Evaluation of isotopic enrichment factors for the biodegradation of chlorinated ethenes using a parameter estimation model: toward an improved quantification of biodegradation

A model was developed to predict the concentrations of chlorinated ethenes and ethene during sequential reductive dechlorination of tetrachloroethene (PCE) from stable carbon isotope values using Rayleigh model principles and specified isotopic enrichment factors for each step of dechlorination. The model was tested using three separate datasets of concentration and isotope values measured during three experiments involving the degradation of PCE to vinyl chloride (VC), trichloroethene (TCE) to ethene, and cis-1,2-dichloroethene (cDCE) to ethene. The model was then coupled to a parameter estimation method to estimate values for the isotopic enrichment factors of TCE, cDCE, and VC when they are intermediates in the dechlorination to ethene. The enrichment factors estimated for TCE and cDCE when they were intermediates in biodegradation experiments were close to or within the published range of enrichment factors determined from experiments where TCE or cDCE were the initial substrates. In contrast, the enrichment factors determined by parameter estimation for experiments in which VC was an intermediate in biodegradation experiments were consistently more negative (by approximately 10 per thousandth) than the most negative published enrichment factor determined from experiments where VC was the initial substrate. This finding suggests that the range of enrichment factors for VC dechlorination may not be as narrow as previously suggested (-21.5 per thousandth to -26.6 per thousandth) and that fractionation during VC dechlorination when VC is an intermediate compound may be significantly larger than when VC is the initial substrate. These findings have important implications both for the current practice of extrapolating laboratory-derived isotopic enrichment factors to quantify biodegradation of chlorinated ethenes in the field and for understanding the details of enzymatic reductive dechlorination.     

Acetylene inhibition of trichloroethene and vinyl chloride reductive dechlorination

Kinetic studies reported here have shown that acetylene is a potent reversible inhibitor of reductive dehalogenation of trichloroethene (TCE) and vinyl chloride (VC) by a mixed dehalogenating anaerobic culture. The mixed culture was enriched from a contaminated site in Corvallis, OR, and exhibited methanogenic, acetogenic, and reductive dehalogenation activities. The H2-fed culture transformed TCE to ethene via cis-dichloroethene (c-DCE) and VC as intermediates. Batch kinetic studies showed acetylene reversibly inhibited reduction of both TCE and VC, and the levels of inhibition were strongly dependent on acetylene concentrations in both cases. Acetylene concentrations of 192 and 12 microM, respectively, were required to achieve 90% inhibition in rates of TCE and VC transformation at an aqueous concentration of 400 microM. Acetylene also inhibited methane production (90% inhibition at 48 microM) but did not inhibit H2-dependent acetate production. Mass balances conducted during the studies of VC inhibition showed that acetogenesis, VC transformation to ethene, and methane production were responsible for 52%, 47%, and 1% of the H2 consumption, respectively. The results indicate that halorespiration is the dominant process responsible for VC and TCE transformation and that dehalorespiring organisms are the target of acetylene inhibition. Acetylene has potential use as a reversible inhibitor to probe the biological activities of reductive dechlorination and methanogenesis. It can be added to inhibit reactions and then removed to permit reactions to proceed. Thus, it can be a powerful tool for investigating intrinsic and enhanced anaerobic remediation of chloroethenes at contaminated sites. The results also suggest that acetylene produced abiotically by reactions of chlorinated ethenes with zero-valent iron could inhibit the biological transformation of VC to ethene.     

