Dual C-Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures.

Brominated organic compounds such as 1,2-dibromoethane (1,2-DBA) are highly toxic groundwater contaminants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C-Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide-respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εbulkC values (-1.8 ± 0.2 and -19.2 ± 3.5‰, respectively) were obtained, whereas their respective εbulkBr values were lower and similar to each other (-1.22 ± 0.08 and -1.2 ± 0.5‰), leading to distinctly different trends (ΛC-Br = Δδ13C/Δδ81Br ≈ εbulkC/εbulkBr) in a dual C-Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2-reaction). The strongly pathway-dependent ΛC-Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εbulkC values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites.

Effect of biostimulation and bioaugmentation on biodegradation of high concentrations of 1,4-dioxane.

1,4-Dioxane is a pervasive and persistent contaminant in numerous aquifers. Although the median concentration in most contaminant plumes is in the microgram per liter range, a subset of sites have contamination in the milligram per liter range. Most prior studies that have examined 1,4-dioxane concentrations in the hundreds of milligrams per liter range have been performed with industrial wastewater. The main objective of this study was to evaluate aerobic biodegradation of 1,4-dioxane in microcosms prepared with soil and groundwater from a site where concentrations range from ~ 1500 mg·L-1 in the source zone, to 450 mg·L-1 at a midpoint of the groundwater plume, and to 6 mg·L-1 at a down-gradient location. Treatments included biostimulation with propane, addition of propane and a propanotrophic enrichment culture (ENV487), and unamended. The highest rates of biodegradation for each location in the plume occurred in the bioaugmented treatments, although indigenous propanotrophs also biodegraded 1,4-dioxane to below 25 µg·L-1. Nutrient additions were required to sustain biodegradation of propane and cometabolism of 1,4-dioxane. Among the unamended treatments, biodegradation of 1,4-dioxane was detected in the mid-gradient microcosms. An isolate was obtained that grows on 1,4-dioxane as a sole source of carbon and energy and identified through whole-genome sequencing as Pseudonocardia dioxivorans BERK-1. In a prior study, the same strain was isolated from an aquifer in the southeastern United States. Monod kinetic parameters for BERK-1 are similar to those for strain CB1190.

Predicting COVID-19 Infected Individuals in a Defined Population from Wastewater RNA Data.

Wastewater surveillance of SARS-CoV-2 RNA has become an important tool for tracking the presence of the virus and serving as an early indicator for the onset of rapid transmission. Nevertheless, wastewater data are still not commonly used to predict the number of infected individuals in a sewershed. The main objective of this study was to calibrate a susceptible-exposed-infectious-recovered (SEIR) model using RNA copy rates in sewage (i.e., gene copies per liter times flow rate) and the number of SARS-CoV-2 saliva-test-positive infected individuals in a university student population that was subject to repeated weekly testing during the Spring 2021 semester. A strong correlation was observed between the RNA copy rates and the number of infected individuals. The parameter in the SEIR model that had the largest impact on calibration was the maximum shedding rate, resulting in a mean value of 7.72 log10 genome copies per gram of feces. Regressing the saliva-test-positive infected individuals on predictions from the SEIR model based on the RNA copy rates yielded a slope of 0.87 (SE=0.11), which is statistically consistent with a 1:1 relationship between the two. These findings demonstrate that wastewater surveillance of SARS-CoV-2 can be used to estimate the number of infected individuals in a sewershed.

Evaluation of natural attenuation of 1,4-dioxane in groundwater using a 14C assay.

Monitored Natural Attenuation (MNA) is a preferred remedy for sites contaminated with 1,4-dioxane due to its low cost and limited environmental impacts compared to active remediation. Having a robust estimate of the rate at which biodegradation occurs is an essential component of assessing MNA. In this study, an assay was developed using 14C-labeled 1,4-dioxane to measure rate constants for biodegradation based on accumulation of 14C products. Purification of the 14C-1,4-dioxane stock solution lowered the level of 14C impurities to below 1% of the total 14C activity. This enabled determination of rate constants in groundwater as low as 0.0021 yr-1, equating to a half-life greater than 300 years. Of the 54 groundwater samples collected from 10 sites in the US, statistically significant rate constants were determined with the 14C assay for 24. The median rate constant was 0.0138 yr-1 (half-life = 50 yr); the maximum rate constant was 0.367 yr-1 (half-life = 1.9 yr). The results confirmed that biodegradation of 1,4-dioxane is occurring at 9 of the 10 sites sampled, albeit with considerable variability in the level of activity. The specificity of the assay was confirmed using acetylene and the absence of oxygen to inhibit monooxygenases.

