Environmental Science & Technology

Mass spectrometry is used to identify chemicals to which humans are exposed, but it cannot directly determine molecular structures. Instead, structures are inferred by matching experimental spectra to libraries of spectra constructed from analyses of pure reference compounds. However, the chemical space of human exposures far exceeds the amount of experimental library spectra. Here, we evaluate a reference-free strategy for confident identification of unknown molecules. Using fentanyl as a case study, we created a suspect library of over 1 billion computationally predicted fentanyl analogs and predicted molecular properties through machine learning, molecular dynamics, and density functional theory. Multi-dimensional spectra from a blinded analysis of a mock fentanyl tablet were matched with the predicted library, yielding an average of three candidate structures per measured analog, with six exact identifications. This work emphasizes the promise of reference-free molecular measurements for assessing human exposure by merging computational predictions with high-dimensional measurements.

Ethylene oxide (EtO) is a highly reactive industrial chemical and classified as a known human carcinogen with a putative mutagenic mode of action (MOA). Its genotoxic potential is primarily mediated through alkylation of DNA, resulting in the formation of the mutagenic adduct O6-(2-hydroxyethyl)-2-deoxyguanosine (O6-HE-dG). The N7-(2-hydroxyethyl)guanine (N7-HE-G) adduct is formed in greater abundance and is generally considered to be non-mutagenic. However, dose-response relationships of these DNA adducts, particularly at low inhalation exposure levels (i. e., below 3 ppm), remain unknown. These data are necessary to inform the biological plausibility of different statistical dose-response models that have been applied to human or animal data used for cancer risk assessment. In the present study, male and female B6C3F1 mice were exposed to EtO (0, 0.05, 0.1, 0.5, 1, 50, 100, and 200 ppm) 6 hours/day for 28 consecutive days. Immediately following the last exposure, DNA was extracted from lung, liver, bone marrow, and mammary gland, and further utilized to measure DNA adduct levels using highly sensitive mass spectrometry platforms. N7-HE-G was detected in all tissues and exposure groups, showing linear dose-response relationships in the low-dose range ([&le;]1 ppm) and increased sharply and exposure-disproportionately in the high-dose range ([&ge;]50 ppm). Despite a very low limit of detection, O6-HE-dG, in contrast, was not detected at exposures <50 ppm in any tissue consistent with at most a shallow linear exposure response. At higher exposures ([&ge;]50 ppm), O6-HE-dG exhibited a dose-response pattern of N7-HE-G. Notably the mammary gland, despite being anatomically distant from the site of inhalation, exhibited the second-highest levels of both adducts at higher doses. This study provides the first reliable quantitative dose-response evidence of DNA adducts in tumor target and non-target (liver) tissues across a wide range of EtO exposures. The two DNA adducts differ markedly in their abundance, repairability and mutagenic potential and together provide a molecular MOA dose-response framework to inform both quantitative cancer risk assessment and genotoxic hazard characterization.

Fluorinated chemicals are increasingly prevalent in pharmaceuticals and agrochemicals, yet their influence on the human gut microbiome and the potential for microbial biotransformation to alter therapeutic and toxicological profiles remain poorly understood. Here, we investigated the bidirectional relationship between 15 structurally diverse fluorinated chemicals and the gut microbiota by using an ex vivo high-throughput fermentation system. Screening revealed that flutamide, fluazinam, and pretomanid were consistently biotransformed across the donor microbiomes, while other compounds showed substantial inter-individual variability in degradation. Furthermore, exposure to fluorinated chemicals induced compound-specific shifts in microbial diversity and community composition, demonstrating their capacity to alter gut microbial ecology. Using a computational workflow combining in silico biotransformation predictions with untargeted LC-MS/MS analysis, we identified nitroreduction as the primary gut microbial transformation across all three compounds. Single-strain experiments confirmed that the nitroreduction of flutamide to flu-6, previously attributed only to hepatic metabolism, is a widespread capacity among gut bacterial strains. Finally, in vitro cytotoxicity assays and in silico modelling further revealed flu-6 to be a less hepatotoxic derivative than the parent compound, suggesting a potential detoxifying role for the gut microbiota. Together, these findings establish an integrated ex vivo, in vitro, and in silico approach for assessing the bidirectional interactions between fluorinated chemicals and the gut microbiome.

