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Bacteria of the phylum Planctomycetota have received much attention over the years due to their unique cell biology and potential for biotechnological application. Within the phylum, bacteria of the class Phycisphaerae have been found in a multitude of environmental datasets. However, only a few species have been brought into culture so far and even enrichments are scarce. Therefore, very little is known about their lifestyle, which has hindered efforts to estimate their environmental relevance. Here, we analysed all medium- and high-quality Phycisphaerae genomes represented in the genome taxonomy database to learn more about their physiology. We combined automatic and manual annotation efforts to provide a bird's eye view of their diverse energy metabolisms. Contrasting previous reports, we did not find indications for the presence of genes for anaerobic ammonium oxidation in any Phycisphaerae genome. Instead, we found that many members of this class are adapted to a facultative anaerobic or strictly fermentative lifestyle and may be specialized in the breakdown of carbon compounds produced by other organisms. Based on these findings, we provide a practical overview of organic carbon substrates predicted to be utilized by Phycisphaerae families., (© 2024. The Author(s).)

Recently, an activity-based labelling protocol for the in vivo detection of ammonia- and alkane-oxidizing bacteria became available. This functional tagging technique enabled targeted studies of these environmentally widespread functional groups, but it failed to capture ammonia-oxidizing archaea (AOA). Since their first discovery, AOA have emerged as key players within the biogeochemical nitrogen cycle, but our knowledge regarding their distribution and abundance in natural and engineered ecosystems is mainly derived from PCR-based and metagenomic studies. Furthermore, the archaeal ammonia monooxygenase is distinctly different from its bacterial counterparts and remains poorly understood. Here, we report on the development of an activity-based labelling protocol for the fluorescent detection of all ammonia- and alkane-oxidizing prokaryotes, including AOA. In this protocol, 1,5-hexadiyne is used as inhibitor of ammonia and alkane oxidation and as bifunctional enzyme probe for the fluorescent labelling of cells via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction. Besides efficient activity-based labelling of ammonia- and alkane-oxidizing microorganisms, this method can also be employed in combination with deconvolution microscopy for determining the subcellular localization of their ammonia- and alkane-oxidizing enzyme systems. Labelling of these enzymes in diverse ammonia- and alkane-oxidizing microorganisms allowed their visualization on the cytoplasmic membranes, the intracytoplasmic membrane stacks of ammonia- and methane-oxidizing bacteria, and, fascinatingly, on vesicle-like structures in one AOA species. The development of this novel activity-based labelling method for ammonia- and alkane-oxidizers will be a valuable addition to the expanding molecular toolbox available for research of nitrifying and alkane-oxidizing microorganisms., Competing Interests: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The Aeolian archipelago is known worldwide for its volcanic activity and hydrothermal emissions, of mainly carbon dioxide and hydrogen sulfide. Hydrogen, methane, and carbon monoxide are minor components of these emissions which together can feed large quantities of bacteria and archaea that do contribute to the removal of these notorious greenhouse gases. Here we analyzed the metagenome of samples taken from the Levante bay on Vulcano Island, Italy. Using a gene-centric approach, the hydrothermal vent community appeared to be dominated by Proteobacteria, and Sulfurimonas was the most abundant genus. Metabolic reconstructions highlight a prominent role of formaldehyde oxidation and the reverse TCA cycle in carbon fixation. [NiFe]-hydrogenases seemed to constitute the preferred strategy to oxidize H 2 , indicating that besides H 2 S, H 2 could be an essential electron donor in this system. Moreover, the sulfur cycle analysis showed a high abundance and diversity of sulfate reduction genes underpinning the H 2 S production. This study covers the diversity and metabolic potential of the microbial soil community in Levante bay and adds to our understanding of the biogeochemistry of volcanic ecosystems., (© 2024. The Author(s).)

Coastal zones account for 75% of marine methane emissions, despite covering only 15% of the ocean surface area. In these ecosystems, the tight balance between methane production and oxidation in sediments prevents most methane from escaping into seawater. However, anthropogenic activities could disrupt this balance, leading to an increased methane escape from coastal sediments. To quantify and unravel potential mechanisms underlying this disruption, we used a suite of biogeochemical and microbiological analyses to investigate the impact of anthropogenically induced redox shifts on methane cycling in sediments from three sites with contrasting bottom water redox conditions (oxic-hypoxic-euxinic) in the eutrophic Stockholm Archipelago. Our results indicate that the methane production potential increased under hypoxia and euxinia, while anaerobic oxidation of methane was disrupted under euxinia. Experimental, genomic, and biogeochemical data suggest that the virtual disappearance of methane-oxidizing archaea at the euxinic site occurred due to sulfide toxicity. This could explain a near 7-fold increase in the extent of escape of benthic methane at the euxinic site relative to the hypoxic one. In conclusion, these insights reveal how the development of euxinia could disrupt the coastal methane biofilter, potentially leading to increased methane emissions from coastal zones.