Quantifying the effects of 1,1,1-trichloroethane and 1,1-dichloroethane on chlorinated ethene reductive dehalogenases

Mixtures of chlorinated ethenes and ethanes are often found at contaminated sites. In this study, we undertook a systematic investigation of the inhibitory effects of 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA) on chlorinated ethene dechlorination in three distinct Dehalococcoides-containing consortia. To focus on inhibition acting directly on the reductive dehalogenases, dechlorination assays used cell-free extracts prepared from cultures actively dechlorinating trichloroethene (TCE) to ethene. The dechlorination assays were initiated with TCE, cis-1,2-dichloroethene (cDCE), or vinyl chloride (VC) as substrates and either 1,1,1-TCA or 1,1-DCA as potential inhibitors. 1,1,1-TCA inhibited VC dechlorination similarly in cell suspension and cell-free extract assays, implicating an effect on the VC reductases associated with the dechlorination of VC to nontoxic ethene. Concentrations of 1,1,1-TCA in the range of 30-270 μg/L reduced VC dechlorination rates by approximately 50% relative to conditions without 1,1,1-TCA. 1,1,1-TCA also inhibited reductive dehalogenases involved in TCE and cDCE dechlorination. In contrast, 1,1-DCA had no pronounced inhibitory effects on chlorinated ethene reductive dehalogenases, indicating that removal of 1,1,1-TCA via reductive dechlorination to 1,1-DCA is a viable strategy to relieve inhibition.     

Carbon isotopes as a tool to evaluate the origin and fate of vinyl chloride: laboratory experiments and modeling of isotope evolution

Accumulation of vinyl chloride (VC) is often a main concern at sites contaminated with chlorinated ethenes and ethanes due to its high toxicity. Since there can be several possible sources of VC and ethene at such sites, assessing the origin and fate of VC can be complicated. Aim of this study was to evaluate carbon isotope fractionation associated with various anaerobic processes that lead to the production of VC and ethene in view of using isotopes to evaluate the origin and fate of these compounds in groundwater. Microcosms were constructed using sediments and groundwater from a contaminated site and amended with potential precursors for VC and ethene production. In the microcosms with dichloroethene isomers, sequential reductive dechlorination was observed, and isotopic enrichmentfactors of -19.9 +/- 1.5 per thousand for cis-1,2-dichloroethene, -30.3 +/- 1.9 per thousand for trans-1,2-dichloroethene, and -7.3 +/- 0.4 per thousand for 1,1-dichloroethene were obtained. In microcosms with chlorinated ethanes, 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) were predominantly transformed by dichloroelimination to ethene and VC, respectively, and enrichmentfactors of -32.1 +/- 1.1 per thousand for 1,2OCA and -2.0 +/- 0.2 per thousand for 1,1,2-TCA were observed. Except for 1,1,2-TCA, a strong 13C enrichment in each of the potential precursor of VC was observed, which opens the possibility to trace the origin of VC based on the isotope ratio of potential precursors. Furthermore, it was possible to model the isotope evolution of VC present as substrate or intermediate product as a function of time. The study demonstrates that carbon isotope ratios can potentially be used for qualitative and possibly quantitative evaluation of the origin and fate of VC at sites with complex contaminant mixtures.     

Growth and yields of dechlorinators, acetogens, and methanogens during reductive dechlorination of chlorinated ethenes and dihaloelimination of 1 ,2-dichloroethane

The population dynamics of a mixed microbial culture dechlorinating trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), 1,2-dichloroethane (1,2-DCA), and vinyl chloride (VC) to ethene were studied. Quantitative PCR revealed that Dehalococcoides, Geobacter, Sporomusa, Spirochaetes, and Methanomicrobiales phylotypes grew in short-term experiments. Both Geobacter and Dehalococcoides populations grew during TCE dechlorination to cDCE, but only Dehalococcoides populations grew during further dechlorination to ethene. The cell yields for Dehalococcoides determined in this study were similar on an electron equivalent basis regardless of the chlorinated compound transformed: (0.9+/-0.3) x 10(8)16S rRNA gene copies/microelectron equivalent (microeeq) ethene produced during cDCE dechlorination, (1.5 +/-0.3) x 10(8) copies/microeeq ethene produced during VC dechlorination, and (1.6+/-0.8) x 10(8) copies/ u,eeq ethene produced during 1,2-DCA dihaloelimination. The yield for the Geobacter population on TCE was estimated to be (1+/-0.5) x 10(8) copies/microeeq cDCE produced. Calculations showed that the Geobacter population was likely responsible for approximately 80% of the TCE dechlorinated to cDCE in this experiment. Acetogenesis by a Sporomusa population was the main competition to dechlorination for reducing equivalents. Sporomusa did not transform any chlorinated substrates tested, but was capable of converting methanol to acetate and hydrogen for dechlorination. Understanding the functions of various populations in mixed communities may explain why Dehalococcoides spp. are active at some sites and not others, and may also assist in optimizing the growth of bioaugmentation cultures, both in the laboratory and in the field.     