Source-separated organics (SSO) are widely processed via anaerobic digestion to produce biogas, yet alternative conversion pathways could generate higher-value products. Here, we demonstrate long-term continuous production and recovery of medium-chain carboxylic acids (MCCAs) from SSO via microbial chain elongation using a bench-scale anaerobic bioreactor operated for 911 days. The reactor was fed with SSO samples collected from two full-scale municipal organics processing facilities in Toronto, Canada, capturing facility-specific and seasonal variability in SSO composition. MCCA production depended strongly on the availability of lactate as an electron donor, which varied with SSO preprocessing operations and outdoor collection temperatures. To mitigate product inhibition, an in-line extraction system using hollow-fiber polydimethylsiloxane (PDMS, also known as silicone) membranes was integrated with the anaerobic membrane bioreactor, providing a robust and solvent-free alternative to solvent-based extraction methods. Maximum MCCA yields reached 0.31 g MCCA/ g VSfeed, with notable octanoic acid production (up to 20% of total MCCA), and production rates up to 0.84 g L-1 d-1. Acidification of the alkaline extract produced a phase-separated MCCA-rich oil ([~]95% purity) without addition of downstream separation steps. Microbial community analysis of the reactor revealed enrichment of putative chain-elongating bacteria, including Eubacterium and Pseudoramibacter species, while shifts in SSO feedstock microbiomes influenced substrate availability and product spectra. These results demonstrate the feasibility of sustained MCCA production from municipal organic waste streams and highlight opportunities to integrate chain elongation with existing anaerobic digestion infrastructure.

Endocrine disrupting chemicals (EDCs) are ubiquitous environmental contaminants capable of dysregulating the production of thyroid hormones. Traditional thyroid toxicological assays rely on 2D cell cultures and animal models, both of which fail to accurately recapitulate human thyroid physiology and provide limited mechanistic insight into EDC toxicity. To overcome these limitations, we report a novel thyroid-on-chip platform integrating mouse embryonic stem cell-derived thyroid organoids with advanced organ-on-chip (OoC) technology and downstream multi-omics analysis. The platform leverages a reversibly-sealed microphysiological flow battery (MFB) to allow scale up of dynamic organoid culture and controlled chemical exposure while reducing operational complexity. Upon EDC exposure, transcriptomic and proteomic analysis revealed new molecular signatures of thyroid disruption across four different EDC classes, even at very low EDC concentrations (1nM), validating the capacity of this system to mechanistically dissect EDC-induced responses. This represents an integrated platform consists of an advanced physiologically relevant assay framework for next-generation endocrine toxicity testing, bridging the gap between in vitro screening and in vivo thyroid physiology.

Integrons are genetic elements that drive bacterial adaptation by capturing and expressing mobile gene cassettes. They play a key role in dissemination of antimicrobial resistance (AMR) genes, particularly in Gram-negative bacteria. In addition to known AMR determinants, integron gene cassettes carry a vast reservoir of novel genes whose functions are largely uncharacterised, making it diNicult to assess their full contribution to the resistome. Contributing to this are limitations in current sequence-based prediction methods which often lack the ability to identify unknown AMR or other adaptive genes with novel mechanisms. To address this, we developed an integron gene cassette capture system, a functional screening platform that captures environmental gene cassettes for direct phenotypic testing. Using this system, we recovered previously unknown AMR determinants while also providing insights into the prevalence of known clinical AMR genes in a range of environmental samples, including food items. Here we provide experimental data on multiple novel bleomycin resistance genes and a stress response gene conferring gentamicin and tobramycin resistance. Our sequence analysis of the captured library also highlighted the diversity of the environmental cassette pool, with 656 unique cassettes recovered, the majority of which encoded proteins with unknown functions. The cassette capture system is a powerful tool for accessing hidden elements of the resistome and discovering novel adaptive genes that may go undetected using current sequence-based approaches. Environmental implicationAntimicrobial resistance (AMR) genes are hazardous biological contaminants, yet the vast majority of environmental integron gene cassettes remain functionally uncharacterised. This study addresses this sequence-to-function gap by deploying a novel functional capture platform directly on realistic environmental matrices, including agricultural fertilisers, coastal seawater, and commercial food products. By characterising these cassettes, we uncovered hidden reservoirs of both novel and clinically established AMR genes circulating in critical exposure pathways. This work reveals the true hazardous potential of the mobile environmental resistome, validating a proactive One Health surveillance tool for monitoring emerging biological threats.

