Journal of Hazardous Materials

Meteorite impact structures on earth represent unique terrestrial analogues of extreme planetary environments where fractured lithologies, hydrothermal alteration, elevated concentrations of radionuclides, and prolonged water-rock interactions create ecological niches for specialized microbial communities. The Palaeoproterozoic Dhala impact structure in north-central India is characterized by uranium-bearing impactites and possesses a very high concentration of Uranium (up to 99.5 ppm) that makes it a unique natural laboratory to investigate microbial adaptation to radioactive and metal-rich geological environments. In addition, the Dhala structure harbors specialized radiotoxic hydrothermal mineral phases such as coffinite and pitchblende, which show a globally distinct and biologically unexplored niche. The present investigation is the first in-depth, whole-genome metagenomic study of soil and water samples of uranium-rich sites from the Dhala structure, exploring the whole microbial community (Bacteria, Archaea, Eukaryotes, and Viruses) up to the species level present in the samples. The cross-domain metagenomics analysis identified a highly unique and resilient microbiome that consists of bacteria, archaea, eukaryotes, and viruses, coevolved under prolonged radiological and multiple metal stress. Functional metagenomic analysis revealed numerous metabolic adaptations responsible for their survival, homeostasis, and biomineralization in situ. The deep sequencing method further enabled the identification of 58 functional genes directly involved in uranium bioremediation. Furthermore, this study revealed important pathways for radionuclide reduction, cellular efflux, and biotransformation. Novel findings of the present study point out the in-depth evolutionary relations of biosphere-geosphere interaction within an ancient hypervelocity impact-generated ecosystem. In a nutshell, the Dhala extremophiles with novel genetic makeup present an untapped and promising reservoir for the development of eco-friendly, next-generation microbial bioremediation strategies to efficiently tackle the global anthropogenic contamination of uranium and the challenges of nuclear waste management. Thus, the present study establishes Dhala structure as an unparalleled site for environmental geomicrobiology, microbial evolution, and astrobiology.

Microplastics are pervasive environmental pollutants that pose a growing concern for human health. Oral ingestion is a common route of human microplastic exposure, yet the proteomic response of the gut epithelium to microplastics remains unclear. This study aimed to investigate the cellular effects of pristine and artificially digested microplastic exposure in primary rat duodenal epithelial cells using untargeted proteomics. Cells were exposed to pristine or digested 0.5 um polystyrene microplastics at 10 or 100 ug/mL for 72 hours and were then analyzed by tandem liquid chromatography and mass spectrometry (LC-MS). Proteins that were both significantly different in intensity compared to controls, with a threshold change of 1.3 or greater, were considered to be differentially expressed. This criterion identified 41 differentially expressed proteins after 100 ug/mL pristine MP exposure, with 19 downregulated and 21 upregulated. Following exposure to 100 ug/mL digested MP, only 3 differentially expressed proteins were upregulated and 7 were down regulated, demonstrating the impact of microplastic physicochemistry. FGSEA pathway analysis revealed that 270 Reactome pathways were significantly altered following microplastic exposure in either condition at both concentrations. These pathways contributed to functional domains including protein synthesis, DNA replication, cell cycle control and aerobic respiration. Overall, microplastic exposure was associated with upregulated mitochondrial respiration, and downregulation of nuclear-related processes including DNA synthesis, transcription and cell proliferation. This study provides targets for future investigation (mitochondria and nucleus) and emphasizes the need to consider biological and environmental conditions for in vitro models of microplastic exposure.

