough and irregular and composed of fine-grained clastic sediments enriched with evaporitic minerals. They consisted mainly of clay minerals (including illite and kaolinite), quartz, halite, anhydrite, and gypsum, with additional contributions from carbonate minerals such as calcite and dolomite. These deposits originated from the accumulation of terrigenous material transported into a marine basin and subsequently mixed with evaporitic phases during sedimentation (Środoń, 1984; Garlicki, 2008). The regional geological setting, stratigraphy and evolution of the Bochnia Salt Mine within the Miocene evaporitic succession of the Carpathian Foredeep have been described in detail elsewhere and are therefore not discussed further here (e.g., Poborski, 1952; Garlicki, 1979). A geological cross-section of the deposit and the distribution of the different rock types at the sampling sites are shown in Figure 1.

Figure 1.

Panel A shows a simplified geological cross-section with colored stratigraphic units and indicates the locations of sampling sites BL-2/BZ-2, BL-3/BZ-3, and BL-4/BZ-4. A stratigraphic column on the right shows lithological layers with salt and claystone intervals. Panel B shows rock layers with visible folding; dashed yellow lines separate rock salt from salty claystone. Panel C shows alternating layers of rock salt and salty claystone outlined by dashed yellow lines. Panel D shows a contact between rock salt above and salty claystone below, separated by a dashed yellow line.

Open in a new tab

Geological setting of the Bochnia salt deposit and lithological context of sampled rock surfaces. (A) Simplified geological cross-section and lithostratigraphic profile of the Bochnia salt deposit showing the structure of the Miocene evaporite succession, modified from Puławska et al. (2021b) and originally based on Poborski (1952). (B–D) Representative photographs of underground mine walls illustrating the spatial association of rock salt (RS) and salty claystone (SC) at the sampling sites (BL-2/BZ-2, BL-3/BZ-3, and BL-4/BZ-4). Yellow dashed lines indicate lithological boundaries between halite-dominated layers and clay-rich evaporitic units.

The microbial samples were collected from the surface area limited by a square metal frame of 5x5cm (25 cm2) by scraping the entire surface using sterile collection swabs with a plastic stick and cotton tip (Medlab products, Raszyn, Poland) and immediately transferred into the 2 mL of liquid Halobacterium medium (HBM; 5 g/L yeast extract, 5 g/L casamino acids, 1 g/L Na-glutamate, 2 g/L KCl, 3 g/L Na3-citrate, 20 g/L MgSO4 x 7 H2O, 36 mg/L FeCl2 x 4 H2O, 360 ng/L MnCl2 x 4 H2O) containing 15, 20% or 25% NaCl and to a tube containing 0.85% NaCl. The samples were transferred from the salt mine to the laboratory within 8 h. At each sampling point samples were collected in triplicate.

2.2. Microbial isolation and culture conditions

One hundred μl of the swab samples in HBM media were plated onto solid HBM containing 20 g/L agar and 15, 20% or 25% NaCl and onto Tryptic-Soya Agar (TSA). The plates were incubated under the humid conditions at three temperatures (37 °C, 28 °C and 21 °C) up to 10 days for TSA plates and up to 3 months for HBM plates. After incubation the numbers of Colony Forming Units (CFU) were counted and expressed as CFU/25cm2 rock surface. Single colonies with red/reddish pigments growing on HBM agar were picked and cultured on fresh HBM agar with the same NaCl concentration as for the initial plates until the pure colonies were obtained. Similarly, colonies growing on TSA were picked and transferred to fresh TSA plates until pure colonies were obtained. The pure isolates were frozen in 50% glycerol and stored at −80 °C until further use.

2.3. Identification of halophilic microorganisms

Genomic DNA was extracted by using the Nielsen method (Nielsen et al., 1995). Briefly, the colonies from the HBM medium containing 15, 20 and 25% of NaCl were collected and suspended in TES buffer, centrifuged, re-suspended in the lysozyme solution (Merck) and incubated for 2 h in 37 °C. After incubation proteinase K was added and the samples were incubated for an additional 1.5 h at 37 °C. After addition of 7.5 M of ammonium acetate and incubation for 10 min on ice, the DNA was extracted by phenol:chloroform and isopropanol precipitation. The DNA pellet was then washed twice with 70% ethanol, air dried at room temperature, resuspended in 50-100 μL of the nuclease-free milliQ water and stored at −20° C. The 16S rRNA gene was amplified by PCR using primers 27F (5’-GAGTTTGATCCTGGCTCAG-3′) and 1525R (5’-AGAAAGGAGGTGATCCAGCC-3′) for bacteria and primers 21F (5’-TTCCGGTTGATCCTGCCGGA-3′) and 1492R (5’-TACGGYTACCTTGTTACG-3′) for archaea. The 16S rRNA PCR products were purified by using NZYGelpure (NZYtech) according to the manufacturer’s protocol and were stored at −20 °C until analyzed. The 16S rRNA gene sequence (~700 bp) was determined by Sanger sequencing (Stab Vida, Portugal) and used for the taxonomic affiliation of each isolate according to the online EzBioCloud database (Yoon et al., 2017). The DDBJ/ENA/GenBank accession numbers for the 16S rRNA gene sequence of strains BL28, BL31, BL35, BL36, BL37, BL38, BZ19, BZ34, BZ28, BZ39, BZ68, BZ1, BZ15, BZ125, BZ70, BZ6, BZ21, BZ27, BZ44, BZ56 and BZ66 are PZ221422, PZ221423, PZ221424, PZ221425, PZ221426, PZ221427, PZ221428, PZ221429, PZ221430, PZ221431, PZ221432, PZ221433, PZ221434, PZ221435, PZ221436, PZ221437, PZ221438, PZ221439, PZ221440, PZ221441 and PZ221442, respectively. For phylogenetic analysis, the 16S rRNA gene sequences were aligned with ClustalW (Thompson et al., 1994), and phylogenetic trees were reconstructed neighbor-joining (NJ) algorithm (Saitou and Nei, 1987) with the Jukes-Cantor model (Jukes and Cantor, 1969) using MEGA (version 11; Tamura et al., 2021). Bootstrap analysis based on 1000 replicates evaluated resulting tree topologies (Felsenstein, 1985).

