Enrichment and characterization of a nitric oxide-reducing microbial community in a continuous bioreactor.

Nitric oxide (NO) is a highly reactive and climate-active molecule and a key intermediate in the microbial nitrogen cycle. Despite its role in the evolution of denitrification and aerobic respiration, high redox potential and capacity to sustain microbial growth, our understanding of NO-reducing microorganisms remains limited due to the absence of NO-reducing microbial cultures obtained directly from the environment using NO as a substrate. Here, using a continuous bioreactor and a constant supply of NO as the sole electron acceptor, we enriched and characterized a microbial community dominated by two previously unknown microorganisms that grow at nanomolar NO concentrations and survive high amounts (>6 µM) of this toxic gas, reducing it to N2 with little to non-detectable production of the greenhouse gas nitrous oxide. These results provide insight into the physiology of NO-reducing microorganisms, which have pivotal roles in the control of climate-active gases, waste removal, and evolution of nitrate and oxygen respiration.

Discovery of a new genus of anaerobic ammonium oxidizing bacteria with a mechanism for oxygen tolerance.

In the past 20 years, there has been a major stride in understanding the core mechanism of anaerobic ammonium-oxidizing (anammox) bacteria, but there are still several discussion points on their survival strategies. Here, we discovered a new genus of anammox bacteria in a full-scale wastewater-treating biofilm system, tentatively named "Candidatus Loosdrechtia aerotolerans". Next to genes of all core anammox metabolisms, it encoded and transcribed genes involved in the dissimilatory nitrate reduction to ammonium (DNRA), which coupled to oxidation of small organic acids, could be used to replenish ammonium and sustain their metabolism. Surprisingly, it uniquely harbored a new ferredoxin-dependent nitrate reductase, which has not yet been found in any other anammox genome and might confer a selective advantage to it in nitrate assimilation. Similar to many other microorganisms, superoxide dismutase and catalase related to oxidative stress resistance were encoded and transcribed by "Ca. Loosdrechtia aerotolerans". Interestingly, bilirubin oxidase (BOD), likely involved in oxygen resistance of anammox bacteria under fluctuating oxygen concentrations, was identified in "Ca. Loosdrechtia aerotolerans" and four Ca. Brocadia genomes, and its activity was demonstrated using purified heterologously expressed proteins. A following survey of oxygen-active proteins in anammox bacteria revealed the presence of other previously undetected oxygen defense systems. The novel cbb3-type cytochrome c oxidase and bifunctional catalase-peroxidase may confer a selective advantage to Ca. Kuenenia and Ca. Scalindua that face frequent changes in oxygen concentrations. The discovery of this new genus significantly broadens our understanding of the ecophysiology of anammox bacteria. Furthermore, the diverse oxygen tolerance strategies employed by distinct anammox bacteria advance our understanding of their niche adaptability and provide valuable insight for the operation of anammox-based wastewater treatment systems.

Nitrogen input promotes denitrifying methanotrophs' abundance and contribution to methane emission reduction in coastal wetland and paddy soil.

Denitrifying anaerobic methane oxidation (DAMO) microorganisms, using nitrate/nitrite to oxidize methane, have been proved to be an important microbial methane sink in natural habitats. Increasing nitrogen deposit around the globe brings increased availability of substrates for these microorganisms. However, how elevated nitrogen level affects denitrifying methanotrophs has not been elucidated. In this study, sediment/soil samples from coastal wetland with continuous nitrogen input and paddy field with periodic nitrogen input were collected to investigate the influence of nitrogen input on the abundance and activity of denitrifying methanotrophs. The results indicated that nitrogen input significantly promoted DAMO microorganisms' abundance and contribution to methane emission reduction. In the coastal wetland, the contribution rate of DAMO process to methane removal increased from 12.1% to 33.5% along with continuously elevated nitrogen level in the 3-year tracking study. In the paddy field, the DAMO process accounted for 71.9% of total methane removal when nitrogen fertilizer was applied during the growing season, exceeding the aerobic methane oxidation process. This work would help us better understand the microbial methane cycle and reduce uncertainties in the estimations of the global methane emission.

Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex.

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.

Simultaneous Anaerobic and Aerobic Ammonia and Methane Oxidation under Oxygen Limitation Conditions.