Coastal environments are a major source of marine methane in the atmosphere. Eutrophication and deoxygenation have the potential to amplify the coastal methane emissions. Here, we investigate methane dynamics in the eutrophic Stockholm Archipelago. We cover a range of sites with contrasting water column redox conditions and rates of organic matter degradation, with the latter reflected by the depth of the sulfate-methane transition zone (SMTZ) in the sediment. We find the highest benthic release of methane (2.2-8.6 mmol m -2 d -1 ) at sites where the SMTZ is located close to the sediment-water interface (2-10 cm). A large proportion of methane is removed in the water column via aerobic or anaerobic microbial pathways. At many locations, water column methane is highly depleted in 13 C, pointing toward substantial bubble dissolution. Calculated and measured rates of methane release to the atmosphere range from 0.03 to 0.4 mmol m -2 d -1 and from 0.1 to 1.7 mmol m -2 d -1 , respectively, with the highest fluxes at locations with a shallow SMTZ and anoxic and sulfidic bottom waters. Taken together, our results show that sites suffering most from both eutrophication and deoxygenation are hotspots of coastal marine methane emissions.

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, 15 N 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.

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., (© 2024 The Authors. Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Global riverine nitrous oxide (N 2 O) emissions have increased more than 4-fold in the last century. It has been estimated that the hyporheic zones in small streams alone may contribute approximately 85% of these N 2 O emissions. However, the mechanisms and pathways controlling hyporheic N 2 O production in stream ecosystems remain unknown. Here, we report that ammonia-derived pathways, rather than the nitrate-derived pathways, are the dominant hyporheic N 2 O sources (69.6 ± 2.1%) in agricultural streams around the world. The N 2 O fluxes are mainly in positive correlation with ammonia. The potential N 2 O metabolic pathways of metagenome-assembled genomes (MAGs) provides evidence that nitrifying bacteria contain greater abundances of N 2 O production-related genes than denitrifying bacteria. Taken together, this study highlights the importance of mitigating agriculturally derived ammonium in low-order agricultural streams in controlling N 2 O emissions. Global models of riverine ecosystems need to better represent ammonia-derived pathways for accurately estimating and predicting riverine N 2 O emissions., (© 2024. The Author(s).)

Aquatic ecosystems are large contributors to global methane (CH4) emissions. Eutrophication significantly enhances CH4-production as it stimulates methanogenesis. Mitigation measures aimed at reducing eutrophication, such as the addition of metal salts to immobilize phosphate (PO43-), are now common practice. However, the effects of such remedies on methanogenic and methanotrophic communities-and therefore on CH4-cycling-remain largely unexplored. Here, we demonstrate that Fe(II)Cl2 addition, used as PO43- binder, differentially affected microbial CH4 cycling-processes in field experiments and batch incubations. In the field experiments, carried out in enclosures in a eutrophic pond, Fe(II)Cl2 application lowered in-situ CH4 emissions by lowering net CH4-production, while sediment aerobic CH4-oxidation rates-as found in batch incubations of sediment from the enclosures-did not differ from control. In Fe(II)Cl2-treated sediments, a decrease in net CH4-production rates could be attributed to the stimulation of iron-dependent anaerobic CH4-oxidation (Fe-AOM). In batch incubations, anaerobic CH4-oxidation and Fe(II)-production started immediately after CH4 addition, indicating Fe-AOM, likely enabled by favorable indigenous iron cycling conditions and the present methanotroph community in the pond sediment. 16S rRNA sequencing data confirmed the presence of anaerobic CH4-oxidizing archaea and both iron-reducing and iron-oxidizing bacteria in the tested sediments. Thus, besides combatting eutrophication, Fe(II)Cl2 application can mitigate CH4 emissions by reducing microbial net CH4-production and stimulating Fe-AOM., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Methane emissions present a significant environmental challenge in both natural and engineered aquatic environments. Denitrifying anaerobic methane oxidation (N-DAMO) has the potential for application in wastewater treatment plants. However, our understanding of the N-DAMO process is primarily based on studies conducted on environmental samples or enrichment cultures using metagenomic approaches. To gain deeper insights into N-DAMO, we used antimicrobial compounds to study the function and physiology of ' Candidatus Methanoperedens nitroreducens' and ' Candidatus Methylomirabilis oxyfera' in N-DAMO enrichment cultures. We explored the effects of inhibitors and antibiotics and investigated the potential application of N-DAMO in wastewater contaminated with ammonium and heavy metals. Our results showed that ' Ca. M. nitroreducens' was susceptible to puromycin and 2-bromoethanesulfonate, while the novel methanogen inhibitor 3-nitrooxypropanol had no effect on N-DAMO. Furthermore, ' Ca. M. oxyfera' was shown to be susceptible to the particulate methane monooxygenase inhibitor 1,7-octadiyne and a bacteria-suppressing antibiotic cocktail. The N-DAMO activity was not affected by ammonium concentrations below 10 mM. Finally, the N-DAMO community appeared to be remarkably resistant to lead (Pb) but susceptible to nickel (Ni) and cadmium (Cd). This study provides insights into microbial functions in N-DAMO communities, facilitating further investigation of their application in methanogenic, nitrogen-polluted water systems.

