Hydroxylamine production by Alcaligenes faecalis challenges the paradigm of heterotrophic nitrification.

Heterotrophic nitrifiers continue to be a hiatus in our understanding of the nitrogen cycle. Despite their discovery over 50 years ago, the physiology and environmental role of this enigmatic group remain elusive. The current theory is that heterotrophic nitrifiers are capable of converting ammonia to hydroxylamine, nitrite, nitric oxide, nitrous oxide, and dinitrogen gas via the subsequent actions of nitrification and denitrification. In addition, it was recently suggested that dinitrogen gas may be formed directly from ammonium. Here, we combine complementary high-resolution gas profiles, 15N isotope labeling studies, and transcriptomics data to show that hydroxylamine is the major product of nitrification in Alcaligenes faecalis. We demonstrated that denitrification and direct ammonium oxidation to dinitrogen gas did not occur under the conditions tested. Our results indicate that A. faecalis is capable of hydroxylamine production from an organic intermediate. These results fundamentally change our understanding of heterotrophic nitrification and have important implications for its biotechnological application.

Feeding strategy and feed protein level affect the gut microbiota of common carp (Cyprinus carpio).

Common carp (Cyprinus carpio) were fed food with different protein concentrations following different feeding regimes, which were previously shown to affect growth, nitrogen excretion and amino acid catabolism. 16S rRNA gene amplicon sequencing was performed to investigate the gut microbiota of these fish. Lower dietary protein content increased microbial richness, while the combination of demand feeding and dietary protein content affected the composition of the gut microbiota. Hepatic glutamate dehydrogenase (GDH) activity was correlated to the composition of the gut microbiota in all dietary treatments. We found that demand-fed carp fed a diet containing 39% protein had a significantly higher abundance of Beijerinckiaceae compared to other dietary groups. Network analysis identified this family and two Rhizobiales families as hubs in the microbial association network. In demand-fed carp, the microbial association network had significantly fewer connections than in batch-fed carp. In contrast to the large effects of the feeding regime and protein content of the food on growth and nitrogen metabolism, it had only limited effects on gut microbiota composition. However, correlations between gut microbiota composition and liver GDH activity showed that host physiology and gut microbiota are connected, which warrants functional studies into the role of the gut microbiota in fish physiology.

Influence of filter age on Fe, Mn and NH4+ removal in dual media rapid sand filters used for drinking water production.

Rapid sand filtration is a common method for removal of iron (Fe), manganese (Mn) and ammonium (NH4+) from anoxic groundwaters used for drinking water production. In this study, we combine geochemical and microbiological data to assess how filter age influences Fe, Mn and NH4+ removal in dual media filters, consisting of anthracite overlying quartz sand, that have been in operation for between ∼2 months and ∼11 years. We show that the depth where dissolved Fe and Mn removal occurs is reflected in the filter medium coatings, with ferrihydrite forming in the anthracite in the top of the filters (< 1 m), while birnessite-type Mn oxides are mostly formed in the sand (> 1 m). Removal of NH4+ occurs through nitrification in both the anthracite and sand and is the key driver of oxygen loss. Removal of Fe is independent of filter age and is always efficient (> 97% removal). In contrast, for Mn, the removal efficiency varies with filter age, ranging from 9 to 28% at ∼2-3 months after filter replacement to 100% after 8 months. After 11 years, removal reduces to 60-80%. The lack of Mn removal in the youngest filters (at 2-3 months) is likely the result of a relatively low abundance of mineral coatings that adsorb Mn2+ and provide surfaces for the establishment of a microbial community. 16S rRNA gene amplicon sequencing shows that Gallionella, which are known Fe2+ oxidizers, are present after 2 months, yet Fe2+ removal is mostly chemical. Efficient NH4+ removal (> 90%) establishes within 3 months of operation but leakage occurs upon high NH4+loading (> 160 µM). Two-step nitrification by Nitrosomonas and Candidatus Nitrotoga is likely the most important NH4+ removal mechanism in younger filters during ripening (2 months), after which complete ammonia oxidation by Nitrospira and canonical two-step nitrification occur simultaneously in older filters. Our results highlight the strong effect of filter age on especially Mn2+but also NH4+ removal. We show that ageing of filter medium leads to the development of thick coatings, which we hypothesize leads to preferential flow, and breakthrough of Mn2+. Use of age-specific flow rates may increase the contact time with the filter medium in older filters and improve Mn2+ and NH4+ removal.

