Biochemical cycling in the rhizosphere having an impact on global change

Changes in chemical properties in soil around plant roots influence many microbial processes, including those having an impact on greenhouse gas emissions. To potentially mitigate these emissions according to the Kyoto protocol, knowledge about how and where these gases are produced and consumed in soils is required. In this review, we focus on the greenhouse gases nitrous oxide and methane, which are produced by nitrifying and denitrifying prokaryotes and methanogenic archaea, respectively. After describing the microbial processes involved in production and consumption of nitrous oxide and methane and how they can be affected in the rhizosphere, we give an overview of nitrous oxide and methane emissions from the rhizosphere and soils and sediments with plants. We also discuss strategies to mitigate emissions from the rhizosphere and consider possibilities for carbon sequestration.     

Syntrophic interactions among anode respiring bacteria (ARB) and Non‐ARB in a biofilm anode: electron balances

We demonstrate that the coulombic efficiency (CE) of a microbial electrolytic cell (MEC) fueled with a fermentable substrate, ethanol, depended on the interactions among anode respiring bacteria (ARB) and other groups of micro‐organisms, particularly fermenters and methanogens. When we allowed methanogenesis, we obtained a CE of 60%, and 26% of the electrons were lost as methane. The only methanogenic genus detected by quantitative real‐time PCR was the hydrogenotrophic genus, Methanobacteriales, which presumably consumed all the hydrogen produced during ethanol fermentation (～30% of total electrons). We did not detect acetoclastic methanogenic genera, indicating that acetate‐oxidizing ARB out‐competed acetoclastic methanogens. Current production and methane formation increased in parallel, suggesting a syntrophic interaction between methanogens and acetate‐consuming ARB. When we inhibited methanogenesis with 50 mM 2‐bromoethane sulfonic acid (BES), the CE increased to 84%, and methane was not produced. With no methanogenesis, the electrons from hydrogen were converted to electrical current, either directly by the ARB or channeled to acetate through homo‐acetogenesis. This illustrates the key role of competition among the various H₂ scavengers and that, when the hydrogen‐consuming methanogens were present, they out‐competed the other groups. These findings also demonstrate the importance of a three‐way syntrophic relationship among fermenters, acetate‐consuming ARB, and a H₂ consumer during the utilization of a fermentable substrate. To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current. Biotechnol. Bioeng. 2009;103: 513–523. © 2009 Wiley Periodicals, Inc.     

Microbial mechanism for rice variety control on methane emission from rice field soil

Rice variety is one of the key factors regulating methane (CH₄) production and emission from the paddy fields. However, the relationships between rice varieties and populations of microorganisms involved in CH₄ dynamics are poorly understood. Here we investigated CH₄ dynamics and the composition and abundance of CH₄‐producing archaea and CH₄‐oxidizing bacteria in a Chinese rice field soil planted with three types of rice. Hybrid rice produced 50–60% more of shoot biomass than Indica and Japonica cultivars. However, the emission rate of CH₄ was similar to Japonica and lower than Indica. Furthermore, the dissolved CH₄ concentration in the rhizosphere of hybrid rice was markedly lower than Indica and Japonica cultivars. The rhizosphere soil of hybrid rice showed a similar CH₄ production potential but a higher CH₄ oxidation potential compared with the conventional varieties. Terminal restriction fragment length polymorphism analysis of the archaeal 16S rRNA genes showed that the hydrogenotrophic methanogens dominated in the rhizosphere whereas acetoclastic methanogens mainly inhabited the bulk soil. The abundance of total archaea as determined by quantitative (real‐time) PCR increased in the later stage of rice growth. However, rice variety did not significantly influence the structure and abundance of methanogenic archaea. The analysis of pmoA gene fragments (encoding the α‐subunit of particulate methane monooxygenase) revealed that rice variety also did not influence the structure of methanotrophic proteobacteria, though variable effects of soil layer and sampling time were observed. However, the total copy number of pmoA genes in the rhizosphere of hybrid rice was approximately one order of magnitude greater than the two conventional cultivars. The results suggest that hybrid rice stimulates the growth of methanotrophs in the rice rhizosphere, and hence enhances CH₄ oxidation which attenuates CH₄ emissions from the paddy soil. Hybrid rice is becoming more and more popular in Asian countries. The present study demonstrated that planting of hybrid rice will not enhance CH₄ emissions albeit a higher grain production than the conventional varieties.     

