Microbial Community Biofabrics in a Geothermal Mine Adit

Speleothems such as stalactites and stalagmites are usually considered to be mineralogical in composition and origin; however, microorganisms have been implicated in the development of some speleothems. We have identified and characterized the biological and mineralogical composition of mat-like biofabrics in two novel kinds of speleothems from a 50°C geothermal mine adit near Glenwood Springs, CO. One type of structure consists of 2- to 3-cm-long, 3- to 4-mm-wide, leather-like, hollow, soda straw stalactites. Light and electron microscopy indicated that the stalactites are composed of a mineralized biofabric with several cell morphotypes in a laminated form, with gypsum and sulfur as the dominant mineral components. A small-subunit rRNA gene phylogenetic community analysis along the stalactite length yielded a diverse gradient of organisms, with a relatively simple suite of main constituents: Thermus spp., crenarchaeotes, Chloroflexi, and Gammaproteobacteria. PCR analysis also detected putative crenarchaeal ammonia monooxygenase subunit A (amoA) genes in this community, the majority related to sequences from other geothermal systems. The second type of speleothem, dumpling-like rafts floating on a 50°C pool on the floor of the adit, showed a mat-like fabric of evidently living organisms on the outside of the dumpling, with a multimineral, amorphous, gypsum-based internal composition. These two novel types of biofabrics are examples of the complex roles that microbes can play in mineralization, weathering, and deposition processes in karst environments.     

Molecular and Cellular Fossils of a Mat-Like Microbial Community in Geothermal Boratic Sinters

Silica and travertine deposits, the two most common surface manifestations of terrestrial hot springs, have so far been the only markers universally helpful in locating and interpreting geothermal systems and past lifeforms potentially associated with them. In the current study, we for the first time report microbial fossils from a third type of geothermal sinter, viz. the boron mineral deposits, which are characteristic of the relatively uncommon silicate-poor hot springs. Organic biomarker analyses of the boratic sinters framing the hot springs of Puga valley, Ladakh, identified molecular fossils (viz. respiratory and photosynthetic isoprenoid quinones and photosynthetic pigments) of a mat-like microbial community putatively comprised of algae, fungi, cyanobacteria and other photosynthetic bacteria, some of which may be proteobacteria. So far as microfossil preservation is concerned, mineralized mat-like biofabrics with diverse cellular morphotypes could be identified in scanning electron microscopy of sub-recent sediments as well as hard and consolidated old sinters. Though this putative past community is quite unusual in volcanic niches, its ubiquitous and enduring presence in the Puga geothermal area is highlighted by the discovery of its biosignatures along all of the three dimensions of the explored boratic deposits. These findings usher a new paradigm of looking at past or present geothermal life in as well as outside the Earth.     

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L'Aquila-Italy)

Much is known about the bacterial precipitation of carbonate rocks, but comparatively little is known about the involvement of microbes in the formation of secondary mineral structures in caves. We hypothesized that bacteria isolated from calcareous stalactites, which are able to mediate CaCO₃ precipitation in vitro, play a role in the formation of carbonate speleothems. We collected numerous cultivable calcifying bacteria from calcareous speleothems from Cervo cave, implying that their presence was not occasional. The relative abundance of calcifying bacteria among total cultivable microflora was found to be related to the calcifying activity in the stalactites. We also determined the δ ¹³C and δ ¹⁸ O values of the Cervo cave speleothems from which bacteria were isolated and of the carbonates obtained in vitro to determine whether bacteria were indeed involved in the formation of secondary mineral structures. We identified three groups of biological carbonates produced in vitro at 11°C on the basis of their carbon isotopic composition: carbonates with δ ¹³C values (a) slightly more positive, (b) more negative, and (c) much more negative than those of the stalactite carbonates. The carbonates belonging to the first group, characterized by the most similar δ ¹³C values to stalactites, were produced by the most abundant strains. Most of calcifying isolates belonged to the genus Kocuria. Scanning electron microscopy showed that dominant morphologies of the bioliths were sherulithic with fibrous radiated interiors. We suggest a mechanism of carbonate crystal formation by bacteria.     

Phylogenetic diversity analysis of subterranean hot springs in Iceland

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130 degrees C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-microm-pore-size filter. Cells were observed in wells RG-39 (91.4 degrees C) and MG-18 (71.8 degrees C) and in hot tap water (76 degrees C), but no cells were detected in wells SN-4, SN-5 (95 to 117 degrees C), and RV-5 (130 degrees C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85 degrees C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.     

