Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

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.     

Microbial Activity in Caves−A Geological Perspective

Caves are commonly the home of diverse microbial biotas, the sites of active mineral precipitation, and/or receptacles for the deposition of sediment. Mineral precipitation is commonly considered to be abiogenic despite the fact that microbes are present in caves, especially in the twilight zone. Detailed analysis of cave substrates from a geological perspective shows that microbes can mediate constructive (microbe calcification, trapping and binding, mediation of crystal growth) and destructive (substrate etching and breakdown) processes. Potentially these processes can significantly influence the formation and preservation of any cave deposit. Preservation of microbes is possible if mineralization takes place while the microbe is alive or shortly after its demise. If not, all record of the microbe will be lost to decay. Even if the microbes are preserved, it may be difficult to determine if they played an active or passive role in the formation of the deposits in which they are entombed. For old cave deposits, such an assessment must rely on spatial relationships and comparison of textures with those known to form as a result of microbial activity. Nevertheless, available evidence indicates that microbes can play a major role in the formation and modification of cave deposits. Equally, however, it is apparent that the full scope and impact of microbial activity on cave deposits has yet to be realized. Recognition of microbial activity in old CaCO 3 cave deposits relies on (1) documentation and recognition of mineralized microbes, (2) recognition of stromatolitic structures that formed through microbial activity, and/or (3) the identification of fabrics/textures that are known to be indicative of microbial activity. All of these criteria fundamentally rely on the interpretation of fabrics preserved in the cave deposits. Virtually all of these interpretations are open to debate.     

洞穴微生物组:已知与未知

洞穴是由于岩石受到溶蚀或火山岩浆冷却形成的地下空间和通道。洞穴遍布世界各地,蕴藏着大量的微生物资源。洞穴内黑暗潮湿,温度相对稳定,营养比较贫瘠,是一种极端的生态系统,存在着高度特异性的洞穴微生物。对洞穴微生物组的研究,有助于认知洞穴微生物群体结构、组成及其地理学分布,加深对洞穴生态系统的理解,并为保护和开发洞穴资源提供指导。本文主要介绍了洞穴微生物组的研究方法,总结分析了洞穴微生物的研究进展,并着重阐述了含特殊化合物成分的洞穴中化能自养的特色微生物组。     

Microbial Community Diversity of Moonmilk Deposits at Ballynamintra Cave, Co. Waterford, Ireland

Caves are extreme and specialised habitats for terrestrial life that sometimes contain moonmilk, a fine-grained paste-like secondary mineral deposit that is found in subterranean systems worldwide. While previous studies have investigated the possible role of microorganisms in moonmilk precipitation, the microbial community ecology of moonmilk deposits is poorly understood. Bacterial and fungal community structure associated with four spatially isolated microcrystalline, acicular calcite moonmilk deposits at Ballynamintra Cave (S. Ireland) was investigated during this study. Statistical analyses revealed significant differences in microbial activity, number of bacterial species, bacterial richness and diversity, and fungal diversity (Shannon's diversity) among the moonmilk sites over an area of approximately 2.5 m². However, the number of fungal species and fungal community richness were unaffected by sampling location. SIMPER analysis revealed significant differences in bacterial and fungal community composition among the sampling sites. These data suggest that a rich assemblage of microorganisms exists associated with moonmilk, with some spatial diversity, which may reflect small-scale spatial differences in cave biogeochemistry.     

Neutralization and neoformation: Analogous processes in the atmosphere and in lichen thalli—A review

The present review discusses the concept of mineral neoformation which describes the formation of gypsum as the end-product of analogous processes occurring in the atmosphere, in the lichen thallus and on lithic substrates colonized by lichens. The formation of gypsum occurs as a result of H₂SO₄ reacting with CaCO₃. Climate change, e.g. global warming and desertification led to an increasing number of annual dust storms originating in North African deserts, from 5 days in 1958 to 30 days in 2006, i.e. sixfold in 48 years. Calcium is a major constituent of desert dust. The enlarged dust load in the Eastern Mediterranean coincides with the amount of calcium accumulated in lichens: from 6300 μg g⁻¹ in 1978 to 36,603 ± 7563 μg g⁻¹ in 2002, i.e. about sixfold in 24 years. Desert dust carrying Ca-containing particles reacts with sulfuric acid produced in the atmosphere following both volcanic and anthropogenic activities. Gypsum produced via neoformation in the atmosphere, precipitates in varying formations, e.g. iberulites. A process of neutralization leads to the formation of biominerals. CaCO₃-containing particles entrapped in the lichen thallus react with sulfuric acid to produce gypsum as its end-product, detected in the form of crystals on the outer surface of the thallus. The formation of biogenic gypsum occurs also via the reaction of intra- and extracellular Ca²⁺ cations with SO₄²⁻ anions, penetrating into, or found inside thallial cells. The catalytic capability of lichens is evident in the process of neoformation of gypsum on lithic substrates, e.g. granite.     