Palladium (Pd), a technology critical platinum group metal, is increasingly lost to the environment through anthropogenic activity, yet the molecular basis of Pd uptake and tolerance in plants remains poorly understood. Here, we examine the role of the Arabidopsis thaliana (Arabidopsis) COPT2 copper transporter in Pd transport and plant stress responses. Expression of COPT2 in Saccharomyces cerevisiae conferred Pd sensitivity, indicating a capability for Pd transport. Arabidopsis copt2 loss-of-function mutants accumulated less Pd in shoots and exhibited reduced reactive oxygen species (ROS) production, supporting a direct role for COPT2 in Pd distribution and associated oxidative stress. Our subsequent transcriptome profiling revealed extensive changes under Pd exposure, including downregulation of HMA2 and aquaporins, and upregulation of HMA7, glutathione transferases, and glutamine synthetase GLN1;1, consistent with the activation of metal detoxification and redox homeostasis pathways. Together, these findings identify COPT2 as a key transport protein mediating the uptake of Pd in Arabidopsis and suggest that Pd induces similar detoxification mechanisms as zinc (Zn), copper (Cu) and cadmium (Cd), providing a foundation for engineering plants for precious metal recovery.

Human metabolism converts inorganic arsenic to the pentavalent methylated species MMA(V) and DMA(V), the forms most people excrete, and the forms long read as the end of a detoxification pathway. Whether a transporter sets how much of these metabolites reaches the genome has not been tested in a mammalian cell. We expressed human AQP3, AQP7, AQP9 or AQP10 in HEK293T and MRC5-SV40 cells and measured gamma-H2AX by flow cytometry across dose series of As(V), MMA(V) and DMA(V), pairing every aquaporin with a GFP-Tubulin control and an untransfected mock acquired in the same replicate. As(V) was inactive in HEK293T cells and only weakly active in MRC5-SV40 cells to 20 micromolar, and both methylated species damaged DNA only in the millimolar range, DMA(V) being the more potent of the two in both cell lines. Against that weak baseline, AQP9 and AQP10 raised DMA(V)-induced gamma-H2AX in HEK293T cells by roughly 17 percentage points over the matched control, more than doubling the damage the same exposure produced in control cells, whereas AQP3 and AQP7 changed it not at all. AQP9 alone remained active with MMA(V). The ranking held in MRC5-SV40 fibroblasts at one-sixth the size, and within single wells the damage rose with the amount of AQP9 a cell carried while the control was flat. Aquaglyceroporins therefore discriminate among arsenic species, and AQP9 and AQP10 turn a weakly genotoxic metabolite into a substantially more genotoxic one.

Characterizing the enzymatic accommodation of circular agricultural fertilizers is critical for informing strategies to remediate widespread soil sulfur hunger. Here, we present an exploratory in-silico investigation evaluating the active-site cleft of sulfate thiohydrolase (SoxB) across representative soil Proteobacteria. Following the crystallographic precedent of uncomplexed thiosulfate in PDB 2WDE, site-directed molecular docking indicated that the free thiosulfate polyanion binds favorably within the catalytic pocket (predicted affinity: -3.506 kcal/mol), yielding a substantially lower empirical energy barrier than hydrophobic elemental sulfur (S8, -1.409 kcal/mol). Comparative evaluation against the alkaline-adapted Thiobacillus denitrificans homolog revealed an elevated predicted binding affinity of -4.610 kcal/mol, suggesting a potential structural accommodation in high-pH calcareous soils (pH > 8.0). Unrestrained 5.0 ns all-atom molecular dynamics in explicit TIP3P solvent demonstrated initial structural stability of the unliganded host backbone (RMSD = 1.10 +/- 0.15 Angstrom). Energetic decomposition indicated that ligand association is governed predominantly by electrostatic interactions (delta-E_elec ~ -40 kcal/mol) with basic residues (His146, His269, Trp147; RMSF < 0.60 Angstrom), with a single-trajectory unbinding event observed at 3.2 ns. These computational observations provide an exploratory baseline characterizing the active-site electrostatic landscape of SoxB, generating working hypotheses for downstream empirical soil microcosm and in-planta trials.

