Integrated Advanced Molecular Tools Predict In Situ cVOC Degradation Rates: Field Demonstration.

Chlorinated volatile organic compound (cVOC) degradation rate constants are crucial information for site management. Conventional approaches generate rate estimates from the monitoring and modeling of cVOC concentrations. This requires time series data collected along the flow path of the plume. The estimates of rate constants are often plagued by confounding issues, making predictions cumbersome and unreliable. Laboratory data suggest that targeted quantitative analysis of Dehalococcoides mccartyi (Dhc) biomarker genes (qPCR) and proteins (qProt) can be directly correlated with reductive dechlorination activity. To assess the potential of qPCR and qProt measurements to predict rates, we collected data from cVOC-contaminated aquifers. At the benchmark study site, the rate constant for degradation of cis-dichloroethene (cDCE) extracted from monitoring data was 11.0 ± 3.4 yr-1, and the rate constant predicted from the abundance of TceA peptides was 6.9 yr-1. The rate constant for degradation of vinyl chloride (VC) from monitoring data was 8.4 ± 5.7 yr-1, and the rate constant predicted from the abundance of TceA peptides was 5.2 yr-1. At the other study sites, the rate constants for cDCE degradation predicted from qPCR and qProt measurements agreed within a factor of 4. Under the right circumstances, qPCR and qProt measurements can be useful to rapidly predict rates of cDCE and VC biodegradation, providing a major advance in effective site management.

Microbial Reductive Dechlorination by a Commercially Available Dechlorinating Consortium Is Not Inhibited by Perfluoroalkyl Acids (PFAAs) at Field-Relevant Concentrations.

Perfluoroalkyl acids (PFAAs) have been shown to inhibit biodegradation (i.e., organohalide respiration) of chlorinated ethenes. The potential negative impacts of PFAAs on microbial species performing organohalide respiration, particularly Dehalococcoides mccartyi (Dhc), and the efficacy of in situ bioremediation are a critical concern for comingled PFAA-chlorinated ethene plumes. Batch reactor (no soil) and microcosm (with soil) experiments, containing a PFAA mixture and bioaugmented with KB-1, were completed to assess the impact of PFAAs on chlorinated ethene organohalide respiration. In batch reactors, PFAAs delayed complete biodegradation of cis-1,2-dichloroethene (cis-DCE) to ethene. Maximum substrate utilization rates (a metric for quantifying biodegradation rates) were fit to batch reactor experiments using a numerical model that accounted for chlorinated ethene losses to septa. Fitted values for cis-DCE and vinyl chloride biodegradation were significantly lower (p < 0.05) in batch reactors containing ≥50 mg/L PFAAs. Examination of reductive dehalogenase genes implicated in ethene formation revealed a PFAA-associated change in the Dhc community from cells harboring the vcrA gene to those harboring the bvcA gene. Organohalide respiration of chlorinated ethenes was not impaired in microcosm experiments with PFAA concentrations of 38.7 mg/L and less, suggesting that a microbial community containing multiple strains of Dhc is unlikely to be inhibited by PFAAs at lower, environmentally relevant concentrations.

Western diet unmasks transient low-level vinyl chloride-induced tumorigenesis; potential role of the (epi-)transcriptome.

BACKGROUND & AIMS: Vinyl chloride (VC) monomer is a volatile organic compound commonly used in industry. At high exposure levels, VC causes liver cancer and toxicant-associated steatohepatitis. However, lower exposure levels (i.e., sub-regulatory exposure limits) that do not directly damage the liver, enhance injury caused by Western diet (WD). It is still unknown if the long-term impact of transient low-concentration VC enhances the risk of liver cancer development. This is especially a concern given that fatty liver disease is in and of itself a risk factor for the development of liver cancer. METHODS: C57Bl/6 J mice were fed WD or control diet (CD) for 1 year. During the first 12 weeks of feeding only, mice were also exposed to VC via inhalation at sub-regulatory limit concentrations (<1 ppm) or air for 6 h/day, 5 days/week. RESULTS: Feeding WD for 1 year caused significant hepatic injury, which was exacerbated by VC. Additionally, VC increased the number of tumors which ranged from moderately to poorly differentiated hepatocellular carcinoma (HCC). Transcriptomic analysis demonstrated VC-induced changes in metabolic but also ribosomal processes. Epitranscriptomic analysis showed a VC-induced shift of the modification pattern that has been associated with metabolic disease, mitochondrial dysfunction, and cancer. CONCLUSIONS: These data indicate that VC sensitizes the liver to other stressors (e.g., WD), resulting in enhanced tumorigenesis. These data raise concerns about potential interactions between VC exposure and WD. It also emphasizes that current safety restrictions may be insufficient to account for other factors that can influence hepatotoxicity.