Establishing the prevalence and relative rates of 1,4-dioxane biodegradation in groundwater to improve remedy evaluations.

Options for remediating 1,4-dioxane at groundwater sites are limited due to the physical-chemical properties of this compound. The relevance of natural attenuation processes for 1,4-dioxane was investigated through data from field, lab, and modeling efforts. The objectives were to use multiple lines of evidence for 1,4-dioxane biodegradation to understand the prevalence of this activity and evaluate convergence between lines of evidence. A 14C-1,4-dioxane assay confirmed 1,4-dioxane biodegradation at 9 of 10 sites (median rate constant of 0.0105 yr-1 across wells). Site-wide rate constants were established using a calibrated fate and transport model at 8 sites (median = 0.075 yr-1). The 14C assay constants are likely more conservative, and variability in rates suggested that biodegradation at sites may be localized. Stable isotope fractionation was observed at 7 of 10 sites and served as another direct line of evidence of in situ biodegradation of 1,4-dioxane. This includes sites where indirect lines of evidence, including geochemical conditions or genetic biomarkers for degradation, would not necessarily have been supportive. This highlights the importance of collecting multiple lines of evidence to document 1,4-dioxane natural attenuation, and the widespread prevalence of biodegradation suggests that this process should be part of long-term management decisions.

Anaerobic Biodegradation of Chloroform and Dichloromethane with a Dehalobacter Enrichment Culture.

Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. Complete dechlorination of CF has been reported under anaerobic conditions by microbes that respire CF to DCM and others that biodegrade DCM. The objectives of this study were to ascertain if a commercially available bioaugmentation enrichment culture (KB-1 Plus CF) uses an oxidative or fermentative pathway for biodegradation of DCM and to determine if the products from DCM biodegradation can support organohalide respiration of CF to DCM in the absence of an exogenous electron donor. In various treatments with the KB-1 Plus CF culture to which 14C-CF was added, the predominant product was 14CO2, indicating that oxidation is the predominant pathway for DCM. Recovery of 14C-DCM when biodegradation was still in progress confirmed that CF first undergoes reductive dechlorination to DCM. 14C-labeled organic acids, including acetate and propionate, were also recovered, suggesting that synthesis of organic acids provides a sink for the electron equivalents from oxidation of DCM. When the biomass was washed to remove organic acids from prior additions of exogenous electron donor and only CF and DCM were added, the culture completely dechlorinated CF. The total amount of DCM added was not sufficient to provide the electron equivalents needed to reduce CF to DCM. Thus, the additional reducing power came via the DCM generated from CF reduction. Nevertheless, the rate of CF consumption was considerably lower compared to that of treatments that received an exogenous electron donor. IMPORTANCE Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. One way to address this problem is to add microbes to the subsurface that can biodegrade these compounds. While microbes are known that can accomplish this task, less is known about the pathways used under anaerobic conditions. Some use an oxidative pathway, resulting mainly in carbon dioxide. Others use a fermentative pathway, resulting in formation of organic acids. In this study, a commercially available bioaugmentation enrichment culture (KB-1 Plus CF) was evaluated using carbon-14 labeled chloroform. The main product formed was carbon dioxide, indicating the use of an oxidative pathway. The reducing power gained from oxidation was shown to support reductive dechlorination of CF to DCM. The results demonstrate the potential to achieve full dechlorination of CF and DCM to nonhazardous products that are difficult to identify in the field.

COVID-19 wastewater epidemiology: a model to estimate infected populations.