Dominant microbial populations in limestone-corroding stream biofilms, Frasassi cave system, Italy

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 epsilon-proteobacteria in sulfur-cycling clades accounted for > or = 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 epsilon-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.     

Further Readings in Geomicrobiology

Evaporite accumulations produced by artesian waters in the arid zones of southern Tunisia led to the formation of subrounded, gypsiferous mounds consisting of irregular alternations of mineral precipitates and aeolian sand. The joint occurrence of gypsum crusts and plant colonization determined the stabilization of their top surface. Careful examination of the pigmented (green-brown) crusts revealed endolithic microbial communities just below the surface. In previous optical and scanning electron microscope studies cyanobacteria were the dominant component of these communities. Molecular diversity studies based on small subunit ribosomal RNA (SSU rRNA) gene analysis revealed that Flavobacteria, Actinobacteria, Deinococcales, Alpha- and Gamma- Proteobacteria are also important components of the microbial assemblage. Their pigment analyses, determined by high performance liquid chromatography (HPLC), detected the presence of carotenoids and chlorophyll (chl) a and b. Microbial communities that produce pigmentation and display an endolithic lifestyle typify the extreme environments as those found in arid/semiarid and hot desert regions.     

Rock structure drives the taxonomic and functional diversity of endolithic microbial communities in extreme environments

Endolithic (rock-dwelling) microbial communities are ubiquitous in hyper-arid deserts around the world and the last resort for life under extreme aridity. These communities are excellent models to explore biotic and abiotic drivers of diversity because they are of low complexity. Using high-throughput amplicon and metagenome sequencing, combined with X-ray computed tomography, we investigated how water availability and substrate architecture modulated the taxonomic and functional composition of gypsum endolithic communities in the Atacama Desert, Chile. We found that communities inhabiting gypsum rocks with a more fragmented substrate architecture had higher taxonomic and functional diversity, despite having less water available. This effect was tightly linked with community connectedness and likely the result of niche differentiation. Gypsum communities were functionally similar, yet adapted to their unique micro-habitats by modulating their carbon and energy acquisition strategies and their growth modalities. Reconstructed population genomes showed that these endolithic microbial populations encoded potential pathways for anoxygenic phototrophy and atmospheric hydrogen oxidation as supplemental energy sources.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

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. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-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 γ-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.     

Biogenic Evidences of Moonmilk Deposition in the Mawmluh Cave,Meghalaya, India

Moonmilk, a microcrystalline secondary cave deposit, actively forms on the floor of Krem Mawmluh – a limestone cave in Meghalaya, Northeastern India. Due to the abundance of micrite and calcified microbial filaments, we hypothesize that these deposits form as a result of ongoing microbial interactions. Consistent with this idea, we report electron microscopic and microbiological evidences for the biological origin of moonmilk in Krem Mawmluh. Scanning electron microscopy indicated abundant calcified microbial filaments, needle calcite, fibre calcites (micro-fibre and nano-fibre calcite crystals), biofilm and microbial filaments in the moonmilk. The total viable culturable microbes showed high population densities for microbes in the moonmilk and moonmilk pool waters. In vitro culture experiments, confirmed the capability of many of the isolated strains to precipitate calcite and some of the identified isolates belonged to the Bacillus sp. and Actinomycetes. These results clearly support the biogenic nature of the deposits.     

Profiling Bacterial Diversity and Taxonomic Composition on Speleothem Surfaces in Kartchner Caverns, AZ

