Novel uncultured Epsilonproteobacteria dominate a filamentous sulphur mat from the 13 degrees N hydrothermal vent field, East Pacific Rise

Rapid growth of microbial sulphur mats have repeatedly been observed during oceanographic cruises to various deep-sea hydrothermal vent sites. The microorganisms involved in the mat formation have not been phylogenetically characterized, although the production of morphologically similar sulphur filaments by a Arcobacter strain coastal marine has been documented. An in situ collector deployed for 5 days at the 13 degrees N deep-sea hydrothermal vent site on the East Pacific Rise (EPR) was rapidly colonized by a filamentous microbial mat. Microscopic and chemical analyses revealed that the mat consisted of a network of microorganisms embedded in a mucous sulphur-rich matrix. Molecular surveys based on 16S rRNA gene and aclB genes placed all the environmental clone sequences within the Epsilonproteobacteria. Although few 16S rRNA gene sequences were affiliated with that of cultured organisms, the majority was related to uncultured representatives of the Arcobacter group (< or = 95% sequence similarity). A probe designed to target all of the identified lineages hybridized with more than 95% of the mat community. Simultaneous hybridizations with the latter probe and a probe specific to Arcobacter spp. confirmed the numerical dominance of Arcobacter-like bacteria. This study provides the first example of the prevalence and ecological significance of free-living Arcobacter at deep-sea hydrothermal vents.     

Niche differentiation among mat-forming, sulfide-oxidizing bacteria at cold seeps of the Nile Deep Sea Fan (Eastern Mediterranean Sea)

Sulfidic muds of cold seeps on the Nile Deep Sea Fan (NDSF) are populated by different types of mat-forming sulfide-oxidizing bacteria. The predominant sulfide oxidizers of three different mats were identified by microscopic and phylogenetic analyses as (i) Arcobacter species producing cotton-ball-like sulfur precipitates, (ii) large filamentous sulfur bacteria including Beggiatoa species, and (iii) single, spherical Thiomargarita species. High resolution in situ microprofiles revealed different geochemical settings selecting for the different mat types. Arcobacter mats occurred where oxygen and sulfide overlapped above the seafloor in the bottom water interface. Filamentous sulfide oxidizers were associated with steep gradients of oxygen and sulfide in the sediment. A dense population of Thiomargarita was favored by temporarily changing supplies of oxygen and sulfide in the bottom water. These results indicate that the decisive factors in selecting for different mat-forming bacteria within one deep-sea province are spatial or temporal variations in energy supply. Furthermore, the occurrence of Arcobacter spp.-related 16S rRNA genes in the sediments below all three types of mats, as well as on top of brine lakes of the NDSF, indicates that this group of sulfide oxidizers can switch between different life modes depending on the geobiochemical habitat setting.     

In situ analysis of oxygen consumption and diffusive transport in high‐temperature acidic iron‐oxide microbial mats

The role of dissolved oxygen as a principal electron acceptor for microbial metabolism was investigated within Fe(III)‐oxide microbial mats that form in acidic geothermal springs of Yellowstone National Park (USA). Specific goals of the study were to measure and model dissolved oxygen profiles within high‐temperature (65–75°C) acidic (pH = 2.7–3.8) Fe(III)‐oxide microbial mats, and correlate the abundance of aerobic, iron‐oxidizing Metallosphaera yellowstonensis organisms and mRNA gene expression levels to Fe(II)‐oxidizing habitats shown to consume oxygen. In situ oxygen microprofiles were obtained perpendicular to the direction of convective flow across the aqueous phase/Fe(III)‐oxide microbial mat interface using oxygen microsensors. Dissolved oxygen concentrations dropped from ～ 50–60 μM in the bulk‐fluid/mat surface to below detection (< 0.3 μM) at a depth of ～ 700 μm (～ 10% of the total mat depth). Net areal oxygen fluxes into the microbial mats were estimated to range from 1.4–1.6 × 10^?4 μmol cm^?2 s^?1. Dimensionless parameters were used to model dissolved oxygen profiles and establish that mass transfer rates limit the oxygen consumption. A zone of higher dissolved oxygen at the mat surface promotes Fe(III)‐oxide biomineralization, which was supported using molecular analysis of Metallosphaera yellowstonensis 16S rRNA gene copy numbers and mRNA expression of haem Cu oxidases (FoxA) associated with Fe(II)‐oxidation.     