BACKGROUND: Wastewater-based epidemiology provides an opportunity for near real-time, cost-effective monitoring of community-level transmission of SARS-CoV-2. Detection of SARS-CoV-2 RNA in wastewater can identify the presence of COVID-19 in the community, but methods for estimating the numbers of infected individuals on the basis of wastewater RNA concentrations are inadequate. METHODS: This is a wastewater-based epidemiology study using wastewater samples that were collected weekly or twice a week from three sewersheds in South Carolina, USA, between either May 27 or June 16, 2020, and Aug 25, 2020, and tested for SARS-CoV-2 RNA. We developed a susceptible-exposed-infectious-recovered (SEIR) model based on the mass rate of SARS-CoV-2 RNA in the wastewater to predict the number of infected individuals, and have also provided a simplified equation to predict this. Model predictions were compared with the number of confirmed cases identified by the Department of Health and Environmental Control, South Carolina, USA, for the same time period and geographical area. FINDINGS: We plotted the model predictions for the relationship between mass rate of virus release and numbers of infected individuals, and we validated this prediction on the basis of estimated prevalence from individual testing. A simplified equation to estimate the number of infected individuals fell within the 95% confidence limits of the model. The rate of unreported COVID-19 cases, as estimated by the model, was approximately 11 times that of confirmed cases (ie, ratio of estimated infections for every confirmed case of 10·9, 95% CI 4·2-17·5). This rate aligned well with an independent estimate of 15 infections for every confirmed case in the US state of South Carolina. INTERPRETATION: The SEIR model provides a robust method to estimate the total number of infected individuals in a sewershed on the basis of the mass rate of RNA copies released per day. This approach overcomes some of the limitations associated with individual testing campaigns and thereby provides an additional tool that can be used to inform policy decisions. FUNDING: Clemson University, USA.

Chlorinated Ethene Degradation Rate Coefficients Simulated with Intact Sandstone Core Microcosms.

Abiotic transformation of trichloroethene (TCE) in fractured porous rock such as sandstone is challenging to characterize and quantify. The objective of this study was to estimate the pseudo first-order abiotic reaction rate coefficients in diffusion-dominated intact core microcosms. The microcosms imitated clean flow through a fracture next to a contaminated rock matrix by exchanging uncontaminated groundwater, unamended or lactate-amended, in a chamber above a TCE-infused sandstone core. Rate coefficients were assessed using a numerical model of the microcosms that were calibrated to monitoring data. Average initial rate coefficients for complete dechlorination of TCE to acetylene, ethene, and ethane were estimated as 0.019 y-1 in unamended microcosms and 0.024 y-1 in lactate-amended microcosms. Moderately higher values (0.026 y-1 for unamended and 0.035 y-1 for lactate-amended) were obtained based on 13C enrichment data. Abiotic transformation rate coefficients based on gas formation were decreased in unamended microcosms after ∼25 days, to an average of 0.0008 y-1. This was presumably due to depletion of reductive capacity (average values of 0.12 ± 0.10 µeeq/g iron and 18 ± 15 µeeq/g extractable iron). Model-derived rate coefficients and reductive capacities for the intact core microcosms aligned well with results from a previous microcosm study using crushed sandstone from the same site.

Impact of biological wastewater treatment on the reactivity of N-Nitrosodimethylamine precursors.

N-Nitrosodimethylamine (NDMA) is a probable human carcinogen which forms during chloramination of wastewater-impacted drinking waters. Municipal wastewater effluents are considered as major sources of NDMA precursors affecting downstream water quality. To evaluate the deactivation mechanisms and efficiencies of NDMA precursors during secondary treatment with the activated sludge (AS) process, NDMA formation potentials (FPs) of selected model precursor compounds and sewage components (i.e., blackwaters and greywaters) were monitored in batch AS treatment tests. After 24-h incubation with four different types of AS (i.e., domestic rural, domestic urban, textile and lab-grown AS), NDMA FP of trimethylamine (TMA) and minocycline (MNCL) decreased by 77-100%, while there was only 29-46% reduction in NDMA FP of sumatriptan (SMTR). The reduction in NDMA FP associated with ranitidine (RNTD) varied between 34% and 87%. The decrease in NDMA FP of RNTD depended on the AS type, hydraulic retention time (HRT) and solids retention time (SRT). The domestic AS (rural and urban) achieved higher decreases in NDMA FPs of the tested model precursors than the textile AS or lab-grown AS. Increasing the HRT or SRT enhanced NDMA FP decrease for RNTD. Among different processes tested (i.e., biodegradation, biosorption and volatilization), biosorption was the major mechanism responsible for the NDMA FP decrease of RNTD, MNCL and SMTR, while biodegradation was the major NDMA FP reduction mechanism for TMA. The reduction in NDMA FP of RNTD via biodegradation depended on the AS activity which may vary with sampling seasons and SRT. NDMA FPs in all tested sewage components (i.e., blackwaters and greywaters) decreased after 24-h AS treatment. Urine in blackwater was the predominant (i.e., >90%) contributor to NDMA FP in domestic sewage and AS-treated effluents.

