Microbial paracetamol degradation involves a high diversity of novel amidase enzyme candidates.

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.

Genome-Resolved Metaproteomics Decodes the Microbial and Viral Contributions to Coupled Carbon and Nitrogen Cycling in River Sediments.

Rivers have a significant role in global carbon and nitrogen cycles, serving as a nexus for nutrient transport between terrestrial and marine ecosystems. Although rivers have a small global surface area, they contribute substantially to worldwide greenhouse gas emissions through microbially mediated processes within the river hyporheic zone. Despite this importance, research linking microbial and viral communities to specific biogeochemical reactions is still nascent in these sediment environments. To survey the metabolic potential and gene expression underpinning carbon and nitrogen biogeochemical cycling in river sediments, we collected an integrated data set of 33 metagenomes, metaproteomes, and paired metabolomes. We reconstructed over 500 microbial metagenome-assembled genomes (MAGs), which we dereplicated into 55 unique, nearly complete medium- and high-quality MAGs spanning 12 bacterial and archaeal phyla. We also reconstructed 2,482 viral genomic contigs, which were dereplicated into 111 viral MAGs (vMAGs) of >10 kb in size. As a result of integrating gene expression data with geochemical and metabolite data, we created a conceptual model that uncovered new roles for microorganisms in organic matter decomposition, carbon sequestration, nitrogen mineralization, nitrification, and denitrification. We show how these metabolic pathways, integrated through shared resource pools of ammonium, carbon dioxide, and inorganic nitrogen, could ultimately contribute to carbon dioxide and nitrous oxide fluxes from hyporheic sediments. Further, by linking viral MAGs to these active microbial hosts, we provide some of the first insights into viral modulation of river sediment carbon and nitrogen cycling. IMPORTANCE Here we created HUM-V (hyporheic uncultured microbial and viral), an annotated microbial and viral MAG catalog that captures strain and functional diversity encoded in these Columbia River sediment samples. Demonstrating its utility, this genomic inventory encompasses multiple representatives of dominant microbial and archaeal phyla reported in other river sediments and provides novel viral MAGs that can putatively infect these. Furthermore, we used HUM-V to recruit gene expression data to decipher the functional activities of these MAGs and reconstruct their active roles in Columbia River sediment biogeochemical cycling. Ultimately, we show the power of MAG-resolved multi-omics to uncover interactions and chemical handoffs in river sediments that shape an intertwined carbon and nitrogen metabolic network. The accessible microbial and viral MAGs in HUM-V will serve as a community resource to further advance more untargeted, activity-based measurements in these, and related, freshwater terrestrial-aquatic ecosystems.

Distinct comammox Nitrospira catalyze ammonia oxidation in a full-scale groundwater treatment bioreactor under copper limited conditions.

Microbial ammonia oxidation is the initial nitrification step used in biological nitrogen-removal during water treatment processes, and the discovery of complete ammonia-oxidizing (comammox) bacteria added a novel member to this functional group. It is important to identify and understand the predominant microorganisms responsible for ammonium removal in biotechnological process design and optimization. In this study, we used a full-scale bioreactor to treat ammonium in groundwater (9.3 ± 0.5 mg NH4+-N/L) and investigated the key ammonia-oxidizing prokaryotes present. The groundwater ammonium was stably and efficiently oxidized throughout ∼700 days of bioreactor operation. 16S rRNA gene amplicon sequencing of the bioreactor community showed a high abundance of Nitrospira (12.5-45.9%), with the dominant sequence variant (3.5-37.8%) most closely related to Candidatus Nitrospira nitrosa. Furthermore, analyses of amoA, the marker gene for ammonia oxidation, indicated the presence of two distinct comammox Nitrospira populations, however, the relative abundance of only one of these populations was strongly correlated to ammonia oxidation rates and was robustly expressed. After 380 days of operation copper wires were immersed into the reactor at 0.04-0.06 m2/m3 tank, which caused a gradual abundance increase of one discrete comammox Nitrospira population. However, further increase of the copper dosing (0.08 m2/m3 tank) inverted the most abundant ammonia-oxidizing population to Nitrosomonas sp. These results indicate that comammox Nitrospira were capable of efficient ammonium removal in groundwater without exogenous nutrients, but copper addition can stimulate comammox Nitrospira or lead to dominance of Nitrosomonas depending on dosage.

