基于mcrA基因的沁水盆地煤层气田产甲烷菌群与途径分析

【目的】分析沁水盆地煤层气田不同煤层气井产出水样中产甲烷菌群和生物成因气的生成途径。【方法】以甲基辅酶M还原酶基因(mcr A)作为目标基因,采用454焦磷酸高通量测序方法,同时比对NCBI功能基因文库中的mcr A序列,分析不同煤层气井产出水中的产甲烷菌群。【结果】高通量测序表明,5个出水样产甲烷菌群OTUs(Operational taxonomic units)数为64–157个,共有的为22个,各占样品总数14%-34%;样品共检测到4种已知菌属,即甲烷杆菌属(Methanobacterium)、甲烷微菌属(Methanomicrobium)、甲烷叶菌属(Methanolobus)和甲烷螺菌属(Methanospirillum),优势菌属均为Methanobacterium。系统发育分析表明,未明确地位的菌属主要与Methanobacterium、Methanomicrobium、产甲烷球菌属(Methanococcus)和甲烷囊菌属(Methanoculleus)有较近的亲缘关系。5个样品中菌属所占比例不同,检测到的菌属类别大致相同。所有检测样品生物成因煤层气(Coalbed methane,CBM)的生成途径主要为氢营养型产甲烷途径。【结论】沁水盆地不同煤层气田产甲烷菌群菌种差异比较大,但生物成因气生成途径基本相似,与地理位置和煤藏条件没有相关性。     

Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.     

自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测

自然湿地是CH4排放的重要来源之一。产甲烷菌和甲烷氧化菌是介导自然湿地甲烷循环的重要功能菌群。开展产甲烷菌和甲烷氧化菌多样性的检测研究有助于揭示微生物介导的甲烷循环以及自然湿地甲烷排放的时空异质性。传统基于培养的检测方法已被证实无法充分描述产甲烷菌和甲烷氧化菌的多样性,而分子检测方法为自然湿地土壤产甲烷菌和甲烷氧化菌的多样性检测提供了一种更准确和科学的工具。本文综述了自然湿地土壤产甲烷菌和甲烷氧化菌的定性和定量分子检测方法,包括末端限制性片段长度多态性（T-RFLP）、变性梯度凝胶电泳（DGGE）、荧光原位杂交（FISH）和实时定量PCR（real-time qPCR）,重点分析了分子检测中两类重要的标记基因,总结了不同类型自然湿地产甲烷菌和甲烷氧化菌群落多样性的最新成果,提出了我国在该领域今后应深入研究探讨的一些问题及建议。     

Structure and function of methanogens along a short-term restoration chronosequence in the Florida Everglades

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.     

Heavy-machinery traffic impacts methane emissions as well as methanogen abundance and community structure in oxic forest soils

Temperate forest soils are usually efficient sinks for the greenhouse gas methane, at least in the absence of significant amounts of methanogens. We demonstrate here that trafficking with heavy harvesting machines caused a large reduction in CH(4) consumption and even turned well-aerated forest soils into net methane sources. In addition to studying methane fluxes, we investigated the responses of methanogens after trafficking in two different forest sites. Trafficking generated wheel tracks with different impact (low, moderate, severe, and unaffected). We found that machine passes decreased the soils' macropore space and lowered hydraulic conductivities in wheel tracks. Severely compacted soils yielded high methanogenic abundance, as demonstrated by quantitative PCR analyses of methyl coenzyme M reductase (mcrA) genes, whereas these sequences were undetectable in unaffected soils. Even after a year after traffic compression, methanogen abundance in compacted soils did not decline, indicating a stability of methanogens here over time. Compacted wheel tracks exhibited a relatively constant community structure, since we found several persisting mcrA sequence types continuously present at all sampling times. Phylogenetic analysis revealed a rather large methanogen diversity in the compacted soil, and most mcrA gene sequences were mostly similar to known sequences from wetlands. The majority of mcrA gene sequences belonged either to the order Methanosarcinales or Methanomicrobiales, whereas both sites were dominated by members of the families Methanomicrobiaceae Fencluster, with similar sequences obtained from peatland environments. The results show that compacting wet forest soils by heavy machinery causes increases in methane production and release.     

Traditional cattle manure application determines abundance, diversity and activity of methanogenic Archaea in arable European soil

Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchst?dt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the 'cattle manure' itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore.     

Methanogen communities and Bacteria along an ecohydrological gradient in a northern raised bog complex

Mires forming an ecohydrological gradient from nutrient-rich, groundwater-fed mesotrophic and oligotrophic fens to a nutrient-poor ombrotrophic bog were studied by comparing potential methane (CH(4)) production and methanogenic microbial communities. Methane production was measured from different depths of anoxic peat and methanogen communities were detected by detailed restriction fragment length polymorphism (RFLP) analysis of clone libraries, sequencing and phylogenetic analysis. Potential CH(4) production changed along the ecohydrological gradient with the fens displaying much higher production than the ombrotrophic bog. Methanogen diversity also decreased along the gradient. The two fens had very similar diversity of methanogenic methyl-coenzyme M reductase gene (mcrA), but in the upper layer of the bog the methanogen diversity was strikingly lower, and only one type of mcrA sequence was retrieved. It was related to the Fen cluster, a group of novel methanogenic sequences found earlier in Finnish mires. Bacterial 16S rDNA sequences from the fens fell into at least nine phyla, but only four phyla were retrieved from the bog. The most common bacterial groups were Deltaproteobacteria, Verrucomicrobia and Acidobacteria.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.     

