Nitrite-driven anaerobic methane oxidation by oxygenic bacteria.

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

A microbial consortium couples anaerobic methane oxidation to denitrification.

Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale. Freshwater sediments often receive increased downward fluxes of nitrate from agricultural runoff and upward fluxes of methane generated by anaerobic decomposition. In theory, prokaryotes should be capable of using nitrate to oxidize methane anaerobically, but such organisms have neither been observed in nature nor isolated in the laboratory. Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate. Here we show that the direct, anaerobic oxidation of methane coupled to denitrification of nitrate is possible. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitrification in the complete absence of oxygen. This consortium consisted of two microorganisms, a bacterium representing a phylum without any cultured species and an archaeon distantly related to marine methanotrophic Archaea. The detection of relatives of these prokaryotes in different freshwater ecosystems worldwide indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles.

Deciphering the evolution and metabolism of an anammox bacterium from a community genome.

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics--the reconstruction of genomic data directly from the environment--to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organism's special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

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.

Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea.

Recently, a microbial consortium was shown to couple the anaerobic oxidation of methane to denitrification, predominantly in the form of nitrite reduction to dinitrogen gas. This consortium was dominated by bacteria of an as yet uncharacterized division and archaea of the order Methanosarcinales. The present manuscript reports on the upscaling of the enrichment culture, and addresses the role of the archaea in methane oxidation. The key gene of methanotrophic and methanogenic archaea, mcrA, was sequenced. The associated cofactor F(430) was shown to have a mass of 905 Da, the same as for methanogens and different from the heavier form (951 Da) found in methanotrophic archaea. After prolonged enrichment (> 1 year), no inhibition of anaerobic methane oxidation was observed in the presence of 20 mM bromoethane sulfonate, a specific inhibitor of MCR. Optimization of the cultivation conditions led to higher rates of methane oxidation and to the decline of the archaeal population, as shown by fluorescence in situ hybridization and quantitative MALDI-TOF analysis of F(430). Mass balancing showed that methane oxidation was still coupled to nitrite reduction in the total absence of oxygen. Together, our results show that bacteria can couple the anaerobic oxidation of methane to denitrification without the involvement of Archaea.