Enrichment and Molecular Detection of Denitrifying Methanotrophic Bacteria of the NC10 Phylum

Anaerobic methane oxidation coupled to denitrification was recently assigned to bacteria belonging to the uncultured phylum NC10. In this study, we incubated sediment from a eutrophic ditch harboring a diverse community of NC10 bacteria in a bioreactor with a constant supply of methane and nitrite. After 6 months, fluorescence in situ hybridization showed that NC10 bacteria dominated the resulting population. The enrichment culture oxidized methane and reduced nitrite to dinitrogen gas. We assessed NC10 phylum diversity in the inoculum and the enrichment culture, compiled the sequences currently available for this bacterial phylum, and showed that of the initial diversity, only members of one subgroup had been enriched. The growth of this subgroup was monitored by quantitative PCR and correlated to nitrite-reducing activity and the total biomass of the culture. Together, the results indicate that the enriched subgroup of NC10 bacteria is responsible for anaerobic methane oxidation coupled to nitrite reduction. Due to methodological limitations (a strong bias against NC10 bacteria in 16S rRNA gene clone libraries and inhibition by commonly used stopper material) the environmental distribution and importance of these bacteria could be largely underestimated at present.Atmospheric concentrations of methane have risen 2.6-fold since preindustrial times (10). After several years of stagnation, there was again a clear increase in the methane concentration in 2007 (29). Currently, it is uncertain whether an increase in the number of sources and production or a decrease in the number of sinks and consumption is responsible for this reversal of the trend.Freshwater habitats like natural wetlands and rice fields are a major source (38% 9]) of atmospheric methane. In the absence of other documented electron donors, aerobic methane oxidation is assumed to be the most important sink in these habitats, but the role of alternative electron donors is not well understood (19, 30). Anaerobic methane oxidation coupled to denitrification is energetically favorable, but evidence that it occurs is scarce. In marine, methane-containing sediments, nitrate and nitrite are usually not quantitatively important electron acceptors; in freshwater sediments the denitrifying and aerobic zones are in close proximity (3, 22, 35), possibly masking the process from detection. To our knowledge, concomitant methane and nitrate profiles of sediments have never been published.So far, methane oxidation coupled to denitrification has received the most attention in the field of hydrogeology. In groundwater, contamination with nitrate and nitrite occurs frequently, whereas electron donors are limiting. Methane plumes often form around landfills, and their attenuation has sometimes been attributed to denitrification (2, 37). So far, a single previous study unambiguously demonstrated anaerobic oxidation of methane coupled to denitrification in a contaminated freshwater aquifer (32). The first in vitro observation of anaerobic methane oxidation coupled to denitrification came from a laboratory-scale sludge digestor (11). The use of a laboratory enrichment culture also eventually resulted in identification of the organisms involved; bacteria of the NC10 phylum and archaea of the order Methanosarcinales dominated a mixed culture carrying out anaerobic methane oxidation coupled to denitrification (27). This culture was enriched from a freshwater canal sediment after 1 year of continuous supply of methane and nitrite. Subsequently, the archaea were shown to be dispensable, as they disappeared after prolonged incubation of the same culture (7). Mass balance calculations showed that methane oxidation was coupled to the reduction of nitrite with a 3:8 stoichiometry, in accordance with theoretical expectations. The bacteria that dominated the mixed culture and apparently oxidized methane anaerobically are members of the NC10 phylum, one of the many phyla having no members in pure culture (8, 28). The 16S rRNA gene sequences of such organisms, however, have been found in a number of environmental surveys of aquatic environments; e.g., the most closely related sequences have been found in aquifers (1, 23) and lake sediments (13, 17). Sequence similarity and phylogenetic affiliation may indicate similar metabolic capacities of organisms, but by itself this is not sufficient to infer similar metabolism (5). This is especially true for denitrifying methanotrophs, because only a single enrichment culture has been described so far (7, 27).The objective of the present study was to generalize the previous finding that NC10 bacteria were associated with anaerobic methane oxidation, a necessary step forward in addressing the significance of this poorly understood process as a methane sink in freshwater habitats.