Environmental Science & Technology

The accumulation of propionate is a challenge in numerous fermentative industrial processes because its degradation is energetically unfavorable and limited to few microbial species. Here, we report for the first time the oxidation of propionate by the extracellular electron transfer (EET)-capable bacterium Geobacter sulfurreducens in axenic cultures. G. sulfurreducens was capable of utilizing propionate both as electron donor (ED) and source of carbon with fumarate as electron acceptor (EA). In contrast, propionate was metabolized only in the presence of acetate with soluble Fe(III) citrate, and was not oxidized when insoluble iron oxides or glassy carbon electrodes poised at +0.1 V vs. SHE were the EAs. Biomass yield (per mole of electrons available) was lower with propionate alone than with propionate and acetate together, and acetate was preferentially consumed when both were present. Transcriptomic analysis of cultures grown with either propionate or acetate (with fumarate as EA) showed significant gene expression shifts strongly suggesting the methylmalonyl-CoA pathway as the main route for propionate degradation. Furthermore, propionate-consuming cultures exhibited an upregulation of branched chain amino acids (BCAAs) biosynthesis, as well as sulfur, nitrogen, and 2-oxocarboxylic acids metabolism. IMPORTANCEThe accumulation of propionate is a challenge in anaerobic and fermentative processes because it inhibits methanogenesis, and few microbial species within such systems can degrade it. G. sulfurreducens is a model electroactive bacterium widely used in bioelectrochemical systems and is increasingly studied in wastewater treatment and anaerobic digestion because of its ability to enhance syntrophic metabolism via direct interspecies electron transfer. We show for the first time that G. sulfurreducens can oxidize propionate, expanding its known metabolic repertoire, and that this capability is controlled by the nature of the terminal electron acceptor. Transcriptomic analyses strongly suggest that the methylmalonyl-CoA pathway is the main pathway for propionate degradation and reveal additional associated transcriptional changes. These findings, together with insights into propionate degradation kinetics, could inform future strategies aimed at using this bacterium to mitigate propionate buildup and improve the stability of anaerobic treatment systems.

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.

Plastic materials are widely used in engineered systems and increasingly accumulate in natural environments, where their surfaces interact with colloids, microorganisms, and dissolved organic matter. However, the relative roles of plastic surface properties versus particle-specific characteristics in governing organic matter retention remain poorly constrained. Here, attachment efficiency () was used to quantify intrinsic particle-collector affinity on three common thermoplastics (ABS, HDPE, HIPS) and glass beads as an inorganic reference. Surface chemistry, hydrophobicity, roughness, and charge were characterized, and interactions with submicron carbon particles (SCPs) and Escherichia coli were evaluated using column experiments. Extended DLVO (XDLVO) theory was applied to predict interaction energy barriers, and humic acid (HA) adsorption was quantified through batch isotherms. XDLVO modeling predicted higher affinity of particles for plastics relative to glass; however, experimentally measured attachment efficiencies were uniformly low ( < 0.05) across all materials. Attachment was primarily governed by particle size and surface charge rather than collector hydrophobicity, roughness, or surface chemistry. SCP consistently exhibited higher than bacteria, while differences among plastics were minor. Similarly, HA adsorption was weak and near-linear, with uptake following ABS {approx} HIPS > HDPE > glass, indicating reversible, partitioning-like association dominated by polymer-specific functionality rather than electrostatics. The absence of correlation between and XDLVO-predicted energy barriers further demonstrates limitations of classical physicochemical models in describing particle- plastic interactions. Collectively, these results indicate that pristine thermoplastic surfaces exhibit intrinsically low affinity for organic matter and that particle-specific properties dominate retention under low ionic strength conditions. Enhanced accumulation in environmental systems likely requires surface aging or conditioning processes not captured by classical interaction theory.