Caves are relatively accessible subterranean habitats ideal for the study of subsurface microbial dynamics and metabolisms under oligotrophic, non-photosynthetic conditions. A 454-pyrotag analysis of the V6 region of the 16S rRNA gene was used to systematically evaluate the bacterial diversity of ten cave surfaces within Kartchner Caverns, a limestone cave. Results showed an average of 1,994 operational taxonomic units (97 % cutoff) per speleothem and a broad taxonomic diversity that included 21 phyla and 12 candidate phyla. Comparative analysis of speleothems within a single room of the cave revealed three distinct bacterial taxonomic profiles dominated by either Actinobacteria, Proteobacteria, or Acidobacteria. A gradient in observed species richness along the sampling transect revealed that the communities with lower diversity corresponded to those dominated by Actinobacteria while the more diverse communities were those dominated by Proteobacteria. A 16S rRNA gene clone library from one of the Actinobacteria-dominated speleothems identified clones with 99 % identity to chemoautotrophs and previously characterized oligotrophs, providing insights into potential energy dynamics supporting these communities. The robust analysis conducted for this study demonstrated a rich bacterial diversity on speleothem surfaces. Further, it was shown that seemingly comparable speleothems supported divergent phylogenetic profiles suggesting that these communities are very sensitive to subtle variations in nutritional inputs and environmental factors typifying speleothem surfaces in Kartchner Caverns.     

Messinian bottom-grown selenitic gypsum: An archive of microbial life

Primary gypsum deposits, which accumulated in the Mediterranean Basin during the so-called Messinian salinity crisis (5.97–5.33 Ma), represent an excellent archive of microbial life. We investigated the molecular fossil inventory and the corresponding compound-specific δ¹³C values of bottom-grown gypsum formed during the first stage of the crisis in four marginal basins across the Mediterranean (Nijar, Spain; Vena del Gesso, Italy; Heraklion, Crete; and Psematismenos, Cyprus). All studied gypsum samples contain intricate networks of filamentous microfossils, whose phylogenetic affiliation has been debated for a long time. Petrographic analysis, molecular fossil inventories (hydrocarbons, alcohols, and carboxylic acids), and carbon stable isotope patterns suggest that the mazes of filamentous fossils represent benthic microbial assemblages dominated by chemotrophic sulfide-oxidizing bacteria; in some of the samples, the body fossils are accompanied by lipids produced by sulfate-reducing bacteria. Abundant isoprenoid alcohols including diphytanyl glycerol diethers (DGDs) and glycerol dibiphytanyl glycerol tetraethers (GDGTs), typified by highly variable carbon stable isotope composition with δ¹³C values spanning from ?40 to ?14‰, reveal the presence of planktic and benthic archaeal communities dwelling in Messinian paleoenvironments. The compound inventory of archaeal lipids indicates the existence of a stratified water column, with a normal marine to diluted upper water column and more saline deeper waters. This study documents the lipid biomarker inventory of microbial life preserved in ancient gypsum deposits, helping to reconstruct the widely debated conditions under which Messinian gypsum formed.     

The microbe–mineral environment and gypsum neogenesis in a weathered polar evaporite

Evaporitic deposits are a globally widespread habitat for micro‐organisms. The microbe–mineral environment in weathered and remobilized gypsum from exposed mid‐Ordovician marine evaporite beds in the polar desert of Devon Island, Nunavut, Canadian High Arctic was examined. The gypsum is characterized by internal green zones of cyanobacterial colonization (dominated by Gloeocapsa/Aphanothece and Chroococcidiopsis spp. morphotypes) and abundant black zones, visible from the surface, that contain pigmented cyanobacteria and fungi. Bioessential elements in the gypsum are primarily provided by allochthonous material from the present‐day polar desert. The disruption, uplift and rotation of the evaporite beds by the Haughton meteorite impact 39 Ma have facilitated gypsum weathering and its accessibility as a habitat. No cultured cyanobacteria, bacteria and fungi were halophilic consistent with the expectation that halophily is not required to persist in gypsum habitats. Heterotrophic bacteria from the evaporite were slightly or moderately halotolerant, as were heterotrophs isolated from soil near the gypsum outcrop showing that halotolerance is common in arctic bacteria in this location. Psychrotolerant Arthrobacter species were isolated. No psychrophilic organisms were isolated. Two Arthrobacter isolates from the evaporite were used to mediate gypsum neogenesis in the laboratory, demonstrating a potential role for microbial biomineralization processes in polar environments.     

