Molecular Analysis of the Sulfate Reducing and ArchaealCommunity in a Meromictic Soda Lake (Mono Lake, California) by Targeting 16S rRNA, mcrA, apsA, and dsrAB Genes

Sulfate reduction is the most important process involved in the mineralization of carbon in the anoxic bottom waters of Mono Lake, an alkaline, hypersaline, meromictic Lake in California. Another important biogeochemical process in Mono Lake is thought to be sulfate-dependent methane oxidation (SDMO). However little is known about what types of organisms are involved in these processes in Mono Lake. Therefore, the sulfate-reducing and archaeal microbial community in Mono Lake was analyzed by targeting 16S rRNA, methyl-coenzyme M reductase (mcrA), adenosine-5′-phosphosulfate (apsA), and dissimilatory sulfite reductase (dsrAB) genes to investigate the sulfate-reducing and archaeal community with depth. Most of the 16S rRNA gene sequences retrieved from the samples fell into the δ-subdivision of the Proteobacteria. Phylogenetic analyses suggested that the clones obtained represented sulfate-reducing bacteria, which are probably involved in the mineralization of carbon in Mono Lake, many of them belonging to a novel line of descent in the δ-Proteobacteria. Only 6% of the sequences retrieved from the samples affiliated to the domain Euryarchaeota but did not represent Archaea, which is considered to be responsible for SDMO [Orphan et al. 2001: Appl Environ Microbiol 67:1922–1934; Teske et al.: Appl Environ Microbiol 68:1994–2007]. On the basis of our results and thermodynamic arguments, we proposed that SDMO in hypersaline environments is presumably carried out by SRB alone. Polymerase chain reaction (PCR) amplifications of the mcrA-, apsA-, and dsrAB genes in Mono Lake samples were, in most cases, not successful. Only the PCR amplification of the apsA gene was partially successful. The amplification of these functional genes was not successful because there was either insufficient “target” DNA in the samples, or the microorganisms in Mono Lake have divergent functional genes.     

Characterization of methanogenic and prokaryotic assemblages based on mcrA and 16S rRNA gene diversity in sediments of the Kazan mud volcano (Mediterranean Sea)

The diversity of the methyl‐coenzyme reductase A (mcrA) and 16S rRNA genes was investigated in gas hydrate containing sediment from the Kazan mud volcano, eastern Mediterranean Sea. mcrA was detected only at 15 and 20 cm below seafloor (cmbsf) from a 40‐cm long push core, while based on chemical profiles of methane, sulfate, and sulfide, possible anaerobic oxidation of methane (AOM) depth was inferred at 12–15 cmbsf. The phylogenetic relationships of the obtained mcrA, archaeal and bacterial 16S rRNA genes, showed that all the found sequences were found in both depths and at similar relative abundances. mcrA diversity was low. All sequences were related to the Methanosarcinales, with the most dominant (77.2%) sequences falling in group mcrA‐e. The 16S rRNA‐based archaeal diversity also revealed low diversity and clear dominance (72.8% of all archaeal phylotypes) of the Methanosarcinales and, in particular, ANME‐2c. Bacteria showed higher diversity but 83.2% of the retrieved phylotypes from both sediment layers belonged to the δ‐Proteobacteria. These phylotypes fell in the SEEP‐SRB1 putative AOM group. In addition, the rest of the less abundant phylotypes were related to yet‐uncultivated representatives of the Actinobacteria, Spirochaetales, and candidate divisions OP11 and WS3 from gas hydrate‐bearing habitats. These phylotype patterns indicate that AOM is occurring in the 15 and 20 cmbsf sediment layers.     