Anaerobic methanotrophic (ANME) archaea are environmentally important, uncultivated microorganisms that oxidize the potent greenhouse gas methane. During methane oxidation, ANME archaea engage in extracellular electron transfer (EET) with other microbes, metal oxides, and electrodes through unclear mechanisms. Here, we cultivate ANME-2d archaea ('Ca. Methanoperedens') in bioelectrochemical systems and observe strong methane-dependent current (91-93% of total current) associated with high enrichment of 'Ca. Methanoperedens' on the anode (up to 82% of the community), as determined by metagenomics and transmission electron microscopy. Electrochemical and metatranscriptomic analyses suggest that the EET mechanism is similar at various electrode potentials, with the possible involvement of an uncharacterized short-range electron transport protein complex and OmcZ nanowires., (© 2024. The Author(s).)

In coastal waters, methane-oxidizing bacteria (MOB) can form a methane biofilter and mitigate methane emissions. The metabolism of these MOBs is versatile, and the resilience to changing oxygen concentrations is potentially high. It is still unclear how seasonal changes in oxygen availability and water column chemistry affect the functioning of the methane biofilter and MOB community composition. Here, we determined water column methane and oxygen depth profiles, the methanotrophic community structure, methane oxidation potential, and water-air methane fluxes of a eutrophic marine basin during summer stratification and in the mixed water in spring and autumn. In spring, the MOB diversity and relative abundance were low. Yet, MOB formed a methane biofilter with up to 9% relative abundance and vertical niche partitioning during summer stratification. The vertical distribution and potential methane oxidation of MOB did not follow the upward shift of the oxycline during summer, and water-air fluxes remained below 0.6 mmol m-2 d-1. Together, this suggests active methane removal by MOB in the anoxic water. Surprisingly, with a weaker stratification, and therefore potentially increased oxygen supply, methane oxidation rates decreased, and water-air methane fluxes increased. Thus, despite the potential resilience of the MOB community, seasonal water column dynamics significantly influence methane removal., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Methane-dependent nitrate and nitrite removal in anoxic environments is thought to rely on syntrophy between ANME-2d archaea and bacteria in the genus 'Candidatus Methylomirabilis'. Here we enriched and purified a single Methylomirabilis from paddy soil fed with nitrate and methane, which is capable of coupling methane oxidation to nitrate reduction via nitrite to dinitrogen independently. Isotope labelling showed that this bacterium we name 'Ca. Methylomirabilis sinica' stoichiometrically performed methane-dependent complete nitrate reduction to dinitrogen gas. Multi-omics analyses collectively demonstrated that 'M. sinica' actively expressed a well-established pathway for this process, especially including nitrate reductase Nap. Furthermore, 'M. sinica' exhibited a higher nitrate affinity than most denitrifiers, implying its competitive fitness under oligotrophic nitrogen-limited conditions. Our findings revise the paradigm of methane-dependent denitrification performed by two organisms, and the widespread presence of 'M. sinica' in public databases suggests that the coupling of methane oxidation and complete denitrification in single cells substantially contributes to global methane and nitrogen budgets., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The most abundant known nitrite-oxidizing bacteria in the marine water column belong to the phylum Nitrospinota . Despite their importance in marine nitrogen cycling and primary production, there are only few cultured representatives that all belong to the class Nitrospinia . Moreover, although Nitrospinota were traditionally thought to be restricted to marine environments, metagenome-assembled genomes have also been recovered from groundwater. Over the recent years, metagenomic sequencing has led to the discovery of several novel classes of Nitrospinota (UBA9942, UBA7883, 2-12-FULL-45-22, JACRGO01, JADGAW01), which remain uncultivated and have not been analyzed in detail. Here, we analyzed a nonredundant set of 98 Nitrospinota genomes with focus on these understudied Nitrospinota classes and compared their metabolic profiles to get insights into their potential role in biogeochemical element cycling. Based on phylogenomic analysis and average amino acid identities, the highly diverse phylum Nitrospinota could be divided into at least 33 different genera, partly with quite distinct metabolic capacities. Our analysis shows that not all Nitrospinota are nitrite oxidizers and that members of this phylum have the genomic potential to use sulfide and hydrogen for energy conservation. This study expands our knowledge of the phylogeny and potential ecophysiology of the phylum Nitrospinota and offers new avenues for the isolation and cultivation of these elusive bacteria., Competing Interests: The authors declare that they have no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Climate change-driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12 weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-β-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-β-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Anthropogenic activities are influencing aquatic environments through increased chemical pollution and thus are greatly affecting the biogeochemical cycling of elements. This has increased greenhouse gas emissions, particularly methane, from lakes, wetlands, and canals. Most of the methane produced in anoxic sediments is converted into carbon dioxide by methanotrophs before it reaches the atmosphere. Anaerobic oxidation of methane requires an electron acceptor such as sulphate, nitrate, or metal oxides. Here, we explore the anaerobic methanotrophy in sediments of three urban canals in Amsterdam, covering a gradient from freshwater to brackish conditions. Biogeochemical analysis showed the presence of a shallow sulphate-methane transition zone in sediments of the most brackish canal, suggesting that sulphate could be a relevant electron acceptor for anaerobic methanotrophy in this setting. However, sediment incubations amended with sulphate or iron oxides (ferrihydrite) did not lead to detectable rates of methanotrophy. Despite the presence of known nitrate-dependent anaerobic methanotrophs (Methanoperedenaceae), no nitrate-driven methanotrophy was observed in any of the investigated sediments either. Interestingly, graphene oxide stimulated anaerobic methanotrophy in incubations of brackish canal sediment, possibly catalysed by anaerobic methanotrophs of the ANME-2a/b clade. We propose that natural organic matter serving as electron acceptor drives anaerobic methanotrophy in brackish sediments., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Global urbanization of waterways over the past millennium has influenced microbial communities in these aquatic ecosystems. Increased nutrient inputs have turned most urban waters into net sources of the greenhouse gases carbon dioxide (CO2) and methane (CH4). Here, canal walls of five Dutch cities were studied for their biofilm CH4 oxidation potential, alongside field observations of water chemistry, and CO2 and CH4 emissions. Three cities showed canal wall biofilms with relatively high biological CH4 oxidation potential up to 0.48 mmol gDW-1 d-1, whereas the other two cities showed no oxidation potential. Salinity was identified as the main driver of biofilm bacterial community composition. Crenothrix and Methyloglobulus methanotrophs were observed in CH4-oxidizing biofilms. We show that microbial oxidation in canal biofilms is widespread and is likely driven by the same taxa found across cities with distinctly different canal water chemistry. The oxidation potential of the biofilms was not correlated with the amount of CH4 emitted but was related to the presence or absence of methanotrophs in the biofilms. This was controlled by whether there was enough CH4 present to sustain a methanotrophic community. These results demonstrate that canal wall biofilms can directly contribute to the mitigation of greenhouse gases from urban canals., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Methane is a powerful greenhouse gas that is produced in large quantities in marine sediments. Microbially mediated oxidation of methane in sediments, when in balance with methane production, prevents the release of methane to the overlying water. Here, we present a gene-based reactive transport model that includes both microbial and geochemical dynamics and use it to investigate whether the rate of growth of methane oxidizers in sediments impacts the efficiency of the microbial methane filter. We focus on iron- and methane-rich coastal sediments and, with the model, show that at our site, up to 10% of all methane removed is oxidized by iron and manganese oxides, with the remainder accounted for by oxygen and sulfate. We demonstrate that the slow growth rate of anaerobic methane-oxidizing microbes limits their ability to respond to transient perturbations, resulting in periodic benthic release of methane. Eutrophication and deoxygenation decrease the efficiency of the microbial methane filter further, thereby enhancing the role of coastal environments as a source of methane to the atmosphere.

