Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich spring

The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones. Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within the Halobacteriales represented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors. This study demonstrates that members of the Halobacteriales are not restricted to their typical high-salt habitats, and we propose a role for the Halobacteriales in sulfur reduction in natural ecosystems.     

Bacterial Diversity and Sulfur Cycling in a Mesophilic Sulfide-Rich Spring

An artesian sulfide- and sulfur-rich spring in southwestern Oklahoma is shown to sustain an extremely rich and diverse microbial community. Laboratory incubations and autoradiography studies indicated that active sulfur cycling is occurring in the abundant microbial mats at Zodletone spring. Anoxygenic phototrophic bacteria oxidize sulfide to sulfate, which is reduced by sulfate-reducing bacterial populations. The microbial community at Zodletone spring was analyzed by cloning and sequencing 16S rRNA genes. A large fraction (83%) of the microbial mat clones belong to sulfur- and sulfate-reducing lineages within δ-Proteobacteria, purple sulfur γ-Proteobacteria, -Proteobacteria, Chloroflexi, and filamentous Cyanobacteria of the order Oscillatoria as well as a novel group within γ-Proteobacteria. The 16S clone library constructed from hydrocarbon-exposed sediments at the source of the spring had a higher diversity than the mat clone library (Shannon-Weiner index of 3.84 compared to 2.95 for the mat), with a higher percentage of clones belonging to nonphototrophic lineages (e.g., Cytophaga, Spirochaetes, Planctomycetes, Firmicutes, and Verrucomicrobiae). Many of these clones were closely related to clones retrieved from hydrocarbon-contaminated environments and anaerobic hydrocarbon-degrading enrichments. In addition, 18 of the source clones did not cluster with any of the previously described microbial divisions. These 18 clones, together with previously published or database-deposited related sequences retrieved from a wide variety of environments, could be clustered into at least four novel candidate divisions. The sulfate-reducing community at Zodletone spring was characterized by cloning and sequencing a 1.9-kb fragment of the dissimilatory sulfite reductase (DSR) gene. DSR clones belonged to the Desulfococcus-Desulfosarcina-Desulfonema group, Desulfobacter group, and Desulfovibrio group as well as to a deeply branched group in the DSR tree with no representatives from cultures. Overall, this work expands the division-level diversity of the bacterial domain and highlights the complexity of microbial communities involved in sulfur cycling in mesophilic microbial mats.     

Bacterial diversity and sulfur cycling in a mesophilic sulfide-rich spring

An artesian sulfide- and sulfur-rich spring in southwestern Oklahoma is shown to sustain an extremely rich and diverse microbial community. Laboratory incubations and autoradiography studies indicated that active sulfur cycling is occurring in the abundant microbial mats at Zodletone spring. Anoxygenic phototrophic bacteria oxidize sulfide to sulfate, which is reduced by sulfate-reducing bacterial populations. The microbial community at Zodletone spring was analyzed by cloning and sequencing 16S rRNA genes. A large fraction (83%) of the microbial mat clones belong to sulfur- and sulfate-reducing lineages within delta-Proteobacteria, purple sulfur gamma-Proteobacteria, epsilon -Proteobacteria, Chloroflexi, and filamentous Cyanobacteria of the order Oscillatoria as well as a novel group within gamma-Proteobacteria. The 16S clone library constructed from hydrocarbon-exposed sediments at the source of the spring had a higher diversity than the mat clone library (Shannon-Weiner index of 3.84 compared to 2.95 for the mat), with a higher percentage of clones belonging to nonphototrophic lineages (e.g., Cytophaga, Spirochaetes, Planctomycetes, Firmicutes, and Verrucomicrobiae). Many of these clones were closely related to clones retrieved from hydrocarbon-contaminated environments and anaerobic hydrocarbon-degrading enrichments. In addition, 18 of the source clones did not cluster with any of the previously described microbial divisions. These 18 clones, together with previously published or database-deposited related sequences retrieved from a wide variety of environments, could be clustered into at least four novel candidate divisions. The sulfate-reducing community at Zodletone spring was characterized by cloning and sequencing a 1.9-kb fragment of the dissimilatory sulfite reductase (DSR) gene. DSR clones belonged to the Desulfococcus-Desulfosarcina-Desulfonema group, Desulfobacter group, and Desulfovibrio group as well as to a deeply branched group in the DSR tree with no representatives from cultures. Overall, this work expands the division-level diversity of the bacterial domain and highlights the complexity of microbial communities involved in sulfur cycling in mesophilic microbial mats.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

Biodiversity and monitoring of colorless filamentous bacteitrtn 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/1) 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.     