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.     

Filamentous "Epsilonproteobacteria" dominate microbial mats from sulfidic cave springs

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.     

Phototrophic Phylotypes Dominate Mesothermal Microbial Mats Associated with Hot Springs in Yellowstone National Park

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.     

Microbiological and geochemical heterogeneity in an in situ uranium bioremediation field site

The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.     

Characterization of a deep-sea microbial mat from an active cold seep at the Milano mud volcano in the Eastern Mediterranean Sea

A white, filamentous microbial mat at the Milano mud volcano in the Eastern Mediterranean Sea was sampled during the Medinaut cruise of the R/V Nadir in 1998. The composition of the mat community was characterized using a combination of phylogenetic and lipid biomarker methods. The mat sample was filtered through 0.2 and 5-microm filters to coarsely separate unicellular and filamentous bacteria. Analyses of 16S rRNA gene sequences amplified from the total community DNA from these fractions showed that similar archaeal populations were present in both fractions. However, the bacterial populations in the fractions differed from one another, and were more diverse than the archaeal ones. Lipid analysis showed that bacteria were the dominant members of the mat microbial community and the relatively low delta(13)C carbon isotope values of bulk bacterial lipids suggested the occurrence of methane- and sulfide-based chemo(auto)trophy. Consistent with this, the bacterial populations in the fractions were related to Alpha-, Gamma- and Epsilonproteobacteria, most of which were chemoautotrophic bacteria that utilize hydrogen sulfide (or reduced sulfur compounds) and/or methane. The most common archaeal 16S rRNA gene sequences were related to those of previously identified Archaea capable of anaerobic methane oxidation. Although the filamentous organisms observed in the mat were not conclusively identified, our results indicated that the Eastern Mediterranean deep-sea microbial mat community might be sustained on a combination of methane- and sulfide-driven chemotrophy.     

Bacterial sulfur cycling shapes microbial communities in surface sediments of an ultramafic hydrothermal vent field

The ultramafic-hosted Logatchev hydrothermal field (LHF) is characterized by vent fluids, which are enriched in dissolved hydrogen and methane compared with fluids from basalt-hosted systems. Thick sediment layers in LHF are partly covered by characteristic white mats. In this study, these sediments were investigated in order to determine biogeochemical processes and key organisms relevant for primary production. Temperature profiling at two mat-covered sites showed a conductive heating of the sediments. Elemental sulfur was detected in the overlying mat and metal-sulfides in the upper sediment layer. Microprofiles revealed an intensive hydrogen sulfide flux from deeper sediment layers. Fluorescence in situ hybridization showed that filamentous and vibrioid, Arcobacter-related Epsilonproteobacteria dominated the overlying mats. This is in contrast to sulfidic sediments in basalt-hosted fields where mats of similar appearance are composed of large sulfur-oxidizing Gammaproteobacteria. Epsilonproteobacteria (7-21%) and Deltaproteobacteria (20-21%) were highly abundant in the surface sediment layer. The physiology of the closest cultivated relatives, revealed by comparative 16S rRNA sequence analysis, was characterized by the capability to metabolize sulfur components. High sulfate reduction rates as well as sulfide depleted in (34)S further confirmed the importance of the biogeochemical sulfur cycle. In contrast, methane was found to be of minor relevance for microbial life in mat-covered surface sediments. Our data indicate that in conductively heated surface sediments microbial sulfur cycling is the driving force for bacterial biomass production although ultramafic-hosted systems are characterized by fluids with high levels of dissolved methane and hydrogen.     

