GEOMICROBIOLOGY OF MAMMOTH CAVE USING AN ISOTOPE GEOBIOLOGY APPROACH

Caves are accessible conduits into the shallow subsurface biosphere which function as in situ bioreactors resulting from their unique hydrology, ventilation, and macrobiology, cycling carbon and nitrogen in ways that diverge from both surficial and other subsurface locations. To capture the complexity of this biogeochemical cycling we apply an integrative geobiological approach combining field measurements, fluid and sediment geochemistry, DNA sequencing, cultivation, lipid biomarker analysis, and isotopic characterization of inorganic and organic phases. Here we describe our ongoing work to unravel biogeochemical cycling within a classical limestone karst system in the Mammoth Cave region. Caves contain both heterotrophic and autotrophic microbes, but infiltrating surficial organic carbon and nutrients supplied by bats and other trogloxenes are often thought to be the primary drivers of cave microbiology. However, recent lipid isotope evidence suggests abundant carbon fixation within microbial biofilms in lava caves. At Mammoth Cave we can leverage the size and complexity of the system to compare passages with divergent environmental parameters including levels of bat colonization, human visitation, contamination, and water flow trajectories. Preliminary geochemical surveys confirm high levels of nitrate and sulfate in sediments and fluids, varying across sample type and location. However, sedimentary nitrate concentrations do not correspond to the presence of modern or ancient bat colonization, suggesting complexity in the mechanisms of nutrient delivery between sites. Bulk isotopic measurements of inorganic and organic carbon (DIC and DOC) reveal an organic carbon reservoir that is both large (avg. 56 ppm) and highly depleted (avg. δ¹³C_DOC -49‰) relative to DIC, even as it enters the cave. These data suggest abundant subsurface carbon fixation is occurring early in the fluid flow trajectories. Lipid biomarker analyses are underway to determine if this isotopically depleted carbon is directly incorporated into microbial lipids. Collocated samples are also being processed to identify, enumerate, and isolate key microbial players.