Phylogenetic and functional prokaryotic diversity in the Hoito-Gol mesothermal mineral spring (Eastern Sayan Mountains,Buryat Republic)

High-throughput sequencing was used for comparative analysis of microbial communities of the water and mat from the Hoito-Gol mesothermal mineral sulfide spring (Eastern Sayan Mountains, Buryat Republic). Activity of microbial communities was determined. While both spring biotopes were dominated by members of three bacterial phyla—Proteobacteria, Bacteroidetes, and Firmicutes—they differed drastically in the composition of predominant phylotypes (at the genus level). In the water, the organisms widespread in aquatic environments were predominant, mostly aerobic chemoorganotrophs of the genera Acinetobacter, Pedobacter, and Flavobacterium. In the microbial mat, the organisms actively involved in the sulfur cycle predominated, including sulfur-reducing bacteria Sulfurospirillum, sulfate-reducing deltaproteobacteria, sulfuroxidizing chemoautotrophic bacteria, anoxygenic phototrophic bacteria of the phyla Chloroflexi and Chlorobi, as well as purple bacteria belonging to the α-, ß-, and γ-Proteobacteria. Microbial mats of the spring exhibited higher phylogenetic diversity compared to high-temperature mats containing photosynthetic microorganisms.     

Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk thermal spring (Baikal Area,Russia)

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, and salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54°C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected in the samples from both the thermophilic and mesophilic mats. Cultures of nonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolated from the mats developed at high (50.6–49.4°C) and low temperatures (45–20°C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealed in low-temperature mats. Truly thermophilic purple and green sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfur communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophyll a-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20°C) mat is of interest.     

Sulfur-metabolizing bacterial populations in microbial mats of the Nakabusa hot spring, Japan

At the Nakabusa hot spring, Japan, dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65 °C. The microbial community of such mats was analyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Analyses of 16S rRNA and functional genes (aprA, pufM) suggested the importance of three thermophilic bacterial groups: aerobic chemolithotrophic sulfide-oxidizing species of the genus Sulfurihydrogenibium (Aquificae), anaerobic sulfate-reducing species of the genera Thermodesulfobacterium/Thermodesulfatator, and filamentous anoxygenic photosynthetic species of the genus Chloroflexus. A new oligonucleotide probe specific for Sulfurihydrogenibium was designed and optimized for catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). In situ hybridizations of thin mat sections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfurihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume). Physiological experiments monitoring in vitro changes of sulfide concentration indicated slight sulfide production by sulfate-reducing bacteria under anoxic-dark conditions, sulfide consumption by photosynthetic bacteria under anoxic-light conditions and strong sulfide oxidation by chemolithotrophic members of Aquificae under oxic-dark condition. We therefore propose that Sulfurihydrogenibium spp. act as highly efficient scavengers of oxygen from the spring water, thus creating a favorable, anoxic environment for Chloroflexus and Thermodesulfobacterium/Thermodesulfatator in deeper layers.     

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°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°C, and a Chloroflexus mat is formed at 65 to 70°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°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.     

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.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide stream in the North Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide stream (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Spahaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism's population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or "threads" of natural sulfide springs.     

Characterization of Phototrophic Purple Nonsulfur Bacteria Forming Colored Microbial Mats in a Swine Wastewater Ditch

The community structure of pink-colored microbial mats naturally occurring in a swine wastewater ditch was studied by culture-independent biomarker and molecular methods as well as by conventional cultivation methods. The wastewater in the ditch contained acetate and propionate as the major carbon nutrients. Thin-section electron microscopy revealed that the microbial mats were dominated by rod-shaped cells containing intracytoplasmic membranes of the lamellar type. Smaller numbers of oval cells with vesicular internal membranes were also found. Spectroscopic analyses of the cell extract from the biomats showed the presence of bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Ubiquinone-10 was detected as the major quinone. A clone library of the photosynthetic gene, pufM, constructed from the bulk DNA of the biomats showed that all of the clones were derived from members of the genera Rhodobacter and Rhodopseudomonas. The dominant phototrophic bacteria from the microbial mats were isolated by cultivation methods and identified as being of the genera Rhodobacter and Rhodopseudomonas by studying 16S rRNA and pufM gene sequence information. Experiments of oxygen uptake with lower fatty acids revealed that the freshly collected microbial mats and the Rhodopseudomonas isolates had a wider spectrum of carbon utilization and a higher affinity for acetate than did the Rhodobacter isolates. These results demonstrate that the microbial mats were dominated by the purple nonsulfur bacteria of the genera Rhodobacter and Rhodopseudomonas, and the bioavailability of lower fatty acids in wastewater is a key factor allowing the formation of visible microbial mats with these phototrophs.     