Microbial Diversity in Sediments Collected from the Deepest Cold-Seep Area, the Japan Trench

The Japan Trench land slope at a depth of 6,400 m is the deepest cold-seep environment with Calyptogena communities. Sediment samples from inside and beside the Calyptogena communities were collected, and the microbial diversity in the sediment samples was studied by molecular phylogenetic techniques. From DNA extracted directly from the sediment samples, 16S rDNAs were amplified by the polymerase chain reaction method. The sequences of the amplified 16S rDNAs selected by restriction fragment length polymorphism analysis were determined and compared with sequences in DNA databases. The results showed that 33 different bacterial 16S rDNA sequences from the two samples analyzed fell into similar phylogenetic categories, the α-, γ-, δ-, and ɛ-subdivisions of Proteobacteria, Cytophaga, and gram-positive bacteria; some of the 16S rDNA sequences were common to both samples. δ- and ɛ-Proteobacteria-related sequences were abundant in both sediments. These sequences are mostly related to sulfate-reducing or sulfur-reducing bacteria and epibionts, respectively. Eight different archaeal 16S rDNA sequences were cloned from the sediments. The majority of the archaeal 16S rDNA sequences clustered in Crenarchaeota and showed high similarities to marine group I archaeal rDNA. A Methanococcoides burtonii–related sequence obtained from the sediment clustered in the Euryarchaeota indicating that M. burtonii–related strains in the area of Calyptogena communities may contribute to production of methane in this environment. From these results, we propose a possible model of sulfur circulation within the microbial community and that of Calyptogena clams in the cold-seep environment. Received June 15, 1998; accepted November 10, 1998.     

Comparative Geochemical and Microbiological Characterization of Two Thermal Pools in the Uzon Caldera,Kamchatka, Russia

Arkashin Schurf (Arkashin) and Zavarzin Spring (Zavarzin), two active thermal pools in the Uzon Caldera, Kamchatka, Russia, were studied for geochemical and microbiological characterization. Arkashin, the smaller of the two pools, had broader temperature and pH ranges, and the sediments had higher concentrations of total As (4,250 mg/kg) relative to Zavarzin (48.9 mg/kg). Glycerol dialkyl glycerol tetraether profiles represented distinct archaeal communities in each pool and agreed well with previous studies of these pools. Although no archaeal 16S rRNA sequences were recovered from Arkashin, sequences recovered from Zavarzin were mostly representatives of the Crenarchaeota and “Korarchaeota,” and 13% of the sequences were unclassifiable. The bacterial community in Arkashin was dominated by uncultured “Bacteroidetes,” Hydrogenobaculum of the Aquificales and Variovorax of the Betaproteobacteria, and 19% of the sequences remained unclassified. These results were consistent with other studies of As-rich features. The most abundant members of the Zavarzin bacterial community included the Chloroflexi, as well as members of the classes Deltaproteobacteria and Clostridia. In addition, 24% of the sequences were unclassified and at least 5% of those represent new groups among the established Bacterial phyla. Ecological structure in each pool was inferred from taxonomic classifications and bulk stable isotope δ values of C, N, and S. Hydrogenobaculum was responsible for primary production in Arkashin. However, in Zavarzin, the carbon source appeared to be allochthonous to the identified bacterial community members. Additionally, sequences related to organisms expected to participate in N and S cycles were identified from both pools.     

Microbial community structure analysis of produced water from a high-temperature North Sea oil-field

Molecular and culture-based methods were used to investigate the microbial diversity in produced water obtained from the high-temperature Troll oil formation in the North Sea. 16S rRNA gene libraries were generated from total community DNA, using universal archaeal or bacterial oligonucleotide primer sets. Sequence analysis of 88 clones in the bacterial library indicated that they originated from members of Firmicutes (48 sequences), Bacteroidetes (17 sequences), δ-Proteobacteria (15 sequences), Spirochaetes (5 sequences), Thermotogales (2 sequences) and γ-Proteobacteria (1 sequence). Twenty-two sequences in the archaeal library were close relatives to members of the genera Methanococcus (18 sequences), Methanolobus (3 sequences) and Thermococcus (1 sequence). Most of the bacterial sequences shared less than 95% identity with their closest match in GenBank, indicating that the produced water harbours a unique community of novel bacterial species or genera. Members of the thermophilic genera Thermosipho, Thermotoga, Anaerophaga and Thermovirga were isolated. The Troll formations are not injected with sea water. Thus, dramatic changes of the in situ conditions have been avoided, and a common source of continuous contamination from injection water can be excluded. However, the majority of the organisms detected in the gene libraries were most closely related to cultivated organisms with optimum temperatures for growth well below the in situ reservoir temperature (70°C), indicating that produced water from the Troll platform harbours a substantial amount of non-indigenous organisms. This was confirmed by the isolation of a number of mesophilic and moderately thermophilic organisms that were unable to grow at reservoir temperature.     

