Molecular Screening for Cyanobacteria and Their Cyanotoxin Potential in Diverse Habitats.

Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer-PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins-mcyE, saxitoxins-sxtA, cylindrospermopsins-cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments.

Low Bacterial Diversity and Nitrate Levels in Cores from Deep Boreholes in Pristine Karst.

This study investigates the nitrate gradients within the deep biosphere of karst carbonate rocks and their resident microbiota. Samples were taken from borehole cores at depths down to 350 m below the surface, collected during geological site investigations for proposed railway tunnels and analysed using 16S rRNA amplicon sequencing. 16S rRNA amplicon sequencing analysis revealed relatively low microbial diversity, which can serve as a reliable indicator of the pristine nature of deep karst. However, some local hotspots of diversity are independent of depth. Pseudomonadota dominated the samples, with Gammaproteobacteria dominating at the class level. The low nitrate content in deep karst, in contrast to higher values closer to the surface, serves as an additional marker of its undisturbed and unpolluted status. Based on the prediction of functional profiles from 16S rRNA sequencing data, nitrates remain low due to indigenous microbial denitrification and assimilatory nitrate reduction. Pathways related to nitrogen fixation, ammonia assimilation, and nitrification were not confirmed. When elevated nitrate levels are observed in karst, they are most probably related to anthropogenic activities. Environmental factors other than depth and nitrate content play an important role in shaping bacterial communities.

Long-Term Monitoring of Bioaerosols in an Environment without UV and Desiccation Stress, an Example from the Cave Postojnska Jama, Slovenia.

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.

Characteristics and impact of aged coal ash with slag emplaced in a karst cave: the case of Divaska jama, Slovenia.

A mixture of coal bottom ash and slag, with a fraction of fly ash (CAFAS) from steam locomotives, was placed in the cave Divaska 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.