2.4. Identification and biochemical analysis of non-halophilic microorganisms

Pure isolates of bacteria grown on TSA plates were identified using the VITEK 2 COMPACT system based on colorimetric reactions and analyzed using the Biomerieux database. This was outsourced to the diagnostic laboratory “Mikrografia” in Krakow. The bacteria were divided into three groups: non-sporulating GP (Gram-positive), BCL (Gram-positive spore-inducing bacilli), and GN (Gram-negative). In addition, the VITEK 2 system allowed us to characterize enzymatic activity, sugar metabolism and antibiotic resistance of the isolated bacteria.

2.5. Statistical analyses

Statistical analyses were performed with the GraphPad Prism 7 and STATISTICA 12.0 PL program. Data are expressed as means ± SD or medians ± interquartile range. Differences between samples were analyzed by the analysis of variance Kruskal-Wallis non-parametric test and Mann–Whitney U test. p values ≤0.05 were considered significant.

3. Results

3.1. Microbial sampling at different sites in the Bochnia Salt Mine

Microbial samples were taken in July 2022 and in February 2023 from three different sites of the Bochnia Salt Mine: BL-2/BZ-2, an underground tunnel, 1,185 m from the mine entry and 212 m below the ground surface; BL-3/BZ-3, the Wazyn chamber, 248 m deep and 1728 m from mine entry; and BL-4/BZ-4, 230 m deep and 2,671 m from mine entrance. These were the same sites as those described in our previous study and sampled for airborne microorganisms on the same days in the summer (Puławska et al., 2025) and winter (Puławska et al., 2026).

At each site, both RS and SC surfaces were sampled, as they represented the dominant lithologies exposed at the mine walls (Figure 1). 25 cm2 surfaces from each rock type of each sample site were swabbed and analyzed for the presence of halophilic and non-halophilic microorganisms grown on HBM containing either 15, 20% or 25% NaCl and TSA agar plates, respectively, at 21 °C, 28 °C or 37 °C. At each sampling sites, both the SC and RS surfaces were populated with live halophilic and non-halophilic microorganisms at roughly equal proportions between SC and RS surfaces (Figure 2). The most pronounced differences between SC and RS were found for the halophiles in BL-2 in the summer time (p < 0.0001; Figure 2A) and for the non-halophilic microorganisms in BZ-3 in the winter (p = 0.0137; Figure 2B), where the SC surfaces harbored more microorganisms than the RS surfaces. However, when all three sites were taken together, there was a significant difference between the number of halophiles present on the SC and RS surfaces in the summer (p = 0.0253; Figure 2C). No significance was seen between the number of halophiles present on the SC and RS surfaces in the winter (Figure 2C) and for non-halophilic microorganisms for the SC and RS surfaces in the winter and summer seasons (Figures 2C,D).

Figure 2.

Four bar graphs display CFU per twenty-five square centimeters on HBM and TSA agar plates for RS and SC samples across different locations and seasons. Significant differences are indicated with P-values, and bars show mean and standard deviation.

Open in a new tab

Presence of halophilic and non-halophilic microorganisms on the surface of rock salt (RS) and salty claystone (SC) at the three sampling sites. Colonies of halophilic and non-halophilic microorganisms collected from the swabs from the surface of the rocks at the indicated sampling sites in the summer and winter seasons and grown on HBM (panel A,C) or TSA (panel B,D) agar were counted after 3 or 1 month, respectively. The values shown in panel A represent the average CFU counts +/− standard deviations grown at HBM medium all salinities (15%, 20%, 25%) and all temperatures (room temperature 21 °C, 28 °C, 37 °C) together with three samples counted for each condition in each sampling sites in the summer time (BL-2–BL-4) and in the winter time (BZ-2–BZ-4). The values shown in panel B represent the average CFU counts +/− standard deviations grown at TSA medium at all temperatures together (21 °C, 28 °C, 37 °C) with three samples counted for each condition in each sampling sites in the summer time (BL-2–BL-4) and in the winter time (BZ-2–BZ-4). The values shown in panel C represent the average CFU counts +/− standard deviations grown at HBM medium all salinities (15, 20, 25%), all temperatures (21 °C, 28 °C, 37 °C), and all sampling sites (BL-2–BL-4) in the summer and (BZ-2–BZ-4) in the winter time, together with three samples counted for each condition. Values in panel D represent the average CFU counts +/− standard deviations grown on TSA agar all temperatures (21 °C, 28 °C, 37 °C) and all sampling sites (BL-2–BL-4) in the summer and (BZ-2–BZ-4) in the winter, together with three samples counted for each condition.

3.2. Microbial growth at different temperatures

When the collected microbial communities were deconvoluted by growth temperature, no important differences between SC and RS surfaces were found for halophiles and non-halophilic microorganisms grown at room temperature (Figures 3A,B). Some differences between the two types of rock were found after growth at 28 °C and at 37 °C. In BL-2 SC surfaces contained more of either type of microorganisms than RS surfaces that grew at 28 °C (p < 0.0001 and p = 0.0238, Figures 3C,D, respectively), while in BL-4 the RS surface contained more halophiles than SC surface (p < 0.0001; Figure 3C). Significant differences between rock types were also found among microorganisms isolated in the summer that grew at 37 °C, especially in BL-2 for halophiles and BL4 for non-halophilic microorganisms (p < 0.0001 and p = 0.0022, Figures 3E,F, respectively), where SC surfaces contained more microorganisms than RS surfaces. However, when taken together for each temperature, differences were at best minor.

Figure 3.

Six grouped bar graphs labeled panels A through F, each depicting colony-forming units per 25 square centimeters (CFU/25cm²) on the y-axis and different sampling sites (BL-2, BL-3, BL-4 for summer; BLZ2, BLZ3, BLZ4 for winter) on the x-axis. Individual panels compare seasonal and temperature effects on microbial CFU found in room surfaces (RS) and supply ceilings (SC) for two media: HBM (panels A, C, E at room temperature, 28°C, 37°C respectively) and TSA (panels B, D, F at room temperature, 28°C, 37°C respectively). Error bars indicate variation and p-values. Insets in each panel show aggregated seasonal comparisons.