Groundwater contamination by lead and nitrate exceeding the permissible limits of the World Health Organization (WHO) and the Bureau of Indian Standards (BIS) of 10 ppb lead and 45 to 50 ppm nitrate poses a severe health risk of systemic toxicity in humans and animals. Microbial nitrate reduction coupled with microbiologically induced calcite precipitation (MICP) proved to be a novel bioremediation strategy to transform soluble lead nitrate into insoluble lead biominerals. This study presents statistical optimization of operational parameters for nitrate reduction and co-precipitation of biogenic lead carbonate-calcium carbonate by nitrate-reducing bacterial consortia. The SEM, TEM with SEAD, and XRD analysis of the biomineral confirmed lead carbonate co-precipitation with CaCO3. A two-level factorial design was applied to screen significant variables and interactive factors that influence nitrate reduction and biomineral precipitation. The highest nitrate reduction, maximum ammonia production, and biogenic Pb-mineralization efficiency of > 90% were obtained at the optimum conditions (initial nitrate requirement of 600 ppm, temperature 37 oC, pH 8, and carbon content of 5.5 g/L) compared to those obtained under the common process parameters. ANOVA with a high correlation coefficient (R2 > 0.99) corroborated the linear model for the biogenic mineralization process. This study on the biogenic removal of Pb by nitrate-reducing bacterial consortia revealed possible opportunities for large-scale treatment of Pb-contaminated surface and subsurface water bodies.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with the potential to disrupt metabolic processes and contribute to carcinogenesis; however, molecular evidence linking alternative and emerging PFAS to ovarian cancer (OC) remains limited. We conducted a biomarker-based case-control study integrating exposureomics with multi-omics profiling. Plasma samples from 128 patients with OC and 128 controls were analyzed for 26 PFAS, untargeted metabolomics, lipidomics, and clinical phenotypes, while urine samples from an additional 100 patients and 100 controls underwent untargeted metabolomic and clinical profiling. Single-compound, burden-based, and mixture models were integrated with PFAS-omics association scans, cross-matrix metabolite prioritization, observational exposure-omics-clinical association-chain analyses, and repeated internal cross-validation. Higher PFAS burdens were consistently associated with OC case status, and DONA, GenX, PFNA, PFPeS, PFUdA, and PFTrDA constituted an OC-prioritized six-PFAS mixture. DONA and GenX exhibited the broadest lipid-related bridging signals, particularly across phospholipids, lysophospholipids, sphingolipids, neutral lipids, and acylcarnitines. Integrated plasma and urine analyses further identified five shared metabolites-elaidic acid, hippuric acid, betaine, corticosterone, and taurocholic acid-that provided incremental and complementary discrimination beyond reference models based on clinical variables and tumor markers in internal cross-validation. Collectively, these findings demonstrate that both alternative and emerging PFAS are associated with a distinct OC-related molecular phenotype involving coordinated lipidomic, metabolomic, and hematologic alterations, while highlighting DONA and GenX as the most prominent exposure-related signals.

Microplastic pollution from homopolymers such as polypropylene (PP), polystyrene (PS), and polyethylene (PE) poses growing health and environmental concerns, highlighting the need for robust polymer-degrading biocatalysts. We previously identified a naturally occurring cysteine-dependent hydrolase (CsdH) from Rhodococcus opacus, featuring a unique Cys160-Lys127-Asp39 catalytic triad and providing a novel mechanism for polyethylene terephthalate (PET) degradation. In this study, we have determined crystal structures of CsdH in complex with PP, PS, PE, and styrene and demonstrated the potential of the enzyme to directly degrade these homopolymers into their respective low molecular weight degradation products using LC-MS. Structural analysis reveals that the enzyme retains a conserved principal access tunnel while undergoing subtle substrate-dependent changes to accommodate different substrates. The enzyme exhibited broad plastic degradation activity with the highest weight loss observed for PE, followed by PP and PS. SEM analysis reveals surface roughness, cracks and grooves, confirming polymer degradation. Further, these results corroborate through FTIR and TGA analysis. These findings establish a strong foundation for enzyme engineering and aim at developing efficient microplastic degradation.