Recent evaporite deposition associated with microbial mats,Al-Kharrar supratidal–intertidal sabkha,Rabigh area,Red Sea coastal plain of Saudi Arabia

The supratidal–intertidal sabkha of the Al-Kharrar area, Red Sea coast, Saudi Arabia, contains the evaporite minerals gypsum, anhydrite, and halite. Microbial mats flourish adjacent to the sabkha evaporites in tidal flats and pools of the Al Kharrar lagoon. Desiccation and decay of some microbial mats in tidal flat areas have led to precipitation of gypsum and halite there. The evaporite minerals have been precipitated through displacive, inclusive, and replacive growth within mud, sand, gravelly sand, and bioclastic sediment of the sabkha. Gypsum occurs as lenticular and tabular crystals whereas anhydrite occurs as nodular (individual, mosaic, and enterolithic) and pseudomorphs of lenticular gypsum crystals that grew displacively and replacively near the surface of the sabkha. Halite exists as a diagenetic cement within the sabkha sediment, or as primary rafts and skeletal crystals in desiccated tidal pools with salinity over 220‰. Microbial mats are growing on the surface of the upper tidal flat areas and in pools at a salinity range of 80–110‰, and they lead to biostabilization of the sediment. They have induced a range of sedimentary surface structures (MISS) including gas domes, reticulate patterns, tufts, pinnacles, wrinkles, and microbial shrinkage cracks. The occurrence, abundance, and association of evaporite minerals and MISS are controlled by local environmental factors such topography of the sabkha, emergence or submergence of tidal areas, surface area of the evaporite basin, contribution of meteoric water from floods from the adjoining Red Sea Mountains, and water salinity. These factors promote the growth of the microbial mats in the winter months, and deposition of evaporite minerals during summer months. Field and petrographic data indicate that the main recharge to the sabkha area is from tidal flow and water seepage from the Al-Kharrar lagoon. The results of this study indicate that within a small sabkha area of Al-Kharrar (3?×?17 km), a large variation in evaporite mineral types and morphologies grade into and are associated with MISS due to local environmental parameters. The interpretation of this association of evaporite minerals and MISS provides useful data for understanding the mechanisms responsible for precipitation of evaporite minerals and formation of MISS.     

岩画和壁画类文物微生物病害研究进展

古代岩画和壁画是人类文明发展历史进程的重要记录,具有极高的历史价值、艺术价值及科学价值,但他们无时无刻不受到环境因素引起的物理、化学及生物的劣化作用。近年,有关微生物对古代岩/壁画的危害逐渐受到关注。阐述了世界一些著名的岩/壁画文物的微生物的研究进展,如法国拉斯科洞穴岩画(Lascaux Cave)、西班牙阿尔塔米拉洞穴岩画(Altamira Cave)、敦煌石窟壁画、嘉峪关魏晋墓壁画以及我国东北地区公元5世纪的墓葬壁画等。通过对以上所述岩画和壁画微生物群落的分析比较,发现主要的细菌群落是变形菌门和放线菌门,放线菌门以假诺卡氏菌为主。真菌群落主要以虫生真菌为主。进一步分析了岩画和壁画微生物病害的共性和区别,揭示了洞穴、墓室等特殊环境下的微生物群落特点,为防治古代岩/壁画微生物病害提供了以下借鉴。     

Characterization of bacterial diversity associated with microbial mats,gypsum evaporites and carbonate microbialites in thalassic wetlands: Tebenquiche and La Brava,Salar de Atacama,Chile

In this paper, we report the presence of sedimentary microbial ecosystems in wetlands of the Salar de Atacama. These laminated systems, which bind, trap and precipitate mineral include: microbial mats at Laguna Tebenquiche and Laguna La Brava, gypsum domes at Tebenquiche and carbonate microbialites at La Brava. Microbial diversity and key biogeochemical characteristics of both lakes (La Brava and Tebenquiche) and their various microbial ecosystems (non-lithifying mats, flat and domal microbialites) were determined. The composition and abundance of minerals ranged from trapped and bound halite in organic-rich non-lithifying mats to aragonite-dominated lithified flat microbialites and gypsum in lithified domal structures. Pyrosequencing of the V4 region of the 16s rDNA gene showed that Proteobacteria comprised a major phylum in all of the microbial ecosystems studied, with a marked lower abundance in the non-lithifying mats. A higher proportion of Bacteroidetes was present in Tebenquiche sediments compared to La Brava samples. The concentration of pigments, particularly that of Chlorophyll a, was higher in the Tebenquiche than in La Brava. Pigments typically associated with anoxygenic phototrophic bacteria were present in lower amounts. Organic-rich, non-lithifying microbial mats frequently formed snake-like, bulbous structures due to gas accumulation underneath the mat. We hypothesize that the lithified microbialites might have developed from these snake-like microbial mats following mineral precipitation in the surface layer, producing domes with endoevaporitic communities in Tebenquiche and carbonate platforms in La Brava. Whereas the potential role of microbes in carbonate platforms is well established, the contribution of endoevaporitic microbes to formation of gypsum domes needs further investigation.     

