EXPLORING THE MICROBIAL DIVERSITY OF SULFIDIC KARST SPRINGS AND EVALUATION OF MICROBIAL INDICATOR SPECIES

Microorganisms are fundamental aspects to the physical arrangements and biogeochemical transformations of aquatic habitats. Although microbial indicator species (MIS) have been used in watershed science to evaluate pollution, the application of MIS in natural, uncontaminated aquatic habitats has rarely been done. One reason for this is the lack of data from natural settings, like groundwater. Springs, where groundwater discharges at the surface, are useful for uncovering clues about MIS and biogeochemical processes in the subsurface. We are examining the geomicrobiology of natural H₂S-rich (sulfidic) karst springs throughout the US to evaluate MIS in springs and groundwater, as well as subsurface S cycling. Historically, these springs have been known about for centuries because of purported medicinal or therapeutic properties. Currently we have sampled 17 surface- and 7 cave-discharging sulfidic karst springs. Our investigations show that the white material commonly found at the springs consists of microorganisms that can cover sediment as thin films, float on pool surfaces, or form dense filamentous microbial mats within discharge channels. Most waters are Ca- HCO₃-SO4 water type, with pH values from 6.4 - 7.5, temperatures from 8 - 55^oC, and total dissolved sulfide from <0.1 - 21 ppm. As a 1st approximation of MIS, we assembled a unique dataset of >500 near full-length 16S rRNA gene sequences. Comparisons of species richness, diversity, dominance, and shared species were calculated to delineate spring MIS. Although some springs had unique taxonomic groups (rare sequences retrieved from one site), several groups from multiple, geochemically different springs were phylogenetically closely-related to each other (>98% identity). Shared MIS lineages were in the ε-Proteobacteria, γ-Proteobacteria (especially Thiothrix spp., a known sulfur-oxidizing bacterial group), Bacteriodetes, and Thiobacillus. Novel ε-Proteobacteria dominated high H₂S and low O₂ karst springs, whereas Thiothrix spp. dominated high O₂, low H₂S, and higher pH springs. No correlation was observed between MIS and temperature; e.g., ε-Proteobacteria were found from both high and low temperature springs. Results indicate that MIS are definable in natural spring settings and can be used to evaluate groundwater processes.