Molecular and stable isotopic evidence for the occurrence of nitrite-dependent anaerobic methane-oxidizing bacteria in the mangrove sediment of Zhangjiang Estuary, China.

Nitrite-dependent anaerobic methane oxidation (n-damo), which is mediated by "Candidatus Methylomirabilis oxyfera-like" bacteria, is unique in linking the carbon and nitrogen cycles. However, the niche and activity of n-damo bacteria in the mangrove ecosystem have not been confirmed. Here, we report the occurrence of the n-damo process in the mangrove wetland of the Zhangjiang Estuary, China. The widespread occurrence of n-damo bacteria in mangrove wetland was confirmed using real-time quantitative polymerase chain reaction (qPCR) assay, which showed that the abundance of Methylomirabilis oxyfera-like bacterial 16S rRNA and pmoA genes ranged from 2.43 × 106 to 2.09 × 107 and 2.07 × 106 to 3.38 × 107copies per gram of dry soil in the examined sediment cores. The highest amount of targeting genes was all detected in the upper layer (0-20 cm). Phylogenetic analyses of n-damo bacterial 16S rRNA and pmoA genes illustrated the depth-specific distribution and high diversity of n-damo bacteria in the mangrove wetland. Stable isotope experiments further confirmed the occurrence of n-damo in the examined mangrove sediments, and the potential n-damo rates ranged from 25.93 to 704.08 nmol CO2 per gram of dry soil per day at different depths of the sediment cores, with the n-damo being more active in the upper layer of the mangrove sediments. These results illustrate the existence of active M. oxyfera-like bacteria and indicate that the n-damo process is a previously overlooked microbial methane sink in the mangrove wetlands.

Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake.

Anaerobic methane oxidation coupled to denitrification, also known as "nitrate/nitrite-dependent anaerobic methane oxidation" (n-damo), was discovered in 2006. Since then, only a few studies have identified this process and the associated microorganisms in natural environments. In aquatic sediments, the close proximity of oxygen- and nitrate-consumption zones can mask n-damo as aerobic methane oxidation. We therefore investigated the vertical distribution and the abundance of denitrifying methanotrophs related to Candidatus Methylomirabilis oxyfera with cultivation-independent molecular techniques in the sediments of Lake Constance. Additionally, the vertical distribution of methane oxidation and nitrate consumption zones was inferred from high-resolution microsensor profiles in undisturbed sediment cores. M. oxyfera-like bacteria were virtually absent at shallow-water sites (littoral sediment) and were very abundant at deep-water sites (profundal sediment). In profundal sediment, the vertical distribution of M. oxyfera-like bacteria showed a distinct peak in anoxic layers that coincided with the zone of methane oxidation and nitrate consumption, a strong indication for n-damo carried out by M. oxyfera-like bacteria. Both potential n-damo rates calculated from cell densities (660-4,890 µmol CH4⋅m(-2)⋅d(-1)) and actual rates calculated from microsensor profiles (31-437 µmol CH4⋅m(-2)⋅d(-1)) were sufficiently high to prevent methane release from profundal sediment solely by this process. Additionally, when nitrate was added to sediment cores exposed to anoxic conditions, the n-damo zone reestablished well below the sediment surface, completely preventing methane release from the sediment. We conclude that the previously overlooked n-damo process can be the major methane sink in stable freshwater environments if nitrate is available in anoxic zones.

Nitrite-dependent anaerobic methane oxidizing bacteria along the water level fluctuation zone of the Three Gorges Reservoir.

The nitrite-dependent anaerobic methane oxidation (n-damo) mediated by "Candidatus Methylomirabilis oxyfera" connects the biogeochemical carbon and nitrogen cycles in a novel way. Many environments have been reported to harbor such organism being slow-growing and oxygen-sensitive anaerobes. Here, we focused on the population of n-damo bacteria in a fluctuating habitat being the wetland in the water level fluctuation zone (WLFZ) of the Three Gorges Reservoir (TGR) in China. A molecular approach demonstrated positive amplifications when targeting the functional pmoA gene only in the lower sites which endured longer flooding time in an elevation gradient. Only 1 operational taxonomic unit (OTU) in the lower elevation zone targeting the 16S ribosomal RNA (rRNA) gene was clustering into the NC-10 group a, which is presumed to be the true n-damo group. Moreover, a relatively low level of diversity was observed in this study. The abundances were as low as 4.7 × 10(2) to 1.5 × 10(3) copies g(-1) dry soil (ds) in the initial stage, which were almost the lowest reported. However, an increase was observed (3.2 × 10(3) to 5.3 × 10(4) copies g(-1) ds) after nearly 6 months of flooding. Intriguingly, the abundance of n-damo bacteria correlated positively with the accumulated flooding time (AFT). The current study revealed that n-damo bacteria can be detected in a fluctuating environment and the sites with longer flooding time seem to be preferred habitats. The water flooding may be the principal factor in this ecosystem by creating anoxic condition. The wide range of such habitats suggests a high potential of n-damo bacteria to play a key role in natural CH4 consumption.