Phylogenetic Evidence for the Existence of Novel Thermophilic Bacteria in Hot Spring Sulfur-Turf Microbial Mats in Japan

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.Microbial mats develop in a wide variety of aquatic environments, including geothermal hot springs and hydrothermal vents. There are several types of thermophilic microbial mats, e.g., those of cyanobacteria, anoxygenic phototrophic bacteria, and chemotrophic sulfur bacteria, which differ according to the physical and chemical conditions they favor and other environmental factors (10, 38). These microbial mats in thermal habitats have been studied extensively as a peculiar microbial community of the ecosystem, in relation to the phylogeny and evolution of thermophilic prokaryotes, or as a source of new functional enzymes.So-called sulfur-turf microbial mats are macroscopic bundles of white filaments consisting of colorless sulfur bacteria and elemental sulfur particles that form in shallow streams of sulfide-containing high-temperature hot springs. Since first reported by Miyoshi in 1897 (33), this kind of microbial mat has been recorded for several geographically remote hot springs in Japan, although there have been only scattered reports of sulfur-turf microbial mats or chemotrophic sulfur streamers in geothermal springs in other countries (9, 13, 14). The sulfur-turf mats generally develop within a temperature range of 45 to 73°C, within a pH range of 6 to 9, and at discrete sulfide-oxygen interfaces in geothermal springs. These characteristics suggest that the major constituents of the sulfur-turf prokaryotic community are (hyper)thermophilic, neutrophilic, microaerophilic, and chemolithotrophic bacteria. Early studies of these sulfur-turf mats distinguished microscopically three morphotypes of bacteria, two of which were tentatively named Thiovibrio miyoshi and Thiothrix miyoshi (15). Moreover, in situ ecophysiological and microscopic studies have shown that one of these bacteria, the large sausage-shaped “Thiovibrio miyoshi,” predominates in sulfur-turf mats and oxidizes environmental sulfide to elemental sulfur and then to sulfate via thiosulfate (27–31). So far, however, it has not been possible to isolate and cultivate any thermophilic prokaryotes from the sulfur-turf mats predominated by these sausage-shaped bacteria with artificial media, and no attempt has been made to clarify their taxonomic and phylogenetic positions.Determination of 16S rRNA genes is a useful research strategy for identifying uncultivated prokaryotes and is now commonly performed in ecological studies. This technique, involving PCR amplification of 16S rRNA genes or synthesis of cDNAs from bulk 16S rRNAs of natural mixed microbial populations, has been used successfully for the phylogenetic characterization of prokaryotes in hydrothermal environments (6, 7, 34, 40, 41, 47, 48). In the present study, this approach was applied to characterize the sausage-shaped bacteria in sulfur-turf mats without isolating and cultivating them. Here we report that sulfur-turf mats contain novel thermophilic bacteria belonging to the earliest-branching lineage of the domain bacteria.     

Influence of sulfide and temperature on species composition and community structure of hot spring microbial mats

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80 degrees C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90 degrees C, and a Chloroflexus mat is formed at 65 to 70 degrees C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter(-1)) below 70 degrees C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter(-1)) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.     

Biodiversity and monitoring of colorless filamentous bacteria in sulfide aquatic systems of North Caucasus region

Bacterial mats in sulfide aquatic systems of North Caucasus are basically composed by the species of genera Thiothrix and Sphaerotilus. Additionally, several non-filamentous sulfur-oxidizing bacteria were isolated from the mats and several minor 16S rRNA phylotypes were found in clone libraries from these mats. The minor components were affiliated with Proteobacteria, Chlorobia, Cyanobacteria and Firmicutes. Even in an individual mat population heterogeneity of Thiothrix spp. was revealed by analysis of 16S rRNA gene and RAPD-PCR. Five Thiothrix isolates were described as new species Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov. In the Thiothrix-Sphaerotilus type of bacterial mat the proportion of dominant organisms might be influenced by sulfide concentration in the spring water. The higher sulfide concentration (more than 10 mg/l) in the spring water is more favorable for the development of bacterial mats with dominant Thiothrix organisms than for Thiothrix-Sphaerotilus type of sulfur mat.     

