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Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are known: in the hydrogenotrophic pathway H2 and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (Methanosphaera, Methanimicrococcus, Methanomassiliicoccus, Methanonatronarchaeum) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching Methanonatronarchaeum revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of Methanosarcinales strains. Moreover, recently it has been shown that the methanogen Methermicoccus shengliensis can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (N,N,N-trimethylethanolamine) or betaine (N,N,N-trimethylglycine) have been described as substrates for methane production in Methanococcoides and Methanolobus strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways.Key points• Newly discovered methanogenic substrates and pathways are reviewed for the first time.• The review provides an in-depth analysis of unusual methanogenesis pathways.• The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.

Anammox pathway revealed Anammox, or anaerobic ammonium oxidation, is one of two important microbial processes that recycle fixed nitrogen back to the atmosphere. Less well studied than the other process (denitrification), anammox converts ammonia and nitrite into dinitrogen (N 2 ) gas. The complete biochemical and enzymatic mechanism of anammox has now been established. Nitric oxide and hydrazine are intermediate products in a pathway that involves three key enzymes — nitrite reductase, hydrazine synthase and hydrazine oxidoreductase. Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N 2 ) to the atmosphere 1 , 2 , 3 , 4 . Denitrification has been studied for over 100 years and its intermediates and enzymes are well known 5 . Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N 2 H 4 ) 6 , 7 . Here we show that N 2 H 4 is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N 2 H 4 . We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N 2 H 4 synthesis and its oxidation to N 2 . These results present a new biochemical reaction forging an N–N bond and fill a lacuna in our understanding of the biochemical synthesis of the N 2 in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.

ABSTRACT Methanotrophs are an important group of microorganisms that counteract methane emissions to the atmosphere. Methane-oxidising bacteria of the Alpha- and Gammaproteobacteria have been studied for over a century, while methanotrophs of the phylum Verrucomicrobia are a more recent discovery. Verrucomicrobial methanotrophs are extremophiles that live in very acidic geothermal ecosystems. Currently, more than a dozen strains have been isolated, belonging to the genera Methylacidiphilum and Methylacidimicrobium. Initially, these methanotrophs were thought to be metabolically confined. However, genomic analyses and physiological and biochemical experiments over the past years revealed that verrucomicrobial methanotrophs, as well as proteobacterial methanotrophs, are much more metabolically versatile than previously assumed. Several inorganic gases and other molecules present in acidic geothermal ecosystems can be utilised, such as methane, hydrogen gas, carbon dioxide, ammonium, nitrogen gas and perhaps also hydrogen sulfide. Verrucomicrobial methanotrophs could therefore represent key players in multiple volcanic nutrient cycles and in the mitigation of greenhouse gas emissions from geothermal ecosystems. Here, we summarise the current knowledge on verrucomicrobial methanotrophs with respect to their metabolic versatility and discuss the factors that determine their diversity in their natural environment. In addition, key metabolic, morphological and ecological characteristics of verrucomicrobial and proteobacterial methanotrophs are reviewed. This review discusses the metabolic versatility of verrucomicrobial methanotrophs regarding the acidic volcanic ecosystems they thrive in and a comparison is made with the canonical proteobacterial methanotrophs.

Ridding the world of methane Although much speculated on, no microorganisms had been shown capable of anaerobic methane oxidation using nitrate as the sole electron acceptor. Now this reaction has been demonstrated in the laboratory in a microbial community with two members, one a slow-growing bacterium of a type that has not cultured before, and one an archaeal organism. Nucleic acid markers characteristic of both are present in freshwater samples worldwide, suggesting that this reaction is important in the biological methane and nitrogen cycles. It also has the potential to be used to counteract the increases in methane production associated with intensive agriculture. Although much speculated on, this is the first unambiguous report demonstrating the isolation of a consortium of microorganisms capable of anaerobic methane oxidation using nitrate as the sole electron acceptor. Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale 1 , 2 . Freshwater sediments often receive increased downward fluxes of nitrate from agricultural runoff and upward fluxes of methane generated by anaerobic decomposition 3 . 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 4 , 5 , 6 , 7 , 8 . Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate 9 , 10 . 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 11 , 12 , 13 , 14 indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles.