Chromium Adsorption by Three Yeast Strains Isolated from Sediments in Morocco

Kartchner Caverns is an oligotrophic subterranean environment that hosts a wide diversity of actively growing calcite speleothems (secondary mineral deposits). In a previous study, we demonstrated that bacterial communities extracted from these surfaces are quite complex and vary between formations. In the current study, we evaluated the influence of several environmental variables on the superficial bacterial community structure of 10 active formations located in close proximity to one another in a small room of Kartchner Caverns State Park, Arizona, USA. Physical (color, dimensions) and chemical (elemental profile and organic carbon concentration) properties, as well as the DGGE-based bacterial community structure of the formations were analyzed. While elemental concentration was found to vary among the formations, the differences in the community structure could not be correlated with concentrations of either organic carbon or any of the elements evaluated. In contrast, the locations of formations within a distinct region of the cave as well as the relative location of specific formations within a single room were found to have a significant influence on the bacterial community structure of the formations evaluated. Interestingly, Canonical Correspondence Analysis suggests an association between the observed drip pathways (drip lines) feeding the formations (as determined by the patterns of soda straws and small stalactites that reveal water flow patterns) and the bacterial community structure of the respective formations. The results presented here indicate that a broad range of formations fed by a diversity of drip sources must be sampled to fully characterize the community composition of bacteria present on the surfaces of calcite formations in carbonate caves.     

La croissance des spéléothèmes : un enregistrement climatique à haute résolution, analyses d'images, périodes passées et futures, essais d'interprétation

Research's applied to carbonate laminated sequences (stalactites, stalagmites, cave flowstones, drapery, pisolits). Variability and irregularity about the concretions growth and relation with the climatic events.Crystalline development, fabrics and signs used. The interruption during concretionnement processes marks a signifiant errors in the interpretations. Analytic methods for the quantitative evaluation of speleothems laminated carbonate sequences. Discussion and interpretation essays. Imagery treatment applied to high-resolution laminograms. Forecasting models essays are used with results interpretations and boundaries. Validity of detection upon the future warm and dry periods.     

Discovery and description of giant submarine smectite cones on the seafloor in Eyjafjordur, northern Iceland, and a novel thermal microbial habitat

With the submersible JAGO and by scuba diving we discovered three remarkable geothermal cones, rising 33, 25, and 45 m from the seafloor at a depth of 65 m in Eyjafjordur, northern Iceland. The greatest geothermal activity was on the highest cone, which discharged up to 50 liters of freshwater per s at 72 degrees C and pH 10.0. The cones were built up from precipitated smectite, formed by mixing of the hot SiO2-rich geothermal fluid with the cold Mg-rich seawater. By connecting a rubber hose to one outflow, about 240 liters of pure geothermal fluids was concentrated through a 0.2-microm-pore-size filter. Among 50 thermophilic isolates, we found members of Bacillus and Thermonema and a new unidentified low-G+C gram-positive member of the Bacteria as well as one member of the Archaea, Desulfurococcus mobilis. Analysis of small-subunit rRNA genes PCR amplified and cloned directly from environmental DNA showed that 41 out of 45 Bacteria sequences belonged to members of the Aquificales, whereas all of the 10 Archaea sequences belonged to the Korarchaeota. The physiological characteristics of isolates from different parts of the cones indicate a completely freshwater habitat, supporting the possibility of subterranean transmittance of terrestrial organisms.     

Isolation, characterization, and ecology of sulfur-respiring crenarchaea inhabiting acid-sulfate-chloride-containing geothermal springs in Yellowstone National Park

Elemental sulfur (S(0)) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S(0)-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72 degrees C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81 degrees C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S(0), and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S(0) flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.     

Highly diverse community structure in a remote central Tibetan geothermal spring does not display monotonic variation to thermal stress

We report an assessment of whole-community diversity for an extremely isolated geothermal location with considerable phylogenetic and phylogeographic novelty. We further demonstrate, using multiple statistical analyses of sequence data, that the response of community diversity is not monotonic to thermal stress along a gradient of 52-83 degrees C. A combination of domain- and division-specific PCR was used to obtain a broad spectrum of community phylotypes, which were resolved by denaturing gradient gel electrophoresis. Among 58 sequences obtained from microbial mats and streamers, some 95% suggest novel archaeal and bacterial diversity at the species level or higher. Moreover, new phylogeographic and thermally defined lineages among the Cyanobacteria, Chloroflexi, Eubacterium and Thermus are identified. Shannon-Wiener diversity estimates suggest that mats at 63 degrees C supported highest diversity, but when alternate models were applied [Average Taxonomic Distinctness (AvTD) and Variation in Taxonomic Distinctness (VarTD)] that also take into account the phylogenetic relationships between phylotypes, it is evident that greatest taxonomic diversity (AvTD) occurred in streamers at 65-70 degrees C, whereas greatest phylogenetic distance between taxa (VarTD) occurred in streamers of 83 degrees C. All models demonstrated that diversity is not related to thermal stress in a linear fashion.     