Molecular identification of filterable bacteria and archaea in the water of acidic lakes of northern Russia

Wetland ecosystems are the natural centers of freshwater formation in northern Russia lowland landscapes. The humic acidic waters formed in bogs feed the numerous lakes of the northern regions. One milliliter of the water in these lakes contains up to 10⁴ ultrasmall microbial cells that pass through “bacterial” filters with a pore size of 0.22 μm. The vast majority of these cells do not grow on nutrient media and cannot be identified by routine cultivation-based approaches. Their identification was performed by analysis of clone libraries obtained by PCR amplification of archaeal and bacterial 16S rRNA genes from the fraction of cells collected from water filtrates of acidic lakes. Most of the obtained bacterial 16S rRNA gene sequences represented the class Betaproteobacteria and exhibited the highest homology of (94–99%) with 16S rRNA genes of representatives of the genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia. The archaeal 16S rRNA gene clone library comprised genes of Euryarchaeota representatives. One-third of these genes exhibited 97–99% homology to the 16S rRNA genes of taxonomically described organisms of the orders Methanobacteriales and Methanosarcinales. The rest of the cloned archaeal 16S rRNA genes were only distantly related (71–74% homology) to those in all earlier characterized archaea.     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

The Community and Phylogenetic Diversity of Biological Soil Crusts in the Colorado Plateau Studied by Molecular Fingerprinting and Intensive Cultivation

We studied the bacterial communities in biological soil crusts (BSCs) from the Colorado Plateau by enrichment and cultivation, and by statistically analyzed denaturing gradient gel electrophoresis (DGGE) fingerprinting of environmental 16S rRNA genes, and phylogenetic analyses. Three 500-m-long transects, tens of km apart, consisting of 10 equally spaced samples each, were analyzed. BSC communities consistently displayed less richness (10–32 detectable DGGE bands per sample) and Shannon diversity (2.1–3.3) than typical soil communities, with apparent dominance by few members. In spite of some degree of small-scale patchiness, significant differences in diversity and community structure among transects was detectable, probably related to the degree of crust successional maturity. Phylogenetic surveys indicated that the cyanobacterium Microcoleus vaginatus was dominant, with M. steenstrupii second among phototrophs. Among the 48 genera of nonphototrophs detected, Actinobacteria (particularly Streptomyces spp.) were very common and diverse, with 18 genera and an average contribution to the total 16S rDNA amplificate of 11.8%. β-Proteobacteria and Bacteriodetes contributed around 10% each; Low-GC Gram-positives, α-Proteobacteria, Thermomicrobiales, and Acidobacteria were common (2–5%). However, the second largest contribution was made by deep-branching unaffiliated alleles (12.6%), with some of them representing candidate bacterial divisions. Many of the novel strains isolated are likely new taxa, and some were representatives of the phylotypes detected in the field. The mucoid or filamentous nature of many of these isolates speaks for their role in crust formation.     

Microbial diversity in acid mine drainage of Xiang Mountain sulfide mine, Anhui Province, China

To understand the composition and structure of microbial communities in acid (pH 3.0) mine drainage (AMD) associated with pyrite mine tailings in Anhui Province, China, molecular diversities of 16S rRNA and 18S rRNA genes were examined using a PCR-based cloning approach. Bacterial, archaeal and microeukaryotic clone libraries were constructed. In contrast to typical dominance of autotrophic acidophiles, genus Acidiphilium, which consists of mixotrophic acidophiles capable of chemoorganotrophic and photosynthetic metabolisms, was the largest group in the bacterial clone library. These mixotrophic organisms may be advantageous in the oligotrophic AMD environment of the study site (certain amounts of dissolved organic carbon and light) by switching between two modes of metabolisms. Unexpectedly, a large fraction of bacterial clones (12.7%) were related to the neutrophilic genus Legionella, which can cause Legionnaires’ disease, a potentially lethal pneumonia. The eukaryotic 18S rRNA gene sequences were mostly related to Oxytricha, Nuclearia, and Penicillium. In the archaeal clone library, all the sequences were affiliated to the phylum Crenarchaeota, while the Euryarchaeota was not present.     