Open in a new tab

Presence of halophilic and non-halophilic microorganisms in the swabs from the rock salt (RS) and salty claystone (SC) at the three sampling sites in the summer and winter seasons. Colonies of halophilic microorganisms collected from the surface of RS and SC rocks at the indicated sampling sites and grown on HBM agar containing 15%, 20%, or 25% NaCl were counted after 3 months incubation at 21 °C, 28 °C, or 37 °C. The values shown in panel (A) represent the average CFU counts +/− standard deviations grown at all salinities at room temperature (RT; panel (A)), 28 °C (panel (C)), or 37 °C (panel (E)) with three samples counted for each condition. Values in panels (B–F) represent the average CFU counts +/− standard deviations of collected from the surface of RS and SC rocks at the indicated sampling sites non-halophilic microorganisms grown on TSA medium up to 10 days at room temperature (RT; panel (B)), 28 °C (panel (D)), or 37 °C (panel (F)) with three samples counted for each condition. Little squares incorporated in the upper right corner of the figure at each panel show the total numbers of colonies of halophilic and non-halophilic microorganisms grown on HBM (panel (A,C,E)) and TSA (panel (B,D,F)), respectively isolated from the surface of RS and SC rocks.

3.3. Halophile growth at different salinities

SC and RS surfaces were also compared for the presence of halophiles grown at different salinities. In the summer, in BL-2 almost all halophiles grown at either tested salinity came from the SC surfaces, while in BL-3 and BL-4 similar numbers of halophiles grown at either salinity were collected from both SC and RS surfaces (Figure 4A). Similarly, in the winter, no striking differences were seen between the numbers of halophiles collected from SC or RS surfaces of either sampling site after growth at either temperature (Figure 4B).

Figure 4.

Bar graphs comparing CFU per twenty-five square centimeters on different block surfaces in summer (panel A) and winter (panel B) for RS (open bars) and SC (striped bars) at fifteen, twenty, and twenty-five percent conditions. Significant differences between RS and SC are indicated for some groups, with p-values shown above relevant bars. Error bars indicate standard deviation.

Open in a new tab

Presence of halophilic microorganisms isolated from the surface of RS and SC rocks in the summer season (panel A) and in the winter season (panel B) at the three sampling sites (BL-2 to BL-4) and (BZ-2 to BZ-4), respectively. HBM agar containing 15%, 20%, or 25% NaCl were counted after 3 months incubation at 21 °C, 28 °C, or 37 °C. The values shown represent the average CFU counts +/− standard deviations at all temperatures together, with three samples counted for each condition.

3.4. Comparison between the number of halophiles and non-halophilic microorganisms

When the amounts of halophiles and non-halophilic microorganisms collected from the rock surfaces of the three sampling sites were compared, up to more than a hundred-fold more halophiles were collected over non-halophilic microorganisms at almost each sampling site (p < 0.0001 for all differences). The exception was BZ-3, where roughly equal amounts of either microorganism were collected (Figure 5A). When taken together, in both the summer and the winter season, more than 10-times more live halophiles were collected from the rock surfaces than non-halophilic microorganisms (p < 0.0001; Figure 5B). When summer was compared to winter, significantly more microorganisms were collected from the rock surfaces in the summer than in the winter (p < 0.0001). However, these differences did not exceed a factor of two.

Figure 5.

Bar graph with two panels labeled A and B comparing CFU per 25 square centimeters for TSA (black bars) and HBM (gray bars) under summer and winter conditions. Panel A displays data for BL-2, BL-3, BL-4 (summer) and BZ-2, BZ-3, BZ-4 (winter), with HBM showing significantly higher CFU values than TSA in all cases; statistical significance is indicated with p-values of 0.0001. Panel B summarizes CFU for TSA and HBM in each season, again showing higher values for HBM with all comparisons marked as statistically significant (p=0.0001). Error bars indicate standard deviations.

Open in a new tab

Total number of halophilic and non-halophilic microorganisms isolated from the surface of the rocks in the summer and winter seasons at the four sampling sites in the summer time (BL-2–BL-4) and in the winter time (BZ-2–BZ-4; panel A) or isolated from all sampling sites together (panel B). Colonies of halophilic microorganisms collected from the surface of the rocks at the indicated sampling sites and grown on HBM agar containing 15%, 20%, or 25% NaCl were counted after 3 months incubation at 21 °C, 28 °C, or 37 °C, while non-halophilic microorganisms were growing on TSA agar and counted after 10 days of incubation at 21 °C, 28 °C, or 37 °C. The values shown in panel A and B represent the average CFU counts +/− standard deviations with three samples counted for each condition.

3.5. Species identification of halophilic microorganisms

The halophiles collected from the rock surfaces were mostly halophilic archaea. We therefore determined their species identity, found 11 different species (Table 1), established a similarity matrix (Supplementary Table S1) and constructed a phylogenetic tree (Supplementary Figure S1). Five species of archaea were isolated from both SC and RS surfaces, three only from the RS surfaces, and three only from the SC surfaces. Among the archaeal taxa detected, members of the Halococcus genus were dominant, including Halococcus salifodinae, Halococcus hamelinensis, Halococcus morrhuae and Halococcus dombrowskii, together with Natrinema versiforme and Halalkalicoccus paucihalophilus. Haloarcula hispanica, Halococcus sedimicola and Halorubrum amylolyticum were detected only on RS surfaces, whereas Halococcus qingdaonensis and Haloarcula marismortui were detected exclusively on SC surfaces. Although much less abundant than archaea, we could nevertheless identify 10 different halophilic bacterial species (Table 2), established a similarity matrix (Supplementary Table S2) and constructed a phylogenetic tree (Supplementary Figure S2). Four of them came from both types of rocks, while 3 only from the RS surfaces and 3 only from the SC surfaces. Among non-halophilic microorganisms, most were bacteria, and only very few live fungi were detected on the rock surfaces at either season. The bacteria were characterized by the VITEK 2 system (Supplementary Table S3), with a confidence level for all strains of >90% (except for Lysinibacillus sphaericus, for which the confidence level was 86%), but were not further studied here.