Electrochemical advanced oxidation processes (EAOPs) are increasingly investigated for tertiary wastewater treatment, yet their short-term effects on real wastewater effluent remain poorly constrained. This study investigated real wastewater treatment plant (WWTP) effluent following short-term boron-doped diamond (BDD) electrochemical treatment using an interdigitated double-diamond electrode (iDDE), combined with hydrochemical, stable-isotope and shotgun metagenomic analyses. After 10 min at 7.0 V and 1.02 A, physicochemical parameters and bulk carbon pools remained largely unchanged. In contrast, d18O-DO decreased by 31.3 +/-0.28 permille alongside a small increase in dissolved oxygen (0.16 +/-0.03 mmol/L), indicating substantial oxygen consistent with electrochemical oxygen generation. Shotgun metagenomics revealed pronounced restructuring of the recovered microbial DNA pool, including increases in Comamonadaceae (~12.3% vs. ~25.1%) and Zoogloeaceae (~3.3% vs. ~8.4%), whereas the oxidative-stress-defence gene repertoire showed no coherent response. These taxonomic changes cannot be interpreted as selective survival, as metagenomics do not resolve viability or intra-/extracellular DNA contribution. Overall, short-term iDDE treatment affected the microbial component of real WWTP effluent despite comparatively limited changes in bulk chemistry, highlighting that EAOP effects extend beyond conventional disinfection.

Biodiversity is globally threatened by chemical pollution, yet toxicity data remain unavailable for millions of species and tens of thousands of chemicals, severely limiting our ability to assess ecological impacts. Here we present TRIDENT-2, a multimodal artificial intelligence model for predicting chemical toxicity across evolutionarily diverse eukaryotic species. Trained on 560,780 toxicity assays spanning 82,775 chemicals, 6,793 species, and multiple exposure scenarios, TRIDENT-2 accurately predicts toxicity across Eukaryota with an average median absolute error ranging from 1.76 to 3.80. By jointly learning from chemical, biological, and experimental information, it remains accurate across broad chemical and taxonomic distances, allowing for toxicity assessment for species and chemicals beyond the current experimental evidence. Our findings demonstrate that artificial intelligence can help overcome longstanding data limitations in ecotoxicology, paving the way for improved decision-making and reducing chemical impacts on biodiversity and ecosystems.

Inhalation of airborne per- and polyfluoroalkyl substances (PFAS) represents an important yet understudied exposure route. While volatile organic compounds (VOCs) have been widely explored as biomarkers of toxicant exposure, their potential for early detection of PFAS-induced pulmonary toxicity remains largely unexplored. This study investigated VOC profiles in human alveolar (A549) and bronchial (BEAS-2B) epithelial cells following exposure to environmentally relevant PFAS concentrations (1, 5, and 10 ppb) for 12, 24, and 48 h. VOCs were analyzed using headspace solid-phase microextraction coupled with gas chromatography-Orbitrap mass spectrometry. We observed significant concentration- and time-dependent alterations in VOC profiles in both cell models. The principal VOCs associated with PFAS exposure include butanal, acetone, 2-butanone, 2-nonanone, (E)-2-nonenal, (E)-2-octenal, (E)-2-heptenal, 2,4-heptadienal, 2,4-nonadienal, 2,4-decadienal, 1-pentanol, and 1-hexanol. Cell type-specific regulations were observed, reflecting differential metabolic responses between transformed and non-transformed lung epithelial cells. Metabolite set enrichment analysis indicated perturbations in ketone body metabolism, consistent with altered cellular energy metabolism. Complementary qPCR analysis revealed altered expressions of CYP1A1, CYP2E1, ALDH1A1, and ALDH2, supporting a mechanistic link between PFAS exposure and oxidative stress, lipid peroxidation, and impaired aldehyde detoxification. These findings demonstrate that VOC profiling, combined with transcriptional analysis, can reveal PFAS-induced metabolic perturbations in pulmonary epithelial cells.