Methane and ammonia have to be removed from wastewater treatment effluent in order to discharge it to receiving water bodies. A potential solution for this is a combination of simultaneous ammonia and methane oxidation by anaerobic ammonia oxidation (anammox) bacteria and nitrite/nitrate-dependent anaerobic methane oxidation (N-damo) microorganisms. When applied, these microorganisms will be exposed to oxygen, but little is known about the effect of a low concentration of oxygen on a culture containing these microorganisms. In this study, a stable coculture containing anammox and N-damo microorganisms in a laboratory scale bioreactor was established under oxygen limitation. Membrane inlet mass spectrometry (MIMS) was used to directly measure the in situ simultaneous activity of N-damo, anammox, and aerobic ammonia-oxidizing microorganisms. In addition, batch tests revealed that the bioreactor also harbored aerobic methanotrophs and anaerobic methanogens. Together with fluorescence in situ hybridization (FISH) analysis and metagenomics, these results indicate that the combination of N-damo and anammox activity under the continuous supply of limiting oxygen concentrations is feasible and can be implemented for the removal of methane and ammonia from anaerobic digester effluents. IMPORTANCE Nitrogen in wastewater leads to eutrophication of the receiving water bodies, and methane is a potent greenhouse gas; it is therefore important that these are removed from wastewater. A potential solution for the simultaneous removal of nitrogenous compounds and methane is the application of a combination of nitrite/nitrate-dependent methane oxidation (N-damo) and anaerobic ammonia oxidation (annamox). In order to do so, it is important to investigate the effect of oxygen on these two anaerobic processes. In this study, we investigate the effect of a continuous oxygen supply on the activity of an anaerobic methane- and ammonia-oxidizing coculture. The findings presented in this study are important for the potential application of these two microbial processes in wastewater treatment.

Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link.

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

Learning from microorganisms: using new insights in microbial physiology for sustainable nitrogen management.

Diverse nitrogen-transforming microorganisms with a wide variety of physiological properties are employed for biological nitrogen removal from wastewater. There are many technologies that achieve the required nitrogen discharge standards; however, greenhouse gas emissions and energy consumption constitute the bulk of the environmental footprint of wastewater treatment plants. In this review, we highlight current and proposed approaches aiming to achieve more energy-efficient and environment-friendly biological nitrogen removal, discuss whether new discoveries in microbial physiology of nitrogen-transforming microorganisms could be used to reduce greenhouse gas emissions, and summarize recent advances in ammonium recovery from wastewater.

Multiheme hydroxylamine oxidoreductases produce NO during ammonia oxidation in methanotrophs.

Aerobic and nitrite-dependent methanotrophs make a living from oxidizing methane via methanol to carbon dioxide. In addition, these microorganisms cometabolize ammonia due to its structural similarities to methane. The first step in both of these processes is catalyzed by methane monooxygenase, which converts methane or ammonia into methanol or hydroxylamine, respectively. Methanotrophs use methanol for energy conservation, whereas toxic hydroxylamine is a potent inhibitor that needs to be rapidly removed. It is suggested that many methanotrophs encode a hydroxylamine oxidoreductase (mHAO) in their genome to remove hydroxylamine, although biochemical evidence for this is lacking. HAOs also play a crucial role in the metabolism of aerobic and anaerobic ammonia oxidizers by converting hydroxylamine to nitric oxide (NO). Here, we purified an HAO from the thermophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV and characterized its kinetic properties. This mHAO possesses the characteristic P460 chromophore and is active up to at least 80 °C. It catalyzes the rapid oxidation of hydroxylamine to NO. In methanotrophs, mHAO efficiently removes hydroxylamine, which severely inhibits calcium-dependent, and as we show here, lanthanide-dependent methanol dehydrogenases, which are more prevalent in the environment. Our results indicate that mHAO allows methanotrophs to thrive under high ammonia concentrations in natural and engineered ecosystems, such as those observed in rice paddy fields, landfills, or volcanic mud pots, by preventing the accumulation of inhibitory hydroxylamine. Under oxic conditions, methanotrophs mainly oxidize ammonia to nitrite, whereas in hypoxic and anoxic environments reduction of both ammonia-derived nitrite and NO could lead to nitrous oxide (N2O) production.

Diversity, enrichment, and genomic potential of anaerobic methane- and ammonium-oxidizing microorganisms from a brewery wastewater treatment plant.

