Chlorinated Ethenes Research Articles

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.

Phytoscreening as an efficient tool to delineate chlorinated solvent sources at a chlor-alkali facility

Chlorinated ethenes (CE) are among the most common volatile organic compounds (VOC) that contaminate groundwater, currently representing a major source of pollution worldwide. Phytoscreening has been developed and employed through different applications at numerous sites, where it was generally useful for detection of subsurface chlorinated solvents. We aimed at delineating subsurface CE contamination at a chlor-alkali facility using tree core data that we compared with soil data. For this investigation a total of 170 trees from experimental zones was sampled and analyzed for perchloroethene (PCE) and trichloroethene (TCE) concentrations, measured by solid phase microextraction gas chromatography coupled to mass spectrometry. Within the panel of tree genera sampled, Quercus and Ulmus appeared to be efficient biomonitors of subjacent TCE and PCE contamination, in addition to the well known and widely used Populus and Salix genera. Among the 28 trees located above the dense non-aqueous phase liquid (DNAPL) phase zone, 19 tree cores contained detectable amounts of CE, with concentrations ranging from 3 to 3000 μg L−1. Our tree core dataset was found to be well related to soil gas sampling results, although the tree coring data were more informative. Our data further emphasized the need for choosing the relevant tree species and sampling periods, as well as taking into consideration the nature of the soil and its heterogeneity. Overall, this low-invasive screening method appeared useful to delineate contaminants at a small-scale site impacted by multiple sources of chlorinated solvents.

Origin of VC-only plumes from naturally enhanced dechlorination in a peat-rich hydrogeologic setting

The occurrence of vinyl chloride (VC) is often a main concern at sites contaminated with chlorinated solvents due to its high degree of toxicity and carcinogenicity. VC occurrence in aquifers is most often related to the degradation of higher chlorinated ethenes or ethanes and it is generally detected in plumes along with parent contaminants. However, specific combination of stratigraphic, hydrogeologic and geochemical conditions can enhance the degradation of parents and lead to the formation of plumes almost entirely composed of VC (i.e. VC-only plumes). This paper investigates the causes of VC-only plumes in the aquifers below the city of Ferrara (northern Italy) by combining multiple lines of evidence. The City of Ferrara is located on an alluvial lowland, built by the River Po, and is made up of alternating unconsolidated sandy aquifer and silt-clay aquitard deposits of fluvial origin. This region has been strongly impacted by prior industrial activities, with the occurrence of chlorinated compounds at several sites. VC-only plumes with uncertain source location were found at two contaminated sites. The source zone of a third plume composed of chloroethenes from PCE to VC was investigated for high resolution depositional facies architecture and contaminant distribution (contaminant concentration and Compound Specific Isotope Analysis – CSIA). The investigation suggested that degradation of PCE and TCE takes place during contaminant migration through peat-rich (swamp) layers related to the Holocene transgression, which locally act as a “reactor” for stimulating degradation with the accumulation of VC in the strongly reducing environment of the peat. Regional-scale stratigraphic architecture showed the ubiquitous occurrence of swamp layers at distinct stratigraphic levels in the investigated system and their apparent linkage to the in situ creation of the VC-only plumes.

C, Cl and H compound-specific isotope analysis to assess natural versus Fe(0) barrier-induced degradation of chlorinated ethenes at a contaminated site

Compound-specific isotopic analysis of multiple elements (C, Cl, H) was tested to better assess the effect of a zero-valent iron-permeable reactive barrier (ZVI-PRB) installation at a site contaminated with tetrachloroethene (PCE) and trichloroethene (TCE). The focus was on (1) using 13C to evaluate natural chlorinated ethene biodegradation and the ZVI-PRB efficiency; (2) using dual element 13C-37Cl isotopic analysis to distinguish biotic from abiotic degradation of cis-dichloroethene (cis-DCE); and (3) using 13C-37Cl-2H isotopic analysis of cis-DCE and TCE to elucidate different contaminant sources. Both biodegradation and degradation by ZVI-PRB were indicated by the metabolites that were detected and the 13C data, with a quantitative estimate of the ZVI-PRB efficiency of less than 10% for PCE. Dual element 13C-37Cl isotopic plots confirmed that biodegradation was the main process at the site including the ZVI-PRB area. Based on the carbon isotope data, approximately 45% and 71% of PCE and TCE, respectively, were estimated to be removed by biodegradation. 2H combined with 13C and 37Cl seems to have identified two discrete sources contributing to the contaminant plume, indicating the potential of δ2H to discriminate whether a compound is of industrial origin, or whether a compound is formed as a daughter product during degradation.