Biogenic volatile organic compounds (BVOCs) are gases that influence atmospheric chemistry, nutrient cycling, and species interactions, yet the contribution of heterotrophic marine bacteria to marine BVOC emissions remains poorly constrained. In addition, the extent to which the volatilome is linked to bacterial phylogeny is unknown. Here, we characterize the volatilome of 16 heterotrophic bacterial strains isolated from Baltic Sea surface water, spanning Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, Bacteroidota, and Actinomycetes. Headspace BVOCs were quantified under standardized growth conditions using Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-TOF-MS). A broadly overlapping bacterial volatilome was identified, with compound composition and proportional abundance similar across many strains, irrespective of phylogeny. Namely, most strains shared a core set of abundant compounds with a subset of strain-specific, low abundance compounds. Acetone accounted for more than 50% of the emissions in most volatilomes. The remaining fraction of emissions were primarily comprised of other low-molecular-weight oxygenated compounds. Interestingly, two strains demonstrated strain-specific emission patterns, significantly diverging from the group in their emission rate and compound composition. Together, these findings suggest that marine heterotrophic bacteria may contribute a broadly conserved collection of BVOCs to the ocean-atmosphere interface, highlighting their role as a widespread source of trace gases in marine ecosystems.

Bacterial membrane vesicles (BMVs) have emerged as important contributors to the dissemination of antibiotic resistance genes (ARGs) in the environment. Here, we developed a high-performance immunomagnetic isolation method that improves the purity and selectivity of BMV recovery from wastewater, minimizes contamination from eDNA and viruses, and enables differentiation of BMVs originating from Gram-positive versus Gram-negative bacteria with minimal cross-reactivity. Using this approach, we found that ARGs such as the kanamycin resistance gene (kanR) was highly abundant in BMVs from both raw and treated wastewater, exhibited persistence following treatment, and retained the ability to generate antibiotic-resistant bacteria via transformation. Metagenomic sequencing further revealed that tetracycline resistance genes were the most abundant ARG class across all wastewater samples, while the composition of BMV-associated ARGs differed from the bulk ARG profile. These findings highlight the critical yet underrecognized role of BMVs in the spread of antimicrobial resistance and underscore the need to address BMV-mediated pathways within a One Health framework linking environmental and human health.

Wastewater treatment plants act as convergence zones for antibiotic residues, antibiotic-resistant bacteria (ARB), and antimicrobial resistance genes (ARGs), yet conventional processes are not designed to mitigate resistance dissemination from their effluents. While chemical disinfectants are generally effective, soluble quaternary ammonium compounds (QACs) can generate subinhibitory exposure gradients that promote resistance selection and co-selection both during treatment and after release into receiving waters. Here, we evaluate a contact-restricted alternative: benzyldimethyldodecyl ammonium chloride (BDMDAC) immobilised onto hydroxyapatite microparticles as a reusable and retainable post-treatment polishing strategy. Across single-strain assays, treated wastewater exposure, and experimental community evolution, immobilised BDMDAC-functionalised particles (BDMDAC-FPs) achieved concentration-dependent antimicrobial activity without detectable biocide leaching. Optimal exposure (200 mg/L, 4 h) resulted in a ~5.5 log reduction in total bacterial abundance and removal of clinically relevant ARGs. Antimicrobial efficacy was retained after one reuse cycle, supporting operational stability. Plasmid-borne QAC ARGs did not confer protection, and no enrichment of qac-associated or non-QAC ARGs was observed. Conjugation assays demonstrated suppression of horizontal gene transfer even under suboptimal exposure, and mobility-associated markers remained stable or declined during long-term community incubation. Collectively, the data support a contact-restricted mechanism in which antimicrobial pressure is spatially confined to the particle interface, generating high local lethality while limiting diffuse subinhibitory exposure. This spatial confinement decouples antimicrobial efficacy from classical disinfectant-driven resistance selection and mobility amplification. Immobilised BDMDAC-FPs therefore provide a mechanistically distinct and evolution-conscious framework for wastewater polishing technologies.