Comparison of the gill and gut microbiomes of common carp (Cyprinus carpio) and zebrafish (Danio rerio) and their RAS environment.

Recirculating aquaculture systems (RAS) are increasingly being used to grow fish, as intensive water reuse reduces water consumption and environmental impact. RAS use biofilters containing nitrogen-cycling microorganisms that remove ammonia from the aquaculture water. Knowledge of how RAS microbial communities relate to the fish-associated microbiome is limited, as is knowledge of fish-associated microbiota in general. Recently, nitrogen-cycling bacteria have been discovered in zebrafish and carp gills and shown to detoxify ammonia in a manner similar to the RAS biofilter. Here, we compared RAS water and biofilter microbiomes with fish-associated gut and gill microbial communities in laboratory RAS housing either zebrafish (Danio rerio) or common carp (Cyprinus carpio) using 16S rRNA gene amplicon sequencing. The phylogeny of ammonia-oxidizing bacteria in the gills and the RAS environment was investigated in more detail by phylogenetic analysis of the ammonia monooxygenase subunit A (amoA). The location from which the microbiome was sampled (RAS compartments and gills or gut) had a stronger effect on community composition than the fish species, but species-specific differences were also observed. We found that carp- and zebrafish-associated microbiomes were highly distinct from their respective RAS microbiomes, characterized by lower overall diversity and a small core microbiome consisting of taxa specifically adapted to the respective organ. The gill microbiome was also defined by a high proportion of unique taxa. Finally, we found that amoA sequences from the gills were distinct from those from the RAS biofilter and water. Our results showed that the gut and gill microbiomes of carp and zebrafish share a common and species-specific core microbiome that is distinct from the microbially-rich RAS environment.

Effects of demand-feeding and dietary protein level on nitrogen metabolism and symbiont dinitrogen gas production of common carp (Cyprinus carpio, L.).

Ammonia accumulation is a major challenge in intensive aquaculture, where fish are fed protein-rich diets in large rations, resulting in increased ammonia production when amino acids are metabolized as energy source. Ammonia is primarily excreted via the gills, which have been found to harbor nitrogen-cycle bacteria that convert ammonia into dinitrogen gas (N2) and therefore present a potential in situ detoxifying mechanism. Here, we determined the impact of feeding strategies (demand-feeding and batch-feeding) with two dietary protein levels on growth, nitrogen excretion, and nitrogen metabolism in common carp (Cyprinus carpio, L.) in a 3-week feeding experiment. Demand-fed fish exhibited significantly higher growth rates, though with lower feed efficiency. When corrected for feed intake, nitrogen excretion was not impacted by feeding strategy or dietary protein, but demand-fed fish had significantly more nitrogen unaccounted for in the nitrogen balance and less retained nitrogen. N2 production of individual fish was measured in all experimental groups, and production rates were in the same order of magnitude as the amount of nitrogen unaccounted for, thus potentially explaining the missing nitrogen in the balance. N2 production by carp was also observed when groups of fish were kept in metabolic chambers. Demand feeding furthermore caused a significant increase in hepatic glutamate dehydrogenase activities, indicating elevated ammonia production. However, branchial ammonia transporter expression levels in these animals were stable or decreased. Together, our results suggest that feeding strategy impacts fish growth and nitrogen metabolism, and that conversion of ammonia to N2 by nitrogen cycle bacteria in the gills may explain the unaccounted nitrogen in the balance.

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria.

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.

A Novel Laboratory-Scale Mesocosm Setup to Study Methane Emission Mitigation by Sphagnum Mosses and Associated Methanotrophs.

Degraded peatlands are often rewetted to prevent oxidation of the peat, which reduces CO2 emission. However, the created anoxic conditions will boost methane (CH4) production and thus emission. Here, we show that submerged Sphagnum peat mosses in rewetted-submerged peatlands can reduce CH4 emission from peatlands with 93%. We were able to mimic the field situation in the laboratory by using a novel mesocosm set-up. By combining these with 16S rRNA gene amplicon sequencing and qPCR analysis of the pmoA and mmoX genes, we showed that submerged Sphagnum mosses act as a niche for CH4 oxidizing bacteria. The tight association between Sphagnum peat mosses and methane oxidizing bacteria (MOB) significantly reduces CH4 emissions by peatlands and can be studied in more detail in the mesocosm setup developed in this study.