Until now, the oxidation steps necessary for complete nitrification had always been observed to occur in two separate microorganisms in a cross-feeding interaction; here, together with the study by Daims et al ., van Kessel et al . report the enrichment and characterization of Nitrospira species that encode all of the enzymes necessary to catalyse complete nitrification, a phenotype referred to as ‘comammox’ (for complete ammonia oxidation). Time to rethink nitrification Two groups this week report the enrichment and characterization of Nitrospira species that encode all of the enzymes necessary to catalyse complete nitrification, a phenotype referred to as 'comammox' (for complete ammonia oxidation). Until now, this two-step reaction was thought to involve two organisms in a cross-feeding interaction. Phylogenetic analyses suggest that comammox Nitrospira are present in a number of diverse environments, so these findings have the potential to fundamentally change our view of the nitrogen cycle and open a new frontier in nitrification research. Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890 1 , this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle 2 . Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms 3 . Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

Evolution of novel enzyme activity Many extremophilic organisms require unusual enzymes to help them survive in harsh environments. For example, acid-loving hyperthermophilic Archaea found in the bubbling mud of volcanic solfataras are able to oxidize reduced sulphur compounds. The X-ray crystal structure of a carbon disulphide (CS 2 ) hydrolase from an Acidianus strain isolated from the Solfatara volcano near Naples, Italy, has now been determined. The enzyme, which converts CS 2 into hydrogen sulphide and carbon dioxide, has a typical carbonic anhydrase fold and active site, although CO 2 is not a substrate for the enzyme. This suggests that CS 2 hydrolase is an example of divergent evolution, where a new enzyme has emerged through the evolution of a new quaternary structure rather than through mutations of the active site. Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H 2 S) and carbon disulphide (CS 2 ) 1 , 2 . The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS 2 into H 2 S and carbon dioxide (CO 2 ), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS 2 hydrolase from Acidianus A1-3. The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO 2 is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme’s substrate specificity for CS 2 , which is hydrophobic. The transposon sequences that surround the gene encoding this CS 2 hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient β-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS 2 metabolism.

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. An early route to oxygen A previously unknown pathway producing oxygen during anaerobic methane oxidation linked to nitrite and nitrate reduction has been found in microbes isolated from freshwater sediments in Dutch drainage ditches. The complete genome of the bacterium responsible for this reaction has been assembled, and found to contain genes for aerobic methane oxidation. The bacterium reduces nitrite via the recombination of two molecules of nitric oxide into nitrogen and oxygen, bypassing the familiar denitrification intermediate nitrous oxide. This discovery is relevant to nitrogen and methane cycling in the environment and, since nitrogen oxides arose early on Earth, raises the possibility that oxygen was available to microbes before the advent of oxygen-producing photosynthesis. In certain microbes, the anaerobic oxidation of methane can be linked to the reduction of nitrates and nitrites. Here it is shown that this occurs through the intermediate production of oxygen. This brings the number of known biological pathways for oxygen production to four, with implications for our understanding of life on the early Earth.

Living without oxygen Several foraminiferal protozoa species grow in anoxic zones in marine sediment, but the type of anaerobic respiration that sustains them was not known. These organisms have now been found to accumulate nitrate intracellularly at concentrations more than 500 times the environmental values. The nitrate substitutes for oxygen in these anoxic habitats. The large amounts of nitrate that accumulate may even allow them to 'hold their breath' for more than a month. Denitrification, the biological conversion of nitrate to nitrogen, was believed to be restricted to bacteria and archaea. It has now been shown that highly abundant benthic foraminifers are also capable of denitrification, suggesting that much remains to be learned about the global nitrogen cycle. Benthic foraminifera are unicellular eukaryotes found abundantly in many types of marine sediments. Many species survive and possibly reproduce in anoxic habitats 1 , but sustainable anaerobic metabolism has not been previously described. Here we demonstrate that the foraminifer Globobulimina pseudospinescens accumulates intracellular nitrate stores and that these can be respired to dinitrogen gas. The amounts of nitrate detected are estimated to be sufficient to support respiration for over a month. In a Swedish fjord sediment where G. pseudospinescens is the dominant foraminifer, the intracellular nitrate pool in this species accounted for 20% of the large, cell-bound, nitrate pool present in an oxygen-free zone. Similarly high nitrate concentrations were also detected in foraminifera Nonionella cf. stella and a Stainforthia species, the two dominant benthic taxa occurring within the oxygen minimum zone of the continental shelf off Chile. Given the high abundance of foraminifera in anoxic marine environments 1 , 2 , 3 , these new findings suggest that foraminifera may play an important role in global nitrogen cycling and indicate that our understanding of the complexity of the marine nitrogen cycle is far from complete.