Contrasting regulatory effects of organic acids on aerobic vinyl chloride biodegradation in etheneotrophs.

Vinyl chloride (VC) is a common groundwater pollutant generated during anaerobic biodegradation of chlorinated solvents (e.g., trichloroethene (TCE) or tetrachloroethene (PCE)). Aerobic VC biodegradation by etheneotrophs can support anaerobic PCE and TCE bioremediation to achieve complete removal in situ. However, anaerobic bioremediation strategies necessitate biostimulation with electron donors that are fermented in situ, generating organic acids that could influence aerobic VC biodegradation processes. We examined the effect of organic acids (lactate, acetate, propionate, and butyrate) on aerobic VC biodegradation by VC-assimilating etheneotrophs Mycobacterium strain JS60 and Nocardioides strain JS614. Strain JS60 grew on all organic acids tested, while strain JS614 did not respond to lactate. VC-grown strain JS60 fed VC and one or more organic acids showed carbon catabolite repression (CCR) behavior where VC biodegradation occurred only after organic acids were depleted. In contrast, CCR was not evident in VC-grown strain JS614, which degraded VC and organic acids simultaneously. Acetate-grown JS60 showed similar CCR behavior when fed VC and a single organic acid, except that extended lag periods (5-12 days) occurred before VC oxidation ensued. Acetate-grown JS614 fed VC and either acetate or butyrate displayed 5-8 day lag periods before simultaneous VC and organic acid biodegradation. In contrast, acetate-grown JS614 degraded VC and propionate without a significant lag, suggesting a regulatory link between propionate and VC oxidation in JS614. Different global regulatory mechanisms controlling VC biodegradation in the presence of organic acids in etheneotrophs have implications for developing combined anaerobic-aerobic bioremediation strategies at chlorinated ethene-contaminated sites. KEY POINTS: • With organic acids present, VC utilization was repressed in JS60, but not in JS614 • Strain JS60 grew readily on lactate, while strain JS614 did not • Propionate alleviated lag periods for VC utilization in acetate-grown JS614.

Prediction of Dichloroethene Concentration in the Groundwater of a Contaminated Site Using XGBoost and LSTM.

Chlorinated aliphatic hydrocarbons (CAHs) are widely used in agriculture and industries and have become one of the most common groundwater contaminations. With the excellent performance of the deep learning method in predicting, LSTM and XGBoost were used to forecast dichloroethene (DCE) concentrations in a pesticide-contaminated site undergoing natural attenuation. The input variables included BTEX, vinyl chloride (VC), and five water quality indicators. In this study, the predictive performances of long short-term memory (LSTM) and extreme gradient boosting (XGBoost) were compared, and the influences of variables on models' performances were evaluated. The results indicated XGBoost was more likely to capture DCE variation and was robust in high values, while the LSTM model presented better accuracy for all wells. The well with higher DCE concentrations would lower the model's accuracy, and its influence was more evident in XGBoost than LSTM. The explanation of the SHapley Additive exPlanations (SHAP) value of each variable indicated high consistency with the rules of biodegradation in the real environment. LSTM and XGBoost could predict DCE concentrations through only using water quality variables, and LSTM performed better than XGBoost.

Microbial community response to a bioaugmentation test to degrade trichloroethylene in a fractured rock aquifer, Trenton, N.J.