Microbiology of the stalactites from Grotta dei Cervi, Porto Badisco, Italy.

The active stalactites from Grotta dei Cervi, Porto Badisco, southeastern Italy, were sampled to investigate the microbial communities present in these speleothems. Sampling was carried out in a transect about 150 m long in the central gallery, where numerous Gram-positive bacteria were isolated. Actinomycetes of the genus Streptomyces were the most abundant, followed by members of the genus Bacillus. Further isolates were assigned to the genera Amycolatopsis, Arthrobacter; Agromyces. Micrococcus, Nocardiopsis and Rhodococcus of the order Actinomycetales. The ability of actinomycetes to colonize subterranean environments is discussed.     

Legionella species diversity in an acidic biofilm community in Yellowstone National Park

Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30 degrees C) and higher (35 to 38 degrees C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.     

Methanotrophs of the psychrophilic microbial community of the Russian Arctic tundra

In tundra, at a low temperature, there exists a slowly developing methanotrophic community. Methane-oxidizing bacteria are associated with plants growing at high humidity, such as sedge and sphagnum; no methonotrophs were found in polytrichous and aulacomnious mosses and lichens, typical of more arid areas. The methanotrophic bacterial community inhabits definite soil horizons, from moss dust to peat formed from it. Potential ability of the methanotrophic community to oxidize methane at 5 degrees C enhances with the depth of the soil profile in spite of the decreasing soil temperature. The methanotrophic community was found to gradually adapt to various temperatures due to the presence of different methane-oxidizing bacteria in its composition. Depending on the temperature and pH, different methanotrophs occupy different econiches. Within a temperature range from 5 to 15 degrees C, three morphologically distinct groups of methanotrophs could be distinguished. At pH 5-7 and 5-15 degrees C, forms morphologically similar to Methylobacter psychrophilus predominated, whereas at the acidic pH 4-6 and 10-15 degrees C, bipolar cells typical of Methylocella palustris were mostly found. The third group of methanotrophic bacteria growing at pH 5-7 and 5-10 degrees C was represented by a novel methanotroph whole large coccoid cells had a thick mucous capsule.     

Soil microbial community structure across a thermal gradient following a geothermal heating event

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.     

Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.     

Influence of crude oil on changes of bacterial communities in Arctic sea-ice

The danger of a petroleum hydrocarbon spillage in the polar, ice-covered regions is increasing due to oil exploration in Arctic offshore areas and a growing interest in using the Northern Sea Route (NSR) as an alternative transportation route for Arctic oil and gas. However, little is known about the potential impact of accidental oil spills on this environment. We investigated the impact of crude oil on microbial community composition in six different Arctic sea-ice samples incubated with crude oil at 1 degrees C in microcosms for one year. Alterations in the composition of bacterial communities were analyzed with the culture-independent molecular methods DGGE (denaturing gradient gel electrophoresis) and FISH (fluorescence in situ hybridization). DGGE, FISH and cultivation methods revealed a strong shift in community composition toward the gamma-proteobacteria in sea-ice and melt pool samples incubated with crude oil. Marinobacter spp., Shewanella spp. and Pseudomonas spp. were the predominant phylotypes in the oil-treated microcosms. The ability of indigenous sea-ice bacteria to degrade hydrocarbons at low temperature (1 degrees C) was tested using four representative strains cultivated from sea-ice enriched with crude oil. [14C]Hexadecane was degraded by the sea-ice isolates at 20-50% capacity of the mesophilic type strain Marinobacter hydrocarbonoclasticus, a known hydrocarbon degrader, incubated at 22 degrees C.     

Carbon sequestration and community composition of ectomycorrhizal fungi across a geothermal warming gradient in an Icelandic spruce forest

Soil warming (0–5.5 °C above controls) effects on ectomycorrhizal growth, carbon sequestration and community composition were examined in a Picea sitchensis forest spanning a geothermal gradient in Iceland. Fungal communities were assayed with sand-filled ingrowth meshbags incubated in the soil for 5 months. Meshbags amended with compost made from maize leaves (a C4 plant enriched in ¹³C) were incubated for 5 or 12 months and used to estimate C sequestration by the fungal community. Despite increases in tree growth, moderate warming only slightly reduced or had no effect on mycelial growth and had no effect on fungal carbon sequestration or overall ectomycorrhizal community composition. Warming was associated with increased abundance of ascomycetes, particularly pyronemataceous ectomycorrhizal fungi, and altered saprotrophic community composition. Increased nitrate availability and root turnover may explain the lack of a positive ectomycorrhizal growth response to increased tree growth and observed shifts in community composition with warming.     

