The sulfidic Frasassi cave system affords a unique opportunity to investigate niche relationships among sulfur-oxidizing bacteria, including epsilonproteobacterial clades with no cultivated representatives. Oxygen and sulfide concentrations in the cave waters range over more than two orders of magnitude as a result of seasonally and spatially variable dilution of the sulfidic groundwater. A full-cycle rRNA approach was used to quantify dominant populations in biofilms collected in both diluted and undiluted zones. Sulfide concentration profiles within biofilms were obtained in situ using microelectrode voltammetry. Populations in rock-attached streamers depended on the sulfide/oxygen supply ratio of bulk water (r ¼ 0.97; Po0.0001). Filamentous epsilonproteobacteria dominated at high sulfide to oxygen ratios (4150), whereas Thiothrix dominated at low ratios (o75). In contrast, Beggiatoa was the dominant group in biofilms at the sediment-water interface regardless of sulfide and oxygen concentrations or supply ratio. Our results highlight the versatility and ecological success of Beggiatoa in diffusion-controlled niches, and demonstrate that high sulfide/oxygen ratios in turbulent water are important for the growth of filamentous epsilonproteobacteria.

Highly acidic (pH 0-1) biofilms, known as 'snottites', form on the walls and ceilings of hydrogen sulfide-rich caves. We investigated the population structure, physiology and biogeochemistry of these biofilms using metagenomics, rRNA methods and lipid geochemistry. Snottites from the Frasassi cave system (Italy) are dominated (470% of cells) by Acidithiobacillus thiooxidans, with smaller populations including an archaeon in the uncultivated 'G-plasma' clade of Thermoplasmatales (415%) and a bacterium in the Acidimicrobiaceae family (45%). Based on metagenomic evidence, the Acidithiobacillus population is autotrophic (ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO), carboxysomes) and oxidizes sulfur by the sulfide-quinone reductase and sox pathways. No reads matching nitrogen fixation genes were detected in the metagenome, whereas multiple matches to nitrogen assimilation functions are present, consistent with geochemical evidence, that fixed nitrogen is available in the snottite environment to support autotrophic growth. Evidence for adaptations to extreme acidity include Acidithiobacillus sequences for cation transporters and hopanoid synthesis, and direct measurements of hopanoid membrane lipids. Based on combined metagenomic, molecular and geochemical evidence, we suggest that Acidithiobacillus is the snottite architect and main primary producer, and that snottite morphology and distributions in the cave environment are directly related to the supply of C, N and energy substrates from the cave atmosphere.

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H 2 and CO 2 , which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H 2 -utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H 2 -utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.

The Frasassi and Acquasanta Terme cave systems in Italy host isolated lithoautotrophic ecosystems characterized by sulfur-oxidizing biofilms with up to 50% S(0) by mass. The net contributions of microbial taxa in the biofilms to production and consumption of S(0) are poorly understood and have implications for understanding the formation of geological sulfur deposits as well as the ecological niches of sulfur-oxidizing autotrophs. Filamentous Epsilonproteobacteria are among the principal biofilm architects in Frasassi and Acquasanta Terme streams, colonizing high-sulfide, low-oxygen niches relative to other major biofilm-forming populations. Metagenomic sequencing of eight biofilm samples indicated the presence of diverse and abundant Epsilonproteobacteria. Populations of Sulfurovum-like organisms were the most abundant Epsilonproteobacteria regardless of differences in biofilm morphology, temperature, or water chemistry. After assembling and binning the metagenomic data, we retrieved four nearly-complete genomes of Sulfurovum-like organisms as well as a Sulfuricurvum spp. Analyses of the binned and assembled metagenomic data indicate that the Epsilonproteobacteria are autotrophic and therefore provide organic carbon to the isolated subsurface ecosystem. Multiple homologs of sulfide-quinone oxidoreductase (Sqr), together with incomplete or absent Sox pathways, suggest that cave Sulfurovum-like Epsilonproteobacteria oxidize sulfide incompletely to S(0) using either O2 or nitrate as a terminal electron acceptor, consistent with previous evidence that they are most successful in niches with high dissolved sulfide to oxygen ratios. In contrast, we recovered homologs of the complete complement of Sox proteins affiliated Gammaproteobacteria and with less abundant Sulfuricurvum spp. and Arcobacter spp., suggesting that these populations are capable of the complete oxidation of sulfide to sulfate. These and other genomic data presented here offer new clues into the physiology and genetic potential of the largely uncultivated and ecologically successful cave Sulfurovum-like populations, and suggest that they play an integral role in subsurface S(0) formation.

Symbioses involving animals and chemoautotrophic bacteria form the foundation of entire ecosystems at deep-sea hydrothermal vents and cold seeps, but have so far not been reported in terrestrial or freshwater environments. A rare example of a terrestrial ecosystem sustained by chemoautotrophy is found within the sulfide-rich Frasassi limestone cave complex of central Italy. In this study, we report the discovery of abundant filamentous bacteria on the exoskeleton of Niphargus ictus, a macroinvertebrate endemic to Frasassi. Using 16S rDNA sequencing and fluorescence in situ hybridization (FISH), we show that N. ictus throughout the large cave complex are colonized by a single phylotype of bacteria in the sulfur-oxidizing clade Thiothrix. The epibiont phylotype is distinct from Thiothrix phylotypes that form conspicuous biofilms in the cave streams and pools inhabited by N. ictus. Using a combination of 13 C labeling, FISH, and secondary ion mass spectrometry (SIMS), we show that the epibiotic Thiothrix are autotrophic, establishing the first known example of a non-marine chemoautotroph-animal symbiosis. Conditions supporting chemoautotrophy, and the N. ictus-Thiothrix association, likely commenced in the Frasassi cave complex between 350 000 and 1 million years ago. Therefore, the N. ictus-Thiothrix symbiosis is probably significantly younger than marine chemoautotrophic symbioses, many of which have been evolving for tens to hundreds of million years.