Abstract
The newly discovered Candidatus ‘Methanoperedens nitroreducens’ (M. nitroreducens), mediating nitrate-dependent anaerobic oxidation of methane, is an important microorganism in linking carbon and nitrogen cycles. In order to explore the diversity of M. nitroreducens-like archaea in various environmental niches with advanced high-throughput sequencing, new primers based on alpha subunit of methyl-coenzyme M reductase gene were designed. The PCR results demonstrated that the new primers could effectively detect M. nitroreducens-like archaea from an enrichment culture dominated by M. nitroreducens as well as samples collected from a natural freshwater lake and a full-scale wastewater treatment plant (WWTP). By high-throughput sequencing, more than 30,000 M. nitroreducens-like sequences were obtained. Phylogenetic analysis of these sequences along with published sequences showed that M. nitroreducens-like archaea could be divided into three sub-branches (named as Group A, Group B and Group C in this study). Clear geographical difference was observed, with Group A and Group B dominating samples in Queensland (Australia) and in European ecosystems, respectively. Further quantitative PCR revealed that the M. nitroreducens-like archaea were more abundant in WWTP than the freshwater lake. The study provided a large number of sequences for M. nitroreducens-like archaeal communities, thus expanded our understanding on the ecological diversity of M. nitroreducens-like archaea.
Highlights
Microbial methane oxidation is an effective way to reduce the potential methane emission into the atmosphere[3]
M. nitroreducens-like sequences was successfully amplified with the primers McrA169F and McrA1360R the fragment (1191 bp) is too long to be sequenced with the high-throughput sequencing technology (Figure S1)
When using the new nested polymerase chain reaction (PCR) strategy developed in this study, a dominant and bright PCR band at the expected size (363 bp) was identified, while no amplification was shown in the negative control sample (NC) (Fig. 1a and Figure S2)
Summary
Microbial methane oxidation is an effective way to reduce the potential methane emission into the atmosphere[3]. In order to detect the M. nitroreducens-like archaea from different environmental niches, specific primers targeting 16 S rRNA and mcrA genes have been designed recently[20,21]. Using these primers, M. nitroreducens-like archaea were detected in wastewater treatment plant (WWTP), river sediment, channel sediment, paddy soil and sea sediment[20,21]. M. nitroreducens-like archaea were detected in wastewater treatment plant (WWTP), river sediment, channel sediment, paddy soil and sea sediment[20,21] In these studies, specific primers were used for polymerase chain reaction (PCR) and the PCR products were cloned before the Sanger sequencing. This study intends to provide a new molecular tool for high-throughput detection of M. nitroreducens-like archaea and expands the sequences database for ecological investigation of M. nitroreducens-like archaea
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