Simulation of in situ biodegradation of 1,4-dioxane under metabolic and cometabolic conditions.

Bioaugmentation is an option for aerobic remediation of groundwater contaminated with 1,4-dioxane. One approach uses microbes that cometabolize 1,4-dioxane following growth on a primary substrate (e.g., propane), whereas another uses microbes (e.g., Pseudonocardia dioxivorans CB1190) capable of using 1,4-dioxane as a sole substrate. The relative merits of these approaches are difficult to distinguish based on field data alone, and theoretical analyses of these processes have yet to be published. The objective of this study was to compare these remediation options using a transport model that incorporates advection, dispersion and biodegradation reactions described by multi-substrate Monod kinetics and co-inhibition effects. The transport model was coupled to an approximate steady-state air sparging simulation used to estimate gas (propane and oxygen) distribution at the field scale. The model was calibrated with field data for 1,4-dioxane and propane concentrations from a previously reported pilot study. The two remediation approaches were evaluated under different conditions that vary the initial concentration of 1,4-dioxane and the loading rates of oxygen, propane, and biomass. The metrics used to evaluate the remediation success were the time to reach an average 1,4-dioxane concentration of 1 µg L-1 and the percent of 1,4-dioxane biodegraded after 10 years of simulation. Results indicate that the initial concentration of 1,4-dioxane strongly influences which remediation approach is more effective. When initial concentrations were <10 mg L-1, propane-driven cometabolism led to faster remediation; whereas metabolic biodegradation was faster when initial concentrations were 10 mg L-1 or higher. Below 0.25 mg L-1, the viability of metabolic biodegradation improved, although cometabolism by propanotrophs still required less time to reach 1 µg L-1. Biomass injection rates had a strong effect on the rate of metabolism but not cometabolism because continuous input of primary substrate supported growth of propanotrophs. The performance of both cultures was negatively affected by a decrease in oxygen injection rate. The endogenous decay coefficient and the dispersion rate for biomass had a significant impact on cometabolic and metabolic biodegradation of 1,4-dioxane. The maximum specific rate for cometabolism of 1,4-dioxane, the dispersion rate for 1,4-dioxane, and effective porosity also had significant effects on the time to achieve remediation with propanotrophs.

Diffusion-Coupled Degradation of Chlorinated Ethenes in Sandstone: An Intact Core Microcosm Study.

Matrix diffusion must be considered when assessing natural attenuation and remediation of chlorinated ethenes in fractured porous bedrock aquifers. In this study, intact sandstone rock and groundwater from a trichloroethene (TCE)-contaminated site were used in microcosms (maintained for approximately 600 days) to simulate a single fracture-matrix system with a chamber at the top of the core allowing advection to represent fracture flow. Diffusion-coupled degradation with and without biostimulation were evaluated and compared to crushed-rock, batch microcosms. In the diffusion-transport microcosms, lactate stimulated reductive dechlorination of TCE to cis-1,2-dichloroethene (cDCE) and sulfate reduction. Reduction of TCE to cDCE led to a higher rate of chlorinated ethene removal from the cores, likely due to higher concentration gradients, along with lower sorption and a higher diffusion coefficient for cDCE relative to TCE. Reduction of cDCE to vinyl chloride or ethene did not occur as in crushed rock microcosms, inferring an absence of Dehalococcoides in the intact cores. Abiotic transformation was evident in the core microcosms based on the appearance of acetylene and enrichment in δ13C-TCE and δ13C-cDCE. Core microcosms permit a more realistic representation of the behavior of chlorinated ethenes in water-saturated fractured porous rock by incorporating the combined influence of fracture flow and matrix diffusion on transport and transformation.

