Microbiology and geochemistry in a hydrogen-sulphide-rich karst environment, 2000, Hose Louise D. , Palmer Arthur N. , Palmer Margaret V. , Northup Diana E. , Boston Penelope J. , Duchene Harvey R. ,

Cueva de Villa Luz, a hypogenic cave in Tabasco, Mexico, offers a remarkable opportunity to observe chemotrophic microbial interactions within a karst environment. The cave water and atmosphere are both rich in hydrogen sulphide. Measured H2S levels in the cave atmosphere reach 210 ppm, and SO2 commonly exceeds 35 ppm. These gases, plus oxygen from the cave air, are absorbed by freshwater that accumulates on cave walls from infiltration and condensation. Oxidation of sulphur and hydrogen sulphide forms concentrated sulphuric acid. Drip waters contain mean pH values of 1.4, with minimum values as low as 0.1.The cave is fed by at least 26 groundwater inlets with a combined flow of 200-300 l/s. Inlet waters fall into two categories: those with high H2S content (300-500 mg/l), mean PCO2=0.03-0.1 atm, and no measurable O2; and those with less than 0.1 mg/l H2S, mean PCO2=0.02 atm, and modest O2 content (up to 4.3 mg/l). Both water types have a similar source, as shown by their dissolved solid content. However, the oxygenated water has been exposed to aerated conditions upstream from the inlets so that original H2S has been largely lost due to outgassing and oxidation to sulphate, increasing the sulphate concentration by about 4%. Chemical modelling of the water shows that it can be produced by the dissolution of common sulphate, carbonate, and chloride minerals.Redox reactions in the cave appear to be microbially mediated. Sequence analysis of small subunit (16S) ribosomal RNA genes of 19 bacterial clones from microbial colonies associated with water drips revealed that 18 were most similar to three Thiobacilli spp., a genus that often obtains its energy from the oxidation of sulphur compounds. The other clone was most similar to Acidimicrobium ferrooxidans, a moderately thermophilic, mineral-sulphide-oxidizing bacterium. Oxidation of hydrogen sulphide to sulphuric acid, and hence the cave enlargement, is probably enhanced by these bacteria.Two cave-enlarging processes were identified. (1) Sulphuric acid derived from oxidation of the hydrogen sulphide converts subaerial limestone surfaces to gypsum. The gypsum falls into the cave stream and is dissolved. (2) Strongly acidic droplets form on the gypsum and on microbial filaments, dissolving limestone where they drip onto the cave floors.The source of the H2S in the spring waters has not been positively identified. The Villahermosa petroleum basin within 50 km to the northwest, or the El Chichon volcano [small tilde]50 km to the west, may serve as source areas for the rising water. Depletion of 34S values (-11.7[per mille sign] for sulphur stabilized from H2S in the cave atmosphere), along with the hydrochemistry of the spring waters, favour a basinal source

Filamentous 'Epsilonproteobacteria' dominate microbial mats from sulfidic cave springs, 2003, Engel As, Lee N, Porter Ml, Stern La, Bennett Pc, Wagner M,

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the 'Epsilonproteobacteria' and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of 'Epsilonproteobacteria' in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems

Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic 'Epsilonproteobacteria', 2004, 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

The distribution and life history of Arrhopalites caecus (Tullberg): Order: Collembola, in Wind Cave, South Dakota, USA., 2005, Moore J. C. , Saunders P. , Selby G. , Horton H. , Chelius M. K. , Chapman A. , Horrocks R. D.

Individuals of the collembolan species Arrhopalites caecus (Tullberg) were collected from drip pools within Wind Cave, South Dakota, at Methodist Church adjacent to the Natural Entrance Tour Route and Room Draculum near survey marker NP-39. Specimens were identified as A. caecus using direct inter­ference and scanning electron microscopy. Molecular analysis of the D2 region of 28S rDNA was per­formed and the sequences were deposited in Genbank (accession number AY239037). We determined that our population of A. caecus reproduced parthenogenetically by successively isolating and rearing eggs through the F4 generation on 9:5 plaster:charcoal media maintained at 21°C, and by the absence of males. Molecular analysis of 16S rDNA for bacterium within our specimens failed to detect the ?-pro-teobacterium (Rickettseales) Wolbachia. Generation times, fecundity, and molt frequency were consistent with other reports for Collembola.