Bioaugmentation is a promising strategy for enhancing trichloroethylene (TCE) degradation in fractured rock. However, slow or incomplete biodegradation can lead to stalling at degradation byproducts such as 1,2-dichloroethene (cis-DCE) and vinyl chloride (VC). Over the course of 7 years, we examined the response of groundwater microbial populations in a bioaugmentation test where an emulsified vegetable oil solution (EOS®) and a dechlorinating consortium (KB-1®), containing the established dechlorinator Dehalococcoides (DHC), were injected into a TCE-contaminated fractured rock aquifer. Indigenous microbial communities responded within 2 days to added substrate and outcompeted KB-1®, and over the years of monitoring, several other notable turnover events were observed. Concentrations of ethene, the end product in reductive dechlorination, had the strongest correlations (P< .05) with members of Candidatus Colwellbacteria but their involvement in reductive dechlorination is unknown and warrants further investigation.DHC never exceeded 0.6% relative abundance of groundwater microbial communities, despite its previously presumed importance at the site. Increased concentrations of carbon dioxide, acetic acid, and methane were positively correlated with increasing ethene concentrations; however, concentrations of cis-DCE and VC remained high by the end of the monitoring period suggesting preferential enrichment of indigenous partial dechlorinators over bioaugmented complete dechlorinators. This study highlights the importance of characterizing in situ microbial populations to understand how they can potentially enhance or inhibit augmented TCE degradation.

Dehalogenation of Chlorinated Ethenes to Ethene by a Novel Isolate, "Candidatus Dehalogenimonas etheniformans".

Dehalococcoides mccartyi strains harboring vinyl chloride (VC) reductive dehalogenase (RDase) genes are keystone bacteria for VC detoxification in groundwater aquifers, and bioremediation monitoring regimens focus on D. mccartyi biomarkers. We isolated a novel anaerobic bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of respiratory dechlorination of VC to ethene. This bacterium couples formate and hydrogen (H2) oxidation to the reduction of trichloro-ethene (TCE), all dichloroethene (DCE) isomers, and VC with acetate as the carbon source. Cultures that received formate and H2 consumed the two electron donors concomitantly at similar rates. A 16S rRNA gene-targeted quantitative PCR (qPCR) assay measured growth yields of (1.2 ± 0.2) × 108 and (1.9 ± 0.2) × 108 cells per µmol of VC dechlorinated in cultures with H2 or formate as electron donor, respectively. About 1.5-fold higher cell numbers were measured with qPCR targeting cerA, a single-copy gene encoding a putative VC RDase. A VC dechlorination rate of 215 ± 40 µmol L-1 day-1 was measured at 30°C, with about 25% of this activity occurring at 15°C. Increasing NaCl concentrations progressively impacted VC dechlorination rates, and dechlorination ceased at 15 g NaCl L-1. During growth with TCE, all DCE isomers were intermediates. Tetrachloroethene was not dechlorinated and inhibited dechlorination of other chlorinated ethenes. Carbon monoxide formed and accumulated as a metabolic by-product in dechlorinating cultures and impacted reductive dechlorination activity. The isolation of a new Dehalogenimonas species able to effectively dechlorinate toxic chlorinated ethenes to benign ethene expands our understanding of the reductive dechlorination process, with implications for bioremediation and environmental monitoring. IMPORTANCE Chlorinated ethenes are risk drivers at many contaminated sites, and current bioremediation efforts focus on organohalide-respiring Dehalococcoides mccartyi strains to achieve detoxification. We isolated and characterized the first non-Dehalococcoides bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of metabolic reductive dechlorination of TCE, all DCE isomers, and VC to environmentally benign ethene. In addition to hydrogen, the new isolate utilizes formate as electron donor for reductive dechlorination, providing opportunities for more effective electron donor delivery to the contaminated subsurface. The discovery that a broader microbial diversity can achieve detoxification of toxic chlorinated ethenes in anoxic aquifers illustrates the potential of naturally occurring microbes for biotechnological applications.

Direct aerobic oxidation (DAO) of chlorinated aliphatic hydrocarbons: A review of key DAO bacteria, biometabolic pathways and in-situ bioremediation potential.

