Gill-associated ammonia oxidizers are widespread in teleost fish.

Recent advances in sequencing methods have greatly expanded the knowledge of teleost-associated microorganisms. While fish-gut microbiomes are comparatively well studied, less attention has gone toward other, external organ-microbiome associations. Gills are particularly interesting to investigate due to their functions in gas exchange, osmoregulation, and nitrogen excretion. We recently discovered a branchial symbiosis between nitrogen-cycling bacteria and teleosts (zebrafish and carp), in which ammonia-oxidizing Nitrosomonas and denitrifying bacteria together convert toxic ammonia excreted by the fish into harmless dinitrogen (N2) gas. This symbiosis can function as a "natural biofilter" in fish gills and can potentially occur in all ammonotelic fish species, but it remains unknown how widespread this symbiosis is. In this study, we analyzed all publicly available gill microbiome data sets and checked for the presence of Nitrosomonas. We discovered that more than half of the described fish gill microbiomes contain 16S rRNA gene sequences of ammonia-oxidizing bacteria (AOB). The presence of gill-specific AOB was shown in both wild and aquacultured fish, as well as in marine and freshwater fish species. Based on these findings, we propose that ammonia oxidizers are widespread in teleost fish gills. These gill-associated AOB can significantly affect fish nitrogen excretion, and the widespread nature of this association suggests that the gill-associated AOB can have similar impacts on more fish species. Future research should address the contribution of these microorganisms to fish nitrogen metabolism and the fundamental characteristics of this novel symbiosis.IMPORTANCERecent advances in sequencing have increased our knowledge of teleost-associated microbiota, but the gill microbiome has received comparatively little attention. We recently discovered a consortium of nitrogen-cycling bacteria in the gills of common carp and zebrafish, which are able to convert (toxic) ammonia into harmless dinitrogen gas. These microorganisms thus function as a natural nitrogen biofilter. We analyzed all available gill microbiome data sets to determine how widespread gill-associated ammonia-oxidizing bacteria (AOB) are. More than half of the data sets contained AOB, representing both aquacultured and wild fish from freshwater and marine habitats. In total, 182 amplicon sequencing variants were obtained, of which 115 were found specifically in the gills and not the environmental microbiomes. As gill-associated AOB are apparently widespread in teleost fish, it is important to study their impact on host nitrogen excretion and the potential to reduce ammonia accumulation in (recirculating) aquaculture of relevant fish species.

Activity-based labelling of ammonia- and alkane-oxidizing microorganisms including ammonia-oxidizing archaea.

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.

Gene-centered metagenome analysis of Vulcano Island soil (Aeolian archipelago, Italy) reveals diverse microbial key players in methane, hydrogen and sulfur cycles.

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 H2, indicating that besides H2S, H2 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 H2S 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.

Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress.

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-ß-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-ß-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.

Genomic analysis of the class Phycisphaerae reveals a versatile group of complex carbon-degrading bacteria.

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.

Sulfide Toxicity as Key Control on Anaerobic Oxidation of Methane in Eutrophic Coastal Sediments.

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.

Eutrophication and Deoxygenation Drive High Methane Emissions from a Brackish Coastal System.

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 13C, 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.

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.

Ammonium-derived nitrous oxide is a global source in streams.

Global riverine nitrous oxide (N2O) 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 N2O emissions. However, the mechanisms and pathways controlling hyporheic N2O production in stream ecosystems remain unknown. Here, we report that ammonia-derived pathways, rather than the nitrate-derived pathways, are the dominant hyporheic N2O sources (69.6 ± 2.1%) in agricultural streams around the world. The N2O fluxes are mainly in positive correlation with ammonia. The potential N2O metabolic pathways of metagenome-assembled genomes (MAGs) provides evidence that nitrifying bacteria contain greater abundances of N2O 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 N2O emissions. Global models of riverine ecosystems need to better represent ammonia-derived pathways for accurately estimating and predicting riverine N2O emissions.

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.

Fe(II)Cl2 amendment suppresses pond methane emissions by stimulating iron-dependent anaerobic oxidation of methane.

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.

Probing Denitrifying Anaerobic Methane Oxidation via Antimicrobial Intervention: Implications for Innovative Wastewater Management.

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.

Mechanisms of extracellular electron transfer in anaerobic methanotrophic archaea.

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.

Metabolic and phylogenetic diversity in the phylum Nitrospinota revealed by comparative genome analyses.

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.

Methane-dependent complete denitrification by a single Methylomirabilis bacterium.

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.

Seasonal dynamics of the microbial methane filter in the water column of a eutrophic coastal basin.

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.

Anaerobic methanotrophy is stimulated by graphene oxide in a brackish urban canal sediment.

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.

Methanotrophic potential of Dutch canal wall biofilms is driven by Methylomonadaceae.

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.

Gene-Based Modeling of Methane Oxidation in Coastal Sediments: Constraints on the Efficiency of the Microbial Methane Filter.

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.