Bacterial dynamics in spring water of alpine karst aquifers indicates the presence of stable autochthonous microbial endokarst communities, 2005, Farnleitner Ah, Wilhartitz I, Ryzinska G, Kirschner Akt, Stadler H, Burtscher Mm, Hornek R, Szewzyk U, Herndl G, Mach Rl,

Spring water of two alpine karst aquifers differing in hydrogeology but of nearby catchments were investigated for their bacterial population dynamics. Dolomite karst aquifer spring 1 (DKAS 1) represents a dolomitic-limestone karst aquifer spring showing high average water residence time and relative constant flow. Limestone karst aquifer spring 2 (LKAS 2) constitutes a typical limestone karst aquifer spring with a dynamic hydrological regime and discharge. Dolomite karst aquifer spring 1 yielded constantly lower cell counts and biomasses (median of 15 x 10(6) cells l(-1) and 0.22 mu g C l(-1)) as the LKAS 2 (median of 63 x 10(6) cells l(-1) and 1.1 mu g C l(-1)) and distribution of morphotypes and mean cell volumes was also different between the considered systems, indicating the influence of hydrogeology on microbial spring water quality. Molecular bacterial V3 16S-rDNA profiles revealed remarkable constancy within each spring water throughout the investigation period. Time course analysis of a flood event in LKAS 2 further supported the trend of the temporal constancy of the microbial community. Except for one case, retrieval of partial and full length 16S rDNA gene sequences from the relative constant DKAS 1 revealed similarities to presently known sequences between 80% to 96%, supporting the discreteness of the microbial populations. The gathered results provide first evidence for the presence of autochthonous microbial endokarst communities (AMEC). Recovery of AMEC may be considered of relevance for the understanding of alpine karst aquifer biogeochemistry and ecology, which is of interest as many alpine and mountainous karst springs are important water resources throughout the world

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy, 2006, Macalady Jennifer L. , Lyon Ezra H. , Koffman Bess, Albertson Lindsey K. , Meyer Katja, Galdenzi Sandro, Mariani Sandro,

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. {beta}-, {gamma}-, {delta}-, and {varepsilon}-proteobacteria in sulfur-cycling clades accounted for [≥]75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating {delta}-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 {gamma}-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of {varepsilon}-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

Biotic versus abiotic calcite formation on prehistoric cave paintings: the Arcy-sur-Cure 'Grande Grotte' (Yonne, France) case, 2007, Chalmin E, D'orlye F, Zinger L, Charlet L, Geremia Ra, Orial G, Menu M, Baffier D, Reiche I,

The Grande Grotte' cave at Arcy-sur-Cure (Yonne, France) with its prehistoric paintings shows important calcite concretions. Two types of calcite have been observed on the wall: translucent yellowish layers and opaque white or grey layers that completely obstruct the paintings. Other calcite types are present in the lakes of the cave (floating calcite rafts at the surface of the lake and soft calcite at the bottom of the lake). The morphology of the different calcites was observed at different scales by optical microscopy with normal and polarized light, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The elemental composition was measured by using particle-induced X-ray emission (micro-PIXE) and the structure by X-ray diffraction (XRD), infrared (FT-IR) and Raman spectroscopy. The bacterial diversity and its role in calcite formation were assessed by culture and 16S-SSCP in order to distinguish and to assess various abiotic and biotic formation mechanisms. The investigation of calcite characteristics enables conclusions on the formation mechanism and on a biotic or abiotic origin of the calcites. The change of calcite types on the walls reveals changes of the environmental cave parameters. In addition, interactions of calcites with the prehistoric paint layer could be evaluated

How long does evolution of the troglomorphic form take? Estimating divergence times in Astyanax mexicanus, 2007, Porter M. L. , Dittmar K. , Pé, Rezlosada M.

Features including colonization routes (stream capture) and the existence of both epigean and cave-adapted hypogean popula­tions make Astyanax mexicanus an attractive system for investi­gating the subterranean evolutionary time necessary for acqui­sition of the troglomorphic form. Using published sequences, we have estimated divergence times for A. mexicanus using: 1) two different population-level mitochondrial datasets (cyto­chrome b and NADH dehydrogenase 2) with both strict and relaxed molecular clock methods, and 2) broad phylogenetic approaches combining fossil calibrations and with four nuclear (recombination activating gene, seven in absentia, forkhead, and α-tropomyosin) and two mitochondrial (16S rDNA and cytochrome b) genes. Using these datasets, we have estimated divergence times for three events in the evolutionary history of troglomorphic A. mexicanus populations. First, divergence among cave haplotypes occurred in the Pleistocene, possibly correlating with fluctuating water levels allowing the coloni­zation and subsequent isolation of new subterranean habitats. Second, in one lineage, A. mexicanus cave populations expe­rienced introgressive hybridization events with recent surface populations (0.26-2.0 Ma), possibly also correlated with Pleis­tocene events. Finally, using divergence times from surface populations in the lineage without evidence of introgression as an estimate, the acquisition of the troglomorphic form in A. mexicanus is younger than 2.2 (fossil calibration estimates) – 5.2 (cytb estimate) Ma (Pliocene).