Contamination of aquifers and vadose zones with chlorinated aliphatic hydrocarbons (CAH) is a world-wide issue. Unlike other reactions, direct aerobic oxidation (DAO) of CAHs does not require growth substrates and avoids the generation of toxic by-products. Here, we critically review the current understanding of chlorinated aliphatic hydrocarbons-DAO and its application in bioreactors and at the field scale. According to reports on chlorinated aliphatic hydrocarbons-DAO bacteria, isolates mainly consisted of Methylobacterium and Proteobacterium. Chlorinated aliphatic hydrocarbons-DAO bacteria are characterized by tolerance to a high concentration of CAHs and highly efficient removal of CAHs. Trans-1,2-dichloroethylene (t-DCE) is easily transformed biomass for bacteria, followed by 1,2-dichloroethane (1,2-DCA), dichloromethane (DCM), vinyl chloride (VC) and cis-1,2-dichloroethylene (c-DCE). Significant differences in the maximum specific growth rates were observed with different CAHs and biometabolic pathways for DCM, 1,2-DCA, VC and c-DCE degradation have been successfully parsed. Detection of the functional genes etnC and etnE is useful for the determination of active VC DAO bacteria. Additionally, DAO bacteria have been successfully applied to CAHs in new types of bioreactors with satisfactory results. To the best of the authors' knowledge, only one study on DAO-CAHs was conducted in-situ and resulted in 99% CAH removal. Lastly, we put forward future development prospect of chlorinated aliphatic hydrocarbons-DAO.

Natural Biodegradation of Vinyl Chloride and cis-Dichloroethene in Aerobic and Suboxic Conditions.

Chlorinated ethene (CE) groundwater contamination is commonly treated through anaerobic biodegradation (i.e., reductive dechlorination) either as part of an engineered system or through natural attenuation. Aerobic biodegradation has also been recognized as a potentially significant pathway for the removal of the lower CEs cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC). However, the role of aerobic biodegradation under low oxygen conditions typical of contaminated groundwater is unclear. Bacteria capable of aerobic VC biodegradation appear to be common in the environment, while aerobic biodegradation of cDCE is less common and little is known regarding the organisms responsible. In this study, we investigate the role of aerobic cDCE and VC biodegradation in a mixed contaminant plume (including CEs, BTEX, and ketones) at Naval Air Station North Island, Installation Restoration Site 9. Sediment and groundwater collected from the plume source area, mid-plume, and shoreline were used to prepare microcosms under fully aerobic (8 mg/L dissolved oxygen (DO)) and suboxic (< 1 mg/L DO) conditions. In the shoreline microcosms, VC and cDCE were rapidly degraded under suboxic conditions (100% and 77% removal in < 62 days). In the suboxic VC microcosms, biodegradation was associated with a > 5 order of magnitude increase in the abundance of functional gene etnE, part of the aerobic VC utilization pathway. VC and cDCE were degraded more slowly under fully aerobic conditions (74% and 30% removal) in 110 days. High-throughput 16S rRNA and etnE sequencing suggest the presence of novel VC- and cDCE-degrading bacteria. These results suggest that natural aerobic biodegradation of cDCE and VC is occurring at the site and provide new evidence that low (< 1 mg/L) DO levels play a significant role in natural attenuation of cDCE and VC.

Dehalococcoides mccartyi NIT01, a novel isolate, dechlorinates high concentrations of chloroethenes by expressing at least six different reductive dehalogenases.

This study presents the isolation of a novel strain of Dehalococcoides mccartyi, NIT01, which can completely dechlorinate up to 4.0 mM of trichloroethene to ethene via 1,2-cis-dichroroethene and vinyl chloride within 25 days. Strain NIT01 dechlorinated chloroethenes (CEs) at a temperature range of 25-32 °C and pH range of 6.5-7.8. The activity of the strain was inhibited by salt at more than 1.3% and inactivated by 1 h exposure to 2.0% air or 0.5 ppm hypochlorous acid. The genome of NIT01 was highly similar to that of the Dehalococcoides strains DCMB5, GT, 11a5, CBDB1, and CG5, and all included identical 16S rRNA genes. Moreover, NIT01 had 19 rdhA genes including NIT01-rdhA7 and rdhA13, which are almost identical to vcrA and pceA that encode known dehalogenases for tetrachloroethene and vinyl chloride, respectively. We also extracted RdhAs from the membrane fraction of NIT01 using 0.5% n-dodecyl-ß-d-maltoside and separated them by anion exchange chromatography to identify those involved in CE dechlorination. LC/MS identification of the LDS-PAGE bands and RdhA activities in the fractions indicated cellular expression of six RdhAs. NIT01-RdhA7 (VcrA) and NIT01-RdhA15 were highly detected and NIT01-RdhA6 was the third-most detected. Among these three RdhAs, NIT01-RdhA15 and NIT01-RdhA6 had no biochemically identified relatives and were suggested to be novel functional dehalogenases for CEs. The expression of multiple dehalogenases may support bacterial tolerance to high concentrations of CEs.