Abstract The anaerobic ammonium-oxidizing (anammox) and nitrite-dependent anaerobic methane-oxidizing (n-damo) bacteria in a paddy soil core (0–100 cm) were investigated with newly designed primers targeting the hydrazine synthase β-subunit (hzsB) of anammox bacteria and the recently published primers targeting the pmoA and 16S rRNA genes of n-damo bacteria. The hzsB gene was identified as a proper biomarker to explore the anammox bacterial biodiversity and abundance in soil. The anammox bacteria were present throughout the soil core with the highest abundance of 2.7 × 106hzsB copies g−1 dry soil at 40–50 cm and were not detectable below 70 cm. Sequences related to at least three species of known anammox bacteria, ‘Brocadia anammoxidans’, ‘Brocadia fulgida’, and ‘Jettenia asiatica’ were detected. By combining the analysis of pmoA and 16S rRNA genes, the n-damo bacteria were observed to be present in 30–70 cm with abundance from 6.5 × 103 (60–70 cm) to 7.5 × 104 (30–40 cm) copies g−1 dry soil. The pmoA sequences retrieved from different depths closely related to each other and formed a unique clade. Our results showed that anammox and n-damo bacteria co-occurred in the paddy soil. Both of them were abundant in deep layers (30–60 cm) and the community structures changed along depths in the soil core.

Passing the acid test Bacteria that consume the greenhouse gas methane are potentially important players in the atmospheric budget, with the potential to sop up methane from the Earth's crust that would otherwise contribute to the atmospheric budget. Two new methane-utilizing bacteria have been isolated independently and both break new ground: unlike previous methanotrophic isolates, which are proteobacteria, they belong to the widely distributed Verrucomicrobia phylum. And both isolates display optimum growth and methane oxidation in remarkably acidic conditions, at pHs as low as 0.8 to 2.5. Acidimethylosilex fumarolicum SolV was isolated from a fuming vent on the Solfatara volcano near Naples, Italy, and Methylokorus infernorum from hot soil in the Hell's Gate (Tikitere) geothermal area of New Zealand. The isolation of the acidophilic bacterium Acidimethylosilex fumarolicum SolV from a fumarole is described. Unlike all previous methanotrophic isolates, which belong to the Alpha- or Gammaproteobacteria, it belongs to the widely distributed Verrumicrobia. Mud volcanoes, mudpots and fumaroles are remarkable geological features characterized by the emission of gas, water and/or semi-liquid mud matrices 1 with significant methane fluxes to the atmosphere (10 -1 to 10 3  t y -1 ) 2 , 3 , 4 . Environmental conditions in these areas vary from ambient temperature and neutral pH to high temperatures and low pH. Although there are strong indications for biological methane consumption in mud volcanoes 4 , 5 , no methanotrophic bacteria are known that would thrive in the hostile conditions of fumaroles (temperatures up to 70 °C and pH down to 1.8) 2 . The first step in aerobic methane oxidation is performed by a soluble or membrane-bound methane mono-oxygenase. Here we report that pmoA (encoding the β-subunit of membrane-bound methane mono-oxygenase) clone libraries, made by using DNA extracted from the Solfatara volcano mudpot and surrounding bare soil near the fumaroles, showed clusters of novel and distant pmoA genes. After methanotrophic enrichment at 50 °C and pH 2.0 the most distant cluster, sharing less than 50% identity with any other described pmoA gene, was represented in the culture. Finally we isolated an acidiphilic methanotrophic bacterium Acidimethylosilex fumarolicum SolV belonging to the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum 6 , ‘outside’ the subphyla of the Alpha- and Gammaproteobacteria containing the established methanotrophs. This bacterium grows under oxygen limitation on methane as the sole source of energy, down to pH 0.8—far below the pH optimum of any previously described methanotroph. A. fumarolicum SolV has three different pmoA genes, with two that are very similar to sequences retrieved from the mudpot. Highly homologous environmental 16S rRNA gene sequences from Yellowstone Park show that this new type of methanotrophic bacteria may be a common inhabitant of extreme environments. This is the first time that a representative of the widely distributed Verrucomicrobia phylum, of which most members remain uncultivated 6 , is coupled to a geochemically relevant reaction.