Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. ␤-, ␥-, ␦-, and -proteobacteria in sulfur-cycling clades accounted for >75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating ␦-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous ␥-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.Sulfidic caves form in carbonate rocks where sulfide-rich waters interact with oxygen at the water table or at subterranean springs. The caves form as a result of sulfuric acid production (equation 1) from microbial or abiotic sulfur oxidation. The sulfuric acid reacts with carbonate host rock to form gypsum and carbonic acid (equation 2).(1)Some of the longest caves known are thought to have formed by this process, including Lechugilla Cave in New Mexico, with 184 km of passages (17). Actively forming sulfidic caves are uncommon but intensely valuable as natural laboratories to understand factors influencing cave formation and resulting biological, geochemical, and isotopic signatures. Active sulfidic caves can host biogeochemically isolated ecosystems based entirely on microbial lithoautotrophic primary productivity (16,38). These ecosystems are aphotic, terrestrial, subsurface environments comparable to sulfureta at hot springs and deep sea vents (10) and are of considerable interest as analogs for microbially dominated, early earth biotic communities such as those that might have developed after the initial rise of oxygen in the early Proterozoic era.Available information from culturing, fluorescence in situ hybridization (FISH), and 16S rDNA libraries suggests that ε-and ␥-proteobacteria are important biofilm-forming groups in the sulfidic cave waters studied to date. Microbial biofilms in Lower Kane Cave (Wyoming) springs and streams are dominated by filamentous ε-prot...

The sulfide-rich Frasassi cave system hosts an aphotic, subsurface microbial ecosystem including extremely acidic (pH 0-1), viscous biofilms (snottites) hanging from the cave walls. We investigated the diversity and population structure of snottites from three locations in the cave system using full cycle rRNA methods and culturing. The snottites were composed primarily of bacteria related to Acidithiobacillus species. Other populations present in the snottites included Thermoplasmata group archaea, bacteria related to Sulfobacillus, Acidimicrobium, and the proposed bacterial lineage TM6, protists, and filamentous fungi. Based on fluorescence in situ hybridization population counts, Acidithiobacillus are key members of the snottite communities, accompanied in some cases by smaller numbers of archaea related to Ferroplasma and other Thermoplasmata. Diversity estimates show that the Frasassi snottites are among the lowest-diversity natural microbial communities known, with one to six prokaryotic phylotypes observed depending on the sample. This study represents the first in-depth molecular survey of cave snottite microbial diversity and population structure, and contributes to understanding of rapid limestone dissolution and cave formation by microbially mediated sulfuric acid speleogenesis.