In the enzymatic toolbox of depollution, hydrolytic dehalogenases are of particular interest for the bioremediation of organohalogenated compounds. To better identify and characterize these enzymes, it is important to develop appropriate sensing methods. Fluorescent probes for dehalogenase detection have been developed in the past years, however, to this date none were available for application using the common FITC parameters (i.e. excitation laser at 488 nm). To address this limitation, we herein report a new fluorescent probe, COU4-Cl, excitable in the violet (405 nm) and blue (488 nm) wavelengths adapted for the detection and characterization of haloalkane dehalogenases. Its applicability is discussed and evaluated in comparison to the previously described COU2-Cl probe only excitable in the violet (405 nm). This study shows the advantages and complementarity of the two probes, and further demonstrate their application for the discrimination of dehalogenase activity in whole cell assays.

Introduction. Polycyclic aromatic hydrocarbon (PAH) toxicology has been primarily defined by host metabolic bioactivation to reactive intermediates. Yet ingested PAHs and biliary-delivered metabolites also enter the intestinal lumen, where they encounter the gut microbiota, an additional but understudied metabolic compartment that may alter toxicant fate. Although PAH interactions are well characterized in environmental microbes, resulting in defined enzymatic pathways for their catabolism, whether gut commensal organisms express related machinery in vivo remains poorly understood. To address this gap, we used an oral murine-exposure model of naphthalene and integrated compartment-resolved 16S rRNA gene profiling, global metaproteomics, and chemoproteomics to resolve the PAH-processing repertoire of the gut microbiota and its spatial distribution. Results. Naphthalene, a common environmental toxicant and representative PAH, elicited a compartment-dependent functional response that was most pronounced in the large intestine. Distally, exposure was associated with 2,386 differential proteins and accounted for 48.4% of metaproteomic variation, compared with 7.9% of 16S rRNA gene compositional variation. The small intestine showed a reduced proteomic response, with 994 differentially abundant proteins. Hydrocarbon catabolic markers showed a 4.8-fold distal expansion (36 to 172 proteins), including the naphthalene-catabolic nahE, proteins associated with phenylacetate, {beta}-ketoadipate, catechol/TOL, and anaerobic CoA-linked redox machinery. The small-intestinal repertoire decreased from 19 to 14 proteins, though aromatic-catabolic markers doubled, indicating a selective expansion. Critically, these homologs concentrated in Lachnospiraceae, a gut family that is prevalent in humans, primarily known for anaerobic fermentation, with no priorly described PAH-processing capacity. An uncultured Acetatifactor showed the largest hydrocarbon hits (14 to 82 protein increase), with additional contributions from Kineothrix, Eisenbergiella, and Petralouisia. Chemoproteomics, using a naphthalene-based probe, revealed enrichment of an orthogonal proteome of proteins interacting with naphthalene, capturing 171 small-intestinal and 67 large-intestinal proteins that were undetected in the global metaproteomes. Conclusions. Gut commensals express homologs of aromatic-hydrocarbon catabolic enzymes characterized in environmental microorganisms, and naphthalene amplifies this repertoire most strongly in the large intestine. The decoupling of taxonomic structure, expressed-protein representation, and probe engagement identifies a functional dimension of PAH exposure that composition-based profiling does not resolve and positions microbiome variation as a candidate modifier of PAH metabolic fate.

The acquisition of sufficient nitrogen for growth, as well as the disposal of highly toxic nitrogen-containing degradation products, represent serious metabolic challenges in all multicellular organisms and microbial ecosystems. Understanding these processes requires a broadly applicable system to accurately quantify the concentration of a wide spectrum of biological amines and inorganic nitrogen-containing compounds. To date, this has been challenging due to limited specificity, selectivity, breadth and/or sensitivity of assays. Here we present a simple, optimized method based on liquid chromatography and high-resolution mass spectrometry, with an accompanying analysis pipeline, for the accurate quantification of more than 30 of these compounds, including ammonia. We demonstrate the effectiveness of this method in quantifying relevant nitrogen-containing compounds in mouse and human gut content, bacterial cultures, algal symbiosis in a cnidarian and freshwater ecosystem samples. As a proof-of-concept, we applied the method to study the influence of the gut microbiome on cecal nitrogen distribution in mice with different gnotobiotic microbiomes. This revealed a profound influence of gut microbiota composition on amino acid, ammonia, and amine concentrations in the upper large intestine. Overall, we provide a broadly applicable approach that can be used across microbial ecology fields to generate novel insights into the biology of nitrogen assimilation, exchange, and disposal.

