Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P_GC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P_GC and optimal growth temperatures (T_opt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.     

Diversity of Archaea in Bottom Sediments of the Discharge Areas With Oil- and Gas-Bearing Fluids in Lake Baikal

Using massively parallel sequencing (the Roche 454 platform) we have studied the diversity of archaeal 16S rRNA gene sequences in oxic and anoxic sediments at six sites in Lake Baikal with oil- and gas-bearing fluids discharge. Archaeal communities appeared to be represented mainly by five phyla: Euryarchaeota, Crenarchaeota, Thaumarchaeota, Bathyarchaeota (miscellaneous Crenarchaeotic group), and Woesearchaeota (deep sea hydrothermal vent group 6). Among them we detected sequences of methanogens of the orders Methanomicrobiales, Methanosarsinales, Methanococcales, as well as representatives of the following uncultured archaeal lineages: Group C3, Marine Benthic Group D, and Terrestrial Miscellaneous Group. We have also identified sequences of ammonia-oxidizing archaea of the phyla Crenarchaeota and Thaumarchaeota. Phylogenetic analysis showed the presence ANME-2d-related sequences. However, the analysis of mcrA genes libraries has not revealed typical representatives of ANME groups. Comparison of amplicon libraries 16S rRNA gene fragments from different samples proved the widespread presence of previously detected Baikal archaeal lineages, which are members of the phylum Crenarchaeota and Thaumarchaeota (formerly Group C3 of Crenarchaeota).     

Analysis of methanogen diversity in a hypereutrophic lake using PCR-RFLP analysis of <Emphasis Type="Italic">mcr</Emphasis> sequences

The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction. A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes. The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies. They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens. The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.     

Methanogenic and Sulphate-Reducing Microbial Communities in Deep Groundwater of Crystalline Rock Fractures in Olkiluoto,Finland

The long-term safety of final disposal of spent nuclear fuel in the deep geosphere is dependent on stability of biogeochemical conditions at the disposal site. Microbial processes, such as sulphate reduction and methanogenesis, may have profound effects on site biogeochemistry. In this study, sulphate-reducing bacteria and methane-producing archaea were investigated at depths ranging from 68 to 545 m in crystalline rock fractures at an intended spent nuclear fuel disposal site in Olkiluoto, Finland. Denaturing gradient gel electrophoresis detected diverse sulphate-reducing bacterial communities in all samples. Although the number of dsrB gene copies was below 10³ copies ml^?1 in all analyzed samples according to real-time quantitative PCR, their abundance was highest in samples that had the highest sulphate concentrations. Several distinct mcrA gene fragments were also recovered from most of the analyzed samples by cloning, although the number of methanogens was lower than that of sulphate-reducing bacteria when measured by mcrA-targeted quantitative PCR. The detected gene fragments were most closely related to sequences obtained from aquatic and deep subsurface environments. Results imply that sulphate reduction, methanogenesis, and anaerobic methane oxidation may all take place in the Olkiluoto deep geobiosphere.     

Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.     

Endophytic Fusarium verticillioides reduces disease severity caused by Ustilago maydis on maize

Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.     

Site History and Edaphic Features Override the Influence of Plant Species on Microbial Communities in Restored Tidal Freshwater Wetlands

Restored wetland soils differ significantly in physical and chemical properties from their natural counterparts even when plant community compositions are similar, but effects of restoration on microbial community composition and function are not well understood. Here, we investigate plant-microbe relationships in restored and natural tidal freshwater wetlands from two subestuaries of the Chesapeake Bay. Soil samples were collected from the root zone of Typha latifolia, Phragmites australis, Peltandra virginica, and Lythrum salicaria. Soil microbial composition was assessed using 454 pyrosequencing, and genes representing bacteria, archaea, denitrification, methanogenesis, and methane oxidation were quantified. Our analysis revealed variation in some functional gene copy numbers between plant species within sites, but intersite comparisons did not reveal consistent plant-microbe trends. We observed more microbial variations between plant species in natural wetlands, where plants have been established for a long period of time. In the largest natural wetland site, sequences putatively matching methanogens accounted for ∼17% of all sequences, and the same wetland had the highest numbers of genes coding for methane coenzyme A reductase (mcrA). Sequences putatively matching aerobic methanotrophic bacteria and anaerobic methane-oxidizing archaea (ANME) were detected in all sites, suggesting that both aerobic and anaerobic methane oxidation are possible in these systems. Our data suggest that site history and edaphic features override the influence of plant species on microbial communities in restored wetlands.     