Soil type links microbial colonization of rice roots to methane emission

Most of the methane (CH₄) emission from rice fields is derived from plant photosynthates, which are converted to CH₄. Rice cluster I (RC-1) archaea colonizing the rhizosphere were found to be the methanogens responsible for this process. Hence, RC-1 methanogens seem to play a crucial role in emission of the greenhouse gas CH₄. We determined the community composition and activity of methanogens colonizing the roots of eight different rice cultivars after growth on both Italian rice soil and river bank soil, which contained different communities of methanogenic archaea. The community composition was analyzed by terminal restriction fragment length polymorphism and cloning/sequencing of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase. When grown on rice field soil, the methanogenic community of the different rice cultivars was always dominated by RC-1 methanogens. In contrast, roots were colonized by Methanomicrobiales when grown on river bank soil, in which RC-1 methanogens were initially not detectable. Roots colonized with Methanomicrobiales compared with RC-1 exhibited lower CH₄ production and CH₄ emission rates. The results show that the type of methanogens colonizing rice roots has a potentially important impact on the global CH₄ cycle.     

The impact of aridification and vegetation type on changes in the community structure of methane-cycling microorganisms in Japanese wetland soils

Over the years, the wetlands covered by Sphagnum in Bibai, Japan have been turning into areas of aridity, resulting in an invasion of Sasa into the bogs. Yet little is known about the methane-cycling microorganisms in such environments. In this study, the methanotrophic, methanogenic, and archaeal community structures within these two types of wetland vegetation were studied by phylogenetic analysis targeting particulate methane monooxygenase (pmoA), methyl coenzyme M reductase (mcrA), and the archaeal 16S rRNA gene. The pmoA library indicated that Methylomonas and Methylocystis predominated in the Sphagnum-covered and Sasa-invaded areas, respectively. The mcrA and 16S rRNA libraries indicated that Methanoregula were abundant methanogens in the Sphagnum-covered area. In the Sasa-invaded area, by contrast, mcrA genes were not detected, and no 16S rRNA clones were affiliated with previously known methanogens. Because the Sasa-invaded area still produced methane, of the various uncultured populations detected, novel euryarchaeotal lineages are candidate methane producers.     

Phylogenetic diversity of methyl-coenzyme M reductase (mcrA) gene and methanogenesis from trimethylamine in hypersaline environments

Methanogens have been reported in complex microbial communities from hypersaline environments, but little is known about their phylogenetic diversity. In this work, methane concentrations in environmental gas samples were determined while methane production rates were measured in microcosm experiments with competitive and non-competitive substrates. In addition, the phylogenetic diversity of methanogens in microbial mats from two geographical locations was analyzed: the well studied Guerrero Negro hypersaline ecosystem, and a site not previously investigated, namely Laguna San Ignacio, Baja California Sur, Mexico. Methanogenesis in these microbial mats was suspected based on the detection of methane (in the range of 0.00086 to 3.204 %) in environmental gas samples. Microcosm experiments confirmed methane production by the mats and demonstrated that it was promoted only by non-competitive substrates (trimethylamine and methanol), suggesting that methylotrophy is the main characteristic process by which these hypersaline microbial mats produce methane. Phylogenetic analysis of amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from natural and manipulated samples revealed various methylotrophic methanogens belonging exclusively to the family Methanosarcinaceae. Moderately halophilic microorganisms of the genus Methanohalophilus were predominant (>60 % of mcrA sequences retrieved). Slightly halophilic and marine microorganisms of the genera Methanococcoides and Methanolobus, respectively, were also identified, but in lower abundances.     

Community analysis of methanogenic archaea within a riparian flooding gradient

Anoxic soils in river floodplains (or riparian soils) are a source of methane emission. However, little is known about the ecology and community structure of archaeal methanogenic microbes, which are a crucial component of methane flux in those habitats. We studied the archaeal community in the vertical profile of four different sites along the River Waal in the Netherlands. These sites differ in their annual flooding regime ranging from never or seldom to permanently flooded. The archaeal community structure has been characterized by terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of the archaeal SSU rRNA gene and the mcrA gene. The latter gene codes for the alpha-subunit of methyl-coenzyme M reductase. Additionally, the potential methanogenic activity was determined by incubation of soil slurries under anoxic conditions. The community composition differed only slightly with the depth of the soil (0-20 cm). However, the diversity of archaeal SSU rRNA genes increased with the frequency of flooding. Terminal restriction fragment length polymorphism analysis of mcrA gene amplicons confirmed the results concerning methanogenic archaea. In the never and rarely flooded soils, crenarchaeotal sequences were the dominant group. In the frequently and permanently flooded soils, Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae and the uncultured Rice Clusters IV and VI (Crenarchaeota) were detectable independently from duration of anoxic conditions. Methanosaetaceae, on the other hand, were only found in the permanently and frequently flooded soils under conditions where concentrations of acetate were < 30 microM. The results indicate that methanogens as well as other archaea occupy characteristic niches according to the flooding conditions in the field. Methanosaetaceae, in particular, seem to be adapted (or proliferate at) to low acetate concentrations.     