Atmospheric pressure ionization of chlorinated ethanes in ion mobility spectrometry and mass spectrometry

This study investigates the APCI mechanisms associated with chlorinated ethanes in an attempt to define conditions under which unique pseudo-molecular adducts, in addition to chloride ion, can be produced for analytical measurements using IMS and MS. The ionization chemistry of chlorinated compounds typically leads to the detection of only the halide ions. Using molecular modeling, which provides insights into the ion formation and relative binding energies, predictions for the formation of pseudo-molecular adducts are postulated. Predicted structures of the chloride ion with multiple hydrogens on the ethane backbone was supported by the observation of specific pseudo-molecular adducts in IMS and MS spectra. With the proper instrumental conditions, such as short reaction times and low temperatures, the molecular species of many chlorinated ethanes can be detected, allowing for identification of specific compounds.

Modeling Long‐Term Trends of Chlorinated Ethene Contamination at a Public Supply Well

AbstractA mass‐balance solute‐transport modeling approach was used to investigate the effects of dense nonaqueous phase liquid (DNAPL) volume, composition, and generation of daughter products on simulated and measured long‐term trends of chlorinated ethene (CE) concentrations at a public supply well. The model was built by telescoping a calibrated regional three‐dimensional MODFLOW model to the capture zone of a public supply well that has a history of CE contamination. The local model was then used to simulate the interactions between naturally occurring organic carbon that acts as an electron donor, and dissolved oxygen (DO), CEs, ferric iron, and sulfate that act as electron acceptors using the Sequential Electron Acceptor Model in three dimensions (SEAM3D) code. The modeling results indicate that asymmetry between rapidly rising and more gradual falling concentration trends over time suggests a DNAPL rather than a dissolved source of CEs. Peak concentrations of CEs are proportional to the volume and composition of the DNAPL source. The persistence of contamination, which can vary from a few years to centuries, is proportional to DNAPL volume, but is unaffected by DNAPL composition. These results show that monitoring CE concentrations in raw water produced by impacted public supply wells over time can provide useful information concerning the nature of contaminant sources and the likely future persistence of contamination.

Modeling the Effects of Naturally Occurring Organic Carbon on Chlorinated Ethene Transport to a Public Supply Well

The vulnerability of public supply wells to chlorinated ethene (CE) contamination in part depends on the availability of naturally occurring organic carbon to consume dissolved oxygen (DO) and initiate reductive dechlorination. This was quantified by building a mass balance model of the Kirkwood-Cohansey aquifer, which is widely used for public water supply in New Jersey. This model was built by telescoping a calibrated regional three-dimensional (3D) MODFLOW model to the approximate capture zone of a single public supply well that has a history of CE contamination. This local model was then used to compute a mass balance between dissolved organic carbon (DOC), particulate organic carbon (POC), and adsorbed organic carbon (AOC) that act as electron donors and DO, CEs, ferric iron, and sulfate that act as electron acceptors (EAs) using the Sequential Electron Acceptor Model in three dimensions (SEAM3D) code. SEAM3D was constrained by varying concentrations of DO and DOC entering the aquifer via recharge, varying the bioavailable fraction of POC in aquifer sediments, and comparing observed and simulated vertical concentration profiles of DO and DOC. This procedure suggests that approximately 15% of the POC present in aquifer materials is readily bioavailable. Model simulations indicate that transport of perchloroethene (PCE) and its daughter products trichloroethene (TCE), cis-dichloroethene (cis-DCE), and vinyl chloride (VC) to the public supply well is highly sensitive to the assumed bioavailable fraction of POC, concentrations of DO entering the aquifer with recharge, and the position of simulated PCE source areas in the flow field. The results are less sensitive to assumed concentrations of DOC in aquifer recharge. The mass balance approach used in this study also indicates that hydrodynamic processes such as advective mixing, dispersion, and sorption account for a significant amount of the observed natural attenuation in this system.