Table 1.

Halophilic archeal species isolated from the rock surfaces of the Bochnia Salt Mine.

Halophilic species RS1 SC2 Accession number % identity3
Halococcus salifodinae DSM 8989T + + AOME01000075 99.14
Haloarcula hispanica ATCC 33960T + − CP002922 98.52
Halococcus hamelinensis 100A6T + + AOMB01000011 99.76
Halococcus qingdaonensis CM5T − + AY243109 99.68
Halococcus morrhuae DSM 1037T + + AOMC01000054 99.48
Haloarcula marismortui ATCC 43049T − + AY596298 99.19
Halococcus sediminicola CBA1101T + − BBMP01000022 99.76
Natrinema versiforme XF10T + + AB023426 98.65
Halorubrum amylolyticum ZC67T + − KX376720 99.45
Halalkalicoccus paucihalophilus DSM 24557T + − LTAZ01000003 99.22
Halococcus dombrowskii H4T − + AJ420376 99.87
Open in a new tab
1

RS, rock salt.

2

SC, salty claystone.

3

Percent of 16S rRNA gene identity with the closest species with validly published names using the online EzBioCloud database (Yoon et al., 2017).

+, isolated, −, not isolated.

Table 2.

Halophilic bacterial species isolated from the rock surfaces of the Bochnia Salt Mine.

Halophilic species RS1 SC2 Accession number % identity3
Lentibacillus persicus AMB31T + + FN376846 99.90
Chromohalobacter canadensis ATCC 43984T + − AJ295143 98.17
Lentibacillus amyloliquefaciens LAM0015T + + CP013862 99.90
Piscibacillus halophilus HS224T + + FM864227 99.69
Virgibacillus kapii KN3-8-4T − + LC041942 100
Alkalibacillus halophilus YIM 012T + + DQ359731 99.90
Gracilibacillus thailandensis TP2-8T + − FJ182214 100
Marinococcus halotolerans NBRC 106070T + − AB682353 99.74
Chromohalobacter beijerinckii ATCC 19372T − + AB021386 98.59
Salibacterium qingdaonense CM1T − + DQ115802 99.80
Open in a new tab
1

RS, rock salt.

2

SC, salty claystone.

3

Percent of 16S rRNA gene identity with the closest species with validly published names using the online EzBioCloud database (Yoon et al., 2017).

+, isolated, −, not isolated.

4. Discussion

4.1. Salt rock surfaces as microbial reservoirs in subterranean saline environments

In this study, we show that exposed salt rock surfaces in the underground Bochnia Salt Mine host abundant and diverse communities of viable halophilic microorganisms, dominated by halophilic archaea. Across all sampled sites and seasons, halophilic archaea were recovered from rock surfaces at approximately 100-fold higher densities than non-halophilic microorganisms, both in summer and winter.

This striking dominance contrasts sharply with airborne microbial communities from the same sites, where non-halophilic bacteria predominated during the winter season, except at the intermediate underground site BZ-2, which is distant from intensive human activity (Puławska et al., 2025; Puławska et al., 2026). A key difference between rock-associated and airborne halophilic communities lies in their seasonal dynamics. While airborne halophilic archaea exhibited pronounced seasonality, with densities up to 13-fold higher in summer than in winter (Puławska et al., 2026), seasonal differences on rock surfaces did not exceed a factor of two.

Although exposed rock surfaces do not provide the long-term environmental buffering characteristic of endolithic or subsurface habitats (Robinson et al., 2015; Meslier et al., 2018; Azua-Bustos et al., 2020; Casero et al., 2021), our results indicate that salt rock surfaces are a more biologically favorable habitat for halophilic archaea than the surrounding mine air. In the Bochnia Salt Mine, relative humidity ranged from approximately 65–77% in summer and 42–63% in winter, conditions that strongly modulate the persistence of saline aerosol microhabitats (Puławska et al., 2025; Puławska et al., 2026). Together, these observations support the interpretation that salt rocks function as spatially stable biological reservoirs for halophilic archaea, whereas the atmosphere appears to be a transient, environmentally filtered compartment that that may receive only a subset of the rock-associated community.

4.2. Salt rock surfaces as a geomicrobiological habitat at the lithosphere–atmosphere interface

Most studies investigating halophilic microorganisms associated with evaporitic rocks have focused on endolithic communities and typically rely on drilling, coring, or mechanical disruption of rock material to access microorganisms residing within the rock interior (e.g., Cycil et al., 2020; Megaw et al., 2019; Gramain et al., 2011; Pecher et al., 2020; Cockell et al., 2020; DasSarma et al., 2021; Schreder-Gomes et al., 2022). In contrast, the surface swabbing approach used in this study specifically targeted exposed rock surfaces and the rock–air interface, which remain comparatively underexplored despite their direct relevance to microbial aerosolization. By focusing on surface-associated communities, our data provide complementary insight into the ecological link between halite-rich substrates and airborne halophilic microorganisms in subterranean saline environments.

Rock surfaces in subterranean salt mines should be regarded as active geomicrobiological habitats rather than inert substrates. Numerous studies have demonstrated that exposed rock surfaces host structured subaerial biofilms (SABs) that occupy the dynamic interface between the lithosphere and the atmosphere, where microbial survival is governed by mineral composition, surface roughness, porosity and microtopography, as well as by fluctuations in humidity and temperature (Gorbushina, 2007; Gorbushina and Broughton, 2009; Ronan et al., 2013; Uroz et al., 2015; Stone et al., 2016; Cuadros, 2017; Meslier et al., 2018; Wierzchos et al., 2018; Fomina and Skorochod, 2020; Casero et al., 2021). These biofilms are highly organized microbial systems that persist under extreme physicochemical stress, including desiccation, nutrient limitation, and high salinity (Azua-Bustos et al., 2020; Dopffel et al., 2021; Medina-Chávez et al., 2023). In saline environments, such as salt mines, these interactions are further modulated by the hygroscopic properties of halite and associated evaporitic minerals, which promote capillary condensation and episodic deliquescence, generating microscale aqueous niches even when bulk air humidity remains below saturation (Davila et al., 2008; Wierzchos et al., 2012; Robinson et al., 2015; Puławska et al., 2021a).