Experimental evidence for a lack of thermodynamic control on hydrogen concentrations during anaerobic degradation of chlorinated ethenes

Hydrogen (H2) concentrations during reductive dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) were investigated with respectto the influence of parameters entering the Gibbs free energy expression of the reactions. A series of laboratory experiments was conducted employing a mixed, Dehalococcoides-containing enrichment culture capable of complete dechlorination of chlorinated ethenes. The objective was to investigate whether a constant energy gain controls H2 levels in dechlorinating systems, thereby evaluating the applicability of the partial equilibrium approach to microbial dechlorination at contaminated sites. Variations in the temperature between 10 and 30 degrees C did not affect the H2 concentration in a fashion that suggested thermodynamic control through a constant energy gain. In another set of experiments, H2 levels at constant ionic strength were independent of the chloride concentration between 10 and 110 mmol chloride per liter. These findings demonstrate that the partial equilibrium approach is not directly applicable to the interpretation of reductive degradation of chlorinated ethenes. We also present recalculated thermodynamic properties of aqueous chlorinated ethene species that allow for calculation of in-situ Gibbs free energy of dechlorination reactions at different temperatures.     

Monitoring biodegradation of ethene and bioremediation of chlorinated ethenes at a contaminated site using compound-specific isotope analysis (CSIA)

Chlorinated ethenes are commonly found in contaminated groundwater. Remediation strategies focus on transformation processes that will ultimately lead to nontoxic products. A major concern with these strategies is the possibility of incomplete dechlorination and accumulation of toxic daughter products (cis-1,2-dichloroethene (cDCE), vinyl chloride (VC)). Ethene mass balance can be used as a direct indicator to assess the effectiveness of dechlorination. However, the microbial processes that affect ethene are not well characterized and poor mass balance may reflect biotransformation of ethene rather than incomplete dechlorination. Microbial degradation of ethene is commonly observed in aerobic systems but fewer cases have been reported in anaerobic systems. Limited information is available on the isotope enrichment factors associated with these processes. Using compound-specific isotope analysis (CSIA) we determined the enrichment factors associated with microbial degradation of ethene in anaerobic microcosms (ε = -6.7‰ ± 0.4‰, and -4.0‰ ± 0.8‰) from cultures collected from the Twin Lakes wetland area at the Savannah River site in Georgia (United States), and in aerobic microcosms (ε = -3.0‰ ± 0.3‰) from Mycobacterium sp. strain JS60. Under anaerobic and aerobic conditions, CSIA can be used to determine whether biotransformation of ethene is occurring in addition to biodegradation of the chlorinated ethenes. Using δ(13)C values determined for ethene and for chlorinated ethenes at a contaminated field site undergoing bioremediation, this study demonstrates how CSIA of ethene can be used to reduce uncertainty and risk at a site by distinguishing between actual mass balance deficits during reductive dechlorination and apparent lack of mass balance that is related to biotransformation of ethene.     

