土壤中甲烷厌氧氧化菌多样性的分子检测

甲烷厌氧氧化作用是减少海洋底泥甲烷释放的重要生物地球化学过程,然而在陆地生态系统中甲烷厌氧氧化作用及其功能菌群的生态功能仍然不确定。对甲烷厌氧氧化菌多样性的研究可为减少甲烷排放提供重要科学依据。与传统的分离培养方法比较,分子检测方法是一种更为快速和高效的研究手段,可直接和全面的反映参与甲烷厌氧氧化作用的功能微生物。以DNA分子标记物为研究对象,重点探讨三类主要的分子标记基因,即16S rRNA,mcr A和pmo A,所采用的相关探针和引物信息,同时从定性和定量两个角度综述土壤甲烷厌氧氧化菌的多样性研究的主要进展,最后提出厌氧甲烷氧化菌多样性研究中存在的一些问题和相应的解决思路。     

Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments

The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera Methylomonas, Methylosinus, and Methylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.     

Weak phylogenetic signal in physiological traits of methane‐oxidizing bacteria

The presence of phylogenetic signal is assumed to be ubiquitous. However, for microorganisms, this may not be true given that they display high physiological flexibility and have fast regeneration. This may result in fundamentally different patterns of resemblance, that is, in variable strength of phylogenetic signal. However, in microbiological inferences, trait similarities and therewith microbial interactions with its environment are mostly assumed to follow evolutionary relatedness. Here, we tested whether indeed a straightforward relationship between relatedness and physiological traits exists for aerobic methane‐oxidizing bacteria (MOB). We generated a comprehensive data set that included 30 MOB strains with quantitative physiological trait information. Phylogenetic trees were built from the 16S rRNA gene, a common phylogenetic marker, and the pmoA gene which encodes a subunit of the key enzyme involved in the first step of methane oxidation. We used a Blomberg's K from comparative biology to quantify the strength of phylogenetic signal of physiological traits. Phylogenetic signal was strongest for physiological traits associated with optimal growth pH and temperature indicating that adaptations to habitat are very strongly conserved in MOB. However, those physiological traits that are associated with kinetics of methane oxidation had only weak phylogenetic signals and were more pronounced with the pmoA than with the 16S rRNA gene phylogeny. In conclusion, our results give evidence that approaches based solely on taxonomical information will not yield further advancement on microbial eco‐evolutionary interactions with its environment. This is a novel insight on the connection between function and phylogeny within microbes and adds new understanding on the evolution of physiological traits across microbes, plants and animals.     

Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing

Arctic lakes are a significant source of the greenhouse gas methane (CH₄), but the role that methane oxidizing bacteria (methanotrophs) play in limiting the overall CH₄ flux is poorly understood. Here, we used stable isotope probing (SIP) techniques to identify the metabolically active aerobic methanotrophs in upper sediments (0–1 cm) from an arctic lake in northern Alaska sampled during ice‐free summer conditions. The highest CH₄ oxidation potential was observed in the upper sediment (0–1 cm depth) with 1.59 µmol g wet weight^?1 day^?1 compared with the deeper sediment samples (1–3 cm, 3–5 cm and 5–10 cm), which exhibited CH₄ oxidation potentials below 0.4 µmol g wet weight^?1 day^?1. Both type I and type II methanotrophs were directly detected in the upper sediment total communities using targeted primer sets based on 16S rRNA genes. Sequencing of 16S rRNA genes and functional genes (pmoA and mxaF) in the ¹³C‐DNA from the upper sediment indicated that type I methanotrophs, mainly Methylobacter, Methylosoma, Methylomonas and Methylovulum miyakonense, dominated the assimilation of CH₄. Methylotrophs, including the genera Methylophilus and/or Methylotenera, were also abundant in the ¹³C‐DNA. Our results show that a diverse microbial consortium acquired carbon from CH₄ in the sediments of this arctic lake.     

Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloud-like conditions

Atmospheric methane is degraded by both photooxidation and, in topsoils, by methanotrophic bacteria, but this may not totally account for the global sink of this greenhouse gas. Topsoils are a prominent source of airborne bacteria, which can degrade some organic atmospheric compounds at rates similar to photooxidation. Although airborne methanotrophs would have direct access to atmospheric methane, their presence and activity in the atmosphere has not been investigated so far. We enriched airborne methanotrophs from air and rainwater and showed that they oxidized methane at atmospheric concentration. The majority of seven OTUs, detected using pmoA gene clone libraries, were affiliated to the type II methanotrophic genera Methylocystis and Methylosinus. Furthermore, 16S rRNA gene clone libraries revealed the presence of OTUs affiliated with the genera Hyphomicrobium and Variovorax, members of which can stimulate methane oxidation by yet unidentified mechanisms. Simulating cloud-like conditions revealed that although both low pH and the presence of common cloud-borne organics negatively affected methane oxidation, airborne methanotrophs were able to degrade atmospheric methane in most cases. We demonstrate here for the first time that viable methanotrophic bacteria are present in air and rain and thus expand our knowledge on the global distribution of methanotrophs to include the atmosphere. The fact that they can degrade methane to below atmospheric concentrations when inoculated into artificial cloud water leads to an important possible effect of these organisms: the atmosphere may not only function as a medium for microbial dissemination, but also as a site of active microbial methane turnover.     