Microbial methane turnover at Marmara Sea cold seeps: a combined 16S rRNA and lipid biomarker investigation

Lipid biomarkers and their stable carbon isotopic composition, as well as 16S rRNA gene sequences, were investigated in sediment cores from active seepage zones in the Sea of Marmara (Turkey) located on the active North Anatolian Fault, to assess processes associated with methane turnover by indigenous microbial communities. Diagnostic ¹³C‐depleted archaeal lipids of anaerobic methane oxidizers were only found in one core from the South of Çinarcik Basin and consist mainly of archaeol, sn‐2 hydroxyarchaeol and various unsaturated pentamethylicosenes. Concurrently, abundant fatty acids (FAs) and a substantial amount of monoalkylglycerolethers (MAGEs), assigned to sulphate‐reducing bacteria, were detected with strong ¹³C‐depletions. Both microbial lipids and their δ¹³C values suggest that anaerobic oxidation of methane with sulphate reduction (AOM/SR) occurs, specially in the 10‐ to 12‐cm depth interval. Lipid biomarker results accompanied by 16S rRNA‐based microbial diversity analyses showed that ANME‐2 (ANME‐2a and ‐2c) archaea and Desulfosarcina/Desulfococcus and Desulfobulbus deltaproteobacterial clades are the major AOM assemblages, which indicate a shallow AOM community at high methane flux. Apart from the typical AOM lipid biomarker pattern, a ¹³C‐depleted diunsaturated hydrocarbon, identified as 7,14‐tricosadiene, occurred in the inferred maximum AOM interval at 10–12 cm depth. Its isotopic fingerprint implies that its microbial precursor occurs in close association with the AOM communities. Interestingly, the presence of 7,14‐tricosadiene coincides with the presence of the so‐far uncultured bacterial Candidate Division JS1, often detected in AOM areas. We propose the hypothesis that the JS1 bacterial group could be the potential source of ¹³C‐depleted tricosadiene. Future testing of this hypothesis is essential to fully determine the role of this bacterial group in AOM.     

Microbial Community in Black Rust Exposed to Hot Ridge Flank Crustal Fluids

During Integrated Ocean Drilling Program Expedition 301, we obtained a sample of black rust from a circulation obviation retrofit kit (CORK) observatory at a borehole on the eastern flank of Juan de Fuca Ridge. Due to overpressure, the CORK had failed to seal the borehole. Hot fluids from oceanic crust had discharged to the overlying bottom seawater and resulted in the formation of black rust analogous to a hydrothermal chimney deposit. Both culture-dependent and culture-independent analyses indicated that the black-rust-associated community differed from communities reported from other microbial habitats, including hydrothermal vents at seafloor spreading centers, while it shared phylotypes with communities previously detected in crustal fluids from the same borehole. The most frequently retrieved sequences of bacterial and archaeal 16S rRNA genes were related to the genera Ammonifex and Methanothermococcus, respectively. Most phylotypes, including phylotypes previously detected in crustal fluids, were isolated in pure culture, and their metabolic traits were determined. Quantification of the dissimilatory sulfite reductase (dsrAB) genes, together with stable sulfur isotopic and electron microscopic analyses, strongly suggested the prevalence of sulfate reduction, potentially by the Ammonifex group of bacteria. Stable carbon isotopic analyses suggested that the bulk of the microbial community was trophically reliant upon photosynthesis-derived organic matter. This report provides important insights into the phylogenetic, physiological, and trophic characteristics of subseafloor microbial ecosystems in warm ridge flank crusts.     