Anaerobic degradation of cis-1,2-dichloroethylene and vinyl chloride by Clostridium sp. strain DC1 isolated from landfill leachate sediment

The bacterial community structure of anaerobic enrichment cultures that are capable of degrading both cis-1,2-dichloroethylene (cis-DCE) and vinyl chloride (VC) and isolation of the organism responsible for the degradation were investigated. Denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified 16S rRNA gene from the cultures showed the possible predominance of Clostridium species. One isolate, designated strain DC1, was closely related to members of Clostridiaceae, based on 16S rRNA gene analysis, and the highest sequence similarity (98.9%) was obtained for Clostridium saccarobutylicum. In culture experiments, strain DC1 was shown to degrade cis-DCE and VC during the stationary phase of growth without accumulation of VC and/or ethene. The bacterial growth was not linked to the degradation of cis-DCE and VC. Stoichiometric analysis revealed that two moles of chloride ions as released from one mole of cis-DCE during the incubation period, indicating that cis-DCE was fully dechlorinated. The results appear consistent with the presence of a mechanism of oxidative dechlorination rather than respiratory reductive dechlorination; the latter is accompanied by transient formation of dechlorinated ethenes from cis-DCE and VC.     

Vinyl chloride and cis-dichloroethene dechlorination kinetics and microorganism growth under substrate limiting conditions

The reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) at contaminated sites often results in the accumulation of cis-1,2-dichloroethene (DCE) and vinyl chloride (VC), rather than the nonhazardous end product ethene. This accumulation may be caused by the absence of appropriate microorganisms, insufficient supply of donor substrate, or reaction kinetic limitations. Here, we address the issue of reaction kinetic limitations by investigating the effect of limiting substrate concentrations (electron donor and acceptor) on DCE and VC dechlorination kinetics and microorganism growth by bacterium VS. For this, a model based on Monod kinetics, but also accounting for competition between electron acceptors and the effect of low electron donor and acceptor concentrations (dual-substrate kinetics), was examined. Competitive coefficients for VC (7.8 +/- 1.5 microM) and DCE (3.6 +/- 1.1 microM) were obtained and included in the model. The half velocity coefficient for hydrogen, the electron donor, was experimentally determined (7 +/- 2 nM) through investigating dechlorination over different substrate concentrations. This complete model was then used, along with experimental data, to determine substrate concentrations at which the dechlorinating microorganisms would be in net decay. Notably, the model indicates net decay will result if the total electron acceptor concentration (DCE plus VC) is below 0.7 microM, regardless of electron donor levels. The ability to achieve sustainable bioremediation to acceptable levels can be greatly influenced by this threshold level.     

Identifying abiotic chlorinated ethene degradation: characteristic isotope patterns in reaction products with nanoscale zero-valent iron

Carbon isotope fractionation is of great interest in assessing chlorinated ethene transformation by nanoscale zero-valent iron at contaminated sites, particularly in distinguishing the effectiveness of an implemented abiotic degradation remediation scheme from intrinsic biotic degradation. Transformation of trichloroethylene (TCE), cis-dichloroethylene (cis-DCE), and vinyl chloride (VC) with two types of nanoscale iron materials showed different reactivity trends, but relatively consistent carbon isotope enrichment factors (epsilon) of -19.4 per thousand +/- 1.8 per thousand (VC), -21.7 per thousand +/- 1.8 per thousand (cis-DCE), and -23.5 per thousand +/- 2.8 per thousand (TCE) with one type of iron (FeBH), and from -20.9 per thousand +/- 1.1 per thousand to -26.5 per thousand +/- 1.5 per thousand (TCE) with the other (FeH2). Products of the dichloroelimination pathway (ethene, ethane, and acetylene) were consistently 10 per thousand more isotopically depleted than those of the hydrogenolysis pathway (cis-DCE from TCE, VC from cis-DCE), displaying a characteristic pattern that may serve as an indicator of abiotic dehalogenation reactions and as a diagnostic parameter for differentiating the effects of abiotic versus biotic degradation. In contrast, the product-related enrichment factors of each respective pathway varied significantly in different experiments. Because such variation would not be expected for independent pathways with constant kinetic isotope effects, our data give preliminary evidence that the two pathways may share an irreversible first reaction step with subsequent isotopically sensitive branching.     