Analysis of Methane Monooxygenase Genes in Mono Lake Suggests That Increased Methane Oxidation Activity May Correlate with a Change in Methanotroph Community Structure

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.     

Molecular biologic techniques applied to the microbial prospecting of oil and gas in the Ban 876 gas and oil field in China

Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth; however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively, were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area.     

Spatiotemporal variations in an assemblage of closely related planktonic aerobic methanotrophs

1. The assemblage of aerobic methane‐oxidising bacteria (MOB) was investigated in different seasons in the water column of a stratified freshwater lake. Species composition was analysed by performing denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes and cloning analysis of the pmoA gene, which encodes the α‐subunit of particulate methane monooxygenase. The relative abundance of MOB to total bacteria was deduced from the copy number of the pmoA gene and 16S rRNA gene using real‐time polymerase chain reaction. 2. The profiles of the DGGE banding patterns changed with water depth, and these changes correlated with oxygen concentration and water temperature. The sequences of the DGGE bands obtained were all associated with the genus Methylobacter. During the analysis of pmoA gene, all clones sequenced were that of the Methylobacter/Methylosarcina group. The relative abundances of pmoA gene peaked around the oxycline, and small peaks of pmoA gene were also observed near the surface when peaks of methane were observed at the corresponding depth. 3. Profiles of the DGGE banding patterns suggested that ecophysiological characteristics differ among members of the genus Methylobacter; this indicates the importance of investigating the MOB assemblage at the species level or lower. Planktonic MOB seemed to be abundant around oxycline.     

Methane oxidation activity and diversity of aerobic methanotrophs in pH-neutral and semi-neutral thermal springs of the Kunashir Island,Russian Far East

Aerobic methane oxidation has been mostly studied in environments with moderate to low temperatures. However, the process also occurs in terrestrial thermal springs, where little research on the subject has been done to date. The potential activity of methane oxidation and diversity of aerobic methanotrophic bacteria were studied in sediments of thermal springs with various chemical and physical properties, sampled across the Kunashir Island, the Kuriles archipelago. Activity was measured by means of the radioisotope tracer technique utilizing ¹⁴C-labeled methane. Biodiversity assessments were based on the particulate methane monooxygenase (pmoA) gene, which is found in all known thermophilic and thermotolerant methanotrophs. We demonstrated the possibility of methane oxidation in springs with temperature exceeding 74 °C, and the most intensive methane uptake was shown in springs with temperatures about 46 °C. PmoA was detected in 19 out of 30 springs investigated and the number of pmoA gene copies varied between 10⁴ and 10⁶ copies per ml of sediment. Phylogenetic analysis of PmoA sequences revealed the presence of methanotrophs from both the Alpha- and Gammaproteobacteria. Our results suggest that methanotrophs inhabiting thermal springs with temperature exceeding 50 °C may represent novel thermophilic and thermotolerant species of the genera Methylocystis and Methylothermus, as well as previously undescribed Gammaproteobacteria.     

Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA and pmoA gene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescence in situ hybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 μM for methane and 8.7 ± 1.5 μM for nitrite.     

Functional Gene Analysis of Freshwater Iron-Rich Flocs at Circumneutral pH and Isolation of a Stalk-Forming Microaerophilic Iron-Oxidizing Bacterium

Iron-rich flocs often occur where anoxic water containing ferrous iron encounters oxygenated environments. Culture-independent molecular analyses have revealed the presence of 16S rRNA gene sequences related to diverse bacteria, including autotrophic iron oxidizers and methanotrophs in iron-rich flocs; however, the metabolic functions of the microbial communities remain poorly characterized, particularly regarding carbon cycling. In the present study, we cultivated iron-oxidizing bacteria (FeOB) and performed clone library analyses of functional genes related to carbon fixation and methane oxidization (cbbM and pmoA, respectively), in addition to bacterial and archaeal 16S rRNA genes, in freshwater iron-rich flocs at groundwater discharge points. The analyses of 16S rRNA, cbbM, and pmoA genes strongly suggested the coexistence of autotrophic iron oxidizers and methanotrophs in the flocs. Furthermore, a novel stalk-forming microaerophilic FeOB, strain OYT1, was isolated and characterized phylogenetically and physiologically. The 16S rRNA and cbbM gene sequences of OYT1 are related to those of other microaerophilic FeOB in the family Gallionellaceae, of the Betaproteobacteria, isolated from freshwater environments at circumneutral pH. The physiological characteristics of OYT1 will help elucidate the ecophysiology of microaerophilic FeOB. Overall, this study demonstrates functional roles of microorganisms in iron flocs, suggesting several possible linkages between Fe and C cycling.     