Anaerobic wastewater treatment offers several advantages; however, the effluent of anaerobic digesters still contains high levels of ammonium and dissolved methane that need to be removed before these effluents can be discharged to surface waters. The simultaneous anaerobic removal of methane and ammonium by denitrifying (N-damo) methanotrophs in combination with anaerobic ammonium-oxidizing (anammox) bacteria could be a potential solution to this challenge. After a molecular survey of a wastewater plant treating brewery effluent, indicating the presence of both N-damo and anammox bacteria, we started an anaerobic bioreactor with a continuous supply of methane, ammonium, and nitrite to enrich these anaerobic microorganisms. After 14 months of operation, a stable enrichment culture containing two types of 'Candidatus Methylomirabilis oxyfera' bacteria and two strains of 'Ca. Brocadia'-like anammox bacteria was achieved. In this community, anammox bacteria converted 80% of the nitrite with ammonium, while 'Ca. Methylomirabilis' contributed to 20% of the nitrite consumption. The analysis of metagenomic 16S rRNA reads and fluorescence in situ hybridization (FISH) correlated well and showed that, after 14 months, 'Ca. Methylomirabilis' and anammox bacteria constituted approximately 30 and 20% of the total microbial community. In addition, a substantial part (10%) of the community consisted of Phycisphaera-related planctomycetes. Assembly and binning of the metagenomic sequences resulted in high-quality draft genome of two 'Ca. Methylomirabilis' species containing the marker genes pmoCAB, xoxF, and nirS and putative NO dismutase genes. The anammox draft genomes most closely related to 'Ca. Brocadia fulgida' included the marker genes hzsABC, hao, and hdh. Whole-reactor and batch anaerobic activity measurements with methane, ammonium, nitrite, and nitrate revealed an average anaerobic methane oxidation rate of 0.12 mmol h-1 L-1 and ammonium oxidation rate of 0.5 mmol h-1 L-1. Together, this study describes the enrichment and draft genomes of anaerobic methanotrophs from a brewery wastewater treatment plant, where these organisms together with anammox bacteria can contribute significantly to the removal of methane and ammonium in a more sustainable way. KEY POINTS: • An enrichment culture containing both N-damo and anammox bacteria was obtained. • Simultaneous consumption of ammonia, nitrite, and methane under anoxic conditions. • In-depth metagenomic biodiversity analysis of inoculum and enrichment culture.

Discovery of a functional, contracted heme-binding motif within a multiheme cytochrome.

Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of c-type cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons. It is unclear how these electrons are delivered to hydrazine synthase. Here, we report the discovery of a functional tetraheme c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis with a naturally-occurring contracted Cys-Lys-Cys-His (CKCH) heme-binding motif, which is encoded in the hydrazine synthase gene cluster. The purified tetraheme protein (named KsTH) exchanged electrons with hydrazine synthase. Complementary spectroscopic techniques revealed that this protein harbors four low-spin hexa-coordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (hemes 3 and 4). A genomic database search revealed that c-type cytochromes with a contracted CXCH heme-binding motif are present throughout the bacterial and archaeal domains in the tree of life, suggesting that this heme recognition site may be employed by many different groups of microorganisms.

A nitric oxide-binding heterodimeric cytochrome c complex from the anammox bacterium Kuenenia stuttgartiensis binds to hydrazine synthase.

Anaerobic ammonium oxidation (anammox) is a microbial process responsible for significant nitrogen loss from the oceans and other ecosystems. The redox reactions at the heart of anammox are catalyzed by large multiheme enzyme complexes that rely on small cytochrome c proteins for electron shuttling. Among the most highly abundant of these cytochromes is a unique heterodimeric complex composed of class I and class II c-type cytochromes called NaxLS, which has distinctive biochemical and spectroscopic properties. Here, we present the 1.7 Å resolution crystal structure of this complex from the anammox organism Kuenenia stuttgartiensis (KsNaxLS). The structure reveals that the heme irons in each subunit exhibit a rare His/Cys ligation, which, as we show by substitution, causes the observed unusual spectral properties. Unlike its individual subunits, the KsNaxLS complex binds nitric oxide (NO) only at the distal heme side, forming 6cNO adducts. This is likely due to steric immobilization of the proximal heme-binding motifs upon complex formation, a finding that may be of functional relevance, because NO is an intermediate in the central anammox metabolism. Pulldown experiments with K. stuttgartiensis cell-free extract showed that the KsNaxLS complex binds specifically to one of the central anammox enzyme complexes, hydrazine synthase, which uses NO as one of its substrates. It is therefore possible that the KsNaxLS complex plays a role in binding the volatile NO to retain it in the cell for transfer to hydrazine synthase. Alternatively, we propose that KsNaxLS may shuttle electrons to this enzyme complex.