Stable carbon isotope fractionation of chloroethenes by dehalorespiring isolates

Stable carbon isotope fractionation during the reductive dechlorination of chloroethenes by two bacterial strains that dechlorinate to ethene, Dehalococcoides ethenogenes 195 and Dehalococcoides sp. strain BAV1 as well as Sulfurospirillum multivorans and Dehalobacter restrictus strain PER-K23, isolates that do not dechlorinate past DCE, are reported. Fractionation by a Dehalococcoides-containing enrichment culture is also measured for comparison to the isolates. All data adequately fit the Rayleigh model and results are presented as enrichment factors. For strain 195, the measured enrichment factors were -9.6 +/- 0.4, -21.1 +/- 1.8, and -5.8 +/- 0.5 when degrading TCE, cDCE, and 1,1-DCE, respectively. Strain BAV1 exhibited enrichment factors of -16.9 +/- 1.4, -8.4 +/- 0.3, -21.4 +/- 0.9, and -24.0 +/- 2.0 for cDCE, 1,1-DCE, tDCE, and VC, respectively. The surprisingly large differences in enrichment factors caused by individual reductases (RDases) reducing different chloroethenes is likely the result of chemical structure differences among the chloroethenes. For TCE reduction, S. multivorans and D. restrictus strain PER-K23 exhibited enrichment factors of -16.4 +/- 1.5 and -3.3 +/- 0.3, respectively. While all of the organisms studied here utilize RDases that require corrinoid cofactors, the biotic TCE enrichment factors varied widely from those reported for the abiotic cobalamin-catalyzed reaction, indicating that additional factors affect the extent of fractionation in these biological systems. The enrichment factors measured for the Dehalococcoides-containing enrichment culture did not match well with those from any of the isolates, demonstrating the inherent difficulties in predicting fractionation factors of undefined communities. Although compound-specific isotope fractionation is a powerful tool for evaluating the progress of in situ bioremediation in the field, given the wide range of enrichment factors associated with functionally similar and phylogenetically diverse organisms, caution must be exercised when applying enrichment factors for the interpretation of dechlorination data.     

Kinetics and inhibition of reductive dechlorination of chlorinated ethylenes by two different mixed cultures

Kinetic studies with two different anaerobic mixed cultures (the PM and the EV cultures) were conducted to evaluate inhibition between chlorinated ethylenes. The more chlorinated ethylenes inhibited the reductive dechlorination of the less chlorinated ethylenes, while the less chlorinated ethylenes weakly inhibited the dechlorination of the more chlorinated ethylenes. Tetrachloroethylene (PCE) inhibited reductive trichloroethylene (TCE) dechlorination but not cis-dichloroethylene (c-DCE) dechlorination, while TCE strongly inhibited c-DCE and VC dechlorination. c-DCE also inhibited vinyl chloride (VC) transformation to ethylene (ETH). When a competitive inhibition model was applied, the inhibition constant (K(I)) for the more chlorinated ethylene was comparable to its respective Michaelis-Menten half-velocity coefficient, K(S). Model simulations using independently derived kinetic parameters matched the experimental results well. k(max) and K(S) values required for model simulations of anaerobic dechlorination reactions were obtained using a multiple equilibration method conducted in a single reactor. The method provided precise kinetic values for each step of the dechlorination process. The greatest difference in kinetic parameters was for the VC transformation step. VC was transformed more slowly by the PM culture (k(max) and K(S) values of 2.4+/-0.4 micromol/mg of protein/day and 602+/-7 microM, respectively) compared to the EV culture (8.1+/-0.9 micromol/mg of protein/day and 62.6+/-2.4 microM). Experimental results and model simulations both illustrate how low K(S) values corresponded to efficient reductive dechlorination for the more highly chlorinated ethylenes but caused strong inhibition of the transformation of the less chlorinated products. Thus, obtaining accurate K(S) values is important for modeling both transformation rates of parent compounds and their inhibition on daughter product transformation.     

