Subterranean calcite dissolution and precipitation are often considered as strictly geochemical processes. The active involvement of microbes in these processes is commonly underestimated in the literature due to general oligotrophic conditions in caves, except in particular cave conditions, such as sulfidic caves and moonmilk deposits, where the presence of microbes likely plays a key role in mineral deposition. Here, we study the possible involvement of microbes from Postojna Cave, Slovenia, in carbonate dissolution (litholysis) and precipitation (lithogenesis). Microbes were sampled from small pools below hydrologically diverse drip sites and incubated on polished limestone tablets at 10 and 20°C for 2 and 14 weeks under cave-analogue conditions. The tablets were then observed under scanning electron microscope to investigate microbe–rock interactions. Our experiments showed the presence of various microbial morphotypes, often associated with extracellular polymeric substances, firmly attached on the surfaces. Unfortunately, our surface sterilization method using 96% and 70% ethanol could not establish the complete aseptic conditions in deep natural cracks in the experimental limestone tablets. Nonetheless, our results emulate the interaction of environmental microbes with limestone rock. Conspicuous calcite dissolution and precipitation were observed in association with these microbes. Furthermore, we show evidence of entombment of microbes in a Si-rich precipitate during nutrient-depleted growth conditions and we suggest that microbial involvement in silica mobilization under ambient conditions may be a widespread and often overlooked phenomenon. Our findings have important implications for microbial-mediation of cave carbonate dissolution and precipitation, including the preservation of past climate proxy signals in speleothems and prehistoric cave art. Improvements to the methodology and further work are suggested to enable more robust ex-situ cultivation experiments in the future, facilitating better and more detailed research into this topic.

During long periods with no precipitation, a sulfidic spring (Smrdljivec) appears in the dry bed of the Reka River before sinking into the karst underground. The study characterizes the area’s geological setting, development of microbial communities and an ecotone, and impact on the vulnerable karst ecosystem. Geological mapping of the area, stable isotopic analyses, field measurements, and physico-chemical and toxicity analyses were applied to elucidate the environmental conditions. The spring’s microbial diversity was assessed using cultivation methods, microscopy, and metagenomics. Sulfur compounds in the spring probably originate from coal layers in the vicinity. Metagenomic analyses revealed 175 distinct operational taxonomic units in spring water and biofilms. Proteobacteria predominated in developed biofilms, and a “core” microbiome was represented by methylotrophs, including Methylobacter, Methylomonas, and Methylotenera. Diatoms represented an important component of biofilm biomass. A combination of environmental factors and climatic conditions allows the formation and accessibility of emerging biodiversity hotspots and ecotones. Details of their dynamic nature, global impact, and distribution should be highlighted further and given more protection.

Escherichia coli, one of the primary intestinal commensal bacteria in humans and endothermic animals, is commonly considered an indicator of faecal pollution. E. coli strains were isolated from karst rivers under different hydrological conditions, from footpaths in tourist caves and from bat guano. Isolates were tested for phenotypic resistance to ampicillin, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline and trimethoprim. The highest percentage of antimicrobial resistant E. coli was found in karst waters, followed by those from surface swabs and from bat guano. Several isolates from rivers and swabs exhibited multidrug-resistant phenotype. Environmental conditions impact the populations of E. coli; a positive correlation between dissolved oxygen and E. coli counts, and a negative correlation between conductivity and E. coli concentrations have been observed for karst rivers. Malenščica (Slovenia), a drinking water resource with an extensive catchment area, contained a relative high percentage of antimicrobial-resistant E. coli strains. None of the isolates from bat guano was resistant to ampicillin, chloramphenicol, and tetracycline. Future monitoring of bats should consider a regular follow-up of indicative microbial disease indicators in fresh guano. Regular cleansing of tourist footpaths in caves and disinfection barriers at the cave entrances reduce the concentration and transmission of E. coli significantly. A future, more detailed, study on characterization of additional E. coli isolates is needed to reveal their pathogeneicity, mechanisms of antibiotic resistance, mobile genetic elements, and gene transfer frequencies to other members of the karst microbiome.

A mixture of coal bottom ash and slag, with a fraction of fly ash (CAFAS) from steam locomotives, was placed in the cave Divaška jama to delimit and level tourist trails. Emplacement began in 1914 and carried on for several decades. The CAFAS mixed with other cave material gradually changed its structure and appearance. Currently the concentration of some elements in the CAFAS (As, Cu, Hg, Ni, Pb, Zn), and also to a lesser extent in cave sediments (Cr, Cu, Ni), indicates a possibly harmful effect on sediment-associated biota based on ecotoxicological assays. Compared to the cave sediment, the CAFAS contains distinctly different mineral phases and presents a different source of radioactivity. Microbial metabolic activity of CAFAS is low, 0.22 μl O2/gDW h, but higher than that of cave sediment. The present environmental hazards from CAFAS are estimated to be low. Whereas the emplacement of CAFAS was seen initially a long-term solution for waste disposal and management of the cave, it turned out that CAFAS enriches the underground environment with inorganic and organic compounds and disperses pollution into the cave ecosystem. After its removal from the cave, the CAFAS should be investigated thoroughly due to its susceptibility to alteration.

A natural cave environment subject to regular human visitation was selected for aerobiological study to minimize the effects of severe temperature fluctuations, UV radiation, and desiccation stress on the aerobiome. The longer sampling period of bioaerosols, up to 22 months, was generally not associated with a proportionally incremental and cumulative increase of microbial biomass. The culture-independent biomass indicator ATP enabled quick and reliable determination of the total microbial biomass. Total airborne microbial biomass was influenced by human visitation to the cave, as confirmed by significantly higher concentrations being observed along tourist footpaths (p < 0.05). Airborne beta-glucans (BG) and lipopolysaccharide (LPS) are present in cave air, but their impact on the cave remains to be evaluated. Staphylococcus spp., as an indicator of human presence, was detected at all sites studied. Their long-term survival decrease is likely due to high relative humidity, low temperature, the material to which they adhere, and potentially natural elevated radon concentration. The most commonly recorded species were: S. saprophyticus, which was identified in 52% of the studied sites, S. equorum in 29%, and S. warneri in 24% of the studied sites. Only a few isolates were assigned to Risk group 2: S. aureus, S. epidermidis, S. haemolyticus, S. pasteuri, and S. saprophyticus.