Interactions between viruses and filter-feeding zooplankton can alter viral persistence in surface waters, with direct implications for water quality and public health risk. However, data on virus-zooplankton interactions and the environmental factors that influence them are still limited. This study evaluated the impact of filter feeding, in the dark and under simulated sunlight, on a bacteriophage (MS2) and a human virus (echovirus11; E11) in the presence of a ciliate (Tetrahymena pyriformis) and rotifer (Brachionus calyciflorus). Dark experiments established organism-dependent baseline removal for each virus, and rotifers showed greater removal of both viruses in comparison to ciliates. Under simulated sunlight, in contrast, experiments with ciliates resulted in greater virus removal compared to experiments with rotifers over a similar timespan (4.2 vs. 2.7 log MS2 in 53-58 h; 3.5 vs. 3.0 log E11 in 24-25 h). Analysis of decay rate constants reveals species-specific shifts in virus removal between dark and light that, depending on viral type and zooplankton species, either accelerate viral attenuation or protect viruses and prolong infectivity. T. pyriformis increases removal under sunlight relative to dark conditions and acts synergistically with sunlight inactivation, whereas rotifers impede sunlight inactivation.

Phthalate plasticizers are contaminants of emerging concern that interfere with the synthesis, secretion, and transport of hormones and receptors, altering the immune response and energy balance. Phthalate metabolites have been detected in marine mammals globally, and while studies on phthalate toxicity in marine mammals are beginning to emerge, a comprehensive understanding of the cellular response to these compounds remains elusive. Here, we investigated the transcriptional and bioenergetic responses to mono-ethylhexyl phthalate (MEHP), the active metabolite of di(2-ethylhexyl) phthalate (DEHP), in primary dermal derived from northern elephant seals (Mirounga angustirostris), common dolphins (Delphinus delphis), and humans. MEHP exposure did not induce cytotoxicity in any species, but triggered distinct, species-specific changes in gene expression and mitochondrial metabolism. Human cells showed the greatest transcriptional response to MEHP, upregulating detoxification, antioxidant, and inflammatory genes, and downregulating lipid metabolism pathways. Although mitochondrial respiration declined only at the highest dose, sustained extracellular acidification rates and increased glycolytic gene expression indicate a metabolic shift toward glycolysis. In contrast, elephant seal cells upregulated antioxidant and immune genes while maintaining mitochondrial respiration until the highest MEHP dose, alongside increased expression of genes involved in oxidative phosphorylation, the TCA cycle, and mitochondrial dynamics, suggesting a delayed shift to glycolysis and a potential evolutionary adaptation to sustain mitochondrial function during energy-demanding conditions such as breath-hold diving. Dolphin cells exhibited fewer transcriptional changes, which were enriched for hormone signaling and mitotic pathways, and showed dose-dependent declines in both oxygen consumption and extracellular acidification rates, even at the lowest MEHP concentration, alongside upregulation of stress and hypoxia-related genes. Together, these findings highlight distinct cellular strategies for coping with phthalate exposure and likely species-specific susceptibility to toxicant-induced stress. This study provides new insights into how marine mammals respond to plastic-derived contaminants at the cellular level, reinforcing the need for species-specific ecotoxicological risk assessments.

PFAS are ubiquitous endocrine-disrupting pollutants that cross the placenta and impact offspring health, but the extent and timing of their transfer to both placental and fetal compartments remain poorly understood. We aimed to characterize the relationship between trimester-specific maternal serum levels of prenatal PFAS and paired placental and cord levels at term. Data came from Glowing, a prospective birth cohort (n=151). Seventeen PFAS were measured in maternal serum, cord serum, and pulverized flash-frozen villous placenta with liquid chromatography-tandem mass spectrometry. Mixed effects models tested transplacental transfer efficiency (TTE) over pregnancy. Regularization models, stochastic intervention, and quantile g-computation models tested the association between maternal and placental or cord PFAS levels. TTE increased linearly across trimesters for all PFAS (p<0.001). Quartile increases in maternal PFAS were strongly associated with placental levels (0.018-0.24 ng/g, p<0.001). Stochastic intervention identified T1 PFNA and PFDA; T2 PFOS, PFOA, PFHxS, and PFNA; and T3 PFHxS as robust predictors (p<0.001) of placental levels, consistent with quantile-based contributions. Quartile increases in maternal and placental PFAS concentrations were associated with cord levels (0.08 ng/g-0.55 ng/g, p<0.001). Stochastic intervention identified T1 PFOS and PFHxS; T2 PFOS and PFNA; T3 PFOA; and placental PFOA as important predictors (p<0.05) of cord levels, consistent with quantile-based contributions. Early-to-mid gestation, especially 2nd trimester PFAS measures, were the strongest sentinels of placental and cord serum levels, apart from PFOA which was best reflected by 3rd trimester or placental levels. Placental PFOS and PFOA strongly influenced cord levels. Our findings underscore the heterogeneity in PFAS transfer or metabolism across pregnancy and the placenta.