Microbial sensitivity to temperature and sulfate deposition modulates greenhouse gas emissions from peat soils

Peatlands are among the largest natural sources of atmospheric methane (CH₄) worldwide. Microbial processes play a key role in regulating CH₄ emissions from peatland ecosystems, yet the complex interplay between soil substrates and microbial communities in controlling CH₄ emissions as a function of global change remains unclear. Herein, we performed an integrated analysis of multi-omics data sets to provide a comprehensive understanding of the molecular processes driving changes in greenhouse gas (GHG) emissions in peatland ecosystems with increasing temperature and sulfate deposition in a laboratory incubation study. We sought to first investigate how increasing temperatures (4, 21, and 35°C) impact soil microbiome–metabolome interactions; then explore the competition between methanogens and sulfate-reducing bacteria (SRBs) with increasing sulfate concentrations at the optimum temperature for methanogenesis. Our results revealed that peat soil organic matter degradation, mediated by biotic and potentially abiotic processes, is the main driver of the increase in CO₂ production with temperature. In contrast, the decrease in CH₄ production at 35°C was linked to the absence of syntrophic communities and the potential inhibitory effect of phenols on methanogens. Elevated temperatures further induced the microbial communities to develop high growth yield and stress tolerator trait-based strategies leading to a shift in their composition and function. On the other hand, SRBs were able to outcompete methanogens in the presence of non-limiting sulfate concentrations at 21°C, thereby reducing CH₄ emissions. At higher sulfate concentrations, however, the prevalence of communities capable of producing sufficient low-molecular-weight carbon substrates for the coexistence of SRBs and methanogens was translated into elevated CH₄ emissions. The use of omics in this study enhanced our understanding of the structure and interactions among microbes with the abiotic components of the system that can be useful for mitigating GHG emissions from peatland ecosystems in the face of global change.     

滨海湿地甲烷产生途径和产甲烷菌研究进展

滨海湿地在全球碳循环中起着重要的作用,其甲烷排放量占全球海洋甲烷排放的75%.本文综述了滨海湿地主要甲烷产生途径、产甲烷菌种类及其影响因子.滨海湿地SO₄^2-含量丰富,乙酸发酵和H₂/CO₂途径产甲烷受抑制,乙酸营养型和氢营养型产甲烷菌丰度较低;而利用甲胺类等“非竞争性”底物的C1甲基化合物歧化途径不受硫酸还原菌竞争底物的限制,兼性营养型产甲烷菌成为产甲烷优势菌.盐度与SO₄^2-含量和植被类型密切相关,影响竞争性电子和产甲烷底物的种类和含量,对甲烷产生途径和产甲烷菌群落结构有重要影响.目前,滨海湿地产甲烷菌群落结构、甲烷产生途径的关键控制因素尚需明确,其对甲烷排放的影响有待进一步研究.     

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。     

Where old meets new: An ecosystem study of methanogenesis in a reflooded agricultural peatland

Reflooding formerly drained peatlands has been proposed as a means to reduce losses of organic matter and sequester soil carbon for climate change mitigation, but a renewal of high methane emissions has been reported for these ecosystems, offsetting mitigation potential. Our ability to interpret observed methane fluxes in reflooded peatlands and make predictions about future flux trends is limited due to a lack of detailed studies of methanogenic processes. In this study we investigate methanogenesis in a reflooded agricultural peatland in the Sacramento Delta, California. We use the stable‐and radio‐carbon isotopic signatures of wetland sediment methane, ecosystem‐scale eddy covariance flux observations, and laboratory incubation experiments, to identify which carbon sources and methanogenic production pathways fuel methanogenesis and how these processes are affected by vegetation and seasonality. We found that the old peat contribution to annual methane emissions was large (~30%) compared to intact wetlands, indicating a biogeochemical legacy of drainage. However, fresh carbon and the acetoclastic pathway still accounted for the majority of methanogenesis throughout the year. Although temperature sensitivities for bulk peat methanogenesis were similar between open‐water (Q₁₀ = 2.1) and vegetated (Q₁₀ = 2.3) soils, methane production from both fresh and old carbon sources showed pronounced seasonality in vegetated zones. We conclude that high methane emissions in restored wetlands constitute a biogeochemical trade‐off with contemporary carbon uptake, given that methane efflux is fueled primarily by fresh carbon inputs.     