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.

Ammonia oxidation at pH 2.5 by a new gammaproteobacterial ammonia-oxidizing bacterium.

Ammonia oxidation was considered impossible under highly acidic conditions, as the protonation of ammonia leads to decreased substrate availability and formation of toxic nitrogenous compounds. Recently, some studies described archaeal and bacterial ammonia oxidizers growing at pH as low as 4, while environmental studies observed nitrification at even lower pH values. In this work, we report on the discovery, cultivation, and physiological, genomic, and transcriptomic characterization of a novel gammaproteobacterial ammonia-oxidizing bacterium enriched via continuous bioreactor cultivation from an acidic air biofilter that was able to grow and oxidize ammonia at pH 2.5. This microorganism has a chemolithoautotrophic lifestyle, using ammonia as energy source. The observed growth rate on ammonia was 0.196 day-1, with a doubling time of 3.5 days. The strain also displayed ureolytic activity and cultivation with urea as ammonia source resulted in a growth rate of 0.104 day-1 and a doubling time of 6.7 days. A high ammonia affinity (Km(app) = 147 ± 14 nM) and high tolerance to toxic nitric oxide could represent an adaptation to acidic environments. Electron microscopic analysis showed coccoid cell morphology with a large amount of intracytoplasmic membrane stacks, typical of gammaproteobacterial ammonia oxidizers. Furthermore, genome and transcriptome analysis showed the presence and expression of diagnostic genes for nitrifiers (amoCAB, hao, nor, ure, cbbLS), but no nirK was identified. Phylogenetic analysis revealed that this strain belonged to a novel bacterial genus, for which we propose the name "Candidatus Nitrosacidococcus tergens" sp. RJ19.

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification.

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named "Candidatus Nitrospira kreftii", and performed a detailed genomic and physiological characterization. The complete genome of "Ca. N. kreftii" allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (Km(app)_NO2- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of "Ca. N. kreftii" indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.

Metagenomic profiling of ammonia- and methane-oxidizing microorganisms in two sequential rapid sand filters.

Elevated concentrations of ammonium and methane in groundwater are often associated with microbiological, chemical and sanitary problems during drinking water production and distribution. To avoid their accumulation, raw water in the Netherlands and many other countries is purified by sand filtration. These drinking water filtration systems select for microbial communities that mediate the biodegradation of organic and inorganic compounds. In this study, the top layers and wall biofilm of a Dutch drinking water treatment plant (DWTP) were sampled from the filtration units of the plant over three years. We used high-throughput sequencing in combination with differential coverage and sequence composition-based binning to recover 56 near-complete metagenome-assembled genomes (MAGs) with an estimated completion of ≥70% and with ≤10% redundancy. These MAGs were used to characterize the microbial communities involved in the conversion of ammonia and methane. The methanotrophic microbial communities colonizing the wall biofilm (WB) and the granular material of the primary rapid sand filter (P-RSF) were dominated by members of the Methylococcaceae and Methylophilaceae. The abundance of these bacteria drastically decreased in the secondary rapid sand filter (S-RSF) samples. In all samples, complete ammonia-oxidizing (comammox) Nitrospira were the most abundant nitrifying guild. Clade A comammox Nitrospira dominated the P-RSF, while clade B was most abundant in WB and S-RSF, where ammonium concentrations were much lower. In conclusion, the knowledge obtained in this study contributes to understanding the role of microorganisms in the removal of carbon and nitrogen compounds during drinking water production. We furthermore found that drinking water treatment plants represent valuable model systems to study microbial community function and interaction.

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.

Microbial nitrogen fixation and methane oxidation are strongly enhanced by light in Sphagnum mosses.

