Abiotic factors affecting the presence of airborne halophilic microorganisms in the extreme atmospheric conditions of an underground salt mine
Authors: Puławska Aleksandra , Kowalewicz-Kulbat Magdalena , Kalinowska Jolanta , Arciszewska Gabriela , Drzewiecka Dominika , Manecki Maciej 
Published: February 2023

Underground salt mines represent the most extreme environments which combine low nutrient availability, darkness, and hypersaline conditions. Halophilic (&#8220;salt-loving&#8221;) microorganisms are known to constitute the natural microbial communities of hypersaline ecosystems around the world (salt rocks, underground brines, saline lakes, etc.). For the first time halophilic microorganisms were detected in the air of the Bochnia Salt Mine, Poland.&#160;The purpose of this study was to determine the impact of various abiotic factors of the atmosphere on the presence and abundance of halophilic microbial communities present in mine air. Samples of aerosol components were collected at four different locations: one on the surface (at the air intake) and three underground at increasing distances from the intake. The inorganic aerosol was collected by dry (filter-based) and wet (scrubber-based) sampling method using portable air pumps. Besides microclimatic conditions, the content of water-soluble constituents, trace elements, carbon, and minerals were determined in aerosols. Simultaneously, the airborne cultivable microorganisms were collected by MAS-100 sampler. Mesophilic microorganisms were cultivated as a control, on general tryptic-soya (TSA) media, at 37&#176;C and 28&#176;C for up to one month. Halophilic ones were grown on a specific HBM medium containing 20% and 25% of NaCl concentration, at 37&#176;C and 28&#176;C for up to three months.The primary component of aerosol was NaCl (1000-3000 &#181;g&#183;m-3). It enters the air mainly in the form of a solution droplets due to the deliquescence of rock salt in humid air (up to 80% of relative humidity). The wet aerosol in salt mine is also composed of SO42- (110-300 &#181;g&#183;m-3), Ca2+ (90-280 &#181;g&#183;m-3), K+ (50-190 &#181;g&#183;m-3), Mg2+ (15-40 &#181;g&#183;m-3), and Fe3+ (10-50 &#181;g&#183;m-3). The dry fraction of aerosol does not exceed 200 &#181;g m-3 and is composed of fragments of natural rock salt (halite), anhydrite, gypsum, and clay minerals. The maximum indoor concentrations of airborne halophilic microorganisms cultivated at 37&#176;C or 28&#176;C reached 1910 CFU (colony-forming units) &#183;m-3 and 1210 CFU&#183;m-3, respectively. Moreover, the content of halophilic microorganisms increased with the increase of the water-soluble constituents and NaCl concentrations. Our results suggest that airborne salt-saturated droplets may be a major factor influencing the abundance of live halophilic microorganisms in the atmosphere while the presence of mesophilic microorganism (which are associated with the outdoor environment) and the presence of humans seem to have no effect on the presence of halophilic microbial community.Our research indicates that halophilic microorganisms can survive in the air of the underground Bochnia Salt Mine. Abiotic factors, like high moisture content in the air and saline aerosol in liquid form may play an important role in their survival in the air. This way some extremophile microorganisms, given favorable environmental conditions, can survive even in such a hostile environment as the atmosphere in the underground mine. This could be important for astrobiology research, since various extremophiles, including halophiles, are considered excellent candidates for life beyond our planet.The study was supported by the Polish National Science Center (NCN) grant No. 2021/41/N/ST10/02751.&#160;