Rapamycin attenuates liver injury caused by vinyl chloride metabolite chloroethanol and lipopolysaccharide in mice.

Vinyl chloride (VC) is a prevalent environmental toxicant that is rapidly metabolized within the liver. Its metabolites have been shown to directly cause hepatic injury at high exposure levels. We have previously reported that VC metabolite, chloroethanol (CE), potentiates liver injury caused by lipopolysaccharide (LPS). Importantly, that study showed that CE alone, while not causing damage per se, was sufficient to alter hepatic metabolism and increase mTOR phosphorylation in mice, suggesting a possible role for the mTOR pathway. Here, we explored the effect of an mTOR inhibitor, rapamycin, in this model. C57BL/6 J mice were administered CE, followed by rapamycin 1 h and LPS 24 h later. As observed previously, the combination of CE and LPS significantly enhanced liver injury, inflammation, oxidative stress, and metabolic dysregulation. Rapamycin attenuated not only inflammation, but also restored the metabolic phenotype and protected against CE + LPS-induced oxidative stress. Importantly, rapamycin protected against mitochondrial damage and subsequent production of reactive oxygen species (ROS). The protective effect on mitochondrial function by rapamycin was mediated, by restoring the integrity of the electron transport chain at least in part, by blunting the deactivation of mitochondrial c-src, which is involved mitochondrial ROS production by electron transport chain leakage. Taken together, these results further demonstrate a significant role of mTOR-mediated pathways in VC-metabolite induced liver injury and provide further insight into VC-associated hepatic damage. As mTOR mediated pathways are very complex and rapamycin is a more global inhibitor, more specific mTOR (i.e. mTORC1) inhibitors should be considered in future studies.

Urinary thiodiglycolic acid is associated with increased risk of non-alcoholic fatty liver disease in children living near a petrochemical complex.

BACKGROUND: Occupational and experimental studies have revealed that high vinyl chloride monomer (VCM) exposure is associated with non-alcoholic fatty liver disease (NAFLD). Epidemiological study reported that children living near a petrochemical complex have elevated exposure levels of urinary thiodiglycolic acid (TDGA), a potential VCM biomarker. However, no studies on the association of urinary TDGA exposure with NAFLD in children are available. AIM: To assess the association of pediatric NAFLD with urinary TDGA exposure in school-aged children living near a petrochemical complex. MATERIALS AND METHODS: In total, 261 school-aged children (aged 6-13 years) living near a petrochemical complex were recruited during October 2013 to September 2014. First morning spot urine was sampled for analyzing urinary TDGA through liquid chromatography-tandem mass spectrometry. Ultrasonography and serum alanine aminotransferase (ALT) were examined in each participant. NAFLD was diagnosed as recommended by the North American and European Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN and ESPGHAN). Risk for NAFLD with urinary TDGA exposure in children was evaluated using a multivariate logistic regression model. RESULTS: The percentage of children with NAFLDNASPGHAN and NAFLDESPGHAN were 9.6% and 11.5%, respectively. Median levels (µg/g creatinine) of urinary TDGA of children with NAFLDNASPGHAN (vs non-NAFLDNASPGHAN) and NAFLDESPGHAN (vs non-NAFLDESPGHAN) were 118.0 (vs 96.6) and 113.1 (vs 96.5), respectively. Participants in the highest urinary TDGA quartile (Q4: ≥160.0 µg/g creatinine) had a significantly increased risk (odds ratio [OR] = 4.95; 95% confidence interval [CI] = 1.15-21.38; P = 0.032) and dose-response trend (Ptrend = 0.045) for NAFLDNASPGHAN compared with those in the lowest urinary TDGA quartile (Q1: <35.4 µg/g creatinine) after adjustment for age, gender, BMI, triglycerides, HOMA-IR and distance of elementary schools from the petrochemical complex. Participants in the Q4 had borderline significantly increased risk (OR = 3.45; 95% CI = 0.89-13.42; P = 0.074) correlated with NAFLDESPGHAN compared with those in the Q1 after adjustment for confounders. CONCLUSION: Our findings support the hypothesis that children exposed to higher urinary TDGA levels significantly increased pediatric NAFLD risk. Serum ALT levels can be a useful predictor for screening children's NAFLD in field studies. Large and longitudinal studies are warranted to elucidate the association.