Structural and Geomicrobiological Characteristics of a Microbial Community from a Cold Sulfide Spring

The Ancaster sulfur spring is a cold (9°C) sulfur spring located near Ancaster, Ontario, Canada, which hosts an abundant and diverse microbial mat community. We conducted an extensive microscopical study of the microbial community of this spring using a number of techniques: phase light, confocal scanning laser microscopy, conventional scanning electron microscopy using both chemical/critical point drying and cryofixation preparative techniques, environmental scanning electron microscopy, and transmission electron microscopy. The latter two techniques were coupled with energy dispersive X-ray spectrometry for elemental analysis to complement wet geochemical data collected on bulk spring water and mat pore water. In the anoxic source of the spring, green and purple sulfur bacteria were found together with a sulfide-utilizing type of cyanobacteria that had the unusual characteristic of storing colloidal sulfur intracellularly. Deeper within the source, the mats were dominated by green sulfur bacteria and thick biofilms of cells that precipitated Fe and Zn sulfide minerals on their surfaces. Downstream from the source, thick, filamentous white mats lined the stream channel, formed by a diverse mass of nonphotosynthetic sulfur oxidizers, which were responsible for forming thick masses of spherical colloidal sulfur. These were distinguished by ESEM-EDS from cells by their simple elemental composition (only S was detected). Aqueous geochemistry analysis by ICP-MS showed that some elements (Fe, C, P, Zn, Mg, Ba) were present at higher levels in mat pore water than in bulk spring water. Our approach allowed us to gain an appreciation of the characteristics of this microbial community and allowed us to develop a good understanding of the types of microorganisms present and infer some of the relationships among the members of the community. In addition, we wish to convey the utility of a thorough microscopical approach in geomicrobiological and microbial ecology studies.     

Pigment profiles and bacterial communities from Thailand thermal mats

Differently colored layers of freshwater hot spring mats at Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Two mats were characterized: a laminated mat with a green and a red layers, and a monolayer, greenish-yellow mat. Bacterial communities in green, red, and yellow layers were investigated using molecular, culturing and pigment analysis methods. Pigment profiles covered a wide spectrum from chlorophylls to carotenoids. A green mat layer showed higher relative content of chlorophyll than yellow and red layers which presented higher proportion of carotenoids. Cyanobacterial isolates grow up to 55–56°C and their pigment profiles showed a relatively high content of chlorophylls suggesting the importance of other bacterial groups in the mat pigment profiles. Bacterial communities were analyzed by 16S rDNA surveys showing Cyanobacteria and Chloroflexi as the mayor components of the community. Other significant members were Candidate Division OP10, Bacteroidetes, Planctomycetes and Actinobacteria. These results highlight a major participation of Cyanobacteria and Chloroflexi in thermal mat communities, and the preferential presence of Candidate Division OP10 in green mat layers. Differently colored mat layers showed characteristic bacterial communities which could be discriminated from pigment profiles and molecular surveys.     

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.     

An integrated study reveals diverse methanogens, Thaumarchaeota, and yet-uncultivated archaeal lineages in Armenian hot springs

Culture-independent and enrichment techniques, with an emphasis on members of the Archaea, were used to determine the composition and structure of microbial communities inhabiting microbial mats in the source pools of two geothermal springs near the towns of Arzakan and Jermuk in Armenia. Amplification of small-subunit rRNA genes using “universal” primers followed by pyrosequencing (pyrotags) revealed highly diverse microbial communities in both springs, with >99 % of pyrosequences corresponding to members of the domain Bacteria. The spring in Arzakan was colonized by a photosynthetic mat dominated by Cyanobacteria, in addition to Proteobacteria, Bacteroidetes, Chloroflexi, Spirochaeta and a diversity of other Bacteria. The spring in Jermuk was colonized by phylotypes related to sulfur, iron, and hydrogen chemolithotrophs in the Betaproteobacteria and Epsilonproteobacteria, along with a diversity of other Bacteria. Analysis of near full-length small subunit rRNA genes amplified using Archaea-specific primers showed that both springs are inhabited by a diversity of methanogens, including Methanomicrobiales and Methanosarcinales and relatives of Methanomassiliicoccus luminyensis, close relatives of the ammonia-oxidizing archaeon (AOA) “Candidatus Nitrososphaera gargensis”, and the yet-uncultivated Miscellaneous Crenarchaeotal Group and Deep Hydrothermal Vent Crenarchaeota group 1. Methanogenic enrichments confirmed the predicted physiological diversity, revealing methylotrophic, acetoclastic, and hydrogenotrophic methanogenesis at 45 and 55 °C, but not 65 °C. This is one of only a few studies combining cultivation-independent and -dependent approaches to study archaea in moderate-temperature (37–73 °C) terrestrial geothermal environments and suggests important roles for methanogenic archaea and AOA in the carbon and nitrogen biogeochemical cycles in these environments.     