Organic micropollutants (OMPs) consist of widely used chemicals such as pharmaceuticals and pesticides that can persist in surface and groundwaters at low concentrations (ng/L to μg/L) for a long time. The presence of OMPs in water can disrupt aquatic ecosystems and threaten the quality of drinking water sources. Wastewater treatment plants (WWTPs) rely on microorganisms to remove major nutrients from water, but their effectiveness at removing OMPs varies. Low removal efficiency might be the result of low concentrations, inherent stable chemical structures of OMPs, or suboptimal conditions in WWTPs. In this review, we discuss these factors, with special emphasis on the ongoing adaptation of microorganisms to degrade OMPs. Finally, recommendations are drawn to improve the prediction of OMP removal in WWTPs and to optimize the design of new microbial treatment strategies. OMP removal seems to be concentration-, compound-, and process-dependent, which poses a great complexity to develop accurate prediction models and effective microbial processes targeting all OMPs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Cornelia Welte reports financial support was provided by Nederlandse Organisatie voor Wetenschappelijk Onderzoek Utrecht. Mike Jetten reports financial support was provided by European Research Council. Mike Jetten reports financial support was provided by Nederlandse Organisatie voor Wetenschappelijk Onderzoek Utrecht., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO 2 , polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA ( V max of 8.4 μmol mg -1  min -1 and K m of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA ( V max of 0.4 μmol mg -1  min -1 and K m of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar V max values for both directions ( V max,acetate of 0.9 μmol mg -1  min -1 versus V max,acetyl-CoA of 0.3 μmol mg -1  min -1 ). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13 C 2 -labeled acetate produced 3 μmol of [ 13 C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight] -1 min -1 ). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d., Competing Interests: The authors declare no conflict of interest.