Hydrocarbons are recalcitrant organic matter that are released into the environment via natural and anthropogenic activities. We hypothesized that abiotic and biotic factors, including salinity, temperature, seasonality, microbial interactions, and functional redundancy, influence the abundance and activity of potential hydrocarbon degraders in the Delaware and Chesapeake Bays. We identified key genes in hydrocarbon degradation pathways in metagenomes, metatranscriptomes, and metagenome assembled genomes (MAGs) from these estuaries. Aerobic aromatic and alkane degradation pathways predominated in both estuaries, with higher gene abundances observed in low-salinity spring and summer samples. Hydrocarbon degrading MAG abundance were significantly structured by salinity, temperature, nitrate, and silicate concentrations. Metatranscriptomic analyses revealed consistently higher expression of aerobic alkane and aromatic degradation genes in the Delaware compared to the Chesapeake Bay, with the highest occurring under low-salinity spring conditions in the former. Catechol degradation pathways exhibited high functional redundancy, whereas the naphthalene degradation pathway showed restricted distribution. Co-expression analysis revealed that Burkholderiales displayed condition dependent metabolic coupling while Pseudomonadales integrated hydrocarbon degradation with fermentation and central metabolism, demonstrating complementary strategies that support multi-scale ecosystem resilience. In conclusion, environmental gradients and taxon-specific metabolic strategies together govern hydrocarbon degradation potential in these estuaries, with implications for predicting ecosystem responses to hydrocarbon inputs under changing conditions. ImportanceCoastal estuaries are among the most contaminated aquatic environments on Earth, receiving continuous hydrocarbon inputs from industrial activity, urban runoff, and natural sources. Microorganisms are the primary agents of hydrocarbon breakdown in these systems yet predicting when and where this capacity is active and how resilient it is to environmental change remains a major challenge. Using paired genomic and transcriptomic data from microbial genomes across two major mid-Atlantic estuaries, we show that hydrocarbon degradation capacity is not uniformly distributed but is instead shaped by salinity, nutrients, and seasonality in pathway-specific ways. Critically, dominant degrader taxa employ fundamentally different metabolic strategies to sustain this function across fluctuating conditions, providing a form of community-level insurance against environmental disturbance. These findings advance our ability to predict microbial hydrocarbon degradation in coastal systems and inform nature-based approaches to bioremediation under increasing climate and anthropogenic pressures.

Chronic contaminant exposure may impose hidden physiological costs long before obvious demographic or health effects become detectable in wildlife populations. Epigenetic clocks quantify biological ageing and may provide sensitive biomarkers of cumulative toxicological stress. Per-and polyfluoroalkyl substances (PFAS) are persistent contaminants that bioaccumulate in marine food webs, yet their long-term physiological consequences for wildlife remain poorly understood. Here, we tested whether PFAS exposure is associated with accelerated biological ageing in common dolphins (Delphinus delphis). We analysed liver PFAS concentrations and skin DNA methylation profiles from 30 stranded or bycaught dolphins from New Zealand waters. Epigenetic age was estimated using a recently developed species-specific epigenetic clock, and age acceleration was calculated as the residual deviation between epigenetic and chronological age. Using an information-theoretic modelling framework, we assessed the effects of total PFAS burden, sex, and their interactions on epigenetic age acceleration. Total PFAS concentrations were positively associated with epigenetic age acceleration, indicating that dolphins with higher PFAS burdens were biologically older than expected for their chronological age. Each 1 ng g{square}{superscript 1} increase in total PFAS was associated with an average increase of 0.031 years in biological age. Sex did not significantly influence age acceleration, suggesting that PFAS-associated ageing effects occur across both sexes. Although modest, this effect is consistent with PFAS acting as a chronic physiological stressor influencing molecular ageing processes. Our findings provide the first evidence linking PFAS exposure to accelerated biological ageing in a wild mammal, highlighting epigenetic ageing as an integrative biomarker of long-term contaminant effects in wildlife.