Draft Genome Sequence of the 1,4-Dioxane-Degrading Bacterium Pseudonocardia dioxanivorans BERK-1.

Pseudonocardia dioxanivorans strain BERK-1 grows aerobically with 1,4-dioxane as its sole substrate. Reported here is its draft genome sequence, with a size of 7.1 Mbp. Key genes are highlighted in this article. BERK-1 exhibits a reduced level of cell aggregation and adherence to surfaces compared to those of P. dioxanivorans CB1190, giving it an apparent advantage for movement through soil.

Aerobic biodegradation kinetics for 1,4-dioxane under metabolic and cometabolic conditions.

Biodegradation of 1,4-dioxane has been studied extensively, however, there is insufficient information on the kinetic characteristics of cometabolism by propanotrophs and a lack of systematic comparisons to metabolic biodegradation. To fill in these gaps, experiments were performed with suspended growth cultures to determine 16 Monod kinetic coefficients that describe metabolic consumption of 1,4-dioxane by Pseudonocardia dioxanivorans CB1190 and cometabolism by the propanotrophic mixed culture ENV487 and the propanotroph Rhodococcus ruber ENV425. Maximum specific growth rates were highest for ENV425, followed by ENV487 and CB1190. Half saturation constants for 1,4-dioxane for the propanotrophs were one-half to one-quarter those for CB1190. Propane was preferentially degraded over 1,4-dioxane, but the reverse did not occur. A kinetic model was used to simulate batch biodegradation of 1,4-dioxane. Propanotrophs decreased 1,4-dioxane from 1000 to 1 µg/L in less time than CB1190 when the initial biomass concentration was 0.74 mg COD/L; metabolic biodegradation was favored at higher initial biomass concentrations and higher initial 1,4-dioxane concentrations. 1,4-Dioxane biodegradation was inhibited when oxygen was below 1.5 mg/L. The kinetic model provides a framework for comparing in situ biodegradation of 1,4-dioxane via bioaugmentation with cultures that use the contaminant as a growth substrate to those that achieve biodegradation via cometabolism.

Remediation of chlorinated ethenes in fractured sandstone by natural and enhanced biotic and abiotic processes: A crushed rock microcosm study.

Biostimulation was identified as a potential technology to treat a fractured sandstone aquifer contaminated with trichloroethene (TCE) and cis-1,2-dichloroethene (cis-DCE). Most of the mass of TCE and cis-DCE resides within the rock matrix and strategies to restore groundwater to pre-existing conditions are severely limited by back diffusion. A microcosm study using crushed rock and groundwater from the site was performed to assess biostimulation and natural attenuation. Lactate, hydrogen release compound® (HRC), and emulsified vegetable oil (EVO) significantly increased the rate of TCE reduction to cis-DCE. Lactate also stimulated dechlorination of cis-DCE to vinyl chloride (VC) and ethene, suggesting the presence of indigenous Dehalococcoides. Illumina sequencing and qPCR analyses suggest that reductive dechlorination of TCE to cis-DCE is mediated by Geobacter spp. while Dehalococcoides spp. perform reduction of cis-DCE to VC and ethene. The rate of VC reduction to ethene was much slower than the reduction of TCE to cis-DCE and cis-DCE to VC, indicating the indigenous Dehalococcoides perform the final step co-metabolically. This was confirmed in enrichment cultures fed with only VC. Consequently, biostimulation may create an elevated risk due to transient accumulation of VC. Abiotic transformation of TCE and cis-DCE was observed based on accumulation of 14C-labeled products from 14C-TCE and 14C-cis-DCE, as well as enrichment in δ13C-cis-DCE in the absence of reductive dechlorination. Based on accumulation rates for 14C-products in unamended microcosms, pseudo-first-order rates for abiotic transformation were 0.038 yr-1 for TCE and 0.044 yr-1 for cis-DCE. These rates within the rock matrix may be sufficient to support natural attenuation in this diffusion controlled system.

Hydrogen Isotope Fractionation during the Biodegradation of 1,2-Dichloroethane: Potential for Pathway Identification Using a Multi-element (C, Cl, and H) Isotope Approach.

