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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

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 CO2, 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 (Vmax of 8.4 µmol mg-1 min-1 and Km 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 (Vmax of 0.4 µmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 µmol mg-1 min-1 versus Vmax,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 13C2-labeled acetate produced 3 µmol of [13C]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.

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

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

Novel and unusual genes for nitrogen and metal cycling in Planctomycetota- and KSB1-affiliated metagenome-assembled genomes reconstructed from a marine subsea tunnel.

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.

Simultaneous sulfide and methane oxidation by an extremophile.

Hydrogen sulfide (H2S) and methane (CH4) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H2S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH4 and H2S simultaneously at equally high rates. By oxidizing H2S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H2S on methanotrophy. Strain SolV adapts to increasing H2S by expressing a sulfide-insensitive ba3-type terminal oxidase and grows as chemolithoautotroph using H2S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H2S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways.

Predicting and improving the microbial removal of organic micropollutants during wastewater treatment: A review.

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.