Variability in carbon isotopic fractionation during biodegradation of chlorinated ethenes: implications for field applications

Stable carbon isotopic analysis has the potential to assess biodegradation of chlorinated ethenes. Significant isotopic shifts, which can be described by Rayleigh enrichment factors, have been observed for the biodegradation of trichloroethlyene (TCE), cis-dichloroethylene (cDCE), and vinyl chloride (VC). However, until this time, no systematic investigation of isotopic fractionation during perchloroethylene (PCE) degradation has been undertaken. In addition, there has been no comparison of isotopic fractionation by different microbial consortia, nor has there been a comparison of isotopic fractionation by consortia generated from the same source, but growing under different conditions. This study characterized carbon isotopic fractionation during reductive dechlorination of the chlorinated ethenes, PCE in particular, for microbial consortia from two different sources growing under different environmental conditions in order to assess the extent to which different microbial consortia result in different fractionation factors. Rayleigh enrichment factors of -13.8@1000, -20.4@1000, and -22.4@1000 were observed for TCE, cDCE, and VC, respectively, for dechlorination by the KB-1 consortium. In contrast, isotopic fractionation during reductive dechlorination of perchloroethylene (PCE) could not always be approximated by a Rayleigh model. Dechlorination by one consortium followed Rayleigh behavior (epsilon = -5.2), while a systematic change in the enrichment factor was observed over the course of PCE degradation by two other consortia. Comparison of all reported enrichment factors for reductive dechlorination of the chlorinated ethenes shows significant variation between experiments. Despite this variability, these results demonstrate that carbon isotopic analysis can provide qualitative evidence of the occurrence and relative extent of microbial reductive dechlorination of the chlorinated ethenes.     

Stable isotope evidence for biodegradation of chlorinated ethenes at a fractured bedrock site

Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site contaminated with trichloroethene (TCE), a dense non-aqueous phase liquid (DNAPL). The site is located in fractured bedrock and had variable groundwater hydraulic gradients during the study due to a local excavation project. Previous attempts to biostimulate a pilot treatment area at the site resulted in the production of cis-1,2-dichloroethene (cis-DCE), the first product of reductive dechlorination of TCE. Cis-DCE concentrations accumulated however, and there was no appreciable production of the breakdown products from further reductive dechlorination, vinyl chloride (VC) and ethene (ETH). Consequently, the pilot treatment area was bioaugmented with a culture of KB-1, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Due to ongoing dissolution of TCE from DNAPL in the fractured bedrock, and to variable hydraulic gradients, concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC, however, demonstrated that biodegradation was occurring in the pilot treatment area. The isotope values of cis-DCE and VC became significantly more enriched in 13C over the last two sampling dates (in one well from -17.6%o to -12.8%o and from -22.5%o to -18.2%o for cis-DCE and VC, respectively). Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that, depending on the well, between 21.3% and 40.7% of the decrease in cis-DCE and between 15.2% and 36.7% of the decrease in VC concentrations can be attributed to the effects of biodegradation during this time period. Within each well, the isotope profile of TCE remained relatively constant due to the continuous input of undegraded TCE due to DNAPL dissolution.     

Comparative evaluation of chloroethene dechlorination to ethene by Dehalococcoides-like microorganisms