Influence of Seasonal and Geochemical Changes on the Geomicrobiology of an Iron Carbonate Mineral Water Spring

Fuschna Spring in the Swiss Alps (Engadin region) is a bicarbonate iron(II)-rich, pH-neutral mineral water spring that is dominated visually by dark green microbial mats at the side of the flow channel and orange iron(III) (oxyhydr)oxides in the flow channel. Gradients of O₂, dissolved iron(II), and bicarbonate establish in the water. Our goals were to identify the dominating biogeochemical processes and to determine to which extent changing geochemical conditions along the flow path and seasonal changes influence mineral identity, crystallinity, and microbial diversity. Geochemical analysis showed microoxic water at the spring outlet which became fully oxygenated within 2.3 m downstream. X-ray diffraction and Mössbauer spectroscopy revealed calcite (CaCO₃) and ferrihydrite [Fe(OH)₃] to be the dominant minerals which increased in crystallinity with increasing distance from the spring outlet. Denaturing gradient gel electrophoresis banding pattern cluster analysis revealed that the microbial community composition shifted mainly with seasons and to a lesser extent along the flow path. 16S rRNA gene sequence analysis showed that microbial communities differ between the flow channel and the flanking microbial mat. Microbial community analysis in combination with most-probable-number analyses and quantitative PCR (qPCR) showed that the mat was dominated by cyanobacteria and the channel was dominated by microaerophilic Fe(II) oxidizers (1.97 × 10⁷ ± 4.36 × 10⁶ 16S rRNA gene copies g⁻¹ using Gallionella-specific qPCR primers), while high numbers of Fe(III) reducers (10⁹ cells/g) were identified in both the mat and the flow channel. Phototrophic and nitrate-reducing Fe(II) oxidizers were present as well, although in lower numbers (10³ to 10⁴ cells/g). In summary, our data suggest that mainly seasonal changes caused microbial community shifts, while geochemical gradients along the flow path influenced mineral crystallinity.     

Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.     

Lipid biomarkers and carbon isotope signatures of a microbial (Beggiatoa) mat associated with gas hydrates in the gulf of Mexico

White and orange mats are ubiquitous on surface sediments associated with gas hydrates and cold seeps in the Gulf of Mexico. The goal of this study was to determine the predominant pathways for carbon cycling within an orange mat in Green Canyon (GC) block GC 234 in the Gulf of Mexico. Our approach incorporated laser-scanning confocal microscopy, lipid biomarkers, stable carbon isotopes, and 16S rRNA gene sequencing. Confocal microscopy showed the predominance of filamentous microorganisms (4 to 5 mum in diameter) in the mat sample, which are characteristic of Beggiatoa. The phospholipid fatty acids extracted from the mat sample were dominated by 16:1omega7c/t (67%), 18:1omega7c (17%), and 16:0 (8%), which are consistent with lipid profiles of known sulfur-oxidizing bacteria, including Beggiatoa. These results are supported by the 16S rRNA gene analysis of the mat material, which yielded sequences that are all related to the vacuolated sulfur-oxidizing bacteria, including Beggiatoa, Thioploca, and Thiomargarita. The delta13C value of total biomass was -28.6 per thousand; those of individual fatty acids were -29.4 to -33.7 per thousand. These values suggested heterotrophic growth of Beggiatoa on organic substrates that may have delta13C values characteristic of crude oil or on their by-products from microbial degradation. This study demonstrated that integrating lipid biomarkers, stable isotopes, and molecular DNA could enhance our understanding of the metabolic functions of Beggiatoa mats in sulfide-rich marine sediments associated with gas hydrates in the Gulf of Mexico and other locations.     

Biodiversity of the microbial mat of the Garga hot spring

Background

Microbial mats are a good model system for ecological and evolutionary analysis of microbial communities. There are more than 20 alkaline hot springs on the banks of the Barguzin river inflows. Water temperature reaches 75 °C and pH is usually 8.0–9.0. The formation of microbial mats is observed in all hot springs. Microbial communities of hot springs of the Baikal rift zone are poorly studied. Garga is the biggest hot spring in this area.