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.

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.

The management of N-nitrosamine impurities challenges pharmaceutical development and regulation worldwide. Because most medicinal exposures are shorter than lifetime and absolute impurity exclusion is impossible, reliable approaches to define duration-specific intake limits are essential. On the premise that carcinogenic risk is proportional to cumulative dose, the Less-Than-Lifetime (LTL) Threshold of Toxicological Concern (TTC) framework defines progressively lower intake limits for mutagenic impurities over longer exposures. However, N-nitrosamines are currently treated as a cohort of concern, necessitating compound-specific evaluation placing reliance on in vivo mutagenicity assays for impurity qualifications. To better understand durational potency relationships and the application domain of the LTL-TTC, we apply benchmark dose (BMD) modelling to cumulative-dose-scaled transgenic rodent (TGR), error-corrected sequencing and rodent carcinogenicity datasets for N-nitrosodimethylamine (NDMA) obtained from the published literature. For TGR, cumulative-dose scaling better resolved liver as the most sensitive organ and reduced interstudy variability: liver BMDs spanned [~]80-fold in daily-dose units but only [~]20-fold when scaled to cumulative dose. Among closely-matched mouse liver gavage studies, cumulative-dose BMDs only varied by [~]2.5-fold across 1 to 28-day treatment regimens. Error-corrected sequencing also demonstrated parity, with acute-dose regimens producing mutation burdens near-identical (< 1.2-fold) to those cumulated from 28-day repeat-dose regimens. Comparable results were obtained from carcinogenicity datasets confirming proportionality-of-effect to cumulative dose. These findings empirically support the validity of the LTL-TTC concept. More broadly, they demonstrate that short-term in vivo mutagenicity assays can serve as reliable surrogates for lifetime carcinogenicity studies, strengthening the scientific and regulatory basis for duration-adjusted acceptable intakes for N-nitrosamine impurities.

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.

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.