Characterization of C1-Metabolizing Prokaryotic Communities in Methane Seep Habitats at the Kuroshima Knoll, Southern Ryukyu Arc, by Analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA Genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (~34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the δ-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 × 10⁶ copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 × 10⁶ copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in γ-Proteobacteria and had a high copy number of mxaF (7.1 × 10⁶ copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 × 10² copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C₁ metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.     

Targeted metagenomics reveals extensive diversity of the denitrifying community in partial nitritation anammox and activated sludge systems

The substantial presence of denitrifiers has already been reported in partial nitritation anammox (PNA) systems using the 16S ribosomal RNA (rRNA) gene, but little is known about the phylogenetic diversity based on denitrification pathway functional genes. Therefore, we performed a metagenomic analysis to determine the distribution of denitrification genes and the associated phylogeny in PNA systems and whether a niche separation between PNA and conventional activated sludge (AS) systems exists. The results revealed a distinct abundance pattern of denitrification pathway genes and their association to the microbial species between PNA and AS systems. In contrast, the taxonomic analysis, based on the 16S rRNA gene, did not detect notable variability in denitrifying community composition across samples. In general, narG and nosZa2 genes were dominant in all samples. While the potential for different stages of denitrification was redundant, variation in species composition and lack of the complete denitrification gene pool in each species appears to confer niche separation between PNA and AS systems. This study suggests that targeted metagenomics can help to determine the denitrifying microbial composition at a fine‐scale resolution while overcoming current biases in quantitative polymerase chain reaction approaches due to a lack of appropriate primers.     

Denitrification with methane as electron donor in oxygen-limited bioreactors

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10⁷ cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10⁷ cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10⁴ cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. “In vivo”¹³C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification. Received: 19 October 1999 / Received revision: 11 January 2000 / Accepted: 14 January 2000     

The influence of hydrogeological disturbance and mining on coal seam microbial communities

The microbial communities present in two underground coal mines in the Bowen Basin, Queensland, Australia, were investigated to deduce the effect of pumping and mining on subsurface methanogens and methanotrophs. The micro‐organisms in pumped water from the actively mined areas, as well as, pre‐ and post‐mining formation waters were analyzed using 16S rRNA gene amplicon sequencing. The methane stable isotope composition of Bowen Basin coal seam indicates that methanogenesis has occurred in the geological past. More recently at the mine site, changing groundwater flow dynamics and the introduction of oxygen in the subsurface has increased microbial biomass and diversity. Consistent with microbial communities found in other coal seam environments, pumped coal mine waters from the subsurface were dominated by bacteria belonging to the genera Pseudomonas and the family Rhodocyclaceae. These environments and bacterial communities supported a methanogen population, including Methanobacteriaceae, Methanococcaceae and Methanosaeta. However, one of the most ubiquitous micro‐organisms in anoxic coal mine waters belonged to the family ‘Candidatus Methanoperedenaceae’. As the Archaeal family ‘Candidatus Methanoperedenaceae’ has not been extensively defined, the one studied species in the family is capable of anaerobic methane oxidation coupled to nitrate reduction. This introduces the possibility that a methane cycle between archaeal methanogenesis and methanotrophy may exist in the anoxic waters of the coal seam after hydrogeological disturbance.     

甲烷氧化菌及其在环境治理中的应用

甲烷的生物氧化包括好氧氧化和厌氧氧化两种,分别由好氧甲烷氧化菌和厌氧甲烷氧化菌完成.由于该过程是减少自然环境中温室气体甲烷排放的重要途径,越来越受到各国学者的重视.本文主要对当前甲烷氧化菌的研究现状进行了综述,对好氧甲烷氧化菌的种类、参与氧化甲烷的关键酶,厌氧甲烷氧化菌的种类、参与的微生物菌种以及氧化机理进行了论述,并对这两类微生物在温室气体减排、污染物治理、废水生物脱氮、硫及金属元素回收等方面的应用现状及前景进行了分析.