Results

In this study, we investigated bacterial and archaeal diversity of the Garga hot spring (Baikal rift zone, Russia) using 16S rRNA metagenomic sequencing. We studied two types of microbial communities: (i) small white biofilms on rocks in the points with the highest temperature (75 °C) and (ii) continuous thick phototrophic microbial mats observed at temperatures below 70 °C. Archaea (mainly Crenarchaeota; 19.8% of the total sequences) were detected only in the small biofilms. The high abundance of Archaea in the sample from hot springs of the Baikal rift zone supplemented our knowledge of the distribution of Archaea. Most archaeal sequences had low similarity to known Archaea. In the microbial mats, primary products were formed by cyanobacteria of the genus Leptolyngbya. Heterotrophic microorganisms were mostly represented by Actinobacteria and Proteobacteria in all studied samples of the microbial mats. Planctomycetes, Chloroflexi, and Chlorobi were abundant in the middle layer of the microbial mats, while heterotrophic microorganisms represented mostly by Firmicutes (Clostridia, strict anaerobes) dominated in the bottom part. Besides prokaryotes, we detect some species of Algae with help of detection their chloroplasts 16 s rRNA.

Conclusions

High abundance of Archaea in samples from hot springs of the Baikal rift zone supplemented our knowledge of the distribution of Archaea. Most archaeal sequences had low similarity to known Archaea. Metagenomic analysis of microbial communities of the microbial mat of Garga hot spring showed that the three studied points sampled at 70 °C, 55 °C, and 45 °C had similar species composition. Cyanobacteria of the genus Leptolyngbya dominated in the upper layer of the microbial mat. Chloroflexi and Chlorobi were less abundant and were mostly observed in the middle part of the microbial mat. We detected domains of heterotrophic organisms in high abundance (Proteobacteria, Firmicutes, Verrucomicrobia, Planctomicetes, Bacteroidetes, Actinobacteria, Thermi), according to metabolic properties of known relatives, which can form complete cycles of carbon, sulphur, and nitrogen in the microbial mat. The studied microbial mats evolved in early stages of biosphere formation. They can live autonomously, providing full cycles of substances and preventing live activity products poisoning.

     

Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur.

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4',6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is "Candidatus Arcobacter sulfidicus." For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO(2) fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO(2) fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by "Candidatus Arcobacter sulfidicus" or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.     

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

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 white 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 had organic carbon isotope values (mean delta13C=-34.7 per thousand, 1sigma=3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean delta13C=-7.4 per thousand for two springs, n=8). Bacterial diversity was 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 from 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 ecosystems through chemolithoautotrophy and expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats.     

Biogeochemistry of an Iron-Rich Hypersaline Microbial Mat (Camargue, France)

In situ microsensor measurements were combined with biogeochemical methods to determine oxygen, sulfur, and carbon cycling in microbial mats growing in a solar saltern (Salin-de-Giraud, France). Sulfate reduction rates closely followed the daily temperature changes and were highest during the day at 25°C and lowest during the night at 11°C, most probably fueled by direct substrate interactions between cyanobacteria and sulfate-reducing bacteria. Sulfate reduction was the major mineralization process during the night and the contribution of aerobic respiration to nighttime DIC production decreased. This decrease of aerobic respiration led to an increasing contribution of sulfide (and iron) oxidation to nighttime O₂ consumption. A peak of elemental sulfur in a layer of high sulfate reduction at low sulfide concentration underneath the oxic zone indicated anoxygenic photosynthesis and/or sulfide oxidation by iron, which strongly contributed to sulfide consumption. We found a significant internal carbon cycling in the mat, and sulfate reduction directly supplied DIC for photosynthesis. The mats were characterized by a high iron content of 56 mol Fe cm^–3, and iron cycling strongly controlled the sulfur cycle in the mat. This included sulfide precipitation resulting in high FeS contents with depth, and reactions of iron oxides with sulfide, especially after sunset, leading to a pronounced gap between oxygen and sulfide gradients and an unusual persistence of a pH peak in the uppermost mat layer until midnight.