Although RS and SC differ in mineralogical composition and surface structure, both rock types contain substantial amounts of halite and provide physicochemical microenvironments that retain moisture through hygroscopicity. Such microscale brine films and associated biofilms are known to protect microbial cells against rapid desiccation and oxidative stress (Davila et al., 2008; Casero et al., 2021; Gorbushina and Broughton, 2009). While surface habitats remain linked to atmospheric humidity, the hygroscopic nature of the substrate likely attenuates the impact of short-term fluctuations compared to the airborne state, supporting the persistence of rock-associated halophilic communities (Wierzchos et al., 2012; Azua-Bustos et al., 2020). Importantly, this rock-surface-associated mode of persistence contrasts with the transient nature of the mine atmosphere, where microbial survival is strongly constrained by humidity-dependent aerosol formation. While elevated relative humidity promotes halite deliquescence and facilitates the aerosolization and dispersal of halophilic microorganisms into the air (Puławska et al., 2026), the rock surface itself represents a far more stable physicochemical compartment. In addition to deliquescence and salt hygroscopy, physical processes may also contribute to the mobilization of microorganisms from rock surfaces into the mine air. Previous aerosol studies in the Bochnia Salt Mine identified geogenic airborne particles derived from weathering and abrasion of rock surfaces, indicating that physical mobilization of salt-derived material occurs in this environment (Puławska et al., 2021b; Puławska et al., 2021a). Ventilation-driven airflow and local turbulence may therefore facilitate the detachment of cell-bearing salt microfragments from moist surface crusts. In contrast, human activity appears to be associated mainly with transient inputs of non-halophilic microorganisms (Puławska et al., 2025; Puławska et al., 2026). In this context, salt rocks function not merely as passive substrates but as long-term microbial reservoirs that sustain halophilic populations and may supply cells to the atmosphere when microclimatic conditions become favorable. This conceptual separation between rocks as stable source habitats and air as a dynamic, environmentally filtered compartment provides a plausible ecological framework linking lithic colonization to the observed patterns of airborne halophilic archaea in the Bochnia Salt Mine.

4.3. Taxonomic continuity between rock-associated and airborne communities

The taxonomic composition of halophilic archaea isolated from rock provides qualitative support for the hypothesis that salt rocks represent an important reservoir, and a likely source habitat, for airborne halophiles in the Bochnia Salt Mine. A total of 11 halophilic archaeal species were identified on rock surfaces, 6 of which, mainly representatives of the genus Halococcus (Hcc. salifodinae, Hcc. hamelinensis, Hcc. morrhuae, and Hcc. dombrowskii), as well as Nnm. versiforme and Hac. paucihalophilus, had previously been detected in airborne samples from the mine (Puławska et al., 2025; Puławska et al., 2026). This extensive overlap (Figure 6A) is consistent with the view that the airborne halophilic community may largely represent a subset of the more diverse rock-associated assemblage.

Figure 6.

Two Venn diagrams labeled A and B compare microbial species found in air, rock, and both environments. Diagram A lists Halalkalicoccus subterraneus GSM28T exclusively in air, several Halalkalicoccus and Halococcus species in both, and Haloarcula and Halorubrum species exclusively in rock. Diagram B shows Alkalibacillus and Salinicoccus species unique to air, several Bacillus and Chromohalobacter species unique to rock, and multiple species found in both habitats.

Open in a new tab

Venn diagram showing the presence of halophilic archaea (A) and bacteria (B) detected in the air and/or rock surfaces, as indicated.

At the same time, several halophilic archaeal species identified on rock surfaces were absent from airborne samples, suggesting selective constraints on aerosolization and atmospheric persistence. Such asymmetry is consistent with the view that the mine atmosphere acts as an ecological filter, allowing only a fraction of the rock-resident community to enter and survive in the aerial compartment. Similar habitat-specific partitioning has been described for halophilic archaea inhabiting halite and other evaporitic substrates, where endolithic or surface-associated populations develop distinct community structures compared with those found in surrounding environments, such as soils (Robinson et al., 2015; Azua-Bustos et al., 2020).

Notably, the dominance of Halococcus species among both rock-associated and airborne halophiles is ecologically coherent, as members of this genus are well known for their exceptional resistance to desiccation and prolonged nutrient limitation thanks to their rigid polysaccharide cell walls, traits that favor survival at the rock–air interface and during atmospheric transport (Stan-Lotter et al., 1999; Fendrihan et al., 2009; Oren, 2012). In addition, Halococcus species exhibit pronounced resistance to oxidative stress and radiation, as demonstrated under simulated space and extraterrestrial conditions (Leuko et al., 2020), further supporting their capacity to persist during aerosolisation and airborne dispersal.

In addition to halophilic archaea, halophilic bacteria also exhibited pronounced taxonomic continuity between rock-associated and airborne communities. Among the 10 halophilic bacterial species identified on rock surfaces, six were likewise detected in airborne samples (Figure 6B), indicating that halophilic bacteria, together with archaea, may participate in microbial connectivity between rock surfaces and air within the mine system. In particular, taxa such as Marinococcus halotolerans, Gracilibacillus thailandensis, Chromohalobacter beijerinckii and Salibacterium qingdaonense were restricted to rock surfaces, whereas other halophilic bacterial species were shared between rock-associated and airborne communities.

Despite species-level differences between rock-associated and airborne communities, the overall abundance of halophiles did not differ substantially between RS and SC surfaces. While RS and SC differ in mineralogical composition and surface microstructure, RS forming relatively smooth crystalline surfaces and SC exhibiting higher roughness and microtopographic heterogeneity, these differences did not translate into major quantitative contrasts in halophilic microbiota abundance. This suggests that, under the saline underground conditions of the Bochnia Salt Mine, the presence of halite and its hygroscopic behavior appear to represent dominant environmental features associated with the persistence of halophilic microbiota across different rock types.