, 2008, Jones O. S. , Lyon E. H. , And Macalady J. L.

Su lfid ic cave walls host abundant, rapid ly-growing micro bia l communities that display a variety o f mo rphologies previously described for verrn iculations. Here we present molecular, microscopic, isotopic, and geochemical data describing the geomicrobiology o f these biovennic ulations from the Frasassi cave system, Italy. The biove rm iculations are compo sed of densely packed prokaryo tic and funga l cells in a mineral-organ ic matrix co ntaining 5 to 25% o rganic carbon. The carbon and nitrogen isoto pe compositions o f the biovermiculations (ti 13e = - 35 to - 43%0, and til 5N = 4 to - 270/00. respectively) indicate that with in sulfidic zo nes, the o rga nic matter o rigina tes from chemolithotrophic bacterial primary productivity. Based on 165 rRNA gene cloning (n=67). the bioverrn ... iculation communitv is extrernelv diverse, incl uding 48 . ~ . ... representative phylotypes (>98% identity) from at least 15 major bacterial lineages. Important lineages include the Betaproteobacteria (1 9.5% of clones). Gammaproteobacteria (1 8%). Acidobacteria (1 0.5%). Nitrospirae (7.5%). and Planctomyces (7.5%). The most abundant phylotype, comprising over 100/0 of the 16S rRNA gene sequences. groups in an unnamed clade within the Gammaproteobacteria. Based on phylogenetic analysis, we have identified potential sulfur- and nitrite-oxidizing bacte ria. as well as both auto- and heterotrophic members of the biovermiculation community. Additionally. many of the clones a re representatives of deeply branching bacterial lineages with no cultivated representatives. The geochemistry and microbial composition of the biovermicula tions suggest that they play a role in acid production and carbonate disso lution. thereby contributing to cave formation.

Geomicrobiology of biovermiculations from the Frasassi Cave System, Italy, 2008, D. S. Jones, E. H. Lyon, And J. L. Macalady

Sulfidic cave walls host abundant, rapidly-growing microbial communities that display a variety of morphologies previously described for vermiculations. Here we present molecular, microscopic, isotopic, and geochemical data describing the geomicrobiology of these biovermiculations from the Frasassi cave system, Italy. The biovermiculations are composed of densely packed prokaryotic and fungal cells in a mineral-organic matrix containing 5 to 25% organic carbon. The carbon and nitrogen isotope compositions of the biovermiculations (d13C 5 235 to 243%, and d15N 5 4 to 227%, respectively) indicate that within sulfidic zones, the organic matter originates from chemolithotrophic bacterial primary productivity. Based on 16S rRNA gene cloning (n567), the biovermiculation community is extremely diverse, including 48 representative phylotypes (.98% identity) from at least 15 major bacterial lineages. Important lineages include the Betaproteobacteria (19.5% of clones), Gammaproteobacteria (18%), Acidobacteria (10.5%), Nitrospirae (7.5%), and Planctomyces (7.5%). The most abundant phylotype, comprising over 10% of the 16S rRNA gene sequences, groups in an unnamed clade within the Gammaproteobacteria. Based on phylogenetic analysis, we have identified potential sulfur- and nitrite-oxidizing bacteria, as well as both auto- and heterotrophic members of the biovermiculation community. Additionally, many of the clones are representatives of deeply branching bacterial lineages with no cultivated representatives. The geochemistry and microbial composition of the biovermiculations suggest that they play a role in acid production and carbonate dissolution, thereby contributing to cave formation.

Productivity-Diversity Relationships from Chemolithoautotrophically Based Sulfidic Karst Systems, 2009, Porter M. L. , Summers Engel A. , Kane T. C. And Kinkle B. K.

Although ecosystems thriving in the absence of photosynthetic processes are no longer considered unique phenomena, we have yet to understand how these ecosystems are energetically sustained via chemosynthesis. Ecosystem energetics were measured in microbial mats from active sulfidic caves (Movile Cave, Romania; Frasassi Caves, Italy; Lower Kane Cave, Wyoming, USA; and Cesspool Cave, Virginia, USA) using radiotracer techniques. We also estimated bacterial diversity using 16S rRNA sequences to relate the productivity measurements to the composition of the microbial communities. All of the microbial communities investigated were dominated by chemolithoautotrophic productivity, with the highest rates from Movile Cave at 281 g C/m2/yr. Heterotrophic productivities were at least one order of magnitude less than autotrophy from all of the caves. We generated 414 new 16S rRNA gene sequences that represented 173 operational taxonomic units (OTUs) with 99% sequence similarity. Although 13% of these OTUs were found in more than one cave, the compositions of each community were significantly different from each other (P?0.001). Autotrophic productivity was positively correlated with overall species richness and with the number of bacterial OTUs affiliated with the Epsilonproteobacteria, a group known for sulfur cycling and chemolithoautotrophy. Higher rates of autotrophy were also strongly positively correlated to available metabolic energy sources, and specifically to dissolved sulfide concentrations. The relationship of autotrophic productivity and heterotrophic cycling rates to bacterial species richness can significantly impact the diversity of higher trophic levels in chemolithoautotrophically-based cave ecosystems, with the systems possessing the highest productivity supporting abundant and diverse macro-invertebrate communities.