Even though multi-element isotope fractionation patterns provide crucial information with which to identify contaminant degradation pathways in the field, those involving hydrogen are still lacking for many halogenated groundwater contaminants and degradation pathways. This study investigates for the first time hydrogen isotope fractionation during both aerobic and anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) using five microbial cultures. Transformation-associated isotope fractionation values (εbulkH) were -115 ± 18‰ (aerobic C-H bond oxidation), -34 ± 4‰ and -38 ± 4‰ (aerobic C-Cl bond cleavage via hydrolytic dehalogenation), and -57 ± 3‰ and -77 ± 9‰ (anaerobic C-Cl bond cleavage via reductive dihaloelimination). The dual-element C-H isotope approach (ΛC-H = Δδ2H/Δδ13C ≈ εbulkH/εbulkC, where Δδ2H and Δδ13C are changes in isotope ratios during degradation) resulted in clearly different ΛC-H values: 28 ± 4 (oxidation), 0.7 ± 0.1 and 0.9 ± 0.1 (hydrolytic dehalogenation), and 1.76 ± 0.05 and 3.5 ± 0.1 (dihaloelimination). This result highlights the potential of this approach to identify 1,2-DCA degradation pathways in the field. In addition, distinct trends were also observed in a multi- (i.e., Δδ2H versus Δδ37Cl versus Δδ13C) isotope plot, which opens further possibilities for pathway identification in future field studies. This is crucial information to understand the mechanisms controlling natural attenuation of 1,2-DCA and to design appropriate strategies to enhance biodegradation.

Bench-scale evaluation of aerosol delivery for biostimulation and bioaugmentation in the vadose zone.

Aerosol delivery was evaluated for distributing biostimulation and bioaugmentation amendments in vadose zones. This technique involves transporting amendments as micron-scale aerosol droplets in injected gas. Microcosm experiments were designed to characterize reductive dechlorination of trichloroethene (TCE) under unsaturated conditions when delivering components as aerosols. Delivering amendments and/or microbes as aqueous aerosols resulted in complete dechlorination of TCE, similar to controls operated under saturated conditions. Reductive dechlorination was achieved with manual injection of a bioaugmentation culture suspended in soybean oil into microcosms. However, aerosol delivery of the culture in soybean oil induced little reductive dechlorination activity. Overall, the results indicate that delivery as aqueous aerosols may be a viable option for delivery of amendments to enhance vadose zone bioremediation at the field-scale.

Biodegradation of high concentrations of halomethanes by a fermentative enrichment culture.

A fermentative enrichment culture (designated DHM-1) that grows on corn syrup was evaluated for its ability to cometabolically biodegrade high concentrations of chloroform (CF), carbon tetrachloride (CT), and trichlorofluoromethane (CFC-11). When provided with corn syrup and vitamin B12 (0.03 mol B12 per mol CF), DHM-1 grew and biodegraded up to 2,000 mg/L of CF in 180 days, with only minor transient accumulation of dichloromethane and chloromethane. CT (15 mg/L) and CFC-11 (25 mg/L) were also biodegraded without significant accumulation of halomethane daughter products. The rate of CF biodegradation followed a Michaelis-Menten-like pattern with respect to the B12 concentration; one-half the maximum rate (66 mg CF/L/d) occurred at 0.005 mol B12 per mol CF. DHM-1 was able to biodegrade 500 mg/L of CF at an inoculum level as low as 10(-8) mg protein/L. The highest rate of CF biodegradation occurred at pH 7.7; activity decreased substantially below pH 6.0. DHM-1 biodegraded mixtures of CT, CFC-11, and CF, although CFC-11 inhibited CF biodegradation. Evidence for compete defluorination of CFC-11 was obtained based on a fluoride mass balance. Overall, the results suggest that DHM-1 may be effective for bioaugmentation in source zones contaminated with thousands of milligrams per liter of CF and tens of milligrams per liter of CT and CFC-11.

Isolation of an aerobic vinyl chloride oxidizer from anaerobic groundwater.