陆地生态系统甲烷产生和氧化过程的微生物机理

陆地生态系统存在许多常年性或季节性缺氧环境,如:湿地、水稻土、湖泊沉积物、动物瘤胃、垃圾填埋场和厌氧生物反应器等。每年有大量有机物质进入这些环境,在缺氧条件下发生厌氧分解。甲烷是有机质厌氧分解的最终产物。产生的甲烷气体可通过缺氧-有氧界面释放到大气,产生温室效应,是重要的温室气体。产甲烷过程是缺氧环境中有机质分解的核心环节,而甲烷氧化是缺氧-有氧界面的重要微生物过程。甲烷的产生和氧化过程共同调控大气甲烷浓度,是全球碳循环不可分割的组成部分。对陆地生态系统甲烷产生和氧化过程的微生物机理研究进展进行了概要回顾和综述。主要内容包括:新型产甲烷古菌即第六和第七目产甲烷古菌和嗜冷嗜酸产甲烷古菌的发现;短链脂肪酸中间产物互营氧化过程与直接种间电子传递机制;新型甲烷氧化菌包括厌氧甲烷氧化菌和疣微菌属好氧甲烷氧化菌的发现;甲烷氧化菌生理生态与环境适应的新机制。这些研究进展显著拓展了人们对陆地生态系统甲烷产生和氧化机理的认识和理解。随着新一代土壤微生物研究技术的发展与应用,甲烷产生和氧化微生物研究领域将面临更多机遇和挑战,对未来发展趋势做了展望。     

基于过程模型的青藏高原湿地甲烷排放格局评估

甲烷(CH₄)是大气中最丰富的碳氢化合物,是仅次于二氧化碳(CO₂)的温室气体。湿地是甲烷的重要来源,在全球碳循环中发挥着重要作用,其排放的甲烷占所有天然甲烷排放源的70%,占全球甲烷排放总量的24.8%。青藏高原平均海拔4000 m以上,占有中国约三分之一的湿地。近几十年来,由于全球气候变暖和降水增加,该地区甲烷排放率和湿地面积都发生着巨大变化,因此,青藏高原湿地CH₄排放的长期变化在很大程度上仍存在较大的不确定性。利用TRIPLEX-GHG模型模拟了青藏高原湿地1978—2008年CH₄排放的动态特征,研究结果表明:(1)1978—2008年青藏高原湿地CH₄排放速率呈逐渐增加趋势。(2)青藏高原大多数湿地区域CH₄排放速率为0—6.13 g CH₄ m^-2 a^-1;东北部分湿地区域CH₄排放速率为6.14—20.19 g CH₄ m^...     

Cattle Manure Enhances Methanogens Diversity and Methane Emissions Compared to Swine Manure under Rice Paddy

Livestock manures are broadly used in agriculture to improve soil quality. However, manure application can increase the availability of organic carbon, thereby facilitating methane (CH₄) production. Cattle and swine manures are expected to have different CH₄ emission characteristics in rice paddy soil due to the inherent differences in composition as a result of contrasting diets and digestive physiology between the two livestock types. To compare the effect of ruminant and non-ruminant animal manure applications on CH₄ emissions and methanogenic archaeal diversity during rice cultivation (June to September, 2009), fresh cattle and swine manures were applied into experimental pots at 0, 20 and 40 Mg fresh weight (FW) ha⁻¹ in a greenhouse. Applications of manures significantly enhanced total CH₄ emissions as compared to chemical fertilization, with cattle manure leading to higher emissions than swine manure. Total organic C contents in cattle (466 g kg⁻¹) and swine (460 g kg⁻¹) manures were of comparable results. Soil organic C (SOC) contents were also similar between the two manure treatments, but dissolved organic C (DOC) was significantly higher in cattle than swine manure. The mcrA gene copy numbers were significantly higher in cattle than swine manure. Diverse groups of methanogens which belong to Methanomicrobiaceae were detected only in cattle-manured but not in swine-manured soil. Methanogens were transferred from cattle manure to rice paddy soils through fresh excrement. In conclusion, cattle manure application can significantly increase CH₄ emissions in rice paddy soil during cultivation, and its pretreatment to suppress methanogenic activity without decreasing rice productivity should be considered.     