Characteristics and metabolic patterns of soil methanogenic archaea communities in the high‐latitude natural forested wetlands of China

Soil methanogenic microorganisms are one of the primary methane‐producing microbes in wetlands. However, we still poorly understand the community characteristic and metabolic patterns of these microorganisms according to vegetation type and seasonal changes. Therefore, to better elucidate the effects of the vegetation type and seasonal factors on the methanogenic community structure and metabolic patterns, we detected the characteristics of the soil methanogenic mcrA gene from three types of natural wetlands in different seasons in the Xiaoxing''an Mountain region, China. The results indicated that the distribution of Methanobacteriaceae (hydrogenotrophic methanogens) was higher in winter, while Methanosarcinaceae and Methanosaetaceae accounted for a higher proportion in summer. Hydrogenotrophic methanogenesis was the dominant trophic pattern in each wetland. The results of principal coordinate analysis and cluster analysis showed that the vegetation type considerably influenced the methanogenic community composition. The methanogenic community structure in the Betula platyphylla–Larix gmelinii wetland was relatively different from the structure of the other two wetland types. Indicator species analysis further demonstrated that the corresponding species of indicator operational taxonomic units from the Alnus sibirica wetland and the Betula ovalifolia wetland were similar. Network analysis showed that cooperative and competitive relationships exist both within and between the same or different trophic methanogens. The core methanogens with higher abundance in each wetland were conducive to the adaptation to environmental disturbances. This information is crucial for the assessment of metabolic patterns of soil methanogenic archaea and future fluxes in the wetlands of the Xiaoxing''an Mountain region given their vulnerability.     

Biogeochemical and Molecular Signatures of Anaerobic Methane Oxidation in a Marine Sediment

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for Archaea. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802–805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera Desulfonema, Desulfococcus, and Desulfosarcina. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.     

An Anaerobic Methane-Oxidizing Community of ANME-1b Archaea in Hypersaline Gulf of Mexico Sediments

Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as mcrA gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 μM coexist with sulfate concentrations around 10 mM. Clone libraries of dsrAB genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.     

甲烷代谢古菌分离培养研究进展

全球变暖是全人类面临的一个巨大挑战,而温室气体排放持续上升是全球变暖的关键因素,并引发一系列生态环境问题。甲烷是第二温室气体,对全球变暖的贡献达20%。然而,在甲烷代谢中发挥重要作用的产甲烷古菌和厌氧甲烷氧化古菌(anaerobic methanotroph,ANME)较难培养,极大地限制了人们对甲烷代谢及其影响碳源-汇关系与机制的研究。本文综述了最新产甲烷古菌和ANME富集、分离和培养方法,包括富集培养、原位培养、共培养、微流控技术、稀释分离和固体分离技术、ANME反应器和培养瓶富集培养,以及宏基因组预测和反向基因组学,并对这些方法的优缺点进行了评估,对未来甲烷代谢古菌的富集、分离和培养提出新的建议。     

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.     