Reductive dehalogenation of tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (DCE), and vinyl chloride (VC) was examined in four cultures containing Dehalococcoides-like microorganisms. Dechlorination and growth kinetics were compared using a Monod growth-rate model for multiple electron acceptor usage with competition. Included were the Victoria mixed culture containing Dehalococcoides species strain VS (from Victoria, TX), the mixed culture KB-1/VC (from southern Ontario), the Pinellas mixed culture (from Pinellas, FL), and D. ethenogenes strain 195. All cultures, with the exception of D. ethenogenes strain 195, grew with VC as catabolic electron acceptor. A dilution method was developed that allows a valid comparison to be made of dehalogenating kinetics between different mixed cultures. Using this procedure, maximum growth rates on VC were found to be similar for strain VS and KB-1/VC (0.42-0.49 +/- 0.02 d(-1)) but slower for the Pinellas culture (0.28 +/- 0.01 d(-1)). The 16S rRNA gene sequences were determined to ensure that no cross contamination between cultures had occurred. Following enrichment of the VC dechlorinating microorganisms on VC, the cultures were amended with DCE, TCE, or PCE. The three mixed cultures failed to dechlorinate PCE or did so very slowly. However, the dilution technique indicated that all experienced growth on TCE and DCE as well as on VC. Maximum growth rates on DCE alone were quite similar (0.43-0.46 d(-1)), while the Pinellas culture grew faster on TCE alone (0.49 d(-1)) than did the other two mixed cultures (0.33-0.35 d(-1)). Half-velocity and inhibition constants for growth on TCE were also determined for the three mixed cultures; both constants were found to be essentially equal and the same for the different cultures, varying between only 8.6 and 10.5 microM. The ability of the strain VS, KB-1/VC, and Pinellas cultures to utilize TCE rapidly with conversion to ethene is quite different from that of any other reported microorganism. It was separately confirmed with more traditional cell-counting techniques that strain VS coupled TCE, as well as DCE and VC, utilization with growth. This is the first report of an organism obtaining energy for growth through every step in the reduction of TCE to ethene. Also, as suggested by the dilution technique, the dehalogenating organisms in the KB-1/VC and Pinellas cultures appear to obtain growth from TCE utilization as well. Such ability to grow while dehalogenating TCE to ethene will be an important advantage for their use in bioaugmentation.     

Acetate versus hydrogen as direct electron donors to stimulate the microbial reductive dechlorination process at chloroethene-contaminated sites

A study to evaluate the dechlorination end points and the most promising electron donors to stimulate the reductive dechlorination process at the chloroethene-contaminated Bachman Road site in Oscoda, MI, was conducted. Aquifer materials were collected from inside the plume and used to establish microcosms under a variety of electron donor conditions using chlorinated ethenes as electron acceptors. All microcosms that received an electron donor showed dechlorination activity, but the end points depended on the sampling location, indicating a heterogeneous distribution of the dechlorinating populations in the aquifer. Interestingly, several microcosms that received acetate as the only electron donor completely dechlorinated PCE to ethene. All acetate-amended microcosms rapidly converted PCE to cis-DCE, whereas PCE dechlorination in H2-fed microcosms only occurred after a pronounced lag time and after acetate had accumulated by H2/CO2 acetogenic activity. The microcosm experiments were corroborated by defined co-culture experiments, which demonstrated that H2 sustained PCE to cis-DCE dechlorination by acetotrophic populations in the presence of H2/CO2 acetogens. In sediment-free nonmethanogenic enrichment cultures derived from ethene-producing microcosms, acetate alone supported complete reductive dechlorination of chloroethenes to ethene, although the addition of H2 resulted in higher cis-DCE and VC dechlorination rates. Measurements of H2 production and consumption suggested that syntrophic acetate-oxidizing population(s) were active in the enrichment cultures. These findings demonstrated that either acetate or H2 alone can be sufficient to promote complete     

Abiotic reductive dechlorination of chlorinated ethylenes by iron-bearing soil minerals. 1. Pyrite and magnetite