Interactions of anaerobic ammonium oxidizers and sulfide-oxidizing bacteria in a substrate-limited model system mimicking the marine environment.

In nature anaerobic ammonium oxidation (anammox) and denitrification processes convert fixed nitrogen to gaseous nitrogen compounds, which are then released to the atmosphere. While anammox bacteria produce N2 from ammonium and nitrite, in the denitrification process nitrate and nitrite are converted to N2 and the greenhouse gas nitrous oxide (N2O). Furthermore, nitrite needed by the anammox bacteria can be supplied by nitrate reduction to nitrite. Consequently, the interplay between nitrogen-transforming microorganisms control the amount of harmless N2 or the greenhouse gas N2O released to the atmosphere. Therefore, it is important to understand the interactions of these microorganisms in the natural environment, where dynamic conditions result in fluctuating substrate concentrations. Here, we studied the interactions between the sulfide-oxidizing denitrifier Sedimenticola selenatireducens and the anammox bacterium Scalindua brodae in a bioreactor mimicking the marine environment by creating sulfide, ammonium and nitrate limitation in distinct operational phases. Through a microbial interaction, Se. selenatireducens reduced nitrate to nitrite, which together with the supplied ammonium was converted to N2 by Sc. Brodae. Using comparative transcriptomics, we determined that Sc. Brodae and Se. selenatireducens had significant responses to ammonium and nitrate limitation, respectively, indicating that the activities of these microorganisms are regulated by different nitrogen compounds.

Complexome analysis of the nitrite-dependent methanotroph Methylomirabilis lanthanidiphila.

The atmospheric concentration of the potent greenhouse gases methane and nitrous oxide (N2O) has increased drastically during the last century. Methylomirabilis bacteria can play an important role in controlling the emission of these two gases from natural ecosystems, by oxidizing methane to CO2 and reducing nitrite to N2 without producing N2O. These bacteria have an anaerobic metabolism, but are proposed to possess an oxygen-dependent pathway for methane activation. Methylomirabilis bacteria reduce nitrite to NO, and are proposed to dismutate NO into O2 and N2 by a putative NO dismutase (NO-D). The O2 produced in the cell can then be used to activate methane by a particulate methane monooxygenase. So far, the metabolic model of Methylomirabilis bacteria was based mainly on (meta)genomics and physiological experiments. Here we applied a complexome profiling approach to determine which of the proposed enzymes are actually expressed in Methylomirabilis lanthanidiphila. To validate the proposed metabolic model, we focused on enzymes involved in respiration, as well as nitrogen and carbon transformation. All complexes suggested to be involved in nitrite-dependent methane oxidation, were identified in M. lanthanidiphila, including the putative NO-D. Furthermore, several complexes involved in nitrate reduction/nitrite oxidation and NO reduction were detected, which likely play a role in detoxification and redox homeostasis. In conclusion, complexome profiling validated the expression and composition of enzymes hypothesized to be involved in the energy, methane and nitrogen metabolism of M. lanthanidiphila, thereby further corroborating their unique metabolism involved in the environmentally relevant process of nitrite-dependent methane oxidation.

Interactions between anaerobic ammonium- and methane-oxidizing microorganisms in a laboratory-scale sequencing batch reactor.

The reject water of anaerobic digestors still contains high levels of methane and ammonium that need to be treated before these effluents can be discharged to surface waters. Simultaneous anaerobic methane and ammonium oxidation performed by nitrate/nitrite-dependent anaerobic methane-oxidizing(N-damo) microorganisms and anaerobic ammonium-oxidizing(anammox) bacteria is considered a potential solution to this challenge. Here, a stable coculture of N-damo archaea, N-damo bacteria, and anammox bacteria was obtained in a sequencing batch reactor fed with methane, ammonium, and nitrite. Nitrite and ammonium removal rates of up to 455 mg N-NO2- L-1 day-1 and 228 mg N-NH4+ L-1 were reached. All nitrate produced by anammox bacteria (57 mg N-NO3- L-1 day-1) was consumed, leading to a nitrogen removal efficiency of 97.5%. In the nitrite and ammonium limited state, N-damo and anammox bacteria each constituted about 30-40% of the culture and were separated as granules and flocs in later stages of the reactor operation. The N-damo archaea increased up to 20% and mainly resided in the granular biomass with their N-damo bacterial counterparts. About 70% of the nitrite in the reactor was removed via the anammox process, and batch assays confirmed that anammox activity in the reactor was close to its maximal potential activity. In contrast, activity of N-damo bacteria was much higher in batch, indicating that these bacteria were performing suboptimally in the sequencing batch reactor, and would probably be outcompeted by anammox bacteria if ammonium was supplied in excess. Together these results indicate that the combination of N-damo and anammox can be implemented for the removal of methane at the expense of nitrite and nitrate in future wastewater treatment systems.