Does richness of emergent plants affect CO2 and CH4 emissions in experimental wetlands?

相似文献   

Methane emissions from wetlands: biogeochemical,microbial, and modeling perspectives from local to global scales

Understanding the dynamics of methane (CH₄) emissions is of paramount importance because CH₄ has 25 times the global warming potential of carbon dioxide (CO₂) and is currently the second most important anthropogenic greenhouse gas. Wetlands are the single largest natural CH₄ source with median emissions from published studies of 164 Tg yr^?1, which is about a third of total global emissions. We provide a perspective on important new frontiers in obtaining a better understanding of CH₄ dynamics in natural systems, with a focus on wetlands. One of the most exciting recent developments in this field is the attempt to integrate the different methodologies and spatial scales of biogeochemistry, molecular microbiology, and modeling, and thus this is a major focus of this review. Our specific objectives are to provide an up‐to‐date synthesis of estimates of global CH₄ emissions from wetlands and other freshwater aquatic ecosystems, briefly summarize major biogeophysical controls over CH₄ emissions from wetlands, suggest new frontiers in CH₄ biogeochemistry, examine relationships between methanogen community structure and CH₄ dynamics in situ, and to review the current generation of CH₄ models. We highlight throughout some of the most pressing issues concerning global change and feedbacks on CH₄ emissions from natural ecosystems. Major uncertainties in estimating current and future CH₄ emissions from natural ecosystems include the following: (i) A number of important controls over CH₄ production, consumption, and transport have not been, or are inadequately, incorporated into existing CH₄ biogeochemistry models. (ii) Significant errors in regional and global emission estimates are derived from large spatial‐scale extrapolations from highly heterogeneous and often poorly mapped wetland complexes. (iii) The limited number of observations of CH₄ fluxes and their associated environmental variables loosely constrains the parameterization of process‐based biogeochemistry models.     

Archaeal communities in High Arctic wetlands at Spitsbergen, Norway (78 degrees N) as characterized by 16S rRNA gene fingerprinting

Emissions of the greenhouse gas methane from Arctic wetlands have been studied extensively, though little is known about the ecology and community structure of methanogenic archaea that catalyze the methane production. As part of a project addressing microbial transformations of methane in Arctic wetlands, we studied archaeal communities in two wetlands (Solvatnet and Stuphallet) at Spitsbergen, Norway (78 degrees N) during two summer seasons. Directly extracted peat community DNA and enrichment cultures of methanogenic archaea were analyzed by nested PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rRNA gene fragments. Sequences affiliated with Methanomicrobiales, Methanobacteriaceae, Methanosaeta and Group I.3b of the uncultured crenarchaeota were detected at both sites. Sequences affiliated with Methanosarcina were recovered only from the site Solvatnet, while sequences affiliated with the euryarchaeotal clusters Rice Cluster II and Sediment 1 were detected only at the site Stuphallet. The phylogenetic affiliation of the recovered sequences suggested a potential of both hydrogenotrophic and acetoclastic methanogenesis at both sites. At Solvatnet, there were clear temporal trends in the archaeal community structure over the Arctic summer season. The archaeal community composition was significantly affected by factors influencing the activity of the overall bacterial community, as measured by in situ emissions of CO2. Methane emissions at both sites were influenced more by peat temperatures and thaw depth than by the archaeal community structure. Enrichment cultures for methanogenic archaea determined that most of the methanogens detected directly in peat could grow in culture at 10 degrees C. Culture based biases were indicated in later enrichment steps by the abundant growth of a Methanosarcina strain that was not detected directly in peat samples.     