Nevertheless, the occurrence of certain archaeal taxa exclusively on RS or SC surfaces suggests that lithological and microstructural heterogeneity may shape community composition at the species level. In this context, Har. hispanica, Hcc. sedimicola and Hrr. amylolyticum, which were recovered only from RS surfaces, may be adapted to chemically more homogeneous but highly saline substrates, whereas Hcc. qingdaonensis and Har. marismortui, detected exclusively on SC surfaces, may benefit from access to a more mineralogically diverse matrix. Such lithology-associated effects appear secondary to halite availability and do not override the likely importance of rock surfaces as long-term reservoirs of halophilic archaea potentially contributing to airborne populations under favorable microclimatic conditions.

4.4. Limitations

While our results provide convergent quantitative and qualitative evidence consistent with the hypothesis that salt rock surfaces act as important reservoirs for airborne halophilic archaea in the Bochnia Salt Mine, several limitations of the present study should be acknowledged. In particular, our analyses were restricted to culturable microorganisms inhabiting exposed rock surfaces and did not address non-culturable taxa or deeper endolithic communities within the rock matrix. Accordingly, the present study does not capture the full microbial diversity associated with the rock surfaces or the mine atmosphere, and the conclusions should be interpreted as referring specifically to the culturable fraction. Moreover, although surface swabbing enabled direct investigation of the rock–air interface most relevant for aerosolization, it does not resolve whether the detected microorganisms represent actively growing populations or long-term dormant assemblages intermittently reactivated by microclimatic changes. Moreover, the observed taxonomic overlap between rock-associated and airborne communities supports, but does not directly demonstrate, the proposed role of rock surfaces as contributors to airborne halophilic microorganisms. These considerations highlight important avenues for future research and frame the scope of the conclusions drawn here.

4.5. Concluding remarks

In summary, this study indicates that exposed salt rock surfaces in the Bochnia Salt Mine constitute stable, long-term reservoirs of viable culturable halophilic archaea, whereas the mine atmosphere represents a transient and environmentally filtered compartment. By combining surface-based cultivation with seasonal sampling, we show that airborne halophilic communities overlap substantially with rock-associated populations and are consistent with the hypothesis that rock surfaces contribute to airborne communities under favorable microclimatic conditions. These findings provide a plausible ecological framework linking lithic microbial persistence to aerosolized microbiota in subterranean salt environments. Beyond their ecological significance, our results highlight the importance of integrating geomicrobiology with microclimatic monitoring to better understand microbial dynamics in salt mines used for recreational and therapeutic purposes.

Acknowledgments

We thank the geologist Michal Flasza for co-organizing the sampling expedition at the Bochnia Salt Mine. For the purpose of Open Access, the author has applied a CC-BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This research was funded by the National Science Centre, Poland under research project number 2021/41/N/ST10/02751 given to AP and partly by ULAM project funded by NAWA granted to CL (Agreement Number: BPN/ULM/2023/1/00090/U/00001).

Footnotes

Edited by: Afef Najjari, Tunis El Manar University, Tunisia

Reviewed by: Mechri Sondes, Centre of Biotechnology of Sfax, Tunisia

Fevziye Işıl Kesbiç, Kastamonu University, Türkiye

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Author contributions

MK-K: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing, Supervision. GA: Investigation, Writing – review & editing. MM: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. DD: Investigation, Writing – review & editing. MR: Investigation, Writing – review & editing. KS: Formal analysis, Visualization, Writing – review & editing. LA: Formal analysis, Resources, Writing – review & editing. CE: Resources, Writing – review & editing. KK: Visualization, Writing – review & editing. CL: Formal analysis, Funding acquisition, Writing – original draft, Writing – review & editing. AP: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Visualization, Writing – original draft, Writing – review & editing, Supervision.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2026.1813537/full#supplementary-material

Data_Sheet_1.PDF (195KB, PDF)
Table_1.XLSX (21KB, XLSX)
Table_2.XLSX (20.1KB, XLSX)
Table_3.DOCX (58.8KB, DOCX)