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria.

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.

DRAM for distilling microbial metabolism to automate the curation of microbiome function.

Microbial and viral communities transform the chemistry of Earth's ecosystems, yet the specific reactions catalyzed by these biological engines are hard to decode due to the absence of a scalable, metabolically resolved, annotation software. Here, we present DRAM (Distilled and Refined Annotation of Metabolism), a framework to translate the deluge of microbiome-based genomic information into a catalog of microbial traits. To demonstrate the applicability of DRAM across metabolically diverse genomes, we evaluated DRAM performance on a defined, in silico soil community and previously published human gut metagenomes. We show that DRAM accurately assigned microbial contributions to geochemical cycles and automated the partitioning of gut microbial carbohydrate metabolism at substrate levels. DRAM-v, the viral mode of DRAM, established rules to identify virally-encoded auxiliary metabolic genes (AMGs), resulting in the metabolic categorization of thousands of putative AMGs from soils and guts. Together DRAM and DRAM-v provide critical metabolic profiling capabilities that decipher mechanisms underpinning microbiome function.

Methane and nitrous oxide porewater concentrations and surface fluxes of a regulated river.

Greenhouse gas (GHG) emissions from rivers are a critical missing component of current global GHG models. Their exclusion is mainly due to a lack of in-situ measurements and a poor understanding of the spatiotemporal dynamics of GHG production and emissions, which prevents optimal model parametrization. We combined simultaneous observations of porewater concentrations along different beach positions and depths, and surface fluxes of methane and nitrous oxide at a plot scale in a large regulated river during three water stages: rising, falling, and low. Our goal was to gain insights into the interactions between hydrological exchanges and GHG emissions and elucidate possible hypotheses that could guide future research on the mechanisms of GHG production, consumption, and transport in the hyporheic zone (HZ). Results indicate that the site functioned as a net source of methane. Surface fluxes of methane during river water stages at three beach positions (shallow, intermediate and deep) correlated with porewater concentrations of methane. However, fluxes were significantly higher in the intermediate position during the low water stage, suggesting that low residence time increased methane emissions. Vertical profiles of methane peaked at different depths, indicating an influence of the magnitude and direction of the hyporheic mixing during the different river water stages on methane production and consumption. The site acted as either a sink or a source of nitrous oxide depending on the elevation of the water column. Nitrous oxide porewater concentrations peaked at the upper layers of the sediment throughout the different water stages. River hydrological stages significantly influenced porewater concentrations and fluxes of GHG, probably by influencing heterotrophic respiration (production and consumption processes) and transport to and from the HZ. Our results highlight the importance of including dynamic hydrological exchanges when studying and modeling GHG production and consumption in the HZ of large rivers.

Uncovering the Diversity and Activity of Methylotrophic Methanogens in Freshwater Wetland Soils.

