Microbial Community Characterizing Vermiculations from Karst Caves and Its Role in Their Formation.

The microbiota associated with vermiculations from karst caves is largely unknown. Vermiculations are enigmatic deposits forming worm-like patterns on cave walls all over the world. They represent a precious focus for geomicrobiological studies aimed at exploring both the microbial life of these ecosystems and the vermiculation genesis. This study comprises the first approach on the microbial communities thriving in Pertosa-Auletta Cave (southern Italy) vermiculations by next-generation sequencing. The most abundant phylum in vermiculations was Proteobacteria, followed by Acidobacteria > Actinobacteria > Nitrospirae > Firmicutes > Planctomycetes > Chloroflexi > Gemmatimonadetes > Bacteroidetes > Latescibacteria. Numerous less-represented taxonomic groups (< 1%), as well as unclassified ones, were also detected. From an ecological point of view, all the groups co-participate in the biogeochemical cycles in these underground environments, mediating oxidation-reduction reactions, promoting host rock dissolution and secondary mineral precipitation, and enriching the matrix in organic matter. Confocal laser scanning microscopy and field emission scanning electron microscopy brought evidence of a strong interaction between the biotic community and the abiotic matrix, supporting the role of microbial communities in the formation process of vermiculations.

Geomicrobiology of a seawater-influenced active sulfuric acid cave.

Fetida Cave is an active sulfuric acid cave influenced by seawater, showing abundant microbial communities that organize themselves under three main different morphologies: water filaments, vermiculations and moonmilk deposits. These biofilms/deposits have different cave distribution, pH, macro- and microelement and mineralogical composition, carbon and nitrogen content. In particular, water filaments and vermiculations had circumneutral and slightly acidic pH, respectively, both had abundant organic carbon and high microbial diversity. They were rich in macro- and microelements, deriving from mineral dissolution, and, in the case of water filaments, from seawater composition. Vermiculations had different color, partly associated with their mineralogy, and unusual minerals probably due to trapping capacities. Moonmilk was composed of gypsum, poor in organic matter, had an extremely low pH (0-1) and low microbial diversity. Based on 16S rRNA gene analysis, the microbial composition of the biofilms/deposits included autotrophic taxa associated with sulfur and nitrogen cycles and biomineralization processes. In particular, water filaments communities were characterized by bacterial taxa involved in sulfur oxidation and reduction in aquatic, aphotic, microaerophilic/anoxic environments (Campylobacterales, Thiotrichales, Arenicellales, Desulfobacterales, Desulforomonadales) and in chemolithotrophy in marine habitats (Oceanospirillales, Chromatiales). Their biodiversity was linked to the morphology of the water filaments and their collection site. Microbial communities within vermiculations were partly related to their color and showed high abundance of unclassified Betaproteobacteria and sulfur-oxidizing Hydrogenophilales (including Sulfuriferula), and Acidiferrobacterales (including Sulfurifustis), sulfur-reducing Desulfurellales, and ammonia-oxidizing Planctomycetes and Nitrospirae. The microbial community associated with gypsum moonmilk showed the strong dominance (>60%) of the archaeal genus Thermoplasma and lower abundance of chemolithotrophic Acidithiobacillus, metal-oxidizing Metallibacterium, Sulfobacillus, and Acidibacillus. This study describes the geomicrobiology of water filaments, vermiculations and gypsum moonmilk from Fetida Cave, providing insights into the microbial taxa that characterize each morphology and contribute to biogeochemical cycles and speleogenesis of this peculiar seawater-influenced sulfuric acid cave.

The influence of human exploration on the microbial community structure and ammonia oxidizing potential of the Su Bentu limestone cave in Sardinia, Italy.