A recently evolved symbiosis between chemoautotrophic bacteria and a cave-dwelling amphipod, 2009, Dattagupta, S. , Schaperdoth, I. , Montanari, A. , Mariani, S. , Kita, N. , Valley, J. W. And Macalady, J. L.

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 13C 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.

Productivity-Diversity Relationships from Chemolithoautotrophically Based Sulfidic Karst Systems, 2009, Porter M. L. , Summers Engel A. , Kane T. C. , Kinkle B. K.

Although ecosystems thriving in the absence of photosynthetic processes are no longer considered unique phenomena, we have yet to understand how these ecosystems are energetically sustained via chemosynthesis. Ecosystem energetics were measured in microbial mats from active sulfidic caves (Movile Cave, Romania; Frasassi Caves, Italy; Lower Kane Cave, Wyoming, USA; and Cesspool Cave, Virginia, USA) using radiotracer techniques. We also estimated bacterial diversity using 16S rRNA sequences to relate the productivity measurements to the composition of the microbial communities. All of the microbial communities investigated were dominated by chemolithoautotrophic productivity, with the highest rates from Movile Cave at 281 g C/m2/yr. Heterotrophic productivities were at least one order of magnitude less than autotrophy from all of the caves. We generated 414 new 16S rRNA gene sequences that represented 173 operational taxonomic units (OTUs) with 99% sequence similarity. Although 13% of these OTUs were found in more than one cave, the compositions of each community were significantly different from each other (P≤0.001). Autotrophic productivity was positively correlated with overall species richness and with the number of bacterial OTUs affiliated with the Epsilonproteobacteria, a group known for sulfur cycling and chemolithoautotrophy. Higher rates of autotrophy were also strongly positively correlated to available metabolic energy sources, and specifically to dissolved sulfide concentrations. The relationship of autotrophic productivity and heterotrophic cycling rates to bacterial species richness can significantly impact the diversity of higher trophic levels in chemolithoautotrophically-based cave ecosystems, with the systems possessing the highest productivity supporting abundant and diverse macro-invertebrate communities.

Community Structure of Subsurface Biofilms in the Thermal Sulfidic Caves of Acquasanta Terme, Italy, 2010, Jones D. S. , Tobler D. J. , Schaperdoth I. , Mainiero M. , Macalady J. L.

We performed a microbial community analysis of biofilms inhabiting thermal (35 to 50°C) waters more than 60m below the ground surface near Acquasanta Terme, Italy. The groundwater hosting the biofilms has 400 to 830 mkM sulfide, <10 mkM O2, pH of 6.3 to 6.7, and specific conductivity of 8,500 to 10,500 mkS/cm. Based on the results of 16S rRNA gene cloning and fluorescent in situ hybridization (FISH), the biofilms have low species richness, and lithoautotrophic (or possibly mixotrophic) Gamma- and Epsilonproteobacteria are the principle biofilm architects. Deltaproteobacteria sequences retrieved from the biofilms have <90% 16S rRNA similarity to their closest relatives in public databases and may represent novel sulfate-reducing bacteria. The Acquasanta biofilms share few species in common with Frasassi cave biofilms (13°C, 80 km distant) but have a similar community structure, with representatives in the same major clades. The ecological success of Sulfurovumales-group Epsilonproteobacteria in the Acquasanta biofilms is consistent with previous observations of their dominance in sulfidic cave waters with turbulent water flow and high dissolved sulfide/oxygen ratios.

Chemoorganotrophic bacteria isolated from biodeteriorated surfaces in cave and catacombs, 2012, De Leo F. , Iero A. , Zammit G. , Urzì C.

The main objective of this work was the comparative analysis of a large number of bacterial strains isolated from biodeteriorated surfaces in three different sites, namely the catacombs of St. Callistus in Rome, Italy, the catacombs dedicated to St. Agatha in Rabat, Malta and the Cave of Bats in Zuheros, Spain. Our results showed that even considering only culturable chemoorganotrophic bacteria the variability is very high, reflecting the great variety of microhabitats present. Hence any strategies to prevent, control or eliminate the biofilm-embedded microbiota from an archeological surface should take into account a number of considerations as stipulated in our study.