Vinyl chloride (VC) is a known human carcinogen and common groundwater contaminant. Reductive dechlorination of VC to non-toxic ethene under anaerobic conditions has been demonstrated at numerous hazardous waste sites. However, VC disappearance without stoichiometric production of ethene has also been observed at some sites and in microcosms. In this study we identify an organism responsible for this observation in presumably anaerobic microcosms and conclude that oxygen was not detectable based on a lack of color change from added resazurin. This organism, a Mycobacterium sp. closely related to known VC oxidizing strains, was present in high numbers in 16S rRNA gene clone libraries from a groundwater microcosm. Although the oxidation/reduction indicator resazurin remained in the clear reduced state in these studies, these results suggest inadvertent oxygen contamination occurred. This study helps to elucidate the dynamic behavior of chlorinated ethenes in contaminated groundwater, through the isolation of a strictly aerobic organism that may be responsible for at least some disappearance of VC without the concomitant production of ethene in groundwater considered anaerobic.

Anaerobic oxidation of ethene coupled to sulfate reduction in microcosms and enrichment cultures.

Ethene is considered recalcitrant under anaerobic conditions, but biological reduction to ethane and oxidation to CO2 have been reported; however, little is known about these processes or the organisms carrying them out. In this report we describe sulfate dependent ethene consumption in microcosms prepared with sediments from a freshwater canal. A first dose of 0.6 mmol/L ethene was consumed within 77 days, and a second dose was largely consumed twelve days later. Material from this microcosm was transferred into growth medium with ethene as the only electron donor (except for trace amounts of vitamins) and sulfate as the electron acceptor. Four doses of ethene were consumed at increasing rates, and the cultures have been transferred at least eight times in this medium. Conversion of [(14)C]ethene primarily to (14)CO2 was demonstrated in fifth and sixth generation cultures, as well as production of sulfide in other cultures, confirming the ethene/sulfate couple. Ovoid cells 1-2 µm in diameter were found in cultures containing ethene and sulfate, and quantitative PCR showed large increases in bacterial 16S rRNA gene copy number. Over half of a 16S rRNA gene clone library from a sixth-generation culture was a phylotype with a sequence ca. 90% identical with a clade of Deltaproteobacteria that includes Desulfovirga adipica and several Syntrophobacter spp. These studies have solidified the concept that deficits in mass balances for chloroethene fate in sulfate reducing zones of contaminated groundwater sites may be due to ethene oxidation, and suggest a unique phylotype is involved in this process.

Anaerobic abiotic transformations of cis-1,2-dichloroethene in fractured sandstone.

A fractured sandstone aquifer at an industrial site is contaminated with trichloroethene to depths greater than 244 m. Field data indicate that trichloroethene is undergoing reduction to cis-1,2-dichloroethene (cDCE); vinyl chloride and ethene are present at much lower concentrations. Transformation of cDCE by pathways other than reductive dechlorination (abiotic and/or biotic) is of interest. Pyrite, which has been linked to abiotic transformation of chlorinated ethenes, is present at varying levels in the sandstone. To evaluate the possible role of pyrite in transforming cDCE, microcosms were prepared with groundwater, ~40 mg L(-1) cDCE+[(14)C]cDCE, and crushed solids (pure pyrite, pyrite-rich sandstone, or typical sandstone). During 120 d of incubation, the highest level of cDCE transformation occurred with typical sandstone (11-14% (14)CO(2), 1-3% (14)C-soluble products), followed by pyrite-rich sandstone (2-4% (14)CO(2), 1% (14)C-soluble products) and even lesser amounts with pure pyrite. These results indicate pyrite is not likely the mineral involved in transforming cDCE. A separate experiment using only typical sandstone compared the rate of cDCE transformation in non-sterilized, autoclaved, and propylene-oxide sterilized treatments, with pseudo-first order rate constants of 8.7, 5.4, and 1.0 yr(-1), respectively; however, transformation stopped after several months of incubation. Autoclaving increased the volume of pores, adsorption pore diameter, and surface area in comparison to non-sterilized typical sandstone. Nevertheless, autoclaving was less disruptive than chemical sterilization. The results provide definitive experimental evidence that cDCE undergoes anaerobic abiotic and biotic transformation in typical sandstone, with formation of CO(2) and soluble products.