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

Isoprene is a ubiquitous biogenic volatile organic compound (VOC) with atmospheric concentrations comparable to those of methane. Its high reactivity in the atmosphere significantly influences methane concentrations, contributing to adverse effects on the climate. Understanding the role of isoprene and its link to methane metabolism is crucial to addressing climate change. The fate of isoprene and its potential microbial degraders in soil, particularly in anaerobic environments, remains poorly investigated, underscoring the need for comprehensive studies. Our study provides physiological evidence for anaerobic microbial isoprene reduction and its influence on methanogenesis in Eucalyptus-leaf sediment. In our anaerobic culture-based studies, we have demonstrated that isoprene is reduced to three products, 3-methyl-1-butene, 2-methyl-1-butene, and 2-methyl-2-butene. Our findings suggest that Pelotomaculum sp. is capable of anaerobic isoprene reduction, as indicated by 16S rRNA analysis and dilution-to-extinction studies. Anaerobic microbial isoprene reduction simultaneously inhibited methane formation. Methanogenesis was completely inhibited in microcosm cultures amended solely with isoprene. In cultures supplemented with isoprene and carbon sources, only hydrogenotrophic methanogenesis was inhibited, indicating competition for hydrogen between the methanogens and isoprene degraders. Using a culture-based approach and molecular analysis, this study provides novel insights into the microbial dynamics of anaerobic isoprene reduction and the interplay between isoprene reducers and methanogens. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/706888v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@9b559corg.highwire.dtl.DTLVardef@11ab71org.highwire.dtl.DTLVardef@ec759org.highwire.dtl.DTLVardef@41db7b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Elucidating the metabolic mechanisms underpinning complex hydrocarbon biodegradation by microbial consortia benefits from integration of community-level genomic and proteomic data. Here, we deployed a combined metagenomics and metaproteomic analysis to characterize functional dynamics within bacterial consortia enriched from hydrocarbon-contaminated soil, using military waste oil as the sole carbon source. Metagenomic profiling revealed a consortium dominated by Stenotrophomonas maltophilia alongside established hydrocarbon-degrading taxa including Pseudomonas fluorescens, Pseudomonas putida, and Delftia acidovorans. Quantitative label-free metaproteomics uncovered dynamic taxonomic restructuring and metabolic reorientation, with Delftia and Achromobacter proteins enriched during active degradation, indicating niche advantages under chronic contamination. Enzymatic mapping resolved expression of degradation pathways targeting aliphatic and aromatic substrates, with one specific consortium, BPS4, exhibiting preferential catabolism of catechol intermediates channeled toward the tricarboxylic acid cycle, supported by elevated protein abundance of oxidoreductases, dehydrogenases, and {beta}-oxidation enzymes. Gene ontology enrichment revealed heightened nitrogen cycling capacity, including upregulation of nitric oxide biosynthesis and nitrate reductase, suggesting adaptive nitrogen metabolism coupled to hydrocarbon mineralization. In-vitro batch-fed adaptation studies demonstrated limited improvements in degradation of recalcitrant fractions, particularly branched aliphatics and organophosphates, revealing intrinsic metabolic constraints within the consortium. These findings underscore the complementary utility of metaproteomic interrogation for resolving both population-level dynamics and functional metabolic capabilities, while highlighting fundamental limitations in expanding the degradative spectrum of enriched consortia through conventional adaptation strategies. This work establishes a foundation for rational ecological engineering of hydrocarbon-remediating consortia informed by metaproteomics. ImportanceWaste oils from transport and industry are complex and toxic mixtures that are difficult and expensive to treat using conventional chemical methods. Microorganisms offer a sustainable alternative, but their performance depends on how different species work together and where their metabolic limits lie. In this study, we show how advanced protein-based analysis using quantitative metaproteomics, can reveal microbial community activities whilst breaking down real-world waste oils. We enriched bacteria from contaminated soils and then identified the enzymes driving effective degradation. We investigated how communities reorganise under chemical stress, and why certain oil components remain resistant even after long-term adaptation. By uncovering both strengths and limitations of naturally enriched microbial consortia, this work provides practical insights for designing more reliable biological treatments for oil-based wastes and even contaminated environments, supporting greener approaches to pollution management and industrial waste recycling.

The human toxicological risk assessment of per- and polyfluoroalkyl substances (PFAS) is challenging, due to their sheer number and structural diversity, but also the paucity of the toxicity data required to characterize them. The development of Next Generation Physiologically Based Kinetic (NG-PBK) models may assist in overcoming this challenge. The mechanistic nature of NG-PBK models allows for their extrapolation from data-rich PFAS, such as perfluorooctanoic acid (PFOA), to data-poor ones, facilitating their application in Next Generation Risk Assessment (NGRA). The present study proposes a NG-PBK model for PFOA in humans, parametrized exclusively using in vitro-, and in silico-derived data. The model describes the toxicokinetic processes of 1) partitioning to plasma and tissue proteins, 2) partitioning to cell membrane lipids, 3a) transporter-mediated entero-hepatic circulation and 3b) renal elimination and reabsorption, and 4) elimination via menstruation. Global sensitivity analysis indicated that the model was most sensitive to the fraction unbound in plasma, active-transport parameters, and tissue-plasma partition coefficients. The model was equivalent to already available validated human PFOA-PBK models, while compared to those, it is not calibrated to observed animal, nor human data, illustrating its strength in being mechanistic. The serum concentrations and half-lives predicted by the NG-PBK model were within the ranges of those reported in human volunteer and biomonitoring (HBM) studies, demonstrating the models capacity to accurately predict PFOA toxicokinetics on exposure estimates. Extrapolation of the NG-PBK model to other PFAS, in conjunction with its integration with HBM data, will facilitate the NGRA of PFAS. This is particularly relevant given the paucity of in vivo data for most PFAS, ensuring compliance with the 3R principles.