The unprecedented rise in greenhouse gases such as carbon dioxide (CO2), and their detrimental effects on the atmosphere have intensified the current climate emergency. This challenge has inspired the development of novel carbon capture and utilisation (CCU) technologies, specifically microbial electrosynthesis (MES), for the bioelectrochemical fixation of CO2 into commodity chemical compounds (CCCs) using microbial biocatalysts. In this study, we systematically enriched and maintained robust mixed microbial communities of electroactive bacteria (EAB) from wastewater treatment sludge to serve as biocatalysts for MES. The MES performance of these biocatalysts under different operational conditions in bioelectrochemical systems (BES) reactors were investigated. BES reactors with enriched biocatalysts incubated and maintained under controlled conditions produced higher CCCs yields than those developed at ambient temperatures, while strict anaerobic operations further improved the MES efficiency compared to aerobic conditions. At an applied potential of -1000 mV vs Ag/AgCl, CO2 conversion reached up to 88.11%, significantly outperforming the lower potential (-600 mV) operation. These findings underscore the importance of systematic development of robust and stable biocatalysts, as well as maintaining strict anaerobic conditions, for achieving efficient MES performance, thereby demonstrating the potential of our approach for scalable industrial CCU applications.

Environmental exposure involves pollutant mixtures. Building on evidence that individual bisphenols alter zebrafish larval physiology and transcriptomic profiles at ng/L concentrations, we assessed the effects of specific mixtures of BPS, BPF, and BPAF without (M1) or with (M2) BPA. Experimentally, M1 and M2 induced 4.16% and 6.54% mortality in zebrafish larvae, despite the concentration addition model predicting no toxicity. Both mixtures reduced swimming-locomotor activities. These behaviors were accompanied by organ-level functional defects, as in vivo optical mapping revealed bradycardia and increased high-frequency (30-80 Hz) brain oscillations for both mixtures, while M2 additionally altered low-frequency (0.2-4 Hz) brain activity. The neurophysiological changes were accompanied by neurovascular remodeling, with in vivo two-photon microscopy revealing decreased vascular length and branching. Finally, transcriptomic analysis revealed common M1- and M2-induced alterations linked to pathways of vascular function, neuronal communication, and metabolism. M2 altered additional pathways of cardiac and neuronal signaling, consistent with the reported organ-physiology effects. We discuss the environmental relevance of mixture exposures and assess whether BPA intensifies mixture-induced responses.

Antimicrobial resistance (AMR) is a major threat to modern medicine and an emerging environmental pollutant. Wastewater-based monitoring offers a scalable alternative to traditional surveillance by enabling population-level monitoring across One Health compartments. However, it remains unclear how well bacterial variants detected in wastewater reflect those causing clinical infections. Here, we assessed the prevalence of carbapenem-resistant Escherichia coli (CREC) in wastewater from Uppsala, Sweden, and compared wastewater isolates with clinical isolates from the same region and period. We observed partial overlap between wastewater and clinical CREC populations, but several common clinical variants were not detected in wastewater and vice versa. CREC prevalence was an order of magnitude higher in wastewater receiving hospital effluent, yet this did not improve concordance with clinical isolates, suggesting that hospital wastewater may artificially inflate CREC prevalence estimates. These findings indicate that wastewater CREC prevalence does not necessarily reflect the regional clinical burden. Furthermore, metagenomic sequencing of wastewater-derived E. coli populations rarely detected CREC resistance genes, suggesting that CREC abundance is below the detection limit of this approach and that targeted enrichment is required for reliable monitoring. Together, our results show that while wastewater-based monitoring can complement AMR surveillance, it should be interpreted cautiously when used as an indicator of clinical risk.