Evaluation of the kinetic oxidation of aqueous volatile organic compounds by permanganate

The use of permanganate solutions for in-situ chemical oxidation (ISCO) is a well-established groundwater remediation technology, particularly for targeting chlorinated ethenes. The kinetics of oxidation reactions is an important ISCO remediation design aspect that affects the efficiency and oxidant persistence. The overall rate of the ISCO reaction between oxidant and contaminant is typically described using a second-order kinetic model while the second-order rate constant is determined experimentally by means of a pseudo first order approach. However, earlier studies of chlorinated hydrocarbons have yielded a wide range of values for the second-order rate constants. Also, there is limited insight in the kinetics of permanganate reactions with fuel-derived groundwater contaminants such as toluene and ethanol. In this study, batch experiments were carried out to investigate and compare the oxidation kinetics of aqueous trichloroethylene (TCE), ethanol, and toluene in an aqueous potassium permanganate solution. The overall second-order rate constants were determined directly by fitting a second-order model to the data, instead of typically using the pseudo-first-order approach. The second-order reaction rate constants (M−1s−1) for TCE, toluene, and ethanol were 8.0×10−1, 2.5×10−4, and 6.5×10−4, respectively. Results showed that the inappropriate use of the pseudo-first-order approach in several previous studies produced biased estimates of the second-order rate constants. In our study, this error was expressed as a function of the extent (P/N) in which the reactant concentrations deviated from the stoichiometric ratio of each oxidation reaction. The error associated with the inappropriate use of the pseudo-first-order approach is negatively correlated with the P/N ratio and reached up to 25% of the estimated second-order rate constant in some previous studies of TCE oxidation. Based on our results, a similar relation is valid for the other volatile organic compounds studied.

Kinetics of dechlorination by Dehalococcoides mccartyi using different carbon sources

Stimulated anaerobic dechlorination is generally considered a valuable step for the remediation of aquifers polluted with chlorinated ethenes (CEs). Correct simulation and prediction of this process in situ, however, require good knowledge of the associated biological reactions. The aim of this study was to evaluate the dechlorination reaction in an aquifer contaminated with trichloroethene (TCE) and its daughter products, discharging into the Zenne River. Different carbon sources were used in batch cultures and these were related to the dechlorination reaction, together with the monitored biomarkers. Appropriate kinetic formulations were assessed. Reductive dechlorination of TCE took place only when external carbon sources were added to microcosms, and occurred concomitant with a pronounced increase in the Dehalococcoides mccartyi cell count as determined by 16S rRNA gene-targeted qPCR. This indicates that native dechlorinating bacteria are present in the aquifer of the Zenne site and that the oligotrophic nature of the aquifer prevents a complete degradation to ethene. The type of carbon source, the cell number of D. mccartyi or the reductive dehalogenase genes, however, did not unequivocally explain the observed differences in degradation rates or the extent of dechlorination. Neither first-order, Michaelis-Menten nor Monod kinetics could perfectly simulate the dechlorination reactions in TCE spiked microcosms. A sensitivity analysis indicated that the inclusion of donor limitation would not significantly enhance the simulations without a clear process understanding. Results point to the role of the supporting microbial community but it remains to be verified how the complexity of the microbial (inter)actions should be represented in a model framework.

Integration of stable carbon isotope, microbial community, dissolved hydrogen gas, and 2HH2O tracer data to assess bioaugmentation for chlorinated ethene degradation in fractured rocks

An in situ bioaugmentation (BA) experiment was conducted to understand processes controlling microbial dechlorination of trichloroethene (TCE) in groundwater at the Naval Air Warfare Center (NAWC), West Trenton, NJ. In the BA experiment, an electron donor (emulsified vegetable oil and sodium lactate) and a chloro-respiring microbial consortium were injected into a well in fractured mudstone of Triassic age. Water enriched in ²H was also injected as a tracer of the BA solution, to monitor advective transport processes. The changes in concentration and the δ¹³C of TCE, cis-dichloroethene (cis-DCE), and vinyl chloride (VC); the δ²H of water; changes in the abundance of the microbial communities; and the concentration of dissolved H₂ gas compared to pre- test conditions, provided multiple lines of evidence that enhanced biodegradation occurred in the injection well and in two downgradient wells. For those wells where the biodegradation was stimulated intensively, the sum of the molar chlorinated ethene (CE) concentrations in post-BA water was higher than that of the sum of the pre-BA background molar CE concentrations. The concentration ratios of TCE/(cis-DCE+VC) indicated that the increase in molar CE concentration may result from additional TCE mobilized from the rock matrix in response to the oil injection or due to desorption/diffusion. The stable carbon isotope mass-balance calculations show that the weighted average ¹³C isotope of the CEs was enriched for around a year compared to the background value in a two year monitoring period, an effective indication that dechlorination of VC was occurring. Insights gained from this study can be applied to efforts to use BA in other fractured rock systems. The study demonstrates that a BA approach can substantially enhance in situ bioremediation not only in fractures connected to the injection well, but also in the rock matrix around the well due to processes such as diffusion and desorption. Because the effect of the BA was intensive only in wells where an amendment was distributed during injection, it is necessary to adequately distribute the amendments throughout the fractured rock to achieve substantial bioremediation. The slowdown in BA effect after a year is due to some extend to the decrease abundant of appropriate microbes, but more likely the decreased concentration of electron donor.