Reductive dechlorination of high concentrations of chloroethenes by a Dehalococcoides mccartyi strain 11G.

Chloroethenes are common groundwater and soil contaminants due to extensive historic utilization and inappropriate discharge. The tendency for chloroethenes to become sequestered as dense non-aqueous phase liquids (DNAPL)-a point source to groundwater contamination and causing high concentrations of chloroethenes in proximal aquifers poses a great challenge for remediation of chloroethene contaminated sites. In this study, we report isolation and characterization of a Dehalococcoides mccartyi strain 11G which couples growth with reductive dechlorination of trichloroethenes (TCE), dichloroethene (DCE) isomers and vinyl chloride (VC) to ethene at a growth yield ranging from 2.47 ± 0.23 × 108 to 5.64 ± 0.43 × 108 cells/µmoles Cl- released and co-metabolically dechlorinates tetrachloroethene (PCE) in the presence of TCE. Compared with previous D. mccartyi strains showing dechlorination of TCE at up to 2.0 mM, strain 11G is distinguished by its capacity to dechlorinate chloroethenes at initial concentrations of DCE isomers as high as 4 mM and TCE as high as 3.5 mM to ethene. Bioaugmentation of a contaminated microcosm with strain 11G resulted in complete detoxification of a mixture of 5 mM chloroethenes (2.5 mM of each TCE and cis-DCE) after 40 days. Strain 11G is a promising candidate for in situ bioremediation of high-concentration-chloroethene contaminated sites.

Feasibility of using urinary TDGA as a biomarker for VCM exposures.

Thiodiglycolic acid (TDGA) is a major metabolite of vinyl chloride monomer (VCM), and it has been suggested as an exposure biomarker for VCM. The validity of this biomarker when the level of VCM is less than 5 ppm, however, is questionable. The objective of this article is to evaluate the feasibility of using urinary TDGA as a biomarker of VCM exposure in a community health risk assessment setting where the concentration of VCM in air is typically very low (likely below 1 ppm). To achieve this objective, we examine the fraction of urinary TDGA associated with different levels of VCM exposures of three studies from different countries, using estimations of the TDGA metabolite predicted by a PBPK model. It is demonstrated that differences in background TDGA have considerable effect on the adequacy of TDGA as a biomarker of VCM. We conclude that, in a community health assessment setting, TDGA should not be used as an exposure biomarker for VCM without having a proper control group, and a PBPK model can be used first to determine whether or not the amount of TDGA in urine is of concern.

Integrated methodological approach reveals microbial diversity and functions in aerobic groundwater microcosms adapted to vinyl chloride.