Wetland soils are one of the largest natural contributors to the emission of methane, a potent greenhouse gas. Currently, microbial contributions to methane emissions from these systems emphasize the roles of acetoclastic and hydrogenotrophic methanogens, while less frequently considering methyl-group substrates (e.g., methanol and methylamines). Here, we integrated laboratory and field experiments to explore the potential for methylotrophic methanogenesis in Old Woman Creek (OWC), a temperate freshwater wetland located in Ohio, USA. We first demonstrated the capacity for methylotrophic methanogenesis in these soils using laboratory soil microcosms amended with trimethylamine. However, subsequent field porewater nuclear magnetic resonance (NMR) analyses to identify methanogenic substrates failed to detect evidence for methylamine compounds in soil porewaters, instead noting the presence of the methylotrophic substrate methanol. Accordingly, our wetland soil-derived metatranscriptomic data indicated that methanol utilization by the Methanomassiliicoccaceae was the likely source of methylotrophic methanogenesis. Methanomassiliicoccaceae relative contributions to mcrA transcripts nearly doubled with depth, accounting for up to 8% of the mcrA transcripts in 25-cm-deep soils. Longitudinal 16S rRNA amplicon and mcrA gene surveys demonstrated that Methanomassiliicoccaceae were stably present over 2 years across lateral and depth gradients in this wetland. Meta-analysis of 16S rRNA sequences similar (>99%) to OWC Methanomassiliicoccaceae in public databases revealed a global distribution, with a high representation in terrestrial soils and sediments. Together, our results demonstrate that methylotrophic methanogenesis likely contributes to methane flux from climatically relevant wetland soils.IMPORTANCE Understanding the sources and controls on microbial methane production from wetland soils is critical to global methane emission predictions, particularly in light of changing climatic conditions. Current biogeochemical models of methanogenesis consider only acetoclastic and hydrogenotrophic sources and exclude methylotrophic methanogenesis, potentially underestimating microbial contributions to methane flux. Our multi-omic results demonstrated that methylotrophic methanogens of the family Methanomassiliicoccaceae were present and active in a freshwater wetland, with metatranscripts indicating that methanol, not methylamines, was the likely substrate under the conditions measured here. However, laboratory experiments indicated the potential for other methanogens to become enriched in response to trimethylamine, revealing the reservoir of methylotrophic methanogenesis potential residing in these soils. Collectively, our approach used coupled field and laboratory investigations to illuminate metabolisms influencing the terrestrial microbial methane cycle, thereby offering direction for increased realism in predictive process-oriented models of methane flux in wetland soils.

Metagenomic Approaches Unearth Methanotroph Phylogenetic and Metabolic Diversity.

Methanotrophic microorganisms utilize methane as an electron donor and a carbon source. To date, the capacity to oxidize methane is restricted to microorganisms from three bacterial and one archaeal phyla. Most of our knowledge of methanotrophic metabolism has been obtained using highly enriched or pure cultures grown in the laboratory. However, many methanotrophs currently evade cultivation, thus metagenomics provides a complementary approach for gaining insight into currently unisolated microorganisms. Here we synthesize the studies using metagenomics to glean information about methanotrophs. We complement this summary with an analysis of methanotroph marker genes from 235 publically available metagenomic datasets. We analyze the phylogenetic and environmental distribution of methanotrophs sampled by metagenomics. We also highlight metabolic insights that methanotroph genomes assembled from metagenomes are illuminating. In summary, metagenomics has increased methanotrophic foliage within the tree of life, as well as provided new insights into methanotroph metabolism, which collectively can guide new cultivation efforts. Lastly, given the importance of methanotrophs for biotechnological applications and their capacity to filter greenhouse gases from a variety of ecosystems, metagenomics will continue to be an important component in the arsenal of tools needed for understanding methanotroph diversity and metabolism in both engineered and natural systems.

Members of the Genus Methylobacter Are Inferred To Account for the Majority of Aerobic Methane Oxidation in Oxic Soils from a Freshwater Wetland.

