Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria

Abstract

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...

A novel denitrifying methanotroph of the NC10 phylum and its microcolony

“…The apparent substrate affinity constants for methane and nitrite were measured in this work, and they were 7.8 ± 1.2 μM and 8.9 ± 2.9 μM, respectively, similar to the results from the previous halophilic NC10 culture (9.8 ± 2.2 μM for methane and 8.7 ± 1.5 μM for nitrite10). The specific cell activity of M. sinica was approximately 0.3 fmol CH 4 day −1 cell −1 in freshwater47 and 0.14 fmol CH 4 day −1 cell −1 in saline water10, higher than that of M. oxyfera (0.09 fmol CH 4 day −1 cell −1 5). It may be explained by the size of the cell; M. sinica is significantly larger than M. oxyfera (0.7–1.2 × 0.7–1.2 μm vs. 0.25–0.5 × 0.8–1.1 μm).…”

Metabolic potential of microbial communities from ferruginous sediments

“…OTUs listed as potential anaerobic methanotrophs were affiliated with Candidate NC10 (He et al, ) and ANME‐1 (Boetius et al, ; Meyerdierks et al, ). Their relative abundance remained constant with depth at ca.…”

“…). However, methane oxidation via NO‐dismutation was only demonstrated for Candidatus Methylomirabilis , the only cultivated member of the candidate phylum NC10 (He et al, ). Taxonomic assignments confirmed the presence of both Candidatus Methylomirabilis and ANME‐1 in the consortia (Fig.…”

Presence of diverse Candidatus Methylomirabilis oxyfera -like bacteria of NC10 phylum in agricultural soils

“…Recently, the NC10 phylum bacteria were clustered into five different groups, including groups A, group B, group C, group D and group E (He et al . ). The group E, which was not detected in this study, was primarily found to exist in marine environments (He et al .…”

“…With the development of molecular biomarkers (the specific primers targeting the 16S rRNA or pmoA genes of M. oxyfera-like bacteria), M. oxyfera-like bacteria of NC10 phylum have been detected in a variety of habitats, primarily including freshwater lake sediments (Deutzmann and Schink 2011; Kojima et al 2012;Deutzmann et al 2014), river sediments (Shen et al 2014a;Zhu et al 2015), estuarine sediments (Shen et al 2014b;Chen et al 2015), marine sediments (Chen et al 2014;He et al 2015), paddy soils (Shen et al 2014c;Zhou et al 2014;Zhu et al 2015) and freshwater wetland soils (Hu et al 2014;Shen et al 2015b,c;Zhu et al 2015). However, because of methodological limitations (e.g.…”