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.

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.

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.

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.

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.

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.

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.

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.

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.

"Candidatus Hydrogenisulfobacillus filiaventi" strain R50 gen. nov. sp. nov., a highly efficient producer of extracellular organic compounds from H2 and CO2.

Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO2 and a cheap energy source, such as H2, CH4, NH3, CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO2 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 H2-oxidizing microorganism, that fixed CO2 and excreted no less than 0.54 mol organic carbon per mole fixed CO2. 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 O2-limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules.

Hot moment of N2O emissions in seasonally frozen peatlands.

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 (N2O) to the atmosphere. Here we provide evidence that seasonally frozen peatlands are important N2O emission sources in the Northern Hemisphere and the thawing periods are the hot moment of annual N2O emissions. The flux during the hot moment of thawing in spring was 1.20 ± 0.82 mg N2O m-2 d-1, significantly higher than that during the other periods (freezing, -0.12 ± 0.02 mg N2O m-2 d-1; frozen, 0.04 ± 0.04 mg N2O m-2 d-1; thawed, 0.09 ± 0.01 mg N2O 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 N2O source. Furthermore, based on soil incubation with 15N and 18O isotope tracing and differential inhibitors, heterotrophic bacterial and fungal denitrification was revealed as the main source of N2O in peatland profiles (0-200 cm). Metagenomic, metatranscriptomic, and qPCR assays further revealed that seasonally frozen peatlands have high N2O emission potential, but thawing significantly stimulates expression of genes encoding N2O-producing protein complexes (hydroxylamine dehydrogenase (hao) and nitric oxide reductase (nor)), resulting in high N2O emissions during spring. This hot moment converts seasonally frozen peatlands into an important N2O 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 N2O yr-1. However, these N2O emissions are still not routinely included in Earth system models and global IPCC assessments.

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

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

Benzimidazole fungicide biotransformation by comammox Nitrospira bacteria: Transformation pathways and associated proteomic responses.

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.

Metascan: METabolic Analysis, SCreening and ANnotation of Metagenomes.

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.