Response of Archaeal Community Structure to Environmental Changes in Lakes on the Tibetan Plateau,Northwestern China

To study how archaeal community responds to environmental changes, we investigated archaeal community structures in waters of three Tibetan saline lakes in northwestern China (Gahai, Xiaochaidan, and Charhan Lakes) with 16S rRNA gene phylogenetic analysis. Temperature, pH, and water chemistry (major anions and cations) of the lakes were measured. Three archaeal clone libraries were constructed with a total of 297 sequences. Incorporating our previous data obtained from other lakes on the Tibetan Plateau, we performed statistical analyses to identify dominant environmental parameters that could account for the observed variations in archaeal community structure. We concluded that salinity and water chemistry (Na and bicarbonate concentration in particular) played an important role in shaping archaeal community. In particular, the relative abundance of archaeal 16S rRNA genes affiliated with the Halobacteriales of the Euryarchaeota increased with salinity, whereas that of crenarchaeotal 16S rRNA gene sequences showed the opposite trend. Crenarchaeotal 16S rRNA gene sequences were retrieved from lake waters with salinity up to 28.3%. These results have important implications for our understanding of response of archaeal community to environmental changes in high-altitude lake ecosystems.     

Distribution of Cren- and Euryarchaeota in Scots Pine Mycorrhizospheres and Boreal Forest Humus

Archaeal 16S rRNA gene sequences have been found in a variety of moderate-temperature habitats including soil and rhizospheres. In this study, the differences of archaeal communities associated with Scots pine (Pinus sylvestris L.) short roots, different types of mycorrhizospheric compartments, and uncolonized boreal forest humus were tested by direct DNA extraction, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE), and sequencing. The results indicated that mycorrhizal colonization of Scots pine roots substantially influence the archaeal community of pine rhizospheres. Colonization of short roots by most mycorrhizal fungi tested increased both archaeal frequency and diversity. Most of the archaeal sequences encountered in mycorrhizas belonged to the phylum Euryarchaeota, order of Halobacteriales. The difference in archaeal diversity between the mycorrhizospheric compartments and humus was profound. Most compartments with fungal components contained euryarchaeotal 16S rRNA gene sequences, whereas a high diversity of crenarchaeotal sequences and no euryarchaeotal sequences were found in forest humus outside mycorrhizospheres.     

A Novel Community of Acidophiles in an Acid Mine Drainage Sediment

A sediment sample (pH 2.5) was collected at an acid mine drainage site in Anhui, China. The present acidophilic microbial community in the sediment was studied with a 16S rRNA gene clone library. Small-subunit rRNA genes were PCR amplified, cloned and screened by amplified rDNA restriction analysis (ARDRA). Subsequently, 10 different clones were identified and they were affiliated with Acidobacteria, β/γ-Proteobacteria, δ-Proteobacteria, Nitrospira, Candidate division TM7, and Low G + C Gram-positives. Phylogenetic analysis of 16S rRNA gene sequences revealed a diversity of acidophiles in the sediment that were mostly novel. Unexpectedly, 16S rRNA gene sequences affiliated with δ-Proteobacteria were found to constitute more than 60% of clone library. To our knowledge, this is the first occasion that bacteria of δ-Proteobacteria have been found dominant in the acidic habitat. Anaerobic sulfate- or metal reduction is the predominant physiological trait of bacteria of this subdivision. The high sulfate, ferric iron and the presence of bioavailable carbon in the anaerobic microenvironment may result in the dominance of bacteria of δ-Proteobacteria.     

Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes

Abstract We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d ⁻¹ with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d ⁻¹ were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d ⁻¹ , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d ⁻¹ . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d ⁻¹ . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.     

Archaeal Communities in Boreal Forest Tree Rhizospheres Respond to Changing Soil Temperatures

