ANALYSIS OF RRNA GENE SEQUENCES TO STUDY DIVERSITY OF MICROORGANISMS AND AQUIFER EVOLUTION OF INTERSTITIAL FLUIDS OF THE SOUTH CENTRAL KENTUCKY KARST AQUIFER

Previous study of interstitial fluid geochemistry within noncarbonate, clastic sediments beneath a flowing stream within the humid-subtropical Mammoth Cave System within the south central Kentucky karst aquifer found acidic conditions and carbon dioxide pressures as much as an order of magnitude higher than the fluids of the cave stream itself. The fluids were undersaturated with respect to calcite, and dissolution rates correspondingly high. These fluids thus appear to significantly influence both conduit growth rates and geometry, since they are in contact with limestone continuously at the sediment/bedrock interface. An obvious question relates to the source of the elevated CO₂ pressures and acidic conditions, which are presumably influenced by microbial activity within the sediment interstitial fluids.

In this study we use 16S rRNA gene sequences as a means for identifying bacteria in cave sediment without the need to culture environmental bacterial strains. DNA was extracted directly from cave sediments and interstitial fluids and bacterial 16S rRNA genes were selectively amplified by polymerase chain reaction (PCR) methods. PCR products were ligated into the pGEM cloning and sequencing vector and circular molecules thus produced were used to transform Escherichia coli cells. Plasmid DNAs carrying 16S rRNA gene sequences from cave bacteria were isolated and used as templates for automated DNA sequencing. Sequences were compared to online databases and closest genetic matches to the cave bacteria were tabulated. A diverse range of bacterial species was found, including common soil bacteria and other more exotic types, which are providing data about the cave ecosystem. Among the genetic matches to the cave bacteria identified so far, the most numerous are soil inhabitants associated with the atmospheric and aqueous nitrogen cycle. Some have close matches to well classified species, while others belong to more diverse and obscure environmental groups. Several bacterial species associated with the biodegradation of petroleum and other organic molecules were identified.

We are working to expand a cave DNA database, which now includes samples from other locations within the aquifer and caves throughout south central Kentucky, as well as from caves in California's Sierra Nevada.