The transition toward animal-free safety assessment of chemicals has accelerated the development of New Approach Methodologies (NAMs) for predicting skin sensitization. However, individual in silico models and experimental NAM assays frequently produce inconsistent or contradictory results, limiting their reliability when used in isolation. To address this challenge, we present a tiered integrated assessment framework implemented through the open source SaferSkin application, which enables systematic comparison and integration of multiple predictive models and experimental data within a transparent weight-of-evidence workflow. In this case study, a diverse set of 21 reference compounds was evaluated using a battery of in silico approaches, including the OECD QSAR Toolbox, VEGA, CASE Ultra and additional machine-learning models implemented within SaferSkin. The platform enables side-by-side comparison of predictions and integration of experimental data through Bayesian network models, allowing probabilistic updating of predictions as new evidence becomes available. Our results demonstrate that reliance on any single predictive model is insufficient for robust hazard identification due to frequent disagreement between models. In contrast, consensus interpretation across multiple modelling approaches combined with targeted experimental evidence substantially improves predictive confidence. The integrated weight-of-evidence framework showed strong concordance with reference classifications and was further supported by independent validation using the Pred-Skin Bayesian model. Importantly, the tiered workflow enables resolution of ambiguous cases. For example, lower-tier predictions for ethyl (2E,4Z)-deca-dienoate were inconsistent across models, whereas targeted third-tier testing using the SENS-IS assay identified the compound as a strong sensitiser (GHS Category 1A). Overall, this study demonstrates how integrated modelling, Bayesian evidence updating and targeted NAM testing can reduce uncertainty in skin sensitization assessment. The SaferSkin framework provides a transparent and reproducible approach for implementing Next Generation Risk Assessment (NGRA) strategies and supports the development of animal-free regulatory toxicology and Safe-and-Sustainable-by-Design chemical innovation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/711911v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@b59ca0org.highwire.dtl.DTLVardef@13de455org.highwire.dtl.DTLVardef@599358org.highwire.dtl.DTLVardef@d87fd1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

Evaluation of unintended immunotoxicity represents an important component of nonclinical safety assessment, as perturbation of immune function may increase susceptibility to infection, impair vaccine responses, and disrupt immune homeostasis. Regulatory guidance, including the ICH S8 Immunotoxicity Guideline, recommends a weight-of-evidence approach in which observations from conventional toxicological endpoints are integrated with functional immune assays to support interpretation of immune system effects. The present study applied an integrated immunotoxicity evaluation framework to examine concordance among structural, functional, and cellular immune endpoints in male Sprague-Dawley rats using a well-characterized immunosuppressive reference compound. Hematological evaluation revealed leukopenia characterized primarily by lymphocyte depletion. Reductions in spleen and thymus weights were accompanied by histopathological evidence of lymphoid depletion in multiple immune tissues, including spleen, thymus, lymph nodes, Peyers patches, and bone marrow. Functional immune competence was assessed through hemagglutination antibody response to sheep red blood cells and delayed-type hypersensitivity assays, both of which demonstrated marked suppression of adaptive immune responses. Flow cytometric immunophenotyping further demonstrated substantial reductions in B-cell populations and decreases in CD4 and CD8 T-cell counts, whereas NK cell populations were comparatively less affected. The concordance of hematological alterations, lymphoid tissue changes, impaired functional immune responses, and lymphocyte subset depletion provides integrated evidence of immune system perturbation. These findings demonstrate that complementary immunotoxicity endpoints collectively support hazard characterization of immune system effects under GLP conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/713556v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@beaf9dorg.highwire.dtl.DTLVardef@fb9f10org.highwire.dtl.DTLVardef@187ff06org.highwire.dtl.DTLVardef@1780dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days under mildly acidic conditions to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (10-20 g/L), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (30-40 g/L; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis indicated that under high-lactate conditions up to 30% of lactate was redirected toward propionate and acetate formation, likely via the acrylate pathway. Microbial community analysis revealed a stable dominance of Caproiciproducens spp., that could be correlated to valerate production. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems. HighlightsValerate was the dominant product, reaching up to 110 mM. Lactate:propionate ratios drive product selectivities. High lactate concentrations activated in situ propionate formation pathways. Caproiciproducens dominance was associated with sustained valerate production.