A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway.

The hydroxylamine oxidoreductase/hydrazine dehydrogenase (HAO/HDH) protein family constitutes an important group of octaheme cytochromes c (OCCs). The majority of these proteins form homotrimers, with their subunits being covalently attached to each other via a rare cross-link between the catalytic heme moiety and a conserved tyrosine residue in an adjacent subunit. This covalent cross-link has been proposed to modulate the active-site heme towards oxidative catalysis by distorting the heme plane. In this study, the crystal structure of a stable complex of an HAO homologue (KsHAOr) with its diheme cytochrome c redox partner (KsDH) from the anammox bacterium Kuenenia stuttgartiensis was determined. KsHAOr lacks the tyrosine cross-link and is therefore tuned to reductive catalysis. The molecular model of the KsHAOr-KsDH complex at 2.6 Šresolution shows a heterododecameric (α6ß6) assembly, which was also shown to be the oligomeric state in solution by analytical ultracentrifugation and multi-angle static light scattering. The 60-heme-containing protein complex reveals a unique extended electron transfer pathway and provides deeper insights into catalysis and electron transfer in reductive OCCs.

Publisher Correction: Nitric oxide-dependent anaerobic ammonium oxidation.

The original HTML version of this Article had an incorrect Published online date of 20 March 2019; it should have been 18 March 2019. This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.

Nitric oxide-dependent anaerobic ammonium oxidation.

Nitric oxide (NO) has important functions in biology and atmospheric chemistry as a toxin, signaling molecule, ozone depleting agent and the precursor of the greenhouse gas nitrous oxide (N2O). Although NO is a potent oxidant, and was available on Earth earlier than oxygen, it is unclear whether NO can be used by microorganisms for growth. Anaerobic ammonium-oxidizing (anammox) bacteria couple nitrite reduction to ammonium oxidation with NO and hydrazine as intermediates, and produce N2 and nitrate. Here, we show that the anammox bacterium Kuenenia stuttgartiensis is able to grow in the absence of nitrite by coupling ammonium oxidation to NO reduction, and produce only N2. Under these growth conditions, the transcription of proteins necessary for NO generation is downregulated. Our work has potential implications in the control of N2O and NO emissions from natural and manmade ecosystems, where anammox bacteria contribute significantly to N2 release to the atmosphere. We hypothesize that microbial NO-dependent ammonium oxidation may have existed on early Earth.

Key Physiology of a Nitrite-Dependent Methane-Oxidizing Enrichment Culture.

Nitrite-dependent methane-oxidizing bacteria couple the reduction of nitrite to the oxidation of methane via a unique oxygen-producing pathway. This process is carried out by members of the genus Methylomirabilis that belong to the NC10 phylum. Contrary to other known anaerobic methane oxidizers, they do not employ the reverse methanogenesis pathway for methane activation but instead a canonical particulate methane monooxygenase similar to those used by aerobic methanotrophs. Methylomirabilis-like bacteria are detected in many natural and manmade ecosystems, but their physiology is not well understood. Here, using continuous cultivation techniques, batch activity assays, and state-of-the-art membrane-inlet mass spectrometry, we determined growth rate, doubling time, and methane and nitrite affinities of the nitrite-dependent methane-oxidizing bacterium "Candidatus Methylomirabilis lanthanidiphila." Our results provide insight into understanding the interactions of these microorganisms with methanotrophs and other nitrite-reducing microorganisms, such as anaerobic ammonium-oxidizing bacteria. Furthermore, our data can be used in modeling studies as well as wastewater treatment plant design.IMPORTANCE Methane is an important greenhouse gas with a radiative forcing 28 times that of carbon dioxide over a 100-year time scale. The emission of methane to the atmosphere is controlled by aerobic and anaerobic methanotrophs, which are microorganisms that are able to oxidize methane to conserve energy. While aerobic methanotrophs have been studied for over a century, knowledge on the physiological characteristics of anaerobic methanotrophs is scarce. Here, we describe kinetic properties of "Candidatus Methylomirabilis lanthanidiphila," a nitrite-dependent methane-oxidizing microorganism, which is ecologically important and can be applied in wastewater treatment.