Genetic variation impacts biological response to chemical and environmental exposures, contributing to differences in resistance or susceptibility. Forward genetic screens of genetically diverse cell populations can be used to identify the precise genetic variants that drive response variation. Cell lines from laboratory mouse genetic reference populations like the Diversity Outbred (DO) population are well powered for genetic mapping. Diverse stem cell panels are especially beneficial for studying early developmental effects of chemical exposures like triphenyl phosphate (TPHP), an organophosphate flame retardant linked to adverse developmental effects, including altered cell cycle in stem cells. Self-renewal and pluripotency are defining properties of stem cells during development; therefore, it is important to understand how chemicals like TPHP may alter these critical characteristics. To better understand the influence of genetic variation on TPHP exposure response, we utilized a genetically diverse panel of DO induced pluripotent stem cells (DO iPSCs). Our analyses characterize interline variation in gene expression through differential gene expression analysis and expression quantitative trait locus (eQTL) mapping. We identified genomic loci that contribute to variation in gene expression following TPHP exposure, including a regulatory hotspot on chromosome 15. Gene set enrichment highlighted several pathways affected by TPHP including lipid metabolism, steroid hormone signaling, and cell cycle. Our study shows that genetic variation modulates the effects of TPHP on gene expression and cell cycle in stem cells. Additionally, our work demonstrates that genetically diverse cell panels like the DO iPSC resource offer a tractable and scalable approach for identifying the genetic determinants of chemical toxicity.

Pyrogenic black carbon, such as biochar and wildfire char, possesses a reversible electron storage capacity (ESC), allowing it to serve as an electron donor or acceptor for microorganisms. However, not all the ESC is bioavailable, and the proportion available varies by culture. We hypothesized microbes capable of producing soluble redox mediators could access the ESC in biochar interior and thus utilize a higher percentage of the ESC than organisms without a mediator. To test this hypothesis, we grew wild-type Shewanella oneidensis (MR-1) and {Delta}bfe MR-1, a mutant incapable of exporting flavins, on air-oxidized wood biochar. Cell counts and redox titration show that both strains of MR-1 grew to similar cell densities and accessed comparable amounts of biochar ESC (~19%). The addition of exogenous flavin mononucleotide (FMN) yielded no measurable increase in final cell density, due to the strong adsorption of FMN to biochar. SEM images show attachment of MR-1 and {Delta}bfe to biochar surfaces and the presence of nanowires. This study demonstrates MR-1 can utilize biochar ESC for growth, through direct physical contact without the involvement of flavins. Understanding how microbes metabolize biochar provides insight into the impacts and biogeochemical roles of pyrogenic carbon.

Nanoplastics (NPs) are widespread environmental contaminants, yet most toxicity studies rely on short-term, single-dose exposures that poorly reflect daily human exposure. The kidney, particularly the proximal tubule, is a likely site of repeated NP exposure given its role in filtration and reabsorption, but chronic exposure effects are largely unexplored. Here, a long-term in vitro model was developed in which human proximal tubule (HK-2) cells were continuously exposed to 20 nm or 100 nm carboxylated polystyrene NPs (100 {micro}g/mL) over 12 days. Continuous 20 nm NP exposure caused progressive loss of viability whereas 100 nm NPs left viable cell numbers largely unaffected. RNA-sequencing showed 20 nm NPs drove extensive, time-dependent transcriptional dysregulation (1,548 differentially expressed genes at day 6, rising to 4,431 at day 9), far exceeding the minimal alterations in gene expression (35 genes) caused by 100 nm NPs. Cell-cycle analysis and pathway enrichment revealed KRAS pathway activation and S-phase arrest in 20 nm-treated cells, with ANGPTL4 the most upregulated gene. Validation with qRT-PCR confirmed increased ANGPTL4, HMOX1 and FN1 expression, genes implicated in renal fibrosis and chronic kidney disease. These findings show that continuous, size-dependent NP exposure elicits progressive cytotoxic and transcriptional responses in kidney proximal tubule cells and indicate ANGPTL4 as a candidate early biomarker of NP-induced renal stress.