Microbial carbon degradation and methanogenesis in wetland soils generate a large proportion of atmospheric methane, a highly potent greenhouse gas. Despite their potential to mitigate greenhouse gas emissions, knowledge about methane-consuming methanotrophs is often limited to lower-resolution single-gene surveys that fail to capture the taxonomic and metabolic diversity of these microorganisms in soils. Here our objective was to use genome-enabled approaches to investigate methanotroph membership, distribution, and in situ activity across spatial and seasonal gradients in a freshwater wetland near Lake Erie. 16S rRNA gene analyses demonstrated that members of the methanotrophic Methylococcales were dominant, with the dominance largely driven by the relative abundance of four taxa, and enriched in oxic surface soils. Three methanotroph genomes from assembled soil metagenomes were assigned to the genus Methylobacter and represented the most abundant methanotrophs across the wetland. Paired metatranscriptomes confirmed that these Old Woman Creek (OWC) Methylobacter members accounted for nearly all the aerobic methanotrophic activity across two seasons. In addition to having the capacity to couple methane oxidation to aerobic respiration, these new genomes encoded denitrification potential that may sustain energy generation in soils with lower dissolved oxygen concentrations. We further show that Methylobacter members that were closely related to the OWC members were present in many other high-methane-emitting freshwater and soil sites, suggesting that this lineage could participate in methane consumption in analogous ecosystems. This work contributes to the growing body of research suggesting that Methylobacter may represent critical mediators of methane fluxes in freshwater saturated sediments and soils worldwide.IMPORTANCE Here we used soil metagenomics and metatranscriptomics to uncover novel members within the genus Methylobacter We denote these closely related genomes as members of the lineage OWC Methylobacter Despite the incredibly high microbial diversity in soils, here we present findings that unexpectedly showed that methane cycling was primarily mediated by a single genus for both methane production ("Candidatus Methanothrix paradoxum") and methane consumption (OWC Methylobacter). Metatranscriptomic analyses revealed that decreased methanotrophic activity rather than increased methanogenic activity possibly contributed to the greater methane emissions that we had previously observed in summer months, findings important for biogeochemical methane models. Although members of this Methylococcales order have been cultivated for decades, multi-omic approaches continue to illuminate the methanotroph phylogenetic and metabolic diversity harbored in terrestrial and marine ecosystems.

Methanogenesis in oxygenated soils is a substantial fraction of wetland methane emissions.

The current paradigm, widely incorporated in soil biogeochemical models, is that microbial methanogenesis can only occur in anoxic habitats. In contrast, here we show clear geochemical and biological evidence for methane production in well-oxygenated soils of a freshwater wetland. A comparison of oxic to anoxic soils reveal up to ten times greater methane production and nine times more methanogenesis activity in oxygenated soils. Metagenomic and metatranscriptomic sequencing recover the first near-complete genomes for a novel methanogen species, and show acetoclastic production from this organism was the dominant methanogenesis pathway in oxygenated soils. This organism, Candidatus Methanothrix paradoxum, is prevalent across methane emitting ecosystems, suggesting a global significance. Moreover, in this wetland, we estimate that up to 80% of methane fluxes could be attributed to methanogenesis in oxygenated soils. Together, our findings challenge a widely held assumption about methanogenesis, with significant ramifications for global methane estimates and Earth system modeling.

Abundant carbon substrates drive extremely high sulfate reduction rates and methane fluxes in Prairie Pothole Wetlands.

Inland waters are increasingly recognized as critical sites of methane emissions to the atmosphere, but the biogeochemical reactions driving such fluxes are less well understood. The Prairie Pothole Region (PPR) of North America is one of the largest wetland complexes in the world, containing millions of small, shallow wetlands. The sediment pore waters of PPR wetlands contain some of the highest concentrations of dissolved organic carbon (DOC) and sulfur species ever recorded in terrestrial aquatic environments. Using a suite of geochemical and microbiological analyses, we measured the impact of sedimentary carbon and sulfur transformations in these wetlands on methane fluxes to the atmosphere. This research represents the first study of coupled geochemistry and microbiology within the PPR and demonstrates how the conversion of abundant labile DOC pools into methane results in some of the highest fluxes of this greenhouse gas to the atmosphere ever reported. Abundant DOC and sulfate additionally supported some of the highest sulfate reduction rates ever measured in terrestrial aquatic environments, which we infer to account for a large fraction of carbon mineralization in this system. Methane accumulations in zones of active sulfate reduction may be due to either the transport of free methane gas from deeper locations or the co-occurrence of methanogenesis and sulfate reduction. If both respiratory processes are concurrent, any competitive inhibition of methanogenesis by sulfate-reducing bacteria may be lessened by the presence of large labile DOC pools that yield noncompetitive substrates such as methanol. Our results reveal some of the underlying mechanisms that make PPR wetlands biogeochemical hotspots, which ultimately leads to their critical, but poorly recognized role in regional greenhouse gas emissions.