Since the start of the Anthropocene, northern seasonally frozen peatlands have been warming at a rate of 0.6 °C per decade, twice that of the Earth's average rate, thereby triggering increased nitrogen mineralization with subsequent potentially large losses of nitrous oxide (N 2 O) to the atmosphere. Here we provide evidence that seasonally frozen peatlands are important N 2 O emission sources in the Northern Hemisphere and the thawing periods are the hot moment of annual N 2 O emissions. The flux during the hot moment of thawing in spring was 1.20 ± 0.82 mg N 2 O m -2 d -1 , significantly higher than that during the other periods (freezing, -0.12 ± 0.02 mg N 2 O m -2 d -1 ; frozen, 0.04 ± 0.04 mg N 2 O m -2 d -1 ; thawed, 0.09 ± 0.01 mg N 2 O m -2 d -1 ) or observed for other ecosystems at the same latitude in previous studies. The observed emission flux is even higher than those of tropical forests, the World's largest natural terrestrial N 2 O source. Furthermore, based on soil incubation with 15 N and 18 O isotope tracing and differential inhibitors, heterotrophic bacterial and fungal denitrification was revealed as the main source of N 2 O in peatland profiles (0-200 cm). Metagenomic, metatranscriptomic, and qPCR assays further revealed that seasonally frozen peatlands have high N 2 O emission potential, but thawing significantly stimulates expression of genes encoding N 2 O-producing protein complexes (hydroxylamine dehydrogenase (hao) and nitric oxide reductase (nor)), resulting in high N 2 O emissions during spring. This hot moment converts seasonally frozen peatlands into an important N 2 O emission source when it is otherwise a sink. Extrapolation of our data to all northern peatland areas reveals that the hot moment emissions could amount to approximately 0.17 Tg of N 2 O yr -1 . However, these N 2 O emissions are still not routinely included in Earth system models and global IPCC assessments., (© 2023. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Hydrogen sulfide (H 2 S) and methane (CH 4 ) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH 4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H 2 S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH 4 and H 2 S simultaneously at equally high rates. By oxidizing H 2 S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H 2 S on methanotrophy. Strain SolV adapts to increasing H 2 S by expressing a sulfide-insensitive ba 3 -type terminal oxidase and grows as chemolithoautotroph using H 2 S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H 2 S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways., (© 2023. The Author(s).)

Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO 2 and a cheap energy source, such as H 2 , CH 4 , NH 3 , CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO 2 and reduced inorganic gasses and therefore habitats where novel chemolithoautotrophic microorganisms for the synthesis of organic compounds could be discovered. Here we describe " Candidatus Hydrogenisulfobacillus filiaventi" R50 gen. nov., sp. nov., a thermoacidophilic, autotrophic H 2 -oxidizing microorganism, that fixed CO 2 and excreted no less than 0.54 mol organic carbon per mole fixed CO 2 . Extensive metabolomics and NMR analyses revealed that Val, Ala and Ile are the most dominant form of excreted organic carbon while the aromatic amino acids Tyr and Phe, and Glu and Lys were present at much lower concentrations. In addition to these proteinogenic amino acids, the excreted carbon consisted of homoserine lactone, homoserine and an unidentified amino acid. The biological role of the excretion remains uncertain. In the laboratory, we noticed the production under high growth rates (0.034 h -1 , doubling time of 20 h) in combination with O 2 -limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO 2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hogendoorn, Pol, de Graaf, White, Mesman, van Galen, van Alen, Cremers, Jansen, Jetten and Op den Camp.)

Benzimidazole fungicides are frequently detected in aquatic environments and pose a serious health risk. Here, we investigated the metabolic capacity of the recently discovered complete ammonia-oxidizing (comammox) Nitrospira inopinata and kreftii to transform a representative set of benzimidazole fungicides (i.e., benzimidazole, albendazole, carbendazim, fuberidazole, and thiabendazole). Ammonia-oxidizing bacteria and archaea, as well as the canonical nitrite-oxidizing Nitrospira exhibited no or minor biotransformation activity towards all the five benzimidazole fungicides. In contrast, the investigated comammox bacteria actively transformed all the five benzimidazole fungicides, except for thiabendazole. The identified transformation products indicated hydroxylation, S-oxidation, and glycosylation as the major biotransformation pathways of benzimidazole fungicides. We speculated that these reactions were catalyzed by comammox-specific ammonia monooxygenase, cytochrome P450 monooxygenases, and glycosylases, respectively. Interestingly, the exposure to albendazole enhanced the expression of the antibiotic resistance gene acrB of Nitrospira inopinata, suggesting that some benzimidazole fungicides could act as environmental stressors that trigger cellular defense mechanisms. Altogether, this study demonstrated the distinct substrate specificity of comammox bacteria towards benzimidazole fungicides and implies their significant roles in the biotransformation of these fungicides in nitrifying environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Competing interests The authors declare no competing financial interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 (N 2 ) 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. N 2 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. N 2 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 N 2 by nitrogen cycle bacteria in the gills may explain the unaccounted nitrogen in the balance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mes, Kersten, Maas, Eding, Jetten, Siepel, Lücker, Gorissen and Van Kessel.)

