COMMUNITY STRUCTURE AND FUNCTION OF EXTREMELY ACIDIC CAVE BIOFILMS

Microbial biofilms known as “snottites” create extremely acidic (pH 0-1) environments in sulfide-rich caves. These biofilms are formed on cave walls and ceilings by sulfur-oxidizing acidophiles that utilize H₂S(g) in the cave atmosphere. Here, we use metagenomics, culturing, and rRNA-based analyses to explore the energy and nutrient metabolisms of snottites from the Frasassi cave system in central Italy. Snottite samples collected throughout the caves are dominated by a phylotype of the bacterium Acidithiobacillus thiooxidans, with smaller populations of Thermoplasmatales-group archaea and a bacterial phylotype related to Acidimicrobium. Rare taxa include TM6-group bacteria, filamentous fungi, and protists. All communities have extremely low richness, ranging from 1 to 7 phylotypes. Twelve megabases of metagenomic DNA was obtained via pyrosequencing from a Frasassi snottite sample collected in August 2005. The metagenome was annotated by assigning reads to COG and Gene Ontology (GO) categories. Overrepresented gene groups encode cation transport and other functions consistent with the extremely low pH snottite matrix. Analysis of phylogenetic marker genes present in the metagenome accurately reproduces the community structure determined by fluorescence in situ hybridization (FISH).

Nearly complete genomic coverage of the Acidithiobacillus phylotype in the metagenome revealed sulfur oxidation and carbon fixation pathways, as well as mechanisms of nitrogen and phosphorous uptake. Acidithiobacillus strains isolated on autotrophic thiosulfate and elemental sulfur media confirm that these species are primary producers, grow at pH values lower than 0.2, and excrete extracellular polymers. Based on metagenomic analysis and 16S rDNA sequencing, the dominant archaea are wall-less members of the ‘G-plasma’ clade in the Thermoplasmatales and are likely heterotrophs. Although sequence coverage of rare phylotypes is low, available metagenomic data suggest that the Acidimicrobium phylotype is heterotrophic and that autotrophs other than Acidithiobacillus are present in the community. Our results to date show that pyrosequencing can link the biogeochemistry of sulfidic caves with the structure and function of microbial communities therein.