Unique Populations of Sulfur-Oxidizing Bacteria in Natural Cold Sulfur Springs in Slovakia

Currently, there are over 200 officially registered sulfur springs in Slovakia, most of which are cold hydrogen sulfide springs with a characteristic strong odor of rotten eggs. Their typical inhabitants are filamentous sulfur-oxidizing bacteria. The aim of this study was to identify the major bacterial populations inhabiting sulfur springs in Slovakia using a combination of non-cultivation and cultivation approaches. Non-cultivation analyses revealed high variability of sulfur-oxidizing bacteria in Slovak springs. Sulfur mats were usually dominated by chemolithotrophic sulfur-oxidizers from the phylum Proteobacteria (more than 87%), followed by photoautotrophic sulfur-oxidizers of the phylum Chlorobi. The group of nonsulfur-oxidizers was composed of typical freshwater species and bacteria with genetic potential for sulfur oxidation. The low similarity in the 16S rRNA gene sequence of the sequenced DGGE bands indicates that about half of the sulfur-oxidizing bacteria identified represent new species/genera of sulfur-oxidizing bacteria. In attempts to cultivate potential new species of sulfur-oxidizing bacteria, a very low overlap was observed between non-cultivation and cultivation analyses, and a new species of the Thiofaba genus was cultivated. Our results expand knowledge on the diversity of sulfur bacteria in cold sulfur springs in Slovakia and indicate that the diversity of sulfur-oxidizing bacteria is significantly underestimated and that sulfur springs could be inhabited by unique bacterial populations.