The Oslofjord subsea road tunnel is a unique environment in which the typically anoxic marine deep subsurface is exposed to oxygen. Concrete biodeterioration and steel corrosion in the tunnel have been linked to the growth of iron- and manganese-oxidizing biofilms in areas of saline water seepage. Surprisingly, previous 16S rRNA gene surveys of biofilm samples revealed microbial communities dominated by sequences affiliated with nitrogen-cycling microorganisms. This study aimed to identify microbial genomes with metabolic potential for novel nitrogen- and metal-cycling reactions, representing biofilm microorganisms that could link these cycles and play a role in concrete biodeterioration. We reconstructed 33 abundant, novel metagenome-assembled genomes (MAGs) affiliated with the phylum Planctomycetota and the candidate phylum KSB1. We identified novel and unusual genes and gene clusters in these MAGs related to anaerobic ammonium oxidation, nitrite oxidation, and other nitrogen-cycling reactions. Additionally, 26 of 33 MAGs also had the potential for iron, manganese, and arsenite cycling, suggesting that bacteria represented by these genomes might couple these reactions. Our results expand the diversity of microorganisms putatively involved in nitrogen and metal cycling, and contribute to our understanding of potential biofilm impacts on built infrastructure., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Pharmaceuticals are relatively new to nature and often not completely removed in wastewater treatment plants (WWTPs). Consequently, these micropollutants end up in water bodies all around the world posing a great environmental risk. One exception to this recalcitrant conversion is paracetamol, whose full degradation has been linked to several microorganisms. However, the genes and corresponding proteins involved in microbial paracetamol degradation are still elusive. In order to improve our knowledge of the microbial paracetamol degradation pathway, we inoculated a bioreactor with sludge of a hospital WWTP (Pharmafilter, Delft, NL) and fed it with paracetamol as the sole carbon source. Paracetamol was fully degraded without any lag phase and the enriched microbial community was investigated by metagenomic and metatranscriptomic analyses, which demonstrated that the microbial community was very diverse. Dilution and plating on paracetamol-amended agar plates yielded two Pseudomonas sp. isolates: a fast-growing Pseudomonas sp. that degraded 200 mg/L of paracetamol in approximately 10 h while excreting 4-aminophenol, and a slow-growing Pseudomonas sp. that degraded paracetamol without obvious intermediates in more than 90 days. Each Pseudomonas sp. contained a different highly-expressed amidase (31% identity to each other). These amidase genes were not detected in the bioreactor metagenome suggesting that other as-yet uncharacterized amidases may be responsible for the first biodegradation step of paracetamol. Uncharacterized deaminase genes and genes encoding dioxygenase enzymes involved in the catabolism of aromatic compounds and amino acids were the most likely candidates responsible for the degradation of paracetamol intermediates based on their high expression levels in the bioreactor metagenome and the Pseudomonas spp. genomes. Furthermore, cross-feeding between different community members might have occurred to efficiently degrade paracetamol and its intermediates in the bioreactor. This study increases our knowledge about the ongoing microbial evolution towards biodegradation of pharmaceuticals and points to a large diversity of (amidase) enzymes that are likely involved in paracetamol metabolism in WWTPs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier Ltd.)

In the Anthropocene, nitrogen pollution is becoming an increasing challenge for both mankind and the Earth system. Microbial nitrogen cycling begins with aerobic nitrification, which is also the key rate-limiting step. For over a century, it has been accepted that nitrification occurs sequentially involving ammonia oxidation, which produces nitrite followed by nitrite oxidation, generating nitrate. This perception was changed by the discovery of comammox Nitrospira bacteria and their metabolic pathway. In addition, this also provided us with new knowledge concerning the complex nitrogen cycle network. In the comammox process, ammonia can be completely oxidized to nitrate in one cell via the subsequent activity of the enzyme complexes, ammonia monooxygenase, hydroxylamine dehydrogenase, and nitrite oxidodreductase. Over the past five years, research on comammox made great progress. However, there still exist a lot of questions, including how much does comammox contribute to nitrification? How large is the diversity and are there new strains to be discovered? Do comammox bacteria produce the greenhouse gas N 2 O, and how or to which extent may they contribute to global climate change? The above four aspects are of great significance on the farmland nitrogen management, aquatic environment restoration, and mitigation of global climate change. As large number of comammox bacteria and pathways have been detected in various terrestrial and aquatic ecosystems, indicating that the comammox process may exert an important role in the global nitrogen cycle., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Large scale next generation metagenomic sequencing of complex environmental samples paves the way for detailed analysis of nutrient cycles in ecosystems. For such an analysis, large scale unequivocal annotation is a prerequisite, which however is increasingly hampered by growing databases and analysis time. Hereto, we created a hidden Markov model (HMM) database by clustering proteins according to their KEGG indexing. HMM profiles for key genes of specific metabolic pathways and nutrient cycles were organized in subsets to be able to analyze each important elemental cycle separately. An important motivation behind the clustered database was to enable a high degree of resolution for annotation, while decreasing database size and analysis time. Here, we present Metascan, a new tool that can fully annotate and analyze deeply sequenced samples with an average analysis time of 11 min per genome for a publicly available dataset containing 2,537 genomes, and 1.1 min per genome for nutrient cycle analysis of the same sample. Metascan easily detected general proteins like cytochromes and ferredoxins, and additional pmoCAB operons were identified that were overlooked in previous analyses. For a mock community, the BEACON (F1) score was 0.72-0.93 compared to the information in NCBI GenBank. In combination with the accompanying database, Metascan provides a fast and useful annotation and analysis tool, as demonstrated by our proof-of-principle analysis of a complex mock community metagenome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cremers, Jetten, Op den Camp and Lücker.)

