Significance Given the current pressing need to more fully understand the methane cycle on Earth, in particular, unidentified sinks for methane, identifying and quantifying novel sinks for methane is fundamental importance. Here, we provide previously unidentified direct evidence for the nitrite-dependent anaerobic methane oxidation (n-damo) process as a previously overlooked microbial methane sink in wetlands by stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. It is estimated that n-damo could consume 4.1–6.1 Tg of CH 4 m −2 per year in wetlands under anaerobic conditions, which is roughly 2–6% of current worldwide CH 4 flux estimates for wetlands. Given the worldwide increase in nitrogen pollution, this methane sink may become more important in the future.

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N 2 g ؊1 (dry weight) day ؊1 and the potential n-damo rates varied from 0.2 to 2.1 nmol CO 2 g ؊1 (dry weight) day ؊1 in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 ؋ 10 5 to 2.0 ؋ 10 6 copies g ؊1 (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 ؋ 10 5 to 6.1 ؋ 10 6 copies g ؊1 (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with "Candidatus Brocadia" and "Candidatus Kuenenia" and n-damo bacteria related to "Candidatus Methylomirabilis oxyfera" were present in the soil cores. It is estimated that a total loss of 50.7 g N m ؊2 per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH 4 m ؊2 per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.

Nitrite-dependent anaerobic methane oxidation (n-damo) process was reported to be mediated by "Candidatus Methylomirabilis oxyfera", which belongs to the candidate phylum NC10. M. oxyfera-like bacteria have been detected in lake ecosystems, while their distribution, diversity and abundance in river ecosystems have not been well studied. In this study, both the 16S rRNA and the pmoA molecular biomarkers confirmed the presence of diverse NC10 phylum bacteria related to M. oxyfera in a river ecosystem-the Qiantang River, Zhejiang Province (China). Phylogenetic analysis of 16S rRNA genes demonstrated that the recovered M. oxyfera-like sequences could be grouped into several distinct clusters that exhibited 89.8% to 98.9% identity to the M. oxyfera 16S rRNA gene. Similarly, several different clusters of pmoA gene sequences were observed, and these clusters displayed 85.1-95.4% sequence identity to the pmoA gene of M. oxyfera. Quantitative PCR showed that the abundance of M. oxyfera-like bacteria varied from 1.32 ± 0.16 × 10(6) to 1.03 ± 0.12 × 10(7) copies g (dry weight)(-1). Correlation analysis demonstrated that the total inorganic nitrogen content, the ammonium content and the organic content of the sediment were important factors affecting the distribution of M. oxyfera-like bacterial groups in the examined sediments. This study demonstrated the distribution of diverse M. oxyfera-like bacteria and their correlation with environmental factors in Qiantang River sediments.

Anaerobic oxidation of methane (AOM) reduces methane emissions from marine ecosystems but we know little about AOM in rivers, whose role in the global carbon cycle is increasingly recognized. We measured AOM potentials driven by different electron acceptors, including nitrite, nitrate, sulfate, and ferric iron, and identified microorganisms involved across contrasting riverbeds. AOM activity was confined to the more reduced, sandy riverbeds, whereas no activity was measured in the less reduced, gravel riverbeds where there were few anaerobic methanotrophs. Nitrite-dependent and nitrate-dependent AOM occurred in all sandy riverbeds, with the maximum rates of 61.0 and 20.0 nmol CO 2 g −1 (dry sediment) d − 1 , respectively, while sulfate-dependent and ferric iron-dependent AOM occurred only where methane concentration was highest and the diversity of AOM pathways greatest. Diverse Candidatus Methylomirabilis oxyfera ( M. oxyfera )-like bacteria and Candidatus Methanoperedens nitroreducens ( M. nitroreducens )-like archaea were detected in the sandy riverbeds (16S rRNA gene abundance of 9.3 × 10 5 to 1.5 × 10 7 and 2.1 × 10 4 to 2.5 × 10 5 copies g − 1 dry sediment, respectively) but no other known anaerobic methanotrophs. Further, we found M. oxyfera -like bacteria and M. nitroreducens -like archaea to be actively involved in nitrite- and nitrate/ferric iron-dependent AOM, respectively. Hence, we demonstrate multiple pathways of AOM in relation to methane, though the activities of M. oxyfera -like bacteria and M. nitroreducens -like archaea are dominant.

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