The Genome Sequence of the Rumen Methanogen Methanobrevibacter ruminantium Reveals New Possibilities for Controlling Ruminant Methane Emissions

Background

Methane (CH₄) is a potent greenhouse gas (GHG), having a global warming potential 21 times that of carbon dioxide (CO₂). Methane emissions from agriculture represent around 40% of the emissions produced by human-related activities, the single largest source being enteric fermentation, mainly in ruminant livestock. Technologies to reduce these emissions are lacking. Ruminant methane is formed by the action of methanogenic archaea typified by Methanobrevibacter ruminantium, which is present in ruminants fed a wide variety of diets worldwide. To gain more insight into the lifestyle of a rumen methanogen, and to identify genes and proteins that can be targeted to reduce methane production, we have sequenced the 2.93 Mb genome of M. ruminantium M1, the first rumen methanogen genome to be completed.

Methodology/Principal Findings

The M1 genome was sequenced, annotated and subjected to comparative genomic and metabolic pathway analyses. Conserved and methanogen-specific gene sets suitable as targets for vaccine development or chemogenomic-based inhibition of rumen methanogens were identified. The feasibility of using a synthetic peptide-directed vaccinology approach to target epitopes of methanogen surface proteins was demonstrated. A prophage genome was described and its lytic enzyme, endoisopeptidase PeiR, was shown to lyse M1 cells in pure culture. A predicted stimulation of M1 growth by alcohols was demonstrated and microarray analyses indicated up-regulation of methanogenesis genes during co-culture with a hydrogen (H₂) producing rumen bacterium. We also report the discovery of non-ribosomal peptide synthetases in M. ruminantium M1, the first reported in archaeal species.

Conclusions/Significance

The M1 genome sequence provides new insights into the lifestyle and cellular processes of this important rumen methanogen. It also defines vaccine and chemogenomic targets for broad inhibition of rumen methanogens and represents a significant contribution to worldwide efforts to mitigate ruminant methane emissions and reduce production of anthropogenic greenhouse gases.     

The role of vegetation in methane flux to the atmosphere: should vegetation be included as a distinct category in the global methane budget?

Currently, the global annual flux of methane (CH₄) to the atmosphere is fairly well constrained at ca. 645 Tg CH₄ year^?1. However, the relative magnitudes of the fluxes generated from different natural (e.g. wetlands, deep seepage, hydrates, ocean sediments) and anthropogenic sources remain poorly resolved. Of the identified natural sources, the contribution of vegetation to the global methane budget is arguably the least well understood. Historically, reviews of the contribution of vegetation to the global methane flux have focused on the role of plants as conduits for soil-borne methane emissions from wetlands, or the aerobic production of methane within plant tissues. Many recent global budgets only include the latter pathway (aerobic methane production) in estimating the importance of terrestrial vegetation to atmospheric CH₄ flux. However, recent experimental evidence suggests several novel pathways through which vegetation can contribute to the flux of this globally important, trace greenhouse gas (GHG), such as plant cisterns that act as cryptic wetlands, heartwood rot in trees, the degradation of coarse woody debris and litter, or methane transport through herbaceous and woody plants. Herein, we synthesize the existing literature to provide a comprehensive estimate of the role of modern vegetation in the global methane budget. This first, albeit uncertain, estimate indicates that vegetation may represent up to 22 % of the annual flux of methane to the atmosphere, contributing ca. 32–143 Tg CH₄ year^?1 to the global flux of this important trace GHG. Overall, our findings emphasize the need to better resolve the role of vegetation in the biogeochemical cycling of methane as an important component of closing the gap in the global methane budget.     