Bacteria in the order 'Candidatus Brocadiales' within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome-assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK-like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Anaerobic methanotrophic (ANME) archaea have recently been reported to be capable of using insoluble extracellular electron acceptors via extracellular electron transfer (EET). In this study, we investigated EET by a microbial community dominated by " Candidatus Methanoperedens" archaea at the anode of a bioelectrochemical system (BES) poised at 0 V vs. standard hydrogen electrode (SHE), in this way measuring current as a direct proxy of EET by this community. After inoculation of the BES, the maximum current density was 274 mA m -2 (stable current up to 39 mA m -2 ). Concomitant conversion of 13 CH 4 into 13 CO 2 demonstrated that current production was methane-dependent, with 38% of the current attributed directly to methane supply. Based on the current production and methane uptake in a closed system, the Coulombic efficiency was about 17%. Polarization curves demonstrated that the current was limited by microbial activity at potentials above 0 V. The metatranscriptome of the inoculum was mined for the expression of c -type cytochromes potentially used for EET, which led to the identification of several multiheme c -type cytochrome-encoding genes among the most abundant transcripts in " Ca . Methanoperedens." Our study provides strong indications of EET in ANME archaea and describes a system in which ANME-mediated EET can be investigated under laboratory conditions, which provides new research opportunities for mechanistic studies and possibly the generation of axenic ANME cultures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ouboter, Berben, Berger, Jetten, Sleutels, Ter Heijne and Welte.)

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., (© 2021. The Author(s).)

Methanotrophs aerobically oxidize methane to carbon dioxide to make a living and are known to degrade various other short chain carbon compounds as well. Volatile organic sulfur compounds such as methanethiol (CH 3 SH) are important intermediates in the sulfur cycle. Although volatile organic sulfur compounds co-occur with methane in various environments, little is known about how these compounds affect methanotrophy. The enzyme methanethiol oxidase catalyzing the oxidation of methanethiol has been known for decades, but only recently the mtoX gene encoding this enzyme was identified in a methylotrophic bacterium. The presence of a homologous gene in verrucomicrobial methanotrophs prompted us to examine how methanotrophs cope with methanethiol. Here, we show that the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV consumes methanethiol and produces H 2 S, which is concurrently oxidized. Consumption of methanethiol is required since methanethiol inhibits methane oxidation. Cells incubated with ∼15 μM methanethiol from the start clearly showed inhibition of growth. After depletion of methanethiol, growth resumed within 1 day. Genes encoding a putative methanethiol oxidase were found in a variety of methanotrophs. Therefore, we hypothesize that methanethiol degradation is a widespread detoxification mechanism in methanotrophs in a range of environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schmitz, Mohammadi, van Erven, Berben, Jetten, Pol and Op den Camp.)

Traditional rice cultivation consumes up to 2500 L of water per kg yield and new strategies such as the 'Alternate Wetting and Drying' (AWD) might be promising water-saving alternatives. However, they might have large impacts on the soil microbiology. In this study, we compared the bacterial and archaeal communities in experimental field plots, cultivated under continuously flooding (CF) and AWD management, by high-throughput sequencing of the 16S rRNA gene. We analysed alpha and beta diversity in bulk soil and on plant roots, in plots cultivated with two different rice cultivars. The strongest difference was found between soil and root communities. Beside others, the anaerobic methanotroph Methanoperedens was abundant in soil, however, we detected a considerable number of ANME-2a-2b on plant roots. Furthermore, root communities were significantly affected by the water management: Differential abundance analysis revealed the enrichment of aerobic and potentially plant-growth-promoting bacteria under AWD treatment, such as Sphingomonadaceae and Rhizobiaceae (both Alphaproteobacteria), and Bacteroidetes families. Microorganisms with an overall anaerobic lifestyle, such as various Delta- and Epsilonproteobacteria, and Firmicutes were depleted. Our study indicates that the bulk soil communities seem overall well adapted and more resistant to changes in the water treatment, whereas the root microbiota seems more vulnerable., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

