In this study, scanning electron microscopy (SEM) and 16S rRNA gene-based phylogenetic approach were applied to reveal the morphological structure and genetic diversity of thermophilic prokaryotic communities of a thermal karst well located in Budapest (Hungary). Bacterial and archaeal diversity of the well water (73.7 °C) and the biofilm developed on the inner surface of an outflow pipeline of the well were studied by molecular cloning method. According to the SEM images calcium carbonate minerals serve as a surface for colonization of bacterial aggregates. The vast majority of the bacterial and archaeal clones showed the highest sequence similarities to chemolithoautotrophic species. The bacterial clone libraries were dominated by sulfur oxidizer Thiobacillus (Betaproteobacteria) in the water and Sulfurihydrogenibium (Aquificae) in the biofilm. A relatively high proportion of molecular clones represented genera Thermus and Bellilinea in the biofilm library. The most abundant phylotypes both in water and biofilm archaeal clone libraries were closely related to thermophilic ammonia oxidizer Nitrosocaldus and Nitrososphaera but phylotypes belonging to methanogens were also detected. The results show that in addition to the bacterial sulfur and hydrogen oxidation, mainly archaeal ammonia oxidation may play a decisive role in the studied thermal karst system.

The Buda Thermal Karst System is an active hypogenic karst area that offers possibility for the analysis of biogenic cave formation. The aim of the present study was to gain information about morphological structure and genetic diversity of bacterial communities inhabiting the Diana-Hygieia Thermal Spring (DHTS). Using scanning electron microscopy, metal accumulating and unusual reticulated filaments were detected in large numbers in the DHTS biofilm samples. The phyla Actinobacteria, Firmicutes and Proteobacteria were represented by both bacterial strains and molecular clones but phyla Acidobacteria, Chlorobi, Chlorofexi, Gemmatimonadetes, Nitrospirae and Thermotogae only by molecular clones which showed the highest similarity to uncultured clone sequences originating from different environmental sources. The biofilm bacterial community proved to be somewhat more diverse than that of the water sample and the distribution of the dominant bacterial clones was different between biofilm and water samples. The majority of biofilm clones was affiliated with Deltaproteobacteria and Nitrospirae while the largest group of water clones was related to Betaproteobacteria. Considering the metabolic properties of known species related to the strains and molecular clones from DHTS, it can be assumed that these bacterial communities may participate in the local sulphur and iron cycles, and contribute to biogenic cave formation.

The Molnár János Cave is part of the northern discharge area of the Buda Thermal Karst System, and is the largest active thermal water cave in the capital of Hungary. To compare the prokaryotic communities, reddish-brown cave wall biofilm, black biogeochemical layers, and thermal water samples from the phreatic mixing zone of the cave were subjected to three investigative approaches, scanning electron microscopy, cultivation, and molecular cloning. According to the SEM images, multilayer network structures were observed in the biofilm formed by iron-accumulating filamentous bacteria and mineral crystals. Cultivated strains belonging to Aeromonadaceae and Enterobacteriaceae were characteristic from both water and subaqueous biofilm samples. The most abundant molecular clones were representatives of the phylum Chloroflexi in the reddish-brown biofilm, the class Gammaproteobacteria in the black biogeochemical layer, and Thiobacillus (Betaproteobacteria) in the thermal water samples. The reddish-brown biofilm and black biogeochemical layer's bacterial communities proved to be somewhat more diverse than that of the thermal water. The archaeal 16S rRNA gene clone libraries were dominated by thermophilic ammonia-oxidizer Nitrosopumilus and Nitrososphaera phylotypes in all three habitats. Considering the metabolic characteristics of known species related to the detected clones, it can be assumed that these communities may participate in the local sulfur and nitrogen cycles and may contribute to microbial mediated sulfuric acid speleogenesis.

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