土壤中甲烷厌氧氧化菌多样性的分子检测

Molecular detection of diversity of anaerobic methanotroph in soil

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。

Anaerobic oxidation of methane is the most important biogeochemical process to reduce methane released into the atmosphere from marine sediments,however,the anaerobic oxidation of methane and related functional microorganisms in soil still remain uncertain. Therefore,the studies on the diversity of anaerobic methanotrophs may be able to assist with reducing methane emissions from soil. Compared with traditional culture-dependent methods, molecular methods independent of culture techniques has vastly improved the knowledge on microbial diversity. This review mainly focused on the recent progress surrounding abundance and diversity of anaerobic methanotrophs in soils with emphasis on the molecular gene markers including 16 S rRNA, mcr A and pmo A used for detecting anaerobic methanotrophs. Furthermore, the questions existing in the present research as well as the related resolution were also discussed. Methane oxidation in anoxic environments is microbially mediated and of global significance. In the last decade,the diversity of anaerobic methane oxidation populations has been studied intensively. Initially,most studies concerning environmental AOM were carried out in anaerobic marine waters and sediments where AOM was coupled to sulfate reduction. It is now known that there are also some microorganisms capable of coupling AOM to denitrification. Fluorescence in situ hybridization with target probes firstly showed that the sulfate dependent AOM archaea were in the absence of close physical association with sulfate reducingbacteria. With the development of probes, different types of AOM consortia were visualized. In addition, most investigations on the diversity of AOM archaea involved in the consortia were based on the 16 S rRNA or mcr A gene phylogeny. Three lineages of the sulfate dependent AOM have been identified that are referred to as ANME-1,ANME-2,ANME-3. The first nitrate dependent methane oxidation cultures were initially enriched anaerobically,which contained a bacterium belonging to the candidate division NC10. ＂Candidatus,Methylomirabilis oxyfera,＂a member of the uncultured NC10 phylum,forms a novel taxonomic group of bacterial methanotrophs. Recently,special primers targeting methane monooxygenase（ p MMO） for detection of anaerobic methanotrophs were developed. Based on these probes and primers,culture independent approaches were used to screen samples from several oxygen-limited habitats for the presence of both sulfate and nitrate dependent methane oxidation bacteria and archaea,e. g. quantitating the abundance of anaerobic methanotrophs by quantity PCR,detecting the community structure by clone library. Although methane oxidation occurs in a variety of different habitats and appears to be performed by different organisms,the distribution of AOM organisms in aquatic and terrestrial ecosystems remains to be fully revealed. Thus,several suggestions for future research on AOM processes and related microorganisms are put forward as follows： 1） to investigate more diverse terrestrial environments where AOM may occur or is known to occur based on genomic and biomarker-related methods. 2） to combine the enrichment culture with molecular method to better understand the mechanism of AOM and related microorganisms. The enrichment or isolation of these organisms will allow for a variety of novel physiological,biochemical,and genomic studies of AOM one or more key organisms. 3） to detect the environmental factors affecting the AOM process or organisms. Future biogeochemical studies also hold the potential to further our understanding of this process. 4） to explore new types of AOM microorganisms coupled with SO2-4,Mn4 ＋,Fe3 ＋,NO-3acting as the electron acceptors. Understanding AOM communities and the environmental conditions under which they consume methane may help to refine computational models for methane cycling on earth and should improve the accuracy of long-term climate change projections.