Peatlands have acted as C-sinks for millennia, storing large amounts of carbon, of which a significant amount is yearly released as methane (CH4). Sphagnum mosses are a key genus in many peat ecosystems and these mosses live in close association with methane-oxidizing and nitrogen-fixing microorganisms. To disentangle mechanisms which may control Sphagnum-associated methane-oxidation and nitrogen-fixation, we applied four treatments to Sphagnum mosses from a pristine peatland in Finland: nitrogen fertilization, phosphorus fertilization, CH4 addition and light. N and P fertilization resulted in nutrient accumulation in the moss tissue, but did not increase Sphagnum growth. While net CO2 fixation rates remained unaffected in the N and P treatment, net CH4 emissions decreased because of enhanced CH4 oxidation. CH4 addition did not affect Sphagnum performance in the present set-up. Light, however, clearly stimulated the activity of associated nitrogen-fixing and methane-oxidizing microorganisms, increasing N2 fixation rates threefold and CH4 oxidation rates fivefold. This underlines the strong connection between Sphagnum and associated N2 fixation and CH4 oxidation. It furthermore indicates that phototrophy is a strong control of microbial activity, which can be directly or indirectly.

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.

Complete Genome Sequence of the Aerobic Facultative Methanotroph Methylocella tundrae Strain T4.

Methylocella tundrae T4T is a facultative aerobic methanotroph which was isolated from an acidic tundra wetland and possesses only a soluble methane monooxygenase. The complete genome, which includes two megaplasmids, was sequenced using a combination of Illumina and Nanopore technologies. One of the megaplasmids carries a propane monooxygenase gene cluster.

Metagenomic recovery of two distinct comammox Nitrospira from the terrestrial subsurface.

The recently discovered comammox process encompasses both nitrification steps, the aerobic oxidation of ammonia and nitrite, in a single organism. All known comammox bacteria are affiliated with Nitrospira sublineage II and can be grouped into two distinct clades, referred to as A and B, based on ammonia monooxygenase phylogeny. In this study, we report high-quality draft genomes of two novel comammox Nitrospira from the terrestrial subsurface, representing one clade A and one clade B comammox organism. The two metagenome-assembled genomes were compared with other representatives of Nitrospira sublineage II, including both canonical and comammox Nitrospira. Phylogenomic analyses confirmed the affiliation of the two novel Nitrospira with comammox clades A and B respectively. Based on phylogenetic distance and pairwise average nucleotide identity values, both comammox Nitrospira were classified as novel species. Genomic comparison revealed high conservation of key metabolic features in sublineage II Nitrospira, including respiratory complexes I-V and the machineries for nitrite oxidation and carbon fixation via the reductive tricarboxylic acid cycle. In addition, the presence of the enzymatic repertoire for formate and hydrogen oxidation in the Rifle clades A and B comammox genomes, respectively, suggest a broader distribution of these metabolic features than previously anticipated.

In Situ Quantification of Biological N2 Production Using Naturally Occurring 15N15N.

We describe an approach for determining biological N2 production in soils based on the proportions of naturally occurring 15N15N in N2. Laboratory incubation experiments reveal that biological N2 production, whether by denitrification or anaerobic ammonia oxidation, yields proportions of 15N15N in N2 that are within 1‰ of that predicted for a random distribution of 15N and 14N atoms. This relatively invariant isotopic signature contrasts with that of the atmosphere, which has 15N15N proportions in excess of the random distribution by 19.1 ± 0.1‰. Depth profiles of gases in agricultural soils from the Kellogg Biological Station Long-Term Ecological Research site show biological N2 accumulation that accounts for up to 1.6% of the soil N2. One-dimensional reaction-diffusion modeling of these soil profiles suggests that subsurface N2 pulses leading to surface emission rates as low as 0.3 mmol N2 m-2 d-1 can be detected with current analytical precision, decoupled from N2O production.

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.

Complete nitrification: insights into the ecophysiology of comammox Nitrospira.

Nitrification, the oxidation of ammonia via nitrite to nitrate, has been considered to be a stepwise process mediated by two distinct functional groups of microorganisms. The identification of complete nitrifying Nitrospira challenged not only the paradigm of labor division in nitrification, it also raises fundamental questions regarding the environmental distribution, diversity, and ecological significance of complete nitrifiers compared to canonical nitrifying microorganisms. Recent genomic and physiological surveys identified factors controlling their ecology and niche specialization, which thus potentially regulate abundances and population dynamics of the different nitrifying guilds. This review summarizes the recently obtained insights into metabolic differences of the known nitrifiers and discusses these in light of potential functional adaptation and niche differentiation between canonical and complete nitrifiers.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.