Spectral Irradiance and Distribution of Pigments in a Highly Layered Marine Microbial Mat

The spectral irradiance from 400 to 1,100 nm was measured with depth in the intertidal sand mats at Great Sippewissett Salt Marsh, Mass. These mats contained at least four distinct layers, composed of cyanobacteria, purple sulfur bacteria containing bacteriochlorophyll a (Bchl a), purple sulfur bacteria containing Bchl b, and green sulfur bacteria. Spectral irradiance was measured directly by layering sections of mat on a cosine receptor. Irradiance was also approximated by using a calibrated fiber-optic tip. With the tip, irradiance measurements could be obtained at depth intervals less than 250 μm. The irradiance spectra were correlated qualitatively and quantitatively with the distribution of the diverse chlorophyll pigments in this mat and were compared with spectra recorded in plain sand lacking pigmented phototrophs. We found that the shorter wavelengths (400 to 550 nm) were strongly attenuated in the top 2 mm of the mat. The longer wavelengths (red and near infrared) penetrated to much greater depths, where they were attenuated by Bchl a, b, and c-containing anoxygenic phototrophic bacteria. The specific attenuation bands in the irradiance spectra correlated with the specific in vivo absorption bands of the Bchl-protein complexes in the bacteria. We concluded that the pigments in the phototrophs had a profound affect on the light environment within the mat. It seems likely that the diverse Bchl-protein complexes found in the anoxygenic phototrophs evolved in dense mat environments as a result of competition for light.     

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.     

Differential microbial communities in hot spring mats from Western Thailand

The microbial communities of freshwater hot spring mats from Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Green-, red-, and yellow colored mat layers were analyzed. In order to detect the major components of the microbial communities constituting the mat as well as the microorganisms showing significant metabolic activity, samples were analyzed using DNA- and RNA-based molecular techniques, respectively. Microbial community fingerprints, performed by denaturing gradient gel electrophoresis (DGGE), revealed clear differences among mat layers. Thermophilic phototrophic microorganisms, Cyanobacteria and Chloroflexi, constituted the major groups in these communities (on average 65 and 51% from DNA and RNA analyses, respectively). Other bacteria detected in the mat were Bacteroidetes, members of the Candidate Division OP10, Actinobacteria, and Planctomycetes. Differently colored mat layers showed characteristic bacterial communities and the major components of the metabolically active fraction of these communities have been identified.     

Functional structure of algal mat (Cladophora glomerata) in a freshwater in western Poland

GAMs (Generalized Additive Models) present a growth pattern for the common filamentous algae (FA) taxa in freshwater ecosystems in Poland. Tribonema, Ulothrix taxa are an early spring species. In comparison with Rhzioclonium, Cladophora and Oedogonium taxa, the algae Zygnemataceae (e.g. Zygnema, Spirogyra) occurs in waters with lower levels of nutrients, total dissolved substances and electrical conductivity. Cladophora glomerata shows different responses to environmental gradients than C. rivularis, C. fracta. Furthermore, the algae Cladophora glomerata (Chlorophyta, Ulvophyceae) forms a thick monoalgae mat (from the surface to the bottom) in the shallow and eutrophic Lake Oporzynskie in Wielkopolska Region in Poland (Central Europe). Detailed algal mat characteristics, physical and chemical parameters of water, and pigments in thalli were measured vertically (surface, middle, bottom) and spatially (marginal and central part) in the stationary mat phase during maximal algal growth. Temperature, dissolved oxygen and PAR irradiation significantly declined as the depth of the mat increased. Water turbidity was greater in the surface layer than in deeper parts, in contrast to the concentrations of N-NO₃, N-NH₄ and TDS. Pigment content in the cells of C. glomerata, cell lengths and diameters of pyrenoids changed throughout the mat, from top to bottom. The obtained data were used for statistical analyses. In conclusion: dense mats of C. glomerata show a vertical stratification while the mats remain spatially homogenous. Our data suggest that mat structure be considered a biological indicator of freshwater eutrophication.