Methane emissions from freshwater riverine wetlands

To better understand methane emissions from freshwater riverine wetlands, seasonal and spatial patterns of methane emissions were measured over a 1-year period from created freshwater marshes and a river division oxbow, and at a river-floodplain edge (riverside) in central Ohio, USA. Plots were distributed from inflow to outflow and from shallow transition edges to deep water zones in the marshes and oxbow. Median values of CH₄ emissions ranged from 0.33 to 85.7 mg-CH₄-C m⁻² h⁻¹, at the riverside sites and 0.02-20.5 mg CH₄-C m⁻² h⁻¹ in the created marshes. The naturally colonizing marsh had more methane emissions (p = 0.047) than did the planted marsh, probably due to a history of higher net primary productivity in the former. A significant dry period and lower productivity in the oxbow may explain its low range of methane emissions of −0.04 to 0.09 mg CH₄-C m⁻² h⁻¹. There were significantly higher rates of methane emissions in deep water zones compared to transition zones in the created marshes. Overall CH₄ emissions had significant relationships with organic carbon and soil temperature and appear to depend on the hydroperiod and vegetation development. Riparian wetlands can be designed to minimize greenhouse gas emissions while providing other ecosystem services.     

Greenhouse gas emissions and carbon sequestration potential in restored wetlands of the Canadian prairie pothole region

North American prairie pothole wetlands are known to be important carbon stores. As a result there is interest in using wetland restoration and conservation programs to mitigate the effects of increasing greenhouse gas concentration in the atmosphere. However, the same conditions which cause these systems to accumulate organic carbon also produce the conditions under which methanogenesis can occur. As a result prairie pothole wetlands are potential hotspots for methane emissions. We examined change in soil organic carbon density as well as emissions of methane and nitrous oxide in newly restored, long-term restored, and reference wetlands across the Canadian prairies to determine the net GHG mitigation potential associated with wetland restoration. Our results indicate that methane emissions from seasonal, semi-permanent, and permanent prairie pothole wetlands are quite high while nitrous oxide emissions from these sites are fairly low. Increases in soil organic carbon between newly restored and long-term restored wetlands supports the conclusion that restored wetlands sequester organic carbon. Assuming a sequestration duration of 33 years and a return to historical SOC densities we estimate a mean annual sequestration rate for restored wetlands of 2.7 Mg C ha⁻¹year⁻¹ or 9.9 Mg CO₂ eq. ha⁻¹ year⁻¹. Even after accounting for increased CH₄ emissions associated with restoration our research indicates that wetland restoration would sequester approximately 3.25 Mg CO₂ eq. ha⁻¹year⁻¹. This research indicates that widescale restoration of seasonal, semi-permanent, and permanent wetlands in the Canadian prairies could help mitigate GHG emissions in the near term until a more viable long-term solution to increasing atmospheric concentrations of GHGs can be found.     

The effect of vascular plants on carbon turnover and methane emissions from a tundra wetland

This paper investigates how vascular plants affect carbon flow and the formation and emission of the greenhouse gas methane (CH₄) in an arctic wet tundra ecosystem in NE Greenland. We present a field experiment where we studied, in particular, how species‐specific root exudation patterns affect the availability of acetate, a hypothesized precursor of CH₄ formation. We found significantly higher acetate formation rates in the root vicinity of Eriophorum scheuchzeri compared with another dominating sedge in the wetland, i.e. Dupontia psilosantha. Furthermore a shading treatment, which reduced net photosynthesis, resulted in significantly decreased formation rates of acetate. We also found that the potential CH₄ production of the peat profile was highly positively correlated to the concentration of acetate at the respective depths, whereas it was negatively correlated to the concentration of total dissolved organic carbon. This suggests that acetate is a substrate of importance to the methanogens in the studied ecosystem and that acetate concentration in this case can serve as a predictor of substrate quality. To further investigate the importance of acetate as a predecessor to CH₄, we brought an intact peat‐plant monolith system collected at the field site in NE Greenland to the laboratory, sealed it hermetically and studied the decomposition of ¹⁴C‐labelled acetate injected at the depth of methanogenic activity. After 4 h, ¹⁴CH₄ emission from the monolith could be observed. In conclusion, allocation of recently fixed carbon to the roots of certain species of vascular plants affects substrate quality and influence CH₄ formation.     

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。