Diversity of NC10 bacteria associated with sediments of submerged Potamogeton crispus (Alismatales: Potmogetonaceae).

BACKGROUND: The nitrite-dependent anaerobic methane oxidation (N-DAMO) pathway, which plays an important role in carbon and nitrogen cycling in aquatic ecosystems, is mediated by "Candidatus Methylomirabilis oxyfera" (M. oxyfera) of the NC10 phylum. M. oxyfera-like bacteria are widespread in nature, however, the presence, spatial heterogeneity and genetic diversity of M. oxyfera in the rhizosphere of aquatic plants has not been widely reported. METHOD: In order to simulate the rhizosphere microenvironment of submerged plants, Potamogeton crispus was cultivated using the rhizobox approach. Sediments from three compartments of the rhizobox: root (R), near-rhizosphere (including five sub-compartments of one mm width, N1-N5) and non-rhizosphere (>5 mm, Non), were sampled. The 16S rRNA gene library was used to investigate the diversity of M. oxyfera-like bacteria in these sediments. RESULTS: Methylomirabilis oxyfera-like bacteria were found in all three sections, with all 16S rRNA gene sequences belonging to 16 operational taxonomic units (OTUs). A maximum of six OTUs was found in the N1 sub-compartment of the near-rhizosphere compartment and a minimum of four in the root compartment (R) and N5 near-rhizosphere sub-compartment. Indices of bacterial community diversity (Shannon) and richness (Chao1) were 0.73-1.16 and 4-9, respectively. Phylogenetic analysis showed that OTU1-11 were classified into group b, while OTU12 was in a new cluster of NC10. DISCUSSION: Our results confirmed the existence of M. oxyfera-like bacteria in the rhizosphere microenvironment of the submerged plant P. crispus. Group b of M. oxyfera-like bacteria was the dominant group in this study as opposed to previous findings that both group a and b coexist in most other environments. Our results indicate that understanding the ecophysiology of M. oxyfera-like bacteria group b may help to explain their existence in the rhizosphere sediment of aquatic plant.

Cooccurrence and potential role of nitrite- and nitrate-dependent methanotrophs in freshwater marsh sediments.

Nitrite- and nitrate-dependent anaerobic methane oxidation are mediated by the NC10 bacteria closely related to "Candidatus Methylomirabilis oxyfera" (M. oxyfera) and the ANME-2d archaea closely related to "Candidatus Methanoperedens nitroreducens" (M. nitroreducens), respectively. Here, we investigated the occurrence and activity of both M. oxyfera-like bacteria and M. nitroreducens-like archaea in the sediment of freshwater marshes in Eastern China. The presence of diverse M. oxyfera-like bacteria (>87% identity to M. oxyfera) and M. nitroreducens-like archaea (>96% identity to M. nitroreducens) was confirmed by using Illumina-based total bacterial and archaeal 16S rRNA gene sequencing, respectively. The recovered M. oxyfera-like bacterial sequences accounted for 1.6-4.3% of the total bacterial 16S rRNA pool, and M. nitroreducens-like archaeal sequences accounted for 0.2-1.8% of the total archaeal 16S rRNA pool. The detected numbers of OTUs of the 16S rRNA genes of M. oxyfera-like bacteria and M. nitroreducens-like archaea were 78 and 72, respectively, based on 3% sequence difference. Quantitative PCR showed that the 16S rRNA gene abundance of M. oxyfera-like bacteria (6.1 × 106-3.2 × 107 copies g-1 sediment) was 2-4 orders of magnitude higher than that of M. nitroreducens-like archaea (1.4 × 103-3.2 × 104 copies g-1 sediment). Stable isotope experiments showed that the addition of both nitrite and nitrate stimulated the anaerobic methane oxidation, while the stimulation by nitrite is more significant than nitrate. Our results provide the first evidence that the M. oxyfera-like bacteria play a more important role than the M. nitroreducens-like archaea in methane cycling in wetland systems.