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.

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 M for methane and 8.7 ؎ 1.5 M for nitrite.A naerobic oxidation of methane (AOM) occurs extensively in natural ecosystems and is a crucial biological sink in the global methane cycle that maintains the balance of greenhouse gas content in the atmosphere (1). To date, five electron acceptors that support AOM, including AOM coupled to sulfate reduction (2), nitrite reduction (3), nitrate reduction (4), iron reduction (5), and manganese reduction (5), have been discovered in natural settings. Moreover, on the basis of bioenergetic calculations, researchers have speculated that several other types of AOM (e.g., AOM coupled to perchlorate reduction, arsenate reduction, and selenate reduction) may exist in nature (6); however, these possible types have not yet been confirmed. In particular, AOM coupled to nitrite reduction has been a predominant research focus in the past several years. AOM coupled to nitrite reduction was also called nitrite-dependent anaerobic methane oxidation (ndamo) in previous reports. It has been demonstrated that AOM coupled to nitrite reduction is mediated by the bacterium "Candidatus Methylomirabilis oxyfera" (denitrifying methanotroph) (7), which is affiliated with the candidate NC10 phylum (8). This candidate division (NC10 phylum) was first defined by classification of environmental sequences retrieved from aquatic microbial formations in flooded caves (9). To date, our knowledge regarding the NC10 phylum has stemmed largely from research into "Ca. Methylomirabilis oxyfera" and "Ca. Methylomirabilis oxyfera"-like bacteria. According to the phylogenetic affiliations of 16S rRNA gene sequ...

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Nitrite-dependent anaerobic methane oxidation (n-damo) is mediated by bacteria that anaerobically oxidize methane coupled with nitrite reduction and is a potential bioprocess for wastewater treatment. In this work, the effect of reactor configuration on n-damo bacterial cultivation was investigated. A magnetically stirred gas lift reactor (MSGLR), a sequencing batch reactor (SBR), and a continuously stirred tank reactor (CSTR) were selected to cultivate the bacteria. Microbial community was monitored by using quantitative PCR, 16S rRNA gene sequencing, pmoA gene sequencing, and fluorescence in situ hybridization (FISH). The effects of substrate inhibition, methane mass transfer, and biomass washout in the three reactors were focused on. The results indicated that the MSGLR had the best performance among the three reactor systems, with the highest total and specific n-damo activities. Its maximum volumetric nitrogen removal rate was up to 76.9 mg N L(-1) day(-1), which was higher than previously reported values (5.1-37.8 mg N L(-1) d(-1)).

In the current study, we investigated nitrite-dependent anaerobic methane oxidation (N-DAMO) as a potential methane sink in the Hangzhou Bay and the adjacent Zhoushan sea area. The potential activity of the N-DAMO process was primarily observed in Hangzhou Bay by means of (13)C-labeling experiments, whereas very low or no potential N-DAMO activity could be detected in the Zhoushan sea area. The measured potential N-DAMO rates ranged from 0.2 to 1.3 nmol (13)CO2 g(-1) (dry sediment) day(-1), and the N-DAMO potentially contributed 2.0-9.4 % to the total microbial methane oxidation in the examined sediments. This indicated that the N-DAMO process may be an alternative pathway in the coastal methane cycle. Phylogenetic analyses confirmed the presence of Candidatus Methylomirabilis oxyfera-like bacteria in all the examined sediments, while the group A members (the dominant bacteria responsible for N-DAMO) were found mainly in Hangzhou Bay. Quantitative PCR showed that the 16S rRNA gene abundance of Candidatus M. oxyfera-like bacteria varied from 5.4 × 10(6) to 5.0 × 10(7) copies g(-1) (dry sediment), with a higher abundance observed in Hangzhou Bay. In addition, the overlying water NO3 (-) concentration and salinity were identified as the most important factors influencing the abundance and potential activity of Candidatus M. oxyfera-like bacteria in the examined sediments. This study showed the evidence of N-DAMO in coastal environments and indicated the importance of N-DAMO as a potential methane sink in coastal environments.

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