Acidic cave-wall biofilms located in the Frasassi Gorge, Italy, 2000,
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Vlasceanu L. , Sarbu S. M. , Engel A. S. , Kinkle B. K. ,

Acidic bioflms present on cave walls in the sulfidic region of the Frasassi Gorge, Italy, were investigated to determine their microbial composition and their potential role in cave formation and ecosystem functioning. All biofilm samples examined had pH values <1.0. Scanning electron microscopy of the biofilms revealed the presence of various filaments and rods associated in large clusters with mineral crystals. Qualitative energy-dispersive x-ray analysis was used to determine that the crystals present on the cave walls, associated with the microbial biofilm, were composed of calcium and barium sulfate. Ribosomal RNA-based methods to determine the microbial composition of these biofilms revealed the presence of at least two strains of potential acidophilic, sulfur-oxidizing bacteria, belonging to the genera Thiobacillus and Sulfobacillus. An acid producing strain of Thiobacillus sp. also was obtained in pure culture. Stable isotope ratio analysis of carbon and nitrogen showed that the wall biofilms are isotopically light, suggesting that in situ chemoautotrophic activity plays an important role in this subsurface ecosystem

Ecological assessment and geological significance of microbial communities from Cesspool Cave, Virginia, 2001,
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Engel As, Porter Ml, Kinkle Bk, Kane Tc,

Microbial mats from hydrogen sulfide-rich waters and cave-wall biofilms were investigated from Cesspool Cave, Virginia, to determine community composition and potential geomicrobiological functioning of acid-producing bacteria. Rates of microbial mat chemoautotrophic productivity were estimated using [C-14]-bicarbonate incorporations and microbial heterotrophy was determined using [C-14]-leucine incubations. Chemoautotrophic fixation was measured at 30.4 12.0 ng C mg dry wt(1) h(1), whereas heterotrophic productivity was significantly less at 0.17 0.02 ng C mg dry wt(1) h(1). The carbon to nitrogen ratios of the microbial mats averaged 13.5, indicating that the mats are not a high quality food source for higher trophic levels. Ribosomal RNA-based methods were used to examine bacterial diversity in the microbial mats, revealing the presence of at least five strains of bacteria. The identity of some of the strains could be resolved to the genus Thiothrix and the Flexibacter-Cytophaga-Bacteriodes phylum, and the identity of the remaining strains was to either the Helicobacter or Thiovulum group. Two of 10 sulfur-oxidizing, chemoautotrophic pure cultures of Thiobacillus spp. (syn. Thiomonas gen. nov.) demonstrated the ability to corrode calcium carbonate, suggesting that the colonization and metabolic activity of these bacteria may be enhancing cave enlargement

Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic 'Epsilonproteobacteria', 2004,
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Engel As, Porter Ml, Stern La, Quinlan S, Bennett Pc,

Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave. Wyoming. were assessed with regard to bacterial diversity, community structure, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and stable carbon isotope ratio analyses. The most prevalent mat morphotype consisted of while filament bundles, with low C:N ratios (3.5-5.4) and high sulfur content (16.1-51.2%). White filament bundles and two other mat morphotypes organic carbon isotope values (mean delta(13)C = -34.7parts per thousand: 1sigma = 3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean delta(13)C = -7.47parts per thousand for two springs, n = 8). Bacterial diversity was as low overall in the clone libraries, and the most abundant taxonomic group was affiliated with the 'Epsilonproteobacteria' (68%) with other bacterial sequences affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Six distinct epsilonproteobacterial taxonomic groups were identified from the microbial mats. Epsilonproteobacterial and bacterial group abundances and community structure shifted front the spring orifices downstream. corresponding to changes in dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the clone sequences for epsilonproteobacterial groups were retrieved from areas with high sulfide and low oxygen concentrations, whereas Thiothrix spp. and Thiobacillus spp. had higher retrieved clone abundances where conditions of low sulfide and high oxygen concentrations were measured. Genetic and metabolic diversity among the 'Epsilonproteobacteria' maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Our results demonstrate that sulfur cycling supports subsurface ecosystem through chemolithoautotrophy and expand the evolutionary and ecological views of 'Epsilonproteobacteria' in terrestrial habitats. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier BY. All rights reserved

Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy, 2007,
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Jennifer L. Macalady, * Daniel S. Jones And Ezra H. Lyon

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 lowestdiversity 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.