Vinyl chloride (VC), a known human carcinogen, is often formed in groundwater (GW) by incomplete reductive dechlorination of chlorinated ethenes. An integrated microbial ecology approach involving bacterial enrichments and isolations, carbon stable-isotope probing (SIP) and metagenome and genome sequencing was applied to ethene-fed GW microcosms that rapidly transitioned to aerobic growth on VC. Actinobacteria, Proteobacteria and Bacteroidetes dominated the microbial communities in ethene- and VC-grown cultures. SIP with 13C2-VC demonstrated that Nocardioides spp. significantly participated in carbon uptake from VC (52.1%-75.7% enriched in heavy fractions). Sediminibacterium, Pedobacter and Pseudomonas spp. also incorporated 13C from VC into genomic DNA. Ethene- and VC-assimilating Nocardioides sp. strain XL1 was isolated. Sequencing revealed a large (∼300 kbp) plasmid harboring genes encoding alkene monooxygenase and epoxyalkane: coenzyme M transferase, enzymes known to participate in aerobic VC and ethene biodegradation. The plasmid was 100% identical to pNOCA01 found in VC-assimilating Nocardioides sp. strain JS614. Metagenomic analysis of enrichment cultures indicated other bacteria implicated in carbon uptake from VC possessed the genetic potential to detoxify epoxides via epoxide hydrolase or glutathione S-transferase (Pseudomonas) and/or metabolize VC epoxide breakdown products and downstream VC metabolites. This study provides new functional insights into aerobic VC metabolism within a GW microbial community.

Contrasting growth properties of Nocardioides JS614 on threedifferent vinyl halides.

Ethene (ETH)-grown inocula of Nocardioides JS614 grow on vinyl chloride (VC), vinyl fluoride (VF), or vinyl bromide (VB) as the sole carbon and energy source, with faster growth rates and higher cell yields on VC and VF than on VB. However, whereas acetate-grown inocula of JS614 grow on VC and VF after a lag period, growth on VB did not occur unless supplemental ethene oxide (EtO) was present in the medium. Despite inferior growth on VB, the maximum rate of VB consumption by ETH-grown cells was ~ 50% greater than the rates of VC and VF consumption, but Br- release during VB consumption was non-stoichiometric with VB consumption (~ 66%) compared to 100% release of Cl- and F- during VC and VF consumption. Evidence was obtained for VB turnover-dependent toxicity of cell metabolism in JS614 with both acetate-dependent respiration and growth being significantly reduced by VB turnover, but no VC or VF turnover-dependent toxicity of growth was detected. Reduced growth rate and cell yield of JS614 on VB probably resulted from a combination of inefficient metabolic processing of the highly unstable VB epoxide (t0.5 = 45 s), accompanied by growth inhibitory effects of VB metabolites on acetate-dependent metabolism. The exact role(s) of EtO in promoting growth of alkene repressed JS614 on VB remains unresolved, with evidence of EtO inducing epoxide consuming activity prior to an increase in alkene oxidizing activity and supplementing reductant supply when VB is the growth substrate.

Natural attenuation of chlorinated ethenes in hyporheic zones: A review of key biogeochemical processes and in-situ transformation potential.

Chlorinated ethenes (CEs) are legacy contaminants whose chemical footprint is expected to persist in aquifers around the world for many decades to come. These organohalides have been reported in river systems with concerning prevalence and are thought to be significant chemical stressors in urban water ecosystems. The aquifer-river interface (known as the hyporheic zone) is a critical pathway for CE discharge to surface water bodies in groundwater baseflow. This pore water system may represent a natural bioreactor where anoxic and oxic biotransformation process act in synergy to reduce or even eliminate contaminant fluxes to surface water. Here, we critically review current process understanding of anaerobic CE respiration in the competitive framework of hyporheic zone biogeochemical cycling fuelled by in-situ fermentation of natural organic matter. We conceptualise anoxic-oxic interface development for metabolic and co-metabolic mineralisation by a range of aerobic bacteria with a focus on vinyl chloride degradation pathways. The superimposition of microbial metabolic processes occurring in sediment biofilms and bulk solute transport delivering reactants produces a scale dependence in contaminant transformation rates. Process interpretation is often confounded by the natural geological heterogeneity typical of most riverbed environments. We discuss insights from recent field experience of CE plumes discharging to surface water and present a range of practical monitoring technologies which address this inherent complexity at different spatial scales. Future research must address key dynamics which link supply of limiting reactants, residence times and microbial ecophysiology to better understand the natural attenuation capacity of hyporheic systems.

Triple-element compound-specific stable isotope analysis of 1,2-dichloroethane for characterization of the underlying dehalogenation reaction in two Dehalococcoides mccartyi strains.