闽江口芦苇沼泽湿地土壤产甲烷菌群落结构的垂直分布

应用PCR-RFLP技术及测序分析对闽江口芦苇湿地土壤产甲烷菌群落结构的垂直分布特征进行了研究。在构建的6个克隆文库中,每个克隆文库随机挑选100个克隆进行菌落PCR验证,共得到591个阳性克隆。PCR产物经限制性内切酶MspⅠ进行RFLP分析后得到37个不同的分类操作单元(OTUs)。对37个克隆子进行了序列测定,与GenBank数据库中的序列进行比对,最近相似性在91%—99%之间。RFLP分析和系统发育分析表明,闽江口芦苇湿地土壤中产甲烷菌群落包括3大类群:甲烷杆菌目(Methanobacteriales)、甲烷微菌目(Methanomirobiales)和甲烷八叠球菌目(Methanosarcinales)。不同土壤深度中产甲烷菌群落的分布呈现出不同的特征。土壤表层(0—10 cm)优势产甲烷菌类群为Methanoregula,约占76%;10—20 cm土层主要的产甲烷菌类群为Methanolinea和Methanoregula,分别约占23%和29%;20—30cm土层优势的产甲烷菌类群为Methanolinea,约占66%。Shannon指数(H’)和Simpson多样性指教(D)表明,10—20cm土层产甲烷菌多样性高于土壤表层(0—10 cm)和20—30 cm土层。37个测序OTUs中有26个OTUs属于不可培养的产甲烷菌序列,表明闽江口芦苇湿地土壤中存在大量不可培养的产甲烷菌。     

Oligonucleotide primers,probes and molecular methods for the environmental monitoring of methanogenic archaea

For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers.     

Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions

The prototypical representatives of the Euryarchaeota—the methanogens—are oxygen sensitive and are thought to occur only in highly reduced, anoxic environments. However, we found methanogens of the genera Methanosarcina and Methanocella to be present in many types of upland soils (including dryland soils) sampled globally. These methanogens could be readily activated by incubating the soils as slurry under anoxic conditions, as seen by rapid methane production within a few weeks, without any additional carbon source. Analysis of the archaeal 16S ribosomal RNA gene community profile in the incubated samples through terminal restriction fragment length polymorphism and quantification through quantitative PCR indicated dominance of Methanosarcina, whose gene copy numbers also correlated with methane production rates. Analysis of the δ¹³C of the methane further supported this, as the dominant methanogenic pathway was in most cases aceticlastic, which Methanocella cannot perform. Sequences of the key methanogenic enzyme methyl coenzyme M reductase retrieved from the soil samples before incubation confirmed that Methanosarcina and Methanocella are the dominant methanogens, though some sequences of Methanobrevibacter and Methanobacterium were also detected. The global occurrence of only two active methanogenic archaea supports the hypothesis that these are autochthonous members of the upland soil biome and are well adapted to their environment.     

Phylogenetic analysis of Archaea in the deep-sea sediments of west Pacific Warm Pool

Archaea are known to play important roles in carbon cycling in marine sediments. The main compositions of archaeal community in five deep-sea sediment samples collected from west Pacific Warm Pool area (WP-0, WP-1, WP-2, WP-3, WP-4), and in five sediment layers (1 cm-, 3 cm-, 6 cm-, 10 cm-, 12 cm- layer) of the 12 cm sediment core of WP-0 were checked and compared by denaturing gradient gel electrophoresis and 16 S rRNA gene sequencing. It was revealed that all the deep-sea sediment samples checked contained members of non-thermophilic marine group I crenarchaeota as the predominant archaeal group. To further detect groups of archaea possibly relating with C1 metabolism, PCR amplification was carried out using primers targeting methane-oxidizing archaea. Although no methane-oxidizing archaea was detected, a group of novel archaea (named as WPA) was instead identified from all these five WP samples by clone analysis. They could be placed in the euryarchaeota kingdom, separated into two distinct groups, the main group was peripherally related with methanogens, the other group related with Thermoplasma. The vertical distributions of WPA, archaea and bacteria along the WP-0 sediment column were determined by quantitative-PCR. It was found that bacteria dominated at all depths, the numbers of bacteria were 10–10⁴ times more than those of archaea. The proportion of archaea versus bacteria had a depth related increasing tendency, it was lowest at the first layer (0.01%), reached highest at the 12 cm- layer (10%). WPA only constituted a small proportion of the archaeal community (0.05% to 5%) of west Pacific Warm Pool sediment.Peng Wang and Xiang Xiao contributed equally to this paper     