Temperature has generally great effects on both the activity and composition of microbial communities in different soils. We tested the impact of soil temperature and three different boreal forest tree species on the archaeal populations in the bulk soil, rhizosphere, and mycorrhizosphere. Scots pine, silver birch, and Norway spruce seedlings were grown in forest humus microcosms at three different temperatures, 7–11.5°C (night–day temperature), 12–16°C, and 16–22°C, of which 12–16°C represents the typical mid-summer soil temperature in Finnish forests. RNA and DNA were extracted from indigenous ectomycorrhiza, non-mycorrhizal long roots, and boreal forest humus and tested for the presence of archaea by nested PCR of the archaeal 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing. Methanogenic Euryarchaeota belonging to Methanolobus sp. and Methanosaeta sp. were detected on the roots and mycorrhiza. The most commonly detected archaeal 16S rRNA gene sequences belonged to group I.1c Crenarchaeota, which are typically found in boreal and alpine forest soils. Interestingly, also one sequence belonging to group I.1b Crenarchaeota was detected from Scots pine mycorrhiza although sequences of this group are usually found in agricultural and forest soils in temperate areas. Tree- and temperature-related shifts in the archaeal population structure were observed. A clear decrease in crenarchaeotal DGGE band number was seen with increasing temperature, and correspondingly, the number of euryarchaeotal DGGE bands, mostly methanogens, increased. The greatest diversity of archaeal DGGE bands was detected in Scots pine roots and mycorrhizas. No archaea were detected from humus samples from microcosms without tree seedling, indicating that the archaea found in the mycorrhizosphere and root systems were dependent on the plant host. The detection of archaeal 16S rRNA gene sequences from both RNA and DNA extractions show that the archaeal populations were living and that they may have significant contribution to the methane cycle in boreal forest soil, especially when soil temperatures rise.     

Diversity and Spatial Distribution of Prokaryotic Communities Along A Sediment Vertical Profile of A Deep-Sea Mud Volcano

We investigated the top 30-cm sediment prokaryotic community structure in 5-cm spatial resolution, at an active site of the Amsterdam mud volcano, East Mediterranean Sea, based on the 16S rRNA gene diversity. A total of 339 and 526 sequences were retrieved, corresponding to 25 and 213 unique (≥98% similarity) phylotypes of Archaea and Bacteria, respectively, in all depths. The Shannon–Wiener diversity index H was higher for Bacteria (1.92–4.03) than for Archaea (0.99–1.91) and varied differently between the two groups. Archaea were dominated by anaerobic methanotrophs ANME-1, -2 and -3 groups and were related to phylotypes involved in anaerobic oxidation of methane from similar habitats. The much more complex Bacteria community consisted of 20 phylogenetic groups at the phylum/candidate division level. Proteobacteria, in particular δ-Proteobacteria, was the dominant group. In most sediment layers, the dominant phylotypes of both the Archaea and Bacteria communities were found in neighbouring layers, suggesting some overlap in species richness. The similarity of certain prokaryotic communities was also depicted by using four different similarity indices. The direct comparison of the retrieved phylotypes with those from the Kazan mud volcano of the same field revealed that 40.0% of the Archaea and 16.9% of the Bacteria phylotypes are common between the two systems. The majority of these phylotypes are closely related to phylotypes originating from other mud volcanoes, implying a degree of endemicity in these systems.     

Cultivation-independent analysis of archaeal and bacterial communities of the formation water in an Indian coal bed to enhance biotransformation of coal into methane

Biogenic origin of the significant proportion of coal bed methane has indicated the role of microbial communities in methanogenesis. By using cultivation-independent approach, we have analysed the archaeal and bacterial community present in the formation water of an Indian coal bed at 600–700 m depth to understand their role in methanogenesis. Presence of methanogens in the formation water was inferred by epifluorescence microscopy and PCR amplification of mcrA gene. Archaeal 16S rRNA gene clone library from the formation water metagenome was dominated by methanogens showing similarity to Methanobacterium, Methanothermobacter and Methanolinea whereas the clones of bacterial 16S rRNA gene library were closely related to Azonexus, Azospira, Dechloromonas and Thauera. Thus, microbial community of the formation water consisted of predominantly hydrogenotrophic methanogens and the proteobacteria capable of nitrogen fixation, nitrate reduction and polyaromatic compound degradation. Methanogenic potential of the microbial community present in the formation water was elucidated by the production of methane in the enrichment culture, which contained 16S rRNA gene sequences showing close relatedness to the genus Methanobacterium. Microcosm using formation water as medium as well as a source of inoculum and coal as carbon source produced significant amount of methane which increased considerably by the addition of nitrite. The dominance of Diaphorobacter sp. in nitrite amended microcosm indicated their important role in supporting methanogenesis in the coal bed. This is the first study indicating existence of methanogenic and bacterial community in an Indian coal bed that is capable of in situ biotransformation of coal into methane.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.