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

Abstract

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 nitritereducing 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 denit...

High rates of anaerobic methane oxidation in freshwater wetlands reduce potential atmospheric methane emissions

“…M. oxyfera and affiliated members of this phylum in FWW 4,5 . However, to date evidence for n-damo originated in freshwater environments with high nitrogen loading and most published rates are from long-term enrichments 5,36,54,55 . The conditions of such experiments do not fully mimic in situ conditions, as they are often conducted after prolonged preincubation phases, with additions of methane and electron acceptors, and with slurried material.…”

Potential roles of anaerobic ammonium and methane oxidation in the nitrogen cycle of wetland ecosystems

“…However, many 16S rRNA gene sequences similar to the sequence of denitrifying methanotrophs have now been retrieved from different anoxic freshwater sediments, indicating that denitrifying methanotrophic bacteria may be more widespread than previously assumed. In a very recent study (Ettwig et al 2009), sediment from a eutrophic fresh water ditch harboring a diverse community of NC10 bacteria was incubated in a bioreactor with efficient biomass retention and a constant supply of methane and nitrite. In this case already after 6 months, the denitrifying methanotrophic bacteria dominated the reactor community as visualized by fluorescence in situ hybridization with probes specifically designed to detect these bacteria.…”

“…After compilation of the currently available sequences of this bacterial phylum, it was shown that only members of one particular subgroup had been enriched. By designing primers suitable for Q-PCR, the growth of this NC10 subgroup was monitored retrospectively and could be correlated to nitrite-reducing activity and total biomass of the culture (Ettwig et al 2009). The availability of suitable primers makes it possible to survey more anoxic freshwater sediments that are suspected to provide suitable environmental conditions for nitrite-dependent anaerobic methane oxidation.…”

“…Estuarine and marine sediments release similar amounts of ammonium and dinitrogen (N 2 ), whereas freshwater sediments are assumed to release mainly N 2 with coupled nitrification-denitrification. Based on the presently known 16S rRNA gene sequences of anammox and denitrifying methanotrophic bacteria, specific oligonucleotide probes and primers have been developed to document the presence of anammox and denitrifying methanotrophic cells in natural and engineered ecosystems Ettwig et al 2009). However, there is relatively little literature about anammox in natural and constructed wetlands (Shipin et al 2005;Paredes et al 2007;Erler et al 2008), although wetlands are an important part of the ecological systems on earth and may harbor enough suitable habitats for anammox bacteria and other nitrogen cycle bacteria (Fig.…”

Current advances in molecular methods for detection of nitrite-dependent anaerobic methane oxidizing bacteria in natural environments

“…Ettwig et al 78 (2009) reported that there was no measurable n-damo activity before 110 days in the 79 enrichment, and then it started to be detectable and increase. The estimated doubling time for 80 n-damo bacteria is one to two weeks under laboratory condition (Ettwig et al 2008) with a 81 methane conversion rate of 1.7 nmol min -1 mg protein -1 (Ettwig et al 2009). Finally, n-damo 82 bacteria are often simultaneously co-cultured with anaerobic ammonium oxidizing (anammox) 83 bacteria (Luesken et al 2011a;Zhu et al 2011), which also used nitrite as electron acceptor,…”