Chlorinated ethanes belong to the most common groundwater and soil contaminants. Of these, 1,2-dichloroethane (1,2-DCA) is a man-made, persistent and toxic contaminant, released due to improper waste treatment at versatile production sites. This study investigated the anaerobic transformation of 1,2-DCA by Dehalococcoides mccartyi strain 195 and strain BTF08 using triple-element compound-specific stable isotope analysis of carbon, chlorine and hydrogen for the first time. Isotope fractionation patterns for carbon (εCBTF08 = -28.4 ± 3.7‰; εC195 = -30.9 ± 3.6‰) and chlorine (εClBTF08 = -4.6 ± 0.7‰; εCl195 = -4.2 ± 0.5‰) within both investigated D. mccartyi strains, as well as the dual-element analysis (ΛBTF08 = 6.9 ± 1.2; Λ195 = 7.1 ± 0.2), supported identical reaction mechanisms for dehalogenation of 1,2-DCA. Hydrogen isotope fractionation analysis revealed dihaloelimination as prevalent reaction mechanism. Vinyl chloride as major intermediate could be excluded by performing the experiment in deuterated aqueous media. Furthermore, evaluation of the derived apparent kinetic isotope effects (AKIECBTF08 = 1.029/AKIEC195 = 1.031; AKIEClBTF08 = 1.005/AKIECl195 = 1.004) pointed towards simultaneous abstraction of both involved chlorine-substituents in a concerted matter. It was shown that D. mccartyi strain BTF08 and strain 195 are capable of complete, direct dihaloelimination of 1,2-DCA to ethene.

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes.

Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55±0.64 × 108 cells were measured per µmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.

Simultaneous anaerobic transformation of carbon tetrachloride to carbon dioxide and tetrachloroethene to ethene in a continuous flow column.

The simultaneous anaerobic transformation of tetrachloroethene (PCE) and carbon tetrachloride (CT) was evaluated in a continuous flow column. The column was packed with quartz sand and bioaugmented with the Evanite culture (EV) that is capable of transforming PCE to ethene. Azizian and Semprini (2016) reported that PCE and CT could be simultaneously transformed in the column, with PCE (0.1mM) transformed mainly to ethene and CT (0.015mM) to chloroform (CF) (20%) and an unknown transformation product, likely carbon dioxide (CO2). The fermentation of propionate, formed from lactate fermentation, was inhibited after the transformation of CT, likely from the exposure to CF. Reported here is the second phase of that study where a second bioaugmentation of the EV culture was made to reintroduce a lactate and propionate fermenting population to the column. Effective lactate and propionate fermentation were restored with a H2 concentration of ~25nM maintained in the column effluent. PCE (0.1mM) was effectively transformed to ethene (~98%) and vinyl chloride (VC) (~2%). Unlabeled CT (0.015 to 0.03mM) was completely transformed with a transient build-up of CF and chloromethane (CM), which were subsequently removed below their detection limits. A series of transient tests were initiated through the addition of carbon-13 labeled CT (13CT), with concentrations gradually increased from 0.03 to 0.10mM. GC-MS analysis of the column effluent showed that 13C labeled CO2 (13CO2) was formed, ranging from 82 to 93% of the 13CT transformed, with the transient increases in 13CO2 associated with the increased concentration of 13CT. A modified COD analysis indicated a lesser amount of 13CT (18%) was transformed to soluble products, while 13CO2 represented 82% the 13CT transformed. In a final transient test, the influent lactate concentration was decreased from 1.1 to 0.67mM. The transformation of both CT and PCE changed dramatically. Only 59% of the 13CT was transformed, primarily to CF. 13CO2 concentrations gradually decreased to background levels, indicating CO2 was no longer a transformation product. PCE transformation resulted in the following percentage of products formed: cDCE (60%), VC (36%), and ethene (4%). Incomplete propionate fermentation was also observed, consistent with the build-up of CF and the decrease in H2 concentrations to approximately 2nM. The results clearly demonstrate that high concentrations of CT were transformed to CO2, and effective PCE dehalogenation to ethene was maintained when excess lactate was fed and propionate was effectively fermented. However, when the lactate concentration was reduced, both PCE and CT transformation and propionate fermentation were negatively impacted.