Abiotic reductive dechlorination of chlorinated ethylenes (tetrachloroethylene (PCE), trichloroethylene (TCE), cis-dichloroethylene (cis-DCE), and vinyl chloride (VC)) by pyrite and magnetite was characterized in a batch reactor system. Dechlorination kinetics was adequately described by a modified Langmuir-Hinshelwood model that includes the effect of a decreasing reductive capacity of soil mineral. The kinetic rate constant for the reductive dechlorination of target organics at reactive sites of soil minerals was in the range of 0.185 (+/- 0.023) to 1.71 (+/- 0.06) day(-1). The calculated specific reductive capacity of soil minerals for target organics was in the range of 0.33 (+/- 0.02) to 2.26 (+/- 0.06) microM/g and sorption coefficient was in the range of 0.181 (+/- 0.006) to 0.7 (+/- 0.022) mM(-1). Surface area-normalized pseudo-first-order initial rate constants for target organics by pyrite were found to be 23.5 to 40.3 times greater than those by magnetite. Target organics were mainly transformed to acetylene and small amount of chlorinated intermediates, which suggests that beta-elimination was the main dechlorination pathway. The dechlorination of VC followed a hydrogenolysis pathway to produce ethylene and ethane. The addition of Fe(II) increased the dechlorination rate of cis-DCE and VC in magnetite suspension by nearly a factor of 10. The results obtained in this research provide basic knowledge to better predict the fate of chlorinated ethylenes and to understand the potential of abiotic processes in natural attenuation.     

1,1-dichloroethene as a predominant intermediate of microbial trichloroethene reduction

A microbial culture derived from a landfill site in Dover, DE consistently reduced trichloroethene (TCE) to ethene through 1,1-dichloroethene (DCE) as a dominant intermediate in the presence of ampicillin. A constant 1,1-DCE-to-cis-DCE ratio of 2.4 +/- 0.3 was observed for more than two years, while trans-DCE was never detected. Without ampicillin, however, TCE was reduced to ethene almost exclusively through cis-DCE, suggesting that the culture contained at leasttwo TCE-dechlorinating populations. Two subcultures, which were established using 1,1-DCE or vinyl chloride as an electron acceptor, exhibited the same 1,1-DCE-to-cis-DCE ratio when TCE was introduced. PCR amplification of 16S rRNA gene followed by sequencing and DGGE analysis indicate that these (sub)cultures contained a Dehalococcoides population(s). TCE dechlorination assays with crude cell extract showed a DCE distribution pattern similar to that with whole cells. The enzyme involved in 1,1-DCE formation was likely a cobalt corrinoid enzyme, as suggested by the inhibitory effect of CH3I and photoreversibility of the inhibition. This study provides a possible biological mechanism forthe occurrence of 1,1-DCE in TCE-contaminated sites.     

Stable carbon isotope evidence for intrinsic bioremediation of tetrachloroethene and trichloroethene at area 6, Dover Air Force Base

Area 6 at Dover Air Force Base (Dover, DE) has been the location of an in-depth study by the RTDF (Remediation Technologies Development Forum Bioremediation of Chlorinated Solvents Action Team) to evaluate the effectiveness of natural attenuation of chlorinated ethene contamination in groundwater. Compound-specific stable carbon isotope measurements for dissolved PCE and TCE in wells distributed throughout the anaerobic portion of the plume confirm that stable carbon isotope values are isotopically enriched in 13C consistent with the effects of intrinsic biodegradation. During anaerobic microbial reductive dechlorination of chlorinated hydrocarbons, the light (12C) versus heavy isotope (13C) bonds are preferentially degraded, resulting in isotopic enrichment of the residual contaminant in 13C. To our knowledge, this study is the first to provide definitive evidence for reductive dechlorination of chlorinated hydrocarbons at a field site based on the delta13C values of the primary contaminants spilled at the site, PCE and TCE. For TCE, downgradient wells show delta13C values as enriched as -18.0/1000 as compared to delta13C values for TCE in the source zone of -25.0 to -26.0/1000. The most enriched delta13C value on the site was observed at well 236, which also contains the highest concentrations of cis-DCE, VC, and ethene, the daughter products of reductive dechlorination. Stable carbon isotope signatures are used to quantify the relative extent of biodegradation between zones of the contaminant plume. On the basis of this approach, it is estimated that TCE in downgradient well 236 is more than 40% biodegraded relative to TCE in the proposed source area.