Respiratory syncytial virus (RSV) transmission via the aerosol route remains poorly understood, particularly with respect to how evolving virus-laden particles (bioaerosols) microenvironments influence viral survival. Bioaerosol particles contain complex mixtures of organic and inorganic components, and their physicochemical properties change dynamically during evaporation as water is lost upon emission from respiratory activities. These changes directly affect the local environment surrounding embedded virus during both the evaporation stage and the subsequent equilibrium state. However, how these microenvironmental conditions under different relative humidity (RH) levels regulate RSV survival remains unclear. In this study, we quantified RSV survival during the evaporation and early equilibrium stages using a flow-tube system with controlled residence times. Bioaerosols were generated from virus medium alone or supplemented with bovine serum albumin (BSA) or mucin and evaluated under low (35%) and intermediate (61%) RH conditions. Viral infectivity was normalized to RNA copy number to account for particle and sampling losses. At 35% RH, RSV infectivity decreased by one to three orders of magnitude, depending on the solution composition. In contrast, survival was significantly higher at intermediate RH, particularly for virus medium and BSA-supplemented aerosols. Scanning electron microscopy revealed that low RH conditions promote efflorescence, whereas intermediate RH results in viscous or semi-solid particles with higher water content. These observations suggest that efflorescence is associated with enhanced RSV inactivation, while viscous or semi-solid phases tend to preserve RSV in the aerosol state for respirable particles. Overall, RSV infectivity depends strongly on particle chemical composition, phase state (effloresced versus semi-solid), and relative humidity. These results highlight the importance of characterizing particle phase behavior and chemical composition during early aerosol processes to improve mechanistic understanding of viral survival relevant to short-range transmission.

Black Soldier Fly larvae (BSFL, Hermetia illucens) are highly effective for the bioconversion of food waste. However, their rearing process often produces substantial ammonia emissions, which are malodorous and environmentally concerning. We investigated the co-cultivation of BSFL with the sulfur-oxidizing bacterium Thiobacillus thioparus as a strategy to mitigate ammonia release. Importantly, under conditions where ammonia emissions were significantly reduced, neither larval growth nor bacterial viability was negatively affected. Furthermore, even when the initial bacterial inoculum was reduced to 3.3*105 CFU/g-food wastes, the bacterium rapidly recovered to functional levels and effectively controlled ammonia emissions. This indicates the absence of harmful interaction or nutrient competition between BSFL and T. thioparus. These findings suggest an efficient method for controlling ammonia in large-scale BSFL waste treatment. By reducing the required bacterial inoculum, this approach enables scalable microbial co-culturing with environmental and production benefits.

2,4,6-Trinitrotoluene (TNT) is a recalcitrant and pervasive environmental pollutant. Although different environmental microbes have demonstrated their ability to degrade or transform TNT, the underlying genetic basis and cellular machinery remain unclear. In this study, we investigated bacterial strategies in response to TNT exposure in Pantoea sp. MT58 and P. putida KT2440 using proteomics and random barcode transposon-site sequencing (RB-TnSeq). Pantoea sp. MT58 was found to utilize TNT as a sole nitrogen source, whereas P. putida KT2440 exhibited only stress tolerance without assimilation. Pantoea sp. MT58 encodes multiple putative nitroreductases that were upregulated, yet deletion of these genes did not affect growth on TNT, revealing pathway redundancy. Furthermore, fitness profiling provided no evidence for genes involved in the canonical Meisenheimer-complex pathway associated with nitrite release. Instead, the data are most consistent with a sequential nitro-group reduction route in which nitrogen is ultimately recovered as ammonium, with nitrogen routed through the GS-GOGAT pathway with purine and urea pools as the candidate buffering architecture for TNT mineralization. Conversely, P. putida KT2440 relied on Ttg/RND efflux pumps and toluene tolerance proteins for survival without nitrogen assimilation from TNT. This work distinguishes routes for productive nitrogen assimilation from those involved in nitroaromatic tolerance, expanding the mechanistic understanding of anthropogenic compound metabolism to inform future bioremediation efforts.

