Microbial methane oxidation is an important process to reduce the emission of the greenhouse gas methane. Anaerobic microorganisms couple the oxidation of methane to the reduction of sulfate, nitrate and nitrite, and possibly oxidized iron and manganese minerals. In this article, we review the recent finding of the intriguing nitrate- and nitrite-dependent anaerobic oxidation of methane (AOM). Nitrate-dependent AOM is catalyzed by anaerobic archaea belonging to the ANME-2d clade closely related to Methanosarcina methanogens. They were named 'Candidatus Methanoperedens nitroreducens' and use reverse methanogenesis with the key enzyme methyl-coenzyme M (methyl-CoM) reductase for methane activation. Their major end product is nitrite which can be taken up by nitrite-dependent methanotrophs. Nitrite-dependent AOM is performed by the NC10 bacterium 'Candidatus Methylomirabilis oxyfera' that probably utilizes an intra-aerobic pathway through the dismutation of NO to N and O for aerobic methane activation by methane monooxygenase, yet being a strictly anaerobic microbe. Environmental distribution, physiological and biochemical aspects are discussed in this article as well as the cooperation of the microorganisms involved.

Paddy fields are a significant source of methane and contribute up to 20% of total methane emissions from wetland ecosystems. These inundated, anoxic soils featuring abundant nitrogen compounds and methane are an ideal niche for nitrate-dependent anaerobic methanotrophs. After 2 years of enrichment with a continuous supply of methane and nitrate as the sole electron donor and acceptor, a stable enrichment dominated by ‘Candidatus Methanoperedens nitroreducens’ archaea and ‘Candidatus Methylomirabilis oxyfera’ NC10 phylum bacteria was achieved. In this community, the methanotrophic archaea supplied the NC10 phylum bacteria with the necessary nitrite through nitrate reduction coupled to methane oxidation. The results of qPCR quantification of 16S ribosomal RNA (rRNA) gene copies, analysis of metagenomic 16S rRNA reads, and fluorescence in situ hybridization (FISH) correlated well and showed that after 2 years, ‘Candidatus Methanoperedens nitroreducens’ had the highest abundance of (2.2 ± 0.4 × 108) 16S rRNA copies per milliliter and constituted approximately 22% of the total microbial community. Phylogenetic analysis showed that the 16S rRNA genes of the dominant microorganisms clustered with previously described ‘Candidatus Methanoperedens nitroreducens ANME2D’ (96% identity) and ‘Candidatus Methylomirabilis oxyfera’ (99% identity) strains. The pooled metagenomic sequences resulted in a high-quality draft genome assembly of ‘Candidatus Methanoperedens nitroreducens Vercelli’ that contained all key functional genes for the reverse methanogenesis pathway and nitrate reduction. The diagnostic mcrA gene was 96% similar to ‘Candidatus Methanoperedens nitroreducens ANME2D’ (WP_048089615.1) at the protein level. The ‘Candidatus Methylomirabilis oxyfera’ draft genome contained the marker genes pmoCAB, mdh, and nirS and putative NO dismutase genes. Whole-reactor anaerobic activity measurements with methane and nitrate revealed an average methane oxidation rate of 0.012 mmol/h/L, with cell-specific methane oxidation rates up to 0.57 fmol/cell/day for ‘Candidatus Methanoperedens nitroreducens’. In summary, this study describes the first enrichment and draft genome of methanotrophic archaea from paddy field soil, where these organisms can contribute significantly to the mitigation of methane emissions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-017-8416-0) contains supplementary material, which is available to authorized users.

Methane is the final product of the anaerobic decomposition of organic matter. The conversion of organic matter to methane (methanogenesis) as a mechanism for energy conservation is exclusively attributed to the archaeal domain. Methane is oxidized by methanotrophic microorganisms using oxygen or alternative terminal electron acceptors. Aerobic methanotrophic bacteria belong to the phyla Proteobacteria and Verrucomicrobia, while anaerobic methane oxidation is also mediated by more recently discovered anaerobic methanotrophs with representatives in both the bacteria and the archaea domains. The anaerobic oxidation of methane is coupled to the reduction of nitrate, nitrite, iron, manganese, sulfate, and organic electron acceptors (e.g., humic substances) as terminal electron acceptors. This review highlights the relevance of methanotrophy in natural and anthropogenically influenced ecosystems, emphasizing the environmental conditions, distribution, function, co-existence, interactions, and the availability of electron acceptors that likely play a key role in regulating their function. A systematic overview of key aspects of ecology, physiology, metabolism, and genomics is crucial to understand the contribution of methanotrophs in the mitigation of methane efflux to the atmosphere. We give significance to the processes under microaerophilic and anaerobic conditions for both aerobic and anaerobic methane oxidizers. In the context of anthropogenically influenced ecosystems, we emphasize the current and potential future applications of methanotrophs from two different angles, namely methane mitigation in wastewater treatment through the application of anaerobic methanotrophs, and the biotechnological applications of aerobic methanotrophs in resource recovery from methane waste streams. Finally, we identify knowledge gaps that may lead to opportunities to harness further the biotechnological benefits of methanotrophs in methane mitigation and for the production of valuable bioproducts enabling a bio-based and circular economy.

The nitrogen and methane cycles are important biogeochemical processes. Recently, ‘Candidatus Methanoperedens nitroreducens,’ archaea that catalyze nitrate-dependent anaerobic oxidation of methane (AOM), were enriched, and their genomes were analyzed. Diagnostic molecular tools for the sensitive detection of ‘Candidatus M. nitroreducens’ are not yet available. Here, we report the design of two novel mcrA primer combinations that specifically target the alpha sub-unit of the methyl-coenzyme M reductase (mcrA) gene of ‘Candidatus M. nitroreducens’. The first primer pair produces a fragment of 186-bp that can be used to quantify ‘Candidatus M. nitroreducens’ cells, whereas the second primer pair yields an 1191-bp amplicon that is with sufficient length and well suited for more detailed phylogenetic analyses. Six different environmental samples were evaluated with the new qPCR primer pair, and the abundances were compared with those determined using primers for the 16S rRNA gene. The qPCR results indicated that the number of copies of the ‘Candidatus M. nitroreducens’ mcrA gene was highest in rice field soil, with 5.6 ± 0.8 × 106 copies g−1 wet weight, whereas Indonesian river sediment had only 4.6 ± 2.7 × 102 copies g−1 wet weight. In addition to freshwater environments, sequences were also detected in marine sediment of the North Sea, which contained approximately 2.5 ± 0.7 × 104 copies g−1 wet weight. Phylogenetic analysis revealed that the amplified 1191-bp mcrA gene sequences from the different environments all clustered together with available genome sequences of mcrA from known ‘Candidatus M. nitroreducens’ archaea. Taken together, these results demonstrate the validity and utility of the new primers for the quantitative and sensitive detection of the mcrA gene sequences of these important nitrate-dependent AOM archaea. Furthermore, the newly obtained mcrA sequences will contribute to greater phylogenetic resolution of ‘Candidatus M. nitroreducens’ sequences, which have been only poorly captured by general methanogenic mcrA primers.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-8065-8) contains supplementary material, which is available to authorized users.

In order to mitigate methane emissions from paddy fields, it is important to understand the sources and sinks. Most paddy fields are heavily fertilized with nitrite and nitrate, which can be used as electron acceptors by anaerobic methanotrophs. Here we show that slurry incubations of Italian paddy field soil with nitrate and C-labelled methane have the potential for nitrate-dependent anaerobic oxidation of methane (79.9 nmol g d). Community analysis based on 16S rRNA amplicon sequencing and qPCR of the water-logged soil and the rhizosphere showed that anaerobic oxidation of methane-associated archaea (AAA), including Methanoperedens nitroreducens, comprised 9% (bulk soil) and 1% (rhizosphere) of all archaeal reads. The NC10 phylum bacteria made up less than 1% of all bacterial sequences. The phylogenetic analysis was complemented by qPCR showing that AAA ranged from 0.28 × 10 to 3.9 × 10 16S rRNA gene copies g in bulk soil and 0.27 × 10 to 2.8 × 10 in the rhizosphere. The abundance of NC10 phylum bacteria was an order of magnitude lower. Revisiting published diversity studies, we found that AAA have been detected, but not linked to methane oxidation, in several paddy fields. Our data suggest an important role of AAA in methane cycling in paddy fields.