The nervous system is highly vulnerable to chemical disruption, yet current regulatory guidelines do not include behavioral endpoints that capture changes in stress-related responses. Zebrafish larvae, up to 5 days old, have emerged as a promising model to bridge this gap, offering genetic and neurochemical similarity to humans together with high throughput potential. In this work, we have developed and evaluated a larval thigmotaxis assay as a new approach methodology (NAM) to detect behavioral alterations caused by neuroactive substances. Thigmotaxis, or edge-preference behavior, was studied in zebrafish larvae exposed to a range of model compounds and challenged with both visual (light/dark) and acoustic (sound/silence) stimuli. We compared 24 round well plates, commonly used in behavioral assays, with 96 square well plates to increase throughput. The two formats showed equivalent results, supporting the use of the higher-capacity system. Classical controls confirmed assay performance with caffeine increasing thigmotaxis, while diazepam decreased it. Additional neuroactive substances with diverse modes of action (chlorpyrifos, nicotine, dexamethasone, ethylenethiourea) produced stimulus-dependent responses, whereas negative controls (saccharin, amoxicillin) had little or no effect. Benchmark dose modeling showed that thigmotaxis was generally more sensitive than traditional locomotor activity endpoints. Overall, this multiplexed visual-acoustic thigmotaxis assay proved reproducible, scalable, and sensitive. In neurotoxicity testing this method could be used both as a stand-alone assay or as part of a broader behavioral NAM battery to assess potential effects on the vertebrate nervous system. This method provides a practical and ethical tool to improve chemical safety assessment both in ecotoxicology and human toxicology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/703464v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ceea53org.highwire.dtl.DTLVardef@17a2a8borg.highwire.dtl.DTLVardef@17f16b1org.highwire.dtl.DTLVardef@aac24f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Azoxystrobin is a widely used fungicide that has been associated with to reproductive, neurological, and developmental defects. This chemical also disrupts gut microbial communities; however, if these perturbations contribute to the harms associated with exposure to azoxystrobin, this remains unclear. In this study, we investigated the effects of acute exposure to a series of concentrations (5-500 mg/kg) of azoxystrobin on the host and gut microbiota in zebrafish. Fecal amplicon and shotgun metagenomic sequencing was integrated with liver gene expression to quantify associations between microbiome disruption azoxystrobin toxicity in the host. Azoxystrobin exposure resulted in significant alteration in microbiome composition and functional potential in a dose- and sex-dependent manner. Microbial communities in exposed animals exhibited an increased abundance of xenobiotic metabolism pathways and decreased bacterial motility and lipopolysaccharide biosynthesis pathway metabolism. At the host level, histopathology identified increased biliary proliferation, most evident in medium- and high-dose fish. We also observed hepatic transcriptional changes consistent with a stress response, including altered redox-associated genes and reduced expression of lipid and small-molecule metabolic genes, with sex-stratified differences. Importantly, alterations in host transcriptional programming correlated with the compositional changes in exposed microbiota. Together, these results suggest concurrent impacts of azoxystrobin on gut microbiota and the liver implicate the microbiome as a potential contributor to changes in liver gene expression during exposure. ImportanceWidespread fungicide use contaminates ecosystems worldwide, but the biological pathways underlying their effects on humans and other animals are not well understood. Using zebrafish (Danio rerio), we found that short-term exposure to the fungicide azoxystrobin was associated with changes in the gut microbiome, liver gene activity, and liver changes. Exposure produced dose- and sex-dependent shifts in microbial communities, including changes in predicted microbial functions involved in chemical metabolism, bacterial motility and defense. Compositional changes in the microbiome correlated with gene-expression changes consistent with stress and altered metabolism in exposed fish, suggesting that exposure induced disruption may contribute to exposure impact to the host. These results highlight a potential role for the microbiome in mediation of the impacts of azoxystrobin on host physiology. As such microbial based interventions could be a viable strategy to mitigate exposure impacts on health.