Transformation of carbonate minerals in a cyano-bacterial mat in the course of laboratory modeling

A laboratory model of a cyano-bacterial mat with mineral layers of carbonates was used to examine the dynamics of the transformation of calcium-magnesium carbonate under the conditions of a soda lake. The activity of various organisms of the cyanobacterial community results in conditions under which the Ca-Mg carbonate precipitate undergoes changes. The crystal lattice of the initial carbonate is restructured; its mineralogical composition changes depending on the conditions of the mat. In magnesium calcites, which are formed under such low-temperature conditions, a rudimentary cation adjustment can occur with the formation of dolomite domains. These experiments confirm the hypothesis that the dolomite found in stromatolites is of a secondary origin and can be formed in the course of transformation of Ca-Mg carbonates under alkaline conditions in an alkaliphilic cyanobacterial community.     

Transformation of carbonate minerals in a cyano-bacterial mat in the course of laboratory modeling

A laboratory model of a cyano-bacterial mat with mineral layers of carbonates was used to examine the dynamics of the transformation of calcium-magnesium carbonate under the conditions of a soda lake. The activity of various organisms of the cyanobacterial community results in conditions under which the Ca-Mg carbonate precipitate undergoes changes. The crystal lattice of the initial carbonate is restructured; its mineralogical composition changes depending on the conditions of the mat. In magnesium calcites, which are formed under such low-temperature conditions, a rudimentary cation adjustment can occur with the formation of dolomite domains. These experiments confirm the hypothesis that the dolomite found in stromatolites is of a secondary origin and can be formed in the course of transformation of Ca-Mg carbonates under alkaline conditions in an alkaliphilic cyanobacterial community.     

Mineralogy Influences Structure and Diversity of Bacterial Communities Associated with Geological Substrata in a Pristine Aquifer

Our understanding of mineralogical influences on subsurface microbial community structure and diversity has been difficult to assess due to difficulties in isolating this variable from others in the subsurface environment. In this study, biofilm coupons were used to isolate specific geological substrata from the surrounding geological matrix during colonization by microorganisms suspended in the surrounding groundwater for an 8-week period. Upon retrieval, the structure and diversity of the microbial community associated with each type of substratum was evaluated using 16S rDNA-based terminal-restriction fragment length polymorphism (T-RFLP). Phylogenetic affiliations of the populations associated with each type of substratum were established based on sequence analysis of near full-length 16S rDNA obtained through construction of a clone library. Hematite, quartz, and saprolite each harbored a community dominated by members of the division Proteobacteria (>67% of community). However, the different substrata selected for different subdivisions of bacteria within the Proteobacteria. After accounting for the influence exerted by substratum type on recovery of DNA from the attached populations, both phylogenetic data and Jaccard and Bray–Curtis similarity indices derived from terminal-restriction fragment (T-RF) profiles suggested a strong mineralogical influence on the structure and composition of the solid phase-associated community. The results suggest that mineralogical heterogeneity influences microbial community structure and diversity in pristine aquifers. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized authors.     

Avicel-adsorbable endoglucanase production by the thermophilic fungus Scytalidium thermophilum type culture Torula thermophila

Scytalidium thermophilum type culture Torula thermophila was isolated from mushroom compost and the total cellulase, endoglucanase, Avicel-adsorbable endoglucanase activities, as well as the fungal biomass generation and cellulose utilisation were analyzed in shake flask cultures with Avicel (microcrystalline cellulose) as the carbon source. Results were compared with an industrial strain of Scytalidium thermophilum type culture Humicola insolens. The pH and temperature optima for endoglucanase activities during enzyme assays were also analyzed for both organisms and determined to be pH 6.0 and 65 degrees C for type culture Torula thermophila, and pH 6.5 and 60 degrees C for type culture Humicola insolens. Analysis of the effect of growth temperature showed that type culture T. thermophila can grow and produce cellulases in the range of 35 to 55 degrees C although 40 to 50 degrees C seemed to favor growth and cellulase production. Although 45 degrees C was found optimal for fungal growth, both the specific endoglucanase and Avicel-adsorbable endoglucanase activities (U/mg protein) as well as the percentage of Avicel-adsorbable endoglucanase activity reached maxima at 50 degrees C and were higher as compared to type culture H. insolens. Results indicate that type culture T. thermophila, with further optimisations, is of potential use in the industrial production of cellulases.