References

  1. Azua-Bustos A., Fairén A. G., González-Silva C., Ascaso C., Carrizo D., Fernández-Martínez M. A., et al. (2020). Inhabited subsurface wet smectites in the hyperarid core of the Atacama Desert as an analog for the search for life on Mars. Sci. Rep. 10:19183. doi: 10.1038/s41598-020-76302-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Casero M. C., Meslier V., DiRuggiero J., Quesada A., Ascaso C., Artieda O., et al. (2021). The composition of endolithic communities in gypcrete is determined by the specific microhabitat architecture. Biogeosciences 18, 993–1007. doi: 10.5194/bg-18-993-2021 [DOI] [Google Scholar]
  3. Cockell C. S., Wilhelm M. B., Perl S., Wadsworth J., Payler S., McMahon S., et al. (2020). 0.25 Ga salt deposits preserve signatures of habitable conditions and ancient lipids. Astrobiology 20, 864–877. doi: 10.1089/ast.2019.2053, [DOI] [PubMed] [Google Scholar]
  4. Cuadros J. (2017). Clay minerals interaction with microorganisms: a review. Clay Miner. 52, 235–261. doi: 10.1180/claymin.2017.052.2.05 [DOI] [Google Scholar]
  5. Cycil L. M., DasSarma S., Pecher W., McDonald R., AbdulSalam M., Hasan F. (2020). Metagenomic insights into the diversity of halophilic microorganisms indigenous to the Karak salt mine, Pakistan. Front. Microbiol. 11:1567. doi: 10.3389/fmicb.2020.01567, [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DasSarma P., Anton B. P., DasSarma S. L., von Ehrenheim H. A. L., Martinez F. L., Guzmán D., et al. (2021). Genome sequence and methylation pattern of Haloterrigena salifodinae BOL5-1, an extremely halophilic archaeon from a Bolivian salt mine. Microbiol. Resour. Announc. 10:e00275-21. doi: 10.1128/mra.00275-21, [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davila A. F., Gomez-Silva B., de los Rios A., Ascaso C., Olivares H., McKay C. P., et al. (2008). Facilitation of endolithic microbial survival in the hyperarid core of the Atacama Desert by mineral deliquescence. J. Geophys. Res. 113:G01028. doi: 10.1029/2007JG000561 [DOI] [Google Scholar]
  8. de Leeuw A., Bukowski K., Krijgsman W., Kuiper K. F. (2010). Age of the Badenian salinity crisis; impact of Miocene climate variability on the circum-Mediterranean region. Geology 38, 715–718. doi: 10.1130/G30982.1 [DOI] [Google Scholar]
  9. Dopffel N., An-Stepec B. A., Bombach P., Wagner M., Passaris E. (2024). Microbial life in salt caverns and their influence on H2 storage – current knowledge and open questions. Int. J. Hydrog. Energy 58, 1478–1485. doi: 10.1016/j.ijhydene.2024.01.334 [DOI] [Google Scholar]
  10. Dopffel N., Jansen S., Gerritse J. (2021). Microbial side effects of underground hydrogen storage – knowledge gaps, risks and opportunities for successful implementation. Int. J. Hydrog. Energy 46, 8594–8606. doi: 10.1016/j.ijhydene.2020.12.058 [DOI] [Google Scholar]
  11. Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783–791. [DOI] [PubMed] [Google Scholar]
  12. Fendrihan S., Bérces A., Lammer H., Musso M., Rontó G., Polacsek T. K., et al. (2009). Investigating the effects of simulated Martian ultraviolet radiation on Halococcus dombrowskii and other extremely halophilic archaebacteria. Astrobiology 9, 104–112. doi: 10.1089/ast.2007.0234, [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fomina M., Skorochod I. (2020). Microbial interaction with clay minerals and its environmental and biotechnological implications. Minerals 10:861. doi: 10.3390/min10100861 [DOI] [Google Scholar]
  14. Garlicki A. (1979). Sedimentation of Miocene salts in Poland. Pr. Geol. PAN 119, 1–67. [Google Scholar]
  15. Garlicki A. (2008). Salt Mines at Bochnia and Wieliczka. Przegl. Geol. 56, 663–669. [Google Scholar]
  16. Gorbushina A. A. (2007). Life on the rocks. Environ. Microbiol. 9, 1613–1631. doi: 10.1111/j.1462-2920.2007.01301.x, [DOI] [PubMed] [Google Scholar]
  17. Gorbushina A. A., Broughton W. J. (2009). Microbiology of the atmosphere-rock interface: how biological interactions and physical stresses modulate a sophisticated microbial rcosystem. Ann. Rev. Microbiol. 63, 431–450. doi: 10.1146/annurev.micro.091208.073349, [DOI] [PubMed] [Google Scholar]
  18. Gramain A., Díaz G. C., Demergasso C., Lowenstein T. K., McGenity T. J. (2011). Archaeal diversity along a subterranean salt core from the Salar Grande (Chile). Environ. Microbiol. 13, 2105–2121. doi: 10.1111/j.1462-2920.2011.02435.x, [DOI] [PubMed] [Google Scholar]
  19. Jaakkola S. T., Ravantti J. J., Oksanen H. M., Bamford D. H. (2016). Buried alive: microbes from ancient halite. Trends Microbiol. 24, 148–160. doi: 10.1016/j.tim.2015.12.002, [DOI] [PubMed] [Google Scholar]
  20. Jukes T. H., Cantor C. R. (1969). “Evolution of protein molecules,” in Mammalian protein metabolism, ed. Munro H. N. (New York: Academic Press; ), 21–132. [Google Scholar]
  21. Kowalewicz-Kulbat M., Krawczyk K. T., Szulc-Kielbik I., Rykowski S., Denel-Bobrowska M., Olejniczak A. B., et al. (2023). Cytotoxic effects of halophilic archaea metabolites on ovarian cancer cell lines. Microb. Cell Factories 22:197. doi: 10.1186/s12934-023-02206-y, [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leuko S., Stan-Lotter H., Lamers G., Sjöström S., Rabbow E., Parpart A., et al. (2020). “Resistance of the archaeon Halococcus morrhuae and the biofilm-forming bacterium Halomonas muralis to exposure to low-earth orbit for 534 days,” in Extremophiles as Astrobiological Models, eds. Seckbach J., Stan-Lotter H. (Hoboken: Wiley-Scrivener; ), 221–236. [Google Scholar]
  23. Lowenstein T. K., Schubert B. A., Timofeeff M. N. (2011). Microbial communities in fluid inclusions and long-term survival in halite. GSA Today 21, 4–9. doi: 10.1130/GSATG81A.1 [DOI] [Google Scholar]
  24. McGenity T. J., Gemmell R. T., Grant W. D., Stan-Lotter H. (2001). Origins of halophilic microorganisms in ancient salt deposits. Environ. Microbiol. 2, 243–250. doi: 10.1046/j.1462-2920.2000.00105.x, [DOI] [PubMed] [Google Scholar]
  25. Medina-Chávez N. O., Torres-Cerda A., Chacón J. M., Harcombe W. R., De la Torre-Zavala S., Travisano M. (2023). Disentangling a metabolic cross-feeding in a halophilic archaea-bacteria consortium. Front. Microbiol. 14:1276438. doi: 10.3389/fmicb.2023.1276438, [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Megaw J., Kelly S. A., Thompson T. P., Skvortsov T., Gilmore B. F. (2019). Profiling the microbial community of a Triassic halite deposit in Northern Ireland: an environment with significant potential for biodiscovery. FEMS Microbiol. Lett. 366:fnz242. doi: 10.1093/femsle/fnz242, [DOI] [PubMed] [Google Scholar]