Bacterial abundance and composition in marine sediments beneath the Ross Ice Shelf,Antarctica

Marine sediments of the Ross Sea, Antarctica, harbor microbial communities that play a significant role in the decomposition, mineralization, and recycling of organic carbon (OC). In this study, the cell densities within a 153‐cm sediment core from the Ross Sea were estimated based on microbial phospholipid fatty acid (PLFA) concentrations and acridine orange direct cell counts. The resulting densities were as high as 1.7 × 10⁷ cells mL^?1 in the top ten centimeters of sediments. These densities are lower than those calculated for most near‐shore sites but consistent with deep‐sea locations with comparable sedimentation rates. The δ¹³C measurements of PLFAs and sedimentary and dissolved carbon sources, in combination with ribosomal RNA (SSU rRNA) gene pyrosequencing, were used to infer microbial metabolic pathways. The δ¹³C values of dissolved inorganic carbon (DIC) in porewaters ranged downcore from ?2.5‰ to ?3.7‰, while δ¹³C values for the corresponding sedimentary particulate OC (POC) varied from ?26.2‰ to ?23.1‰. The δ¹³C values of PLFAs ranged between ?29‰ and ?35‰ throughout the sediment core, consistent with a microbial community dominated by heterotrophs. The SSU rRNA gene pyrosequencing revealed that members of this microbial community were dominated by β‐, δ‐, and γ‐Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Among the sequenced organisms, many appear to be related to known heterotrophs that utilize OC sources such as amino acids, oligosaccharides, and lactose, consistent with our interpretation from δ¹³C_PLFA analysis. Integrating phospholipids analyses with porewater chemistry, δ¹³C_DIC and δ¹³C_POC values and SSU rRNA gene sequences provides a more comprehensive understanding of microbial communities and carbon cycling in marine sediments, including those of this unique ice shelf environment.     

Topographic and soil surface effects on gypsophile plant community patterns in central Mexico

Abstract. Semiarid gypsum karstlands in north central Mexico are characterized by a mosaic of shrubland and endemic gypsophile grassland community types. Proximal physical factors affecting community patterns on a local scale were investigated at an undisturbed site south of Matehuala, San Luis Potosi. Soil surface characteristics associated with depth of calcareous alluvium overlying gypsum base material were strongly correlated with distribution of desert shrub and endemic grass species. Muhlenbergia purpusii was dominant on bare gypsum soils with indurated crusts, while Bouteloua chasei was dominant where a thin veneer of alluvium permitted development of a cryptogamic crust. A high-diversity mixed shrub-succulent community occupied gypsum overlain by 5 - 20 cm of alluvium, whereas Larrea tridentata was the principal species on gypsum overlain by > 20 cm of alluvium. Open sink walls were occupied mostly by species of the Muhlenbergia grassland, while closed sinks supported more mesophytic mixed shrubland vegetation. Physical factors operating mostly at the establishment stage are probably more important in mediating local species patterns on gypsum than factors associated with mineral nutrition. Gypsum material was much less fertile than alluvial material but did not exclude widely distributed species unless exposed at the surface.     

Microbial Nitrogen and Sulfur Cycles at the Gypsum Dunes of White Sands National Monument,New Mexico

The White Sands National Monument from New Mexico (U.S.A) contains one of the largest known gypsum dune fields with unique, rapidly migrating, arid, evaporitic habitats. Deposits from dune sides and interdune areas were collected in order to determine the characteristics of microbial habitat and communities through mineral assemblages, microbial pigments along with investigations of nitrogen and sulfur cycles. The most abundant pigments, scytonemin and carotenoids, were common UV protective pigments. Predominance of nitrite and nitrate over ammonium nitrogen (2.16: 1) implies that nitrification processes might be important in this ecosystem. Ammonium oxidizers from groups of β-, γ-proteobacteria and archaea were detected in all deposits, thereby indicating microbial involvement in nitrification. Additionally, denitrifying organisms with nirS and nirK genes were also present in most of the analyzed samples. The presence of trace carbonate mineral phases in association with biofilm implies possible microbial sulfate reduction. Microbes with metabolic abilities for sulfur cycling (i.e., dissimilatory sulfite reducers, purple sulfur bacteria, green sulfur and non-sulfur bacteria, and organisms with the APS enzyme) were identified in all samples. These particular organisms have the ability to reduce sulfate and to re-oxidize reduced sulfur compounds back to sulfate.     

Acidic Cave-Wall Biofilms Located in the Frasassi Gorge,Italy

Acidic biofilms 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.