Distribution of anaerobic methane-oxidizing and sulfate-reducing communities in the G11 Nyegga pockmark,Norwegian Sea

Pockmarks are seabed geological structures sustaining methane seepage in cold seeps. Based on RNA-derived sequences the active fraction of the archaeal community was analysed in sediments associated with the G11 pockmark, in the Nyegga region of the Norwegian Sea. The anaerobic methanotrophic Archaea (ANME) and sulfate-reducing bacteria (SRB) communities were studied as well. The vertical distribution of the archaeal community assessed by PCR-DGGE highlighted the presence of ANME-2 in surface sediments, and ANME-1 in deeper sediments. Enrichments of methanogens showed the presence of hydrogenotrophic methanogens of the Methanogenium genus in surface sediment layers as well. The active fraction of the archaeal community was uniquely composed of ANME-2 in the shallow sulfate-rich sediments. Functional methyl coenzyme M reductase gene libraries showed that sequences affiliated with the ANME-1 and ANME-3 groups appeared in the deeper sediments but ANME-2 dominated both surface and deeper layers. Finally, dissimilatory sulfite reductase gene libraries revealed a high SRB diversity (i.e. Desulfobacteraceae, Desulfobulbaceae, Syntrophobacteraceae and Firmicutes) in the shallow sulfate-rich sediments. The SRB diversity was much lower in the deeper section. Overall, these results show that the microbial community in sediments associated with a pockmark harbour classical cold seep ANME and SRB communities.     

Archaeal community diversity in municipal waste landfill sites

Despite the pivotal role of archaea in methane production in landfills, the identity, ecology, and functional diversity of these microorganisms and their link to environmental factors remain largely unknown. We collected 11 landfill leachate samples from six geographically distinct landfills of different ages in China and analyzed the archaeal community by bar-coded 454 pyrosequencing. We retrieved 121,797 sequences from a total of 167,583 sequences (average length of 464 bp). The archaeal community was geographically structured, and nonabundant taxa primarily contributed to the observed dissimilarities. Canonical correlation analysis (CCA) suggested that the total phosphorous (TP), nitrate, and conductivity are important drivers for shaping the archaeal community. The hydrogenotrophic methanogens Methanomicrobiales and Methanobacteriales greatly dominated 9 of 11 samples, ranging from 83.7 to 99.5 %. These methanogens also dominated the remaining two samples, accounting for 70.3 and 58.8 %, respectively. Interestingly, for all of the studied Chinese landfills, 16S rRNA analysis indicated the predominance of hydrogenotrophic methanogens.     

Abundance and Diversity of Ammonia-Oxidizing Microorganisms in the Sediments of Jinshan Lake

Community structures of ammonia-oxidizing microorganisms were investigated using PCR primers designed to specifically target the ammonia monooxygenase α-subunit (amoA) gene in the sediment of Jinshan Lake. Relationships between the abundance and diversity of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), and physicochemical parameters were also explored. The AOA abundance decreased sharply from west to east; however, the AOB abundance changed slightly with AOB outnumbering AOA in two of the four sediment samples (JS), JS3 and JS4. The AOA abundance was significantly correlated with the NH₄–N, NO₃–N, and TP. No significant correlations were observed between the AOB abundance and environmental variables. AOB had a higher diversity and richness of amoA genes than AOA. Among the 76 archaeal amoA sequences retrieved, 57.89, 38.16, and 3.95 % fell within the Nitrosopumilus, Nitrososphaera, and Nitrososphaera sister clusters, respectively. The 130 bacterial amoA gene sequences obtained in this study were grouped with known AOB sequences in the Nitrosomonas and Nitrosospira genera, which occupied 72.31 % and 27.69 % of the AOB group, respectively. Compared to the other three sample sites, the AOA and AOB community compositions at JS4 showed a large difference. This work could enhance our understanding of the roles of ammonia-oxidizing microorganisms in freshwater lake environment.