For extending the current collection of axenic cultures of planctomycetes, we describe in this study the isolation and characterisation of strain Pan265 T obtained from a red biofilm in the hydrothermal vent system close to the Lipari Islands in the Tyrrhenian Sea, north of Sicily, Italy. The strain forms light pink colonies on solid medium and grows as a viscous colloid in liquid culture, likely as the result of formation of a dense extracellular matrix observed during electron microscopy. Cells of the novel isolate are spherical, motile and divide by binary fission. Strain Pan265 T is mesophilic (temperature optimum 30-33 °C), neutrophilic (pH optimum 7.0-8.0), aerobic and heterotrophic. The strain has a genome size of 3.49 Mb and a DNA G + C content of 63.9%. Phylogenetically, the strain belongs to the family Phycisphaeraceae, order Phycisphaerales, class Phycisphaerae. Our polyphasic analysis supports the delineation of strain Pan265 T from the known genera in this family. Therefore, we conclude to assign strain Pan265 T to a novel species within a novel genus, for which we propose the name Mucisphaera calidilacus gen. nov., sp. nov. The novel species is the type species of the novel genus and is represented by strain Pan265 T (= DSM 100697 T  = CECT 30425 T ) as type strain., (© 2022. The Author(s).)

Microbial communities are key drivers of carbon, sulfur, and nitrogen cycling in coastal ecosystems, where they are subjected to dynamic shifts in substrate availability and exposure to toxic compounds. However, how these shifts affect microbial interactions and function is poorly understood. Unraveling such microbial community responses is key to understand their environmental distribution and resilience under current and future disturbances. Here, we used metagenomics and metatranscriptomics to investigate microbial community structure and transcriptional responses to prolonged ammonium deprivation, and sulfide and nitric oxide toxicity stresses in a controlled bioreactor system mimicking coastal sediment conditions. Ca. Nitrobium versatile , identified in this study as a sulfide-oxidizing denitrifier, became a rare community member upon ammonium removal. The ANaerobic Methanotroph (ANME) Ca. Methanoperedens nitroreducens showed remarkable resilience to both experimental conditions, dominating transcriptional activity of dissimilatory nitrate reduction to ammonium (DNRA). During the ammonium removal experiment, increased DNRA was unable to sustain anaerobic ammonium oxidation (anammox) activity. After ammonium was reintroduced, a novel anaerobic bacterial methanotroph species that we have named Ca. Methylomirabilis tolerans outcompeted Ca. Methylomirabilis lanthanidiphila , while the anammox Ca. Kuenenia stuttgartiensis outcompeted Ca. Scalindua rubra . At the end of the sulfide and nitric oxide experiment, a gammaproteobacterium affiliated to the family Thiohalobacteraceae was enriched and dominated transcriptional activity of sulfide:quinone oxidoreductases. Our results indicate that some community members could be more resilient to the tested experimental conditions than others, and that some community functions such as methane and sulfur oxidation coupled to denitrification can remain stable despite large shifts in microbial community structure. Further studies on complex bioreactor enrichments are required to elucidate coastal ecosystem responses to future disturbances., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dalcin Martins, Echeveste Medrano, Arshad, Kurth, Ouboter, Op den Camp, Jetten and Welte.)

Urbanised environments have been identified as hotspots of anthropogenic methane emissions. Especially urban aquatic ecosystems are increasingly recognised as important sources of methane. However, the microbiology behind these emissions remains unexplored. Here, we applied microcosm incubations and molecular analyses to investigate the methane-cycling community of the Amsterdam canal system in the Netherlands. The sediment methanogenic communities were dominated by Methanoregulaceae and Methanosaetaceae, with co-occurring methanotrophic Methanoperedenaceae and Methylomirabilaceae indicating the potential for anaerobic methane oxidation. Methane was readily produced after substrate amendment, suggesting an active but substrate-limited methanogenic community. Bacterial 16S rRNA gene amplicon sequencing of the sediment revealed a high relative abundance of Thermodesulfovibrionia. Canal wall biofilms showed the highest initial methanotrophic potential under oxic conditions compared to the sediment. During prolonged incubations the maximum methanotrophic rate increased to 8.08 mmol g DW -1  d -1 that was concomitant with an enrichment of Methylomonadaceae bacteria. Metagenomic analysis of the canal wall biofilm lead to the recovery of a single methanotroph metagenome-assembled genome. Taxonomic analysis showed that this methanotroph belongs to the genus Methyloglobulus. Our results underline the importance of previously unidentified and specialised environmental niches at the nexus of the natural and human-impacted carbon cycle., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)