Microsite-dependent changes in methanogenic populations in a boreal oligotrophic fen

Wetlands, including peatlands, are the main source of natural methane emission. Well-defined fen microsites have different methane emissions rates, but it is not known whether the methane-producing Archaea communities vary at these sites. Possible horizontal variations of communities, in a natural oligotrophic fen, were analysed by characterizing the methanogens from two well-defined microsites: Eriophorum lawn and Hummock. Community structures were studied at two different layers of the fen, showing, respectively, high and low methane production. The structure of methanogen populations was determined using molecular techniques targeting the 16SrRNA gene and combined denaturing gradient gel electrophoresis (DGGE) and restriction fragment length polymorphism (RFLP) analysis. Results subjected to non-metric multidimensional scaling (MDS), diversity indices calculation and phylogenetic analysis revealed that upper layer communities changed with site while deeper layer communities remained the same. Phylogenetic analyses revealed six different clusters of sequences grouping with only two known orders of methanogens. Upper layers of Hummock were dominated by sequences clustering with members of Methanomicrobiales and sequences dominating the upper part of the Eriophorum lawn were related to members of the order Methanosarcinales. Novel methanogenic sequences were found at both sites at both depths. Vegetation characterizing the microsites probably influences the microbial communities in the layers of the fen where methane is produced.     

Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA) genes point to Methanobacteriales

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming.     

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。     

Methane-cycling communities in a permafrost-affected soil on Herschel Island, Western Canadian Arctic: active layer profiling of mcrA and pmoA genes

In Arctic wet tundra, microbial controls on organic matter decomposition are likely to be altered as a result of climatic disruption. Here, we present a study on the activity, diversity and vertical distribution of methane-cycling microbial communities in the active layer of wet polygonal tundra on Herschel Island. We recorded potential methane production rates from 5 to 40?nmol?h(-1) g(-1) wet soil at 10?°C and significantly higher methane oxidation rates reaching values of 6-10?μmol?h(-1) g(-1) wet soil. Terminal restriction fragment length polymorphism (T-RFLP) and cloning analyses of mcrA and pmoA genes demonstrated that both communities were stratified along the active layer vertical profile. Similar to other wet Arctic tundra, the methanogenic community hosted hydrogenotrophic (Methanobacterium) as well as acetoclastic (Methanosarcina and Methanosaeta) members. A pronounced shift toward a dominance of acetoclastic methanogens was observed in deeper soil layers. In contrast to related circum-Arctic studies, the methane-oxidizing (methanotrophic) community on Herschel Island was dominated by members of the type II group (Methylocystis, Methylosinus, and a cluster related to Methylocapsa). The present study represents the first on methane-cycling communities in the Canadian Western Arctic, thus advancing our understanding of these communities in a changing Arctic.     

Estimates of biogenic methane production rates in deep marine sediments at Hydrate Ridge, Cascadia margin

Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.     

Investigation of the methanogen population structure and activity in a brackish lake sediment

The methanogen community in sediment from the edge of a small brackish lake connected to the Beaulieu Estuary (Hampshire, UK) was investigated by analysis of 16S rRNA gene diversity using new methanogen-specific primers plus Archaea-specific primers. 16S rRNA gene primers previously used for polymerase chain reaction (PCR) detection of methanogenic Archaea from a variety of environments were evaluated by in silico testing. The primers displayed variable coverage of the four main orders of methanogens, highlighting the importance of this type of primer evaluation. Three PCR primer sets were designed using novel reverse primers to facilitate specific amplification of the orders Methanomicrobiales/Methanosarcinales, Methanobacteriales and Methanococcales. Diversity of the methanogen functional gene, methyl coenzyme M reductase (mcrA), was also studied. All gene libraries constructed from this sediment indicated that Methanomicrobiales and Methanosarcinales were the only methanogens detected. There was good agreement between the relative sequence abundances in the methanogen-specific 16S rRNA gene library and terminal restriction fragment length polymorphism (T-RFLP) profiling, suggesting that the population was dominated by putative H2 CO2 utilizing Methanomicrobiales, although acetate-utilizing methanogens were also present. The methanogen population analyses were in agreement with methanogenic activity measurements, which indicated that bicarbonate methanogenesis was higher than acetate methanogenesis at all depths measured and overall there was a significant difference (P = 0.001) between the rates of the two pathways. This study demonstrates the utility of new 16S rRNA gene PCR primers targeting specific methanogenic orders, and the combined results suggest that the CO2 reduction pathway dominates methanogenesis in the brackish sediment investigated.