Occurrence of Dehalococcoides and Reductive Dehalogenase Genes in Microcosms, a Constructed Wetland and Groundwater from a Chlorinated Ethene Contaminated Field Site as Indicators for In Situ Reductive Dehalogenation

Thus far, members of the genus Dehalo- coccoides are the only microorganisms known to dehalogenate chlorinated ethenes to ethene and thereby detoxify these common groundwater pollutants. Therefore, it is important to characterize the taxonomic and functional diversity of these key microorganisms and their reductive dehalogenase (RDase) genes in contaminated aquifers for assessing the natural attenuation potential. Little is known about the diversity of RDase genes under field conditions or in laboratory systems under selective pressure during de- chlorination activities. Here, we evaluate the diversity of Dehalococcoidessp. and three RDase genes in groundwater as well as in water from a constructed wetland and micro- cosms setup with contaminated groundwater from the same field site in Bitterfeld (Saxony-Anhalt, Germany). The pres- ence and relative abundance of Pinellas and Cornell sub- groups of Dehalococcoides was evaluated by a novel direct sequencing method, which revealed that all sequences were identical and affiliated to the Pinellas subgroup. Contrarily, our results showed remarkable differences at the functional gene level between the systems. Of the vinyl chloride reductase genes, vcrA was detected in samples from the groundwater, wetland, and microcosms, whereas bvcAwas only found in wetland and microcosm samples. The trichloroethene dehalogenase gene, tceA could not be de- tected at all, although complete dehalogenation activity of higher chlorinated ethenes was observed. Our study dem- onstrates that although the Dehalococcoides 16S rRNA gene sequences retrieved from the investigated systems were identical, the RDase gene diversity varied among the systems, according to the spectrum of the chlorinated eth- enes present.

Use of a reactive transport model to describe reductive dechlorination (RD) as a remediation design tool: application at a CAH-contaminated site

In this paper, a numerical model is presented that is capable of describing the complex set of biochemical processes that occur in chlorinated aliphatic hydrocarbon (CAH)-contaminated groundwater when an exogenous electron donor is added. The reactive pattern is based on the degradation pathways of both chlorinated ethanes and ethenes, and it includes electron donor production (H2 and acetate) from the fermentation of an organic substrate as well as rate-limiting processes related to electron acceptor competition. Coupling of the kinetic model to a convection-dispersion module is described. The calibration phase was carried out using data obtained at a real CAH-contaminated site in the north of Italy. Model simulations of different application scenarios are presented to draw general conclusions on the effectiveness of reductive dechlorination (RD) as a possible cleanup strategy. Early outcomes indicate that cleanup targets can only be achieved if source longevity is reduced. Therefore, metabolic RD is expected to produce beneficial effects because it is known to induce bioenhanced degradation and transformation of CAHs.

A stable isotope approach for source apportionment of chlorinated ethene plumes at a complex multi-contamination events urban site

The stable carbon isotope composition of chlorinated aliphatic compounds such as chlorinated methanes, ethanes and ethenes was examined as an intrinsic fingerprint for apportionment of sources. A complex field site located in Ferrara (Italy), with more than 50years history of use of chlorinated aliphatic compounds, was investigated in order to assess contamination sources. Several contamination plumes were found in a complex alluvial sandy multi-aquifer system close to the river Po; sources are represented by uncontained former industrial and municipal dump sites as well as by spills at industrial areas. The carbon stable isotope signature allowed distinguishing 2 major sources of contaminants. One source of chlorinated aliphatic contaminants was strongly depleted in ¹³C (<-60‰) suggesting production lines which have used depleted methane for synthesis. The other source had typical carbon isotope compositions of >-40‰ which is commonly observed in recent production of chlorinated solvents. The degradation processes in the plumes could be traced interpreting the isotope enrichment and depletion of parent and daughter compounds, respectively. We demonstrate that, under specific production conditions, namely when highly chlorinated ethenes are produced as by-product during chloromethanes production, ¹³C depleted fingerprinting of contaminants can be obtained and this can be used to track sