Archaea catalyze iron-dependent anaerobic oxidation of methane.

Anaerobic oxidation of methane (AOM) is crucial for controlling the emission of this potent greenhouse gas to the atmosphere. Nitrite-, nitrate-, and sulfate-dependent methane oxidation is well-documented, but AOM coupled to the reduction of oxidized metals has so far been demonstrated only in environmental samples. Here, using a freshwater enrichment culture, we show that archaea of the order Methanosarcinales, related to "Candidatus Methanoperedens nitroreducens," couple the reduction of environmentally relevant forms of Fe3+ and Mn4+ to the oxidation of methane. We obtained an enrichment culture of these archaea under anaerobic, nitrate-reducing conditions with a continuous supply of methane. Via batch incubations using [13C]methane, we demonstrated that soluble ferric iron (Fe3+, as Fe-citrate) and nanoparticulate forms of Fe3+ and Mn4+ supported methane-oxidizing activity. CO2 and ferrous iron (Fe2+) were produced in stoichiometric amounts. Our study connects the previous finding of iron-dependent AOM to microorganisms detected in numerous habitats worldwide. Consequently, it enables a better understanding of the interaction between the biogeochemical cycles of iron and methane.

McrA primers for the detection and quantification of the anaerobic archaeal methanotroph 'Candidatus Methanoperedens nitroreducens'.

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.

Enrichment of anaerobic nitrate-dependent methanotrophic 'Candidatus Methanoperedens nitroreducens' archaea from an Italian paddy field soil.

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.

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.

Iron-mediated anaerobic oxidation of methane in brackish coastal sediments.

Methane is a powerful greenhouse gas and its biological conversion in marine sediments, largely controlled by anaerobic oxidation of methane (AOM), is a crucial part of the global carbon cycle. However, little is known about the role of iron oxides as an oxidant for AOM. Here we provide the first field evidence for iron-dependent AOM in brackish coastal surface sediments and show that methane produced in Bothnian Sea sediments is oxidized in distinct zones of iron- and sulfate-dependent AOM. At our study site, anthropogenic eutrophication over recent decades has led to an upward migration of the sulfate/methane transition zone in the sediment. Abundant iron oxides and high dissolved ferrous iron indicate iron reduction in the methanogenic sediments below the newly established sulfate/methane transition. Laboratory incubation studies of these sediments strongly suggest that the in situ microbial community is capable of linking methane oxidation to iron oxide reduction. Eutrophication of coastal environments may therefore create geochemical conditions favorable for iron-mediated AOM and thus increase the relevance of iron-dependent methane oxidation in the future. Besides its role in mitigating methane emissions, iron-dependent AOM strongly impacts sedimentary iron cycling and related biogeochemical processes through the reduction of large quantities of iron oxides.

Autotrophic carbon dioxide fixation via the Calvin-Benson-Bassham cycle by the denitrifying methanotroph "Candidatus Methylomirabilis oxyfera".

Methane is an important greenhouse gas and the most abundant hydrocarbon in the Earth's atmosphere. Methanotrophic microorganisms can use methane as their sole energy source and play a crucial role in the mitigation of methane emissions in the environment. "Candidatus Methylomirabilis oxyfera" is a recently described intra-aerobic methanotroph that is assumed to use nitric oxide to generate internal oxygen to oxidize methane via the conventional aerobic pathway, including the monooxygenase reaction. Previous genome analysis has suggested that, like the verrucomicrobial methanotrophs, "Ca. Methylomirabilis oxyfera" encodes and transcribes genes for the Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. Here we provide multiple independent lines of evidence for autotrophic carbon dioxide fixation by "Ca. Methylomirabilis oxyfera" via the CBB cycle. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme of the CBB cycle, in cell extracts from an "Ca. Methylomirabilis oxyfera" enrichment culture was shown to account for up to 10% of the total methane oxidation activity. Labeling studies with whole cells in batch incubations supplied with either (13)CH4 or [(13)C]bicarbonate revealed that "Ca. Methylomirabilis oxyfera" biomass and lipids became significantly more enriched in (13)C after incubation with (13)C-labeled bicarbonate (and unlabeled methane) than after incubation with (13)C-labeled methane (and unlabeled bicarbonate), providing evidence for autotrophic carbon dioxide fixation. Besides this experimental approach, detailed genomic and transcriptomic analysis demonstrated an operational CBB cycle in "Ca. Methylomirabilis oxyfera." Altogether, these results show that the CBB cycle is active and plays a major role in carbon assimilation by "Ca. Methylomirabilis oxyfera" bacteria. Our results suggest that autotrophy might be more widespread among methanotrophs than was previously assumed and implies that a methanotrophic community in the environment is not necessarily revealed by (13)C-depleted lipids.

Revisiting methanotrophic communities in sewage treatment plants.

The methanotrophic potential in sewage treatment sludge was investigated. We detected a diverse aerobic methanotrophic community that potentially plays a significant role in mitigating methane emission in this environment. The results suggest that community structure was determined by conditions specific to the processes in a sewage treatment plant.

Rare branched fatty acids characterize the lipid composition of the intra-aerobic methane oxidizer "Candidatus Methylomirabilis oxyfera".

The recently described bacterium "Candidatus Methylomirabilis oxyfera" couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of "Ca. Methylomirabilis oxyfera" is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of "Ca. Methylomirabilis oxyfera" to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple "Ca. Methylomirabilis oxyfera" enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC(16:0)). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC(16:1Δ7)), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC(16:0) and 10MeC(16:1Δ7) are key and characteristic components of the lipid profile of "Ca. Methylomirabilis oxyfera." The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.

Anaerobic oxidization of methane in a minerotrophic peatland: enrichment of nitrite-dependent methane-oxidizing bacteria.

The importance of anaerobic oxidation of methane (AOM) as a methane sink in freshwater systems is largely unexplored, particularly in peat ecosystems. Nitrite-dependent anaerobic methane oxidation (n-damo) was recently discovered and reported to be catalyzed by the bacterium "Candidatus Methylomirabilis oxyfera," which is affiliated with the NC10 phylum. So far, several "Ca. Methylomirabilis oxyfera" enrichment cultures have been obtained using a limited number of freshwater sediments or wastewater treatment sludge as the inoculum. In this study, using stable isotope measurements and porewater profiles, we investigated the potential of n-damo in a minerotrophic peatland in the south of the Netherlands that is infiltrated by nitrate-rich ground water. Methane and nitrate profiles suggested that all methane produced was oxidized before reaching the oxic layer, and NC10 bacteria could be active in the transition zone where countergradients of methane and nitrate occur. Quantitative PCR showed high NC10 bacterial cell numbers at this methane-nitrate transition zone. This soil section was used to enrich the prevalent NC10 bacteria in a continuous culture supplied with methane and nitrite at an in situ pH of 6.2. An enrichment of nitrite-reducing methanotrophic NC10 bacteria was successfully obtained. Phylogenetic analysis of retrieved 16S rRNA and pmoA genes showed that the enriched bacteria were very similar to the ones found in situ and constituted a new branch of NC10 bacteria with an identity of less than 96 and 90% to the 16S rRNA and pmoA genes of "Ca. Methylomirabilis oxyfera," respectively. The results of this study expand our knowledge of the diversity and distribution of NC10 bacteria in the environment and highlight their potential contribution to nitrogen and methane cycles.

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.

A new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizing bacterium Candidatus 'Methylomirabilis oxyfera'.

Biological methane oxidation proceeds either through aerobic or anaerobic pathways. The newly discovered bacterium Candidatus 'Methylomirabilis oxyfera' challenges this dichotomy. This bacterium performs anaerobic methane oxidation coupled to denitrification, but does so in a peculiar way. Instead of scavenging oxygen from the environment, like the aerobic methanotrophs, or driving methane oxidation by reverse methanogenesis, like the methanogenic archaea in sulfate-reducing systems, it produces its own supply of oxygen by metabolizing nitrite via nitric oxide into oxygen and dinitrogen gas. The intracellularly produced oxygen is then used for the oxidation of methane by the classical aerobic methane oxidation pathway involving methane mono-oxygenase. The present mini-review summarizes the current knowledge about this process and the micro-organism responsible for it.

Combined anaerobic ammonium and methane oxidation for nitrogen and methane removal.

Anammox (anaerobic ammonium oxidation) is an environment-friendly and cost-efficient nitrogen-removal process currently applied to high-ammonium-loaded wastewaters such as anaerobic digester effluents. In these wastewaters, dissolved methane is also present and should be removed to prevent greenhouse gas emissions into the environment. Potentially, another recently discovered microbial pathway, n-damo (nitrite-dependent anaerobic methane oxidation) could be used for this purpose. In the present paper, we explore the feasibility of simultaneously removing methane and ammonium anaerobically, starting with granules from a full-scale anammox bioreactor. We describe the development of a co-culture of anammox and n-damo bacteria using a medium containing methane, ammonium and nitrite. The results are discussed in the context of other recent studies on the application of anaerobic methane- and ammonia-oxidizing bacteria for wastewater treatment.

pmoA Primers for detection of anaerobic methanotrophs.

Published pmoA primers do not match the pmoA sequence of "Candidatus Methylomirabilis oxyfera," a bacterium that performs nitrite-dependent anaerobic methane oxidation. Therefore, new pmoA primers for the detection of "Ca. Methylomirabilis oxyfera"-like methanotrophs were developed and successfully tested on freshwater samples from different habitats. These primers expand existing molecular tools for the study of methanotrophs in the environment.

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

Anaerobic ammonium oxidation (anammox) and anaerobic methane oxidation (ANME coupled to denitrification) with nitrite as electron acceptor are two of the most recent discoveries in the microbial nitrogen cycle. Currently the anammox process has been relatively well investigated in a number of natural and man-made ecosystems, while ANME coupled to denitrification has only been observed in a limited number of freshwater ecosystems. The ubiquitous presence of anammox bacteria in marine ecosystems has changed our knowledge of the global nitrogen cycle. Up to 50% of N(2) production in marine sediments and oxygen-depleted zones may be attributed to anammox bacteria. However, there are only few indications of anammox in natural and constructed freshwater wetlands. In this paper, the potential role of anammox and denitrifying methanotrophic bacteria in natural and artificial wetlands is discussed in relation to global warming. The focus of the review is to explore and analyze if suitable environmental conditions exist for anammox and denitrifying methanotrophic bacteria in nitrogen-rich freshwater wetlands.

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.

The influence of oxygen and methane on nitrogen fixation in subarctic Sphagnum mosses.

Biological nitrogen fixation is an important source of bioavailable nitrogen in Sphagnum dominated peatlands. Sphagnum mosses harbor a diverse microbiome including nitrogen-fixing and methane (CH4) oxidizing bacteria. The inhibitory effect of oxygen on microbial nitrogen fixation is documented for many bacteria. However, the role of nitrogen-fixing methanotrophs in nitrogen supply to Sphagnum peat mosses is not well explored. Here, we investigated the role of both oxygen and methane on nitrogen fixation in subarctic Sphagnum peat mosses. Five species of Sphagnum mosses were sampled from two mesotrophic and three oligotrophic sites within the Lakkasuo peatland in Orivesi, central Finland. Mosses were incubated under either ambient or low oxygen conditions in the presence or absence of methane. Stable isotope activity assays revealed considerable nitrogen-fixing and methane-assimilating rates at all sites (1.4 ± 0.2 µmol 15N-N2 g-1 DW day-1 and 12.0 ± 1.1 µmol 13C-CH4 g-1 DW day-1, respectively). Addition of methane did not stimulate incorporation of 15N-nitrogen into biomass, whereas oxygen depletion increased the activity of the nitrogen-fixing community. Analysis of the 16S rRNA genes at the bacterial community level showed a very diverse microbiome that was dominated by Alphaproteobacteria in all sites. Bona fide methane-oxidizing taxa were not very abundant (relative abundance less than 0.1%). Based on our results we conclude that methanotrophs did not contribute significantly to nitrogen fixation in the investigated peatlands.

Stratification of Diversity and Activity of Methanogenic and Methanotrophic Microorganisms in a Nitrogen-Fertilized Italian Paddy Soil.

Paddy fields are important ecosystems, as rice is the primary food source for about half of the world's population. Paddy fields are impacted by nitrogen fertilization and are a major anthropogenic source of methane. Microbial diversity and methane metabolism were investigated in the upper 60 cm of a paddy soil by qPCR, 16S rRNA gene amplicon sequencing and anoxic 13C-CH4 turnover with a suite of electron acceptors. The bacterial community consisted mainly of Acidobacteria, Chloroflexi, Proteobacteria, Planctomycetes, and Actinobacteria. Among archaea, Euryarchaeota and Bathyarchaeota dominated over Thaumarchaeota in the upper 30 cm of the soil. Bathyarchaeota constituted up to 45% of the total archaeal reads in the top 5 cm. In the methanogenic community, Methanosaeta were generally more abundant than the versatile Methanosarcina. The measured maximum methane production rate was 444 nmol gdwh-1, and the maximum rates of nitrate-, nitrite-, and iron-dependent anaerobic oxidation of methane (AOM) were 57 nmol, 55 nmol, and 56 nmol gdwh-1, respectively, at different depths. qPCR revealed a higher abundance of 'Candidatus Methanoperedens nitroreducens' than methanotrophic NC10 phylum bacteria at all depths, except at 60 cm. These results demonstrate that there is substantial potential for AOM in fertilized paddy fields, with 'Candidatus Methanoperedens nitroreducens' archaea as a potential important contributor.

Bacterial oxygen production in the dark.

Nitric oxide (NO) and nitrous oxide (N(2)O) are among nature's most powerful electron acceptors. In recent years it became clear that microorganisms can take advantage of the oxidizing power of these compounds to degrade aliphatic and aromatic hydrocarbons. For two unrelated bacterial species, the "NC10" phylum bacterium "Candidatus Methylomirabilis oxyfera" and the γ-proteobacterial strain HdN1 it has been suggested that under anoxic conditions with nitrate and/or nitrite, monooxygenases are used for methane and hexadecane oxidation, respectively. No degradation was observed with nitrous oxide only. Similarly, "aerobic" pathways for hydrocarbon degradation are employed by (per)chlorate-reducing bacteria, which are known to produce oxygen from chlorite [Formula: see text]. In the anaerobic methanotroph M. oxyfera, which lacks identifiable enzymes for nitrogen formation, substrate activation in the presence of nitrite was directly associated with both oxygen and nitrogen formation. These findings strongly argue for the role of NO, or an oxygen species derived from it, in the activation reaction of methane. Although oxygen generation elegantly explains the utilization of "aerobic" pathways under anoxic conditions, the underlying mechanism is still elusive. In this perspective, we review the current knowledge about intra-aerobic pathways, their potential presence in other organisms, and identify candidate enzymes related to quinol-dependent NO reductases (qNORs) that might be involved in the formation of oxygen.