Versatile methanotrophs form an active methane biofilter in the oxycline of a seasonally stratified coastal basin.

The potential and drivers of microbial methane removal in the water column of seasonally stratified coastal ecosystems and the importance of the methanotrophic community composition for ecosystem functioning are not well explored. Here, we combined depth profiles of oxygen and methane with 16S rRNA gene amplicon sequencing, metagenomics and methane oxidation rates at discrete depths in a stratified coastal marine system (Lake Grevelingen, The Netherlands). Three amplicon sequence variants (ASVs) belonging to different genera of aerobic Methylomonadaceae and the corresponding three methanotrophic metagenome-assembled genomes (MOB-MAGs) were retrieved by 16S rRNA sequencing and metagenomic analysis, respectively. The abundances of the different methanotrophic ASVs and MOB-MAGs peaked at different depths along the methane oxygen counter-gradient and the MOB-MAGs show a quite diverse genomic potential regarding oxygen metabolism, partial denitrification and sulphur metabolism. Moreover, potential aerobic methane oxidation rates indicated high methanotrophic activity throughout the methane oxygen counter-gradient, even at depths with low in situ methane or oxygen concentration. This suggests that niche-partitioning with high genomic versatility of the present Methylomonadaceae might contribute to the functional resilience of the methanotrophic community and ultimately the efficiency of methane removal in the stratified water column of a marine basin.

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.

Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping.

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus 'Kuenenia stuttgartiensis' using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor's side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

Nutrient Limitation Causes Differential Expression of Transport- and Metabolism Genes in the Compartmentalized Anammox Bacterium Kuenenia stuttgartiensis.

Anaerobic ammonium-oxidizing (anammox) bacteria, members of the "Candidatus Brocadiaceae" family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-enclosed compartment called the anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic-, and anammoxosome membranes to enter or exit the anammoxosome. The genomes of all anammox species harbor multiple copies of ammonium-, nitrite-, and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite-, and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells showed that four of the seven ammonium transporter homologs and two of the nine nitrite transporter homologs were significantly upregulated during ammonium-limited growth, while another ammonium transporter- and four nitrite transporter homologs were upregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized anammox cell.

Methanol Production by "Methylacidiphilum fumariolicum" SolV under Different Growth Conditions.

Industrial methanol production converts methane from natural gas into methanol through a multistep chemical process. Biological methane-to-methanol conversion under moderate conditions and using biogas would be more environmentally friendly. Methanotrophs, bacteria that use methane as an energy source, convert methane into methanol in a single step catalyzed by the enzyme methane monooxygenase, but inhibition of methanol dehydrogenase, which catalyzes the subsequent conversion of methanol into formaldehyde, is a major challenge. In this study, we used the thermoacidophilic methanotroph "Methylacidiphilum fumariolicum" SolV for biological methanol production. This bacterium possesses a XoxF-type methanol dehydrogenase that is dependent on rare earth elements for activity. By using a cultivation medium nearly devoid of lanthanides, we reduced methanol dehydrogenase activity and obtained a continuous methanol-producing microbial culture. The methanol production rate and conversion efficiency were growth-rate dependent. A maximal conversion efficiency of 63% mol methanol produced per mol methane consumed was obtained at a relatively high growth rate, with a methanol production rate of 0.88 mmol/g (dry weight)/h. This study demonstrates that methanotrophs can be used for continuous methanol production. Full-scale application will require additional increases in the titer, production rate, and efficiency, which can be achieved by further decreasing the lanthanide concentration through the use of increased biomass concentrations and novel reactor designs to supply sufficient gases, including methane, oxygen, and hydrogen.IMPORTANCE The production of methanol, an important chemical, is completely dependent on natural gas. The current multistep chemical process uses high temperature and pressure to convert methane in natural gas to methanol. In this study, we used the methanotroph "Methylacidiphilum fumariolicum" SolV to achieve continuous methanol production from methane as the substrate. The production rate was highly dependent on the growth rate of this microorganism, and high conversion efficiencies were obtained. Using microorganisms for the production of methanol might enable the use of more sustainable sources of methane, such as biogas, rather than natural gas.