Geomicrobiology and hopanoid content of sulfidic subsurface vent biofilms, Little Salt Spring, Florida

Sulfide-rich, oxygen-poor environments are widespread in the subsurface and were prevalent at the earth's surface during critical intervals in the geologic past. Modern microbial communities in sulfidic niches have the potential to shed light on the biogeochemistry and biosignatures of anoxia and euxinia in earth history. Caves and sinkholes provide rare windows into microbially-dominated, sulfidic subsurface environments that are otherwise difficult and expensive to access. Little Salt Spring (Sarasota County, Florida) is a cover-collapse sinkhole lake with oxic surface water and anoxic, sulfidic bottom water (Alvarez Zarikian 2005). The site is famous for excellent preservation of human and animal archaeological remains (Clausen 1979), and its microbiology has never been investigated. Abundant white biofilms develop seasonally at a warm vent that feeds into the anoxic bottom water at 73 m depth below the water surface. The biofilms are of interest both as potential sources of biomarker compounds and because of their likely role in sulfuric acid production and limestone dissolution (speleogenesis). Biofilm samples were collected by expert science divers and investigated using microscopy, nucleic acid, and lipid analytical methods. Microscopy of the live biofilm revealed clusters of microbial filaments with holdfasts and dendritic, sulfur-rich colonial structures similar to those described in the 1960s for Thiobacterium, a sulfur-oxidizing genus with undetermined phylogeny. A 16S rDNA library constructed from the biofilm was split into three main phylotypes, with multiple clones representing (1) a Betaproteobacterial clade with no cultivated representatives, (2) filamentous Epsilonproteobacteria, and (3) a major bacterial lineage without named isolates (OP11/OD2). A full cycle rRNA approach is currently underway to link 16S rDNA phylotypes with specific populations in the biofilm. We confirmed using fluorescence in situ hybridization (FISH) that abundant filamentous cells with holdfasts are Epsilonproteobacteria. Additional FISH experiments will target the Betaproteobacterial and OP11/OD2 phylotypes retrieved by cloning. Based on HPLC-MS analyses, the biofilm contains at least 5 membrane hopanoid structures distinct from the suite of hopanoids present in sinking organic particles from the photic zone of the sinkhole. Future efforts will be aimed at linking hopanoid structures to specific sulfur-oxidizing populations and to geochemical parameters such as sulfide and oxygen concentrations. References Alvarez Zarikian,C. A., P. K. Swart, J. A. Gifford, P. L. Blackwelder, Palaeogeography, Palaeoclimatology, Palaeoecology 225, 134 (2005). Clausen, C. J., A. D. Cohen, C. Emiliani, J. A. Holman, J. J. Stipp, Science 203, 609 (1979).