  27. Meslier V., Casero M. C., Dailey M., Wierzchos J., Ascaso C., Artieda O., et al. (2018). Fundamental drivers for endolithic microbial community assemblies in the hyperarid Atacama Desert. Environ. Microbiol. 20, 1765–1781. doi: 10.1111/1462-2920.14106, [DOI] [PubMed] [Google Scholar]
  28. Moopantakath J., Imchen M., Anju V. T., Busi S., Dyavaiah M., Martínez-Espinosa R. M., et al. (2023). Bioactive molecules from haloarchaea: scope and prospects for industrial and therapeutic applications. Front. Microbiol. 14:1113540. doi: 10.3389/fmicb.2023.1113540, [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nielsen P., Fritze D., Priest F. (1995). Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology 141, 1745–1761. [Google Scholar]
  30. Onstott T. C., Ehlmann B. L., Sapers H., Coleman M., Ivarsson M., Marlow, et al. (2019). Paleo-rock-hosted life on earth and the search on Mars: a review and strategy for exploration. Astrobiology 19, 1230–1262. doi: 10.1089/ast.2018.1960 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Oren A. (2012). “Approaches toward the study of halophilic microorganisms in their natural environments: who are they and what are they doing?” in Advances in Understanding the Biology of Halophilic Microorganisms, ed. Vreeland R. H. (Dordrecht: Springer; ), 1–33. [Google Scholar]
  32. Pecher W. T., Martínez F. L., DasSarma P., Guzmán D., DasSarma S. (2020). 16S rRNA gene diversity in ancient gray and pink salt from san Simón salt mines in Tarija, Bolivia. Microbiol. Resour. Announc. 9:820-20. doi: 10.1128/mra.00820-20, [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Poborski J. (1952). The Bochnia salt deposit on the geological background of region. Biul. Państw. Inst. Geol. 78, 3–160. [Google Scholar]
  34. Puławska A., Kalinowska J., Rachubik M., Drzewiecka D., Albuquerque L., Egas C., et al. (2025). Halophilic and non-halophilic microbial communities in relation to physico-chemical characteristics of salt mine air. Environ. Microbiol. Rep. 17:e70095. doi: 10.1111/1758-2229.70095, [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Puławska A., Manecki M., Flasza M. (2021b). Mineralogical and chemical tracing of dust variation in an underground historic salt mine. Minerals 11:686. doi: 10.3390/min11070686 [DOI] [Google Scholar]
  36. Puławska A., Manecki M., Flasza M., Styszko K. (2021a). Origin, distribution, and perspective health benefits of particulate matter in the air of underground salt mine: a case study from Bochnia, Poland. Environ. Geochem. Health 43, 3533–3556. doi: 10.1007/s10653-021-00832-2, [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Puławska A., Rachubik M., Drzewiecka D., Albuquerque L., Egas C., Kalinowska J., et al. (2026). Airborne halophilic and non-halophilic microbial communities in an underground salt mine affected by seasonal environmental fluctuations. Environ. Microbiol. Rep. 18:e70293. doi: 10.1111/1758-2229.70293, [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Robinson C. K., Wierzchos J., Black C., Crits-Christoph A., Ma B., Ravel J., et al. (2015). Microbial diversity and the presence of algae in halite endolithic communities are correlated to atmospheric moisture in the hyper-arid zone of the Atacama Desert. Environ. Microbiol. 17, 299–315. doi: 10.1111/1462-2920.12364, [DOI] [PubMed] [Google Scholar]
  39. Ronan E., Yeung C. W., Hausner M., Wolfaardt G. M. (2013). Interspecies interaction extends bacterial survival at solid–air interfaces. Biofouling 29, 1087–1096. doi: 10.1080/08927014.2013.829820, [DOI] [PubMed] [Google Scholar]
  40. Saitou N., Nei M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. doi: 10.1093/oxfordjournals.molbev.a040454 [DOI] [PubMed] [Google Scholar]
  41. Schreder-Gomes S. I., Benison K. C., Bernau J. A. (2022). 830-million-year-old microorganisms in primary fluid inclusions in halite. Geology 50, 918–922. doi: 10.1130/G49957.1 [DOI] [Google Scholar]
  42. Środoń J. (1984). Mixed-layer illite-smectite in low-temperature diagenesis: data from the Miocene of the Carpathian Foredeep. Clay Miner. 19, 205–215. [Google Scholar]
  43. Stan-Lotter H., McGenity T. J., Legat A., Denner E. B. M., Glaser K., Stetter K. O., et al. (1999). Very similar strains of Halococcus salifodinae are found in geographically separated Permo-Triassic salt deposits. Microbiology 145, 3565–3574. [DOI] [PubMed] [Google Scholar]
  44. Stone W., Kroukamp O., Korber D. R., McKelvie J., Wolfaardt G. M. (2016). Microbes at surface-sir interfaces: the metabolic harnessing of relative humidity, surface hygroscopicity, and oligotrophy for resilience. Front. Microbiol. 7:1563. doi: 10.3389/fmicb.2016.01563, [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tamura K., Stecher G., Kumar S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38, 3022–3027. doi: 10.1093/molbev/msab120, [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Thompson J. D., Higgins D. G., Gibson T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Uroz S., Kelly L. C., Turpault M. P., Lepleux C., Frey-Klett P. (2015). The mineralosphere concept: mineralogical control of the distribution and function of mineral-associated bacterial communities. Trends Microbiol. 23, 751–762. doi: 10.1016/j.tim.2015.10.004, [DOI] [PubMed] [Google Scholar]
  48. Wierzchos J., Casero M. C., Artieda O., Ascaso C. (2018). Endolithic microbial habitats as refuges for life in polyextreme environment of the Atacama Desert. Curr. Opin. Microbiol. 43, 124–131. doi: 10.1016/j.mib.2018.01.003, [DOI] [PubMed] [Google Scholar]
  49. Wierzchos J., Davila A. F., Sánchez-Almazo I. M., Hajnos M., Swieboda R., Ascaso C. (2012). Novel water source for endolithic life in the hyperarid core of the Atacama Desert. Biogeosciences 9, 2275–2286. doi: 10.5194/bg-9-2275-2012 [DOI] [Google Scholar]
  50. Yoon S. H., Ha S. M., Kwon S., Lim J., Kim Y., Seo H., et al. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613–1617. doi: 10.1099/ijsem.0.001755, [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data_Sheet_1.PDF (195KB, PDF)
Table_1.XLSX (21KB, XLSX)
Table_2.XLSX (20.1KB, XLSX)
Table_3.DOCX (58.8KB, DOCX)

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Articles from Frontiers in Microbiology are provided here courtesy of Frontiers Media SA

RESOURCES