Polyethylene (PE) is the most produced synthetic polymer and as a result, a major source of microplastic waste accumulating globally. Exposure to photo- and thermo-oxidative conditions in the environment can cause PE to degrade into carbonyl-containing compounds, hydrocarbons, and low molecular weight PE (LMWPE). In both marine and freshwater ecosystems, fish, including Atlantic salmon, can ingest PE and its derivatives, creating opportunities for interactions with their gut microbes. Here, we investigated the ability of a bacterial isolate from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, to grow on an LMWPE model substrate for partially depolymerized and oxidized PE. Comparative genomic analyses showed that ASF-10 has a smaller genome than other Rhodococcus species yet retaining conserved functions including those related to utilization of medium- and long-chain hydrocarbons. In-depth characterization of the substrate following growth with ASF-10 confirmed depletion of alkanes and 2-ketones deriving from LMWPE, while the polymeric component remained unchanged. Proteomic analysis identified multiple enzymes that were likely to be involved in the degradation of LMWPE-derivatives, including an alkane 1-monooxygenase, cytochrome P450 hydroxylases and Baeyer-Villiger monooxygenases, as well as proteins for production of biofilm and a surfactant that may enhance accessibility to the substrate. Collectively, our findings advance the understanding of the ecology and enzymatic mechanisms underlying utilization of medium- to long-chain alkanes and oxidized variants thereof, that resemble molecules that can occur from abiotic PE degradation, by a fish gut-associated microbe. This metabolic capacity could be harnessed to develop sustainable strategies for bioremediation of oxidized, LMWPE-derivatives. ImportanceThe widespread presence of plastics in marine and freshwater environments has raised concerns due to their toxicity when ingested by fish. Microbial mechanisms driving breakdown of microplastic components, such as LMWPE and derivatives, in gut systems remain poorly understood. This study reveals how a bacterium isolated from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, metabolizes alkanes and oxidized variants thereof, that can result from abiotic PE decomposition. We identified key enzymes that are potentially involved in this process as well as in the production of biofilm and surfactants that may facilitate access to the substrate. Besides extending the knowledge of the enzymatic basis for degradation of PE-derivatives in gut-associated microbes from aquatic organisms, our results provide a framework that couples advanced compositional characterization of the substrate with omics techniques, offering valuable insight to support future studies aimed at unequivocally identifying microbes and their enzymes implicated in transformation of PE-derivatives.

Enteroviruses (EVs) are ubiquitous contaminants of surface waters, where they can remain infectious for long periods of time. Most methods used for EV monitoring are unable to distinguish between infectious and non-infectious particles or between EV types. Because different types exhibit both distinct environmental persistence and health implications, there is a need for type-resolved infectivity measurements. Here we developed Integrated Cell Culture-Nanopore Sequencing (ICC-NanoporeSeq), a method combining short-term cell culture amplification with Nanopore sequencing of the VP1 gene. The ICC approach was adapted from a previously described ICC-RTqPCR protocol, while the NanoporeSeq workflow was derived from a clinical EV typing protocol and optimized for environmentally circulating EV types. Using samples containing known concentrations of ten EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types, allowing a simultaneous and type-resolved assessment of infectivity in mixed samples. Overall, ICC-NanoporeSeq provides a scalable approach for the parallel analysis of multiple EV types. Compared with the predecessor ICC-RTqPCR method, it eliminates the need for multiple type-specific PCR primers and can therefore be readily expanded to include additional EV types. IMPORTANCECurrent methods used to detect EVs in environmental samples generally measure viral genome copies without determining whether viruses remain infectious, limiting their use in public health risk assessment or water quality monitoring. At the same time, available infectivity assays are often labor-intensive and cannot distinguish between different EV types. Here, we developed ICC-NanoporeSeq, a method combining cell culture and Nanopore sequencing to simultaneously quantify the infectious concentrations of multiple EV types in samples containing mixed EV populations. The method provides an efficient and scalable approach for studying EVs in complex environmental matrices. ICC-NanoporeSeq has potential applications in wastewater-based epidemiology, environmental surveillance, and disinfection studies, where understanding the persistence of different EV types simultaneously is crucial.

Biological nitrification inhibition (BNI) is a plant-mediated process that suppresses nitrification and is widely considered beneficial for reducing nitrous oxide emissions. Here, we show that BNI compounds also inhibit methane oxidation by methanotrophic bacteria, revealing a previously unrecognized trade-off in greenhouse gas regulation. Across soil bioreactor systems and pure cultures of both Type I and Type II methanotrophs, BNI compounds consistently suppressed methane oxidation activity. Kinetic analyses indicated an uncompetitive-like inhibition pattern, characterized by concurrent decreases in Vmax and Km, while reversibility assays showed that inhibition was not associated with loss of cellular viability. Experiments under copper-replete and copper-depleted conditions further showed that inhibition is predominantly associated with particulate methane monooxygenase (pMMO). Transcriptomic analyses demonstrated compound-specific responses, including suppression of methane oxidation pathways and differential regulation of stress-associated genes. These findings suggest that BNI-mediated inhibition of methane oxidation may offset reductions in nitrous oxide emissions, with implications for predicting net greenhouse gas fluxes in agricultural and wetland ecosystems. Incorporating BNI effects into biogeochemical models will be critical for accurately evaluating their role in the global methane budget.