The bacterial diversity in the Su Bentu Cave in Sardinia was investigated by means of 16S rRNA gene-based analysis. This 15 km long cave, carved in Jurassic limestone, hosts a variety of calcite speleothems, and a long succession of subterranean lakes with mixed granite and carbonate sands. The lower level is occasionally flooded by a rising groundwater level, but with only scarce input of organic remains (leaves and charcoal fragments). On the quiet cave pools there are visible calcite rafts, whereas walls are locally coated with manganese deposits. In the drier upper levels, where organic input is much more subdued, moonmilk-a hydrated calcium-magnesium carbonate speleothem-can be found. Relative humidity approaches 100% and the measured mean annual cave air temperature is 14.8°C. Samples were obtained in 2014 from calcite rafts, moonmilk, manganese oxide deposits and soil (limestone and granite grains). Microclimatic conditions in the cave near the sampling sites, sample properties, physico-chemical parameters of water, and sediment composition were determined. The microbial community of this system is predominately composed of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Nitrospirae, and Firmicutes. Sampling sites near the entrance of the cave and in close proximity of the underground campsite-located 500 meters deep into the cave-revealed the highest diversity as well as the highest number of human associated microorganisms. Two samples obtained in very close proximity of each other near the campsite, indicate that the human impact is localized and is not distributed freely within the system. Analysis of the abundance of bacterial and archaeal amoA genes revealed a far greater abundance of archaeal amoA genes compared to bacterial representatives. The results of this study highlight that human impact is confined to locations that are utilized as campsites and that exploration leaves little microbial trails. Furthermore, we uncovered a highly specialized microbiome, which is perfectly adapted to survive and thrive in an environment with low nutrient availability.

Geochemistry and microbial diversity of cave waters in the gypsum karst aquifers of Emilia Romagna region, Italy.

Fifty-seven control points of waters (sinking streams, rivers in caves, and resurgences) hosted in gypsum karst areas in Emilia Romagna region (N-Italy) were sampled in the framework of a Project LIFE+08NAT/IT/000369 "Gypsum" in the period 2010-2014. The microbiology and chemistry of these waters have been analyzed to evaluate the impact of human activities or natural factors, in the gypsum karst systems. Waters have been analyzed for major chemistry (Ca, Mg, Na, K, SO4, HCO3, Cl, NO3) and some minor constituents (F, Br, NH4 and PO4), measuring pH, electric conductivity (EC), total dissolved solids (TDS) and temperature (T) in situ. The same samples have been analyzed with traditional microbiology techniques focused on total microbial count and on fecal microbiota, as index of human and/or animal contamination, and molecular biology techniques (sequencing of 16S rRNA segment and PCR-DGGE), focused on the characterization of microbial populations in the different sampling sites and determination of their variations and/or changes during the five years of the project. As expected, waters tend to be increasingly mineralized from sinking streams to resurgences, with only local and temporarily high contents in nitrates and ammonium, often related to the presence of bat colonies. PCR-DGGE revealed ecological changes, in terms of microbial populations present in the bulk water samples, in different sampling sites within the same cave. Although the impact of fecal microorganisms only rarely exceeded 2 log UFC/ml, the results evidenced fluctuations of these microorganisms mainly correlated to the season and to the biological activity of bats.

Microbial mediation of complex subterranean mineral structures.

Helictites--an enigmatic type of mineral structure occurring in some caves--differ from classical speleothems as they develop with orientations that defy gravity. While theories for helictite formation have been forwarded, their genesis remains equivocal. Here, we show that a remarkable suite of helictites occurring in Asperge Cave (France) are formed by biologically-mediated processes, rather than abiotic processes as had hitherto been proposed. Morphological and petro-physical properties are inconsistent with mineral precipitation under purely physico-chemical control. Instead, microanalysis and molecular-biological investigation reveals the presence of a prokaryotic biofilm intimately associated with the mineral structures. We propose that microbially-influenced mineralization proceeds within a gliding biofilm which serves as a nucleation site for CaCO3, and where chemotaxis influences the trajectory of mineral growth, determining the macroscopic morphology of the speleothems. The influence of biofilms may explain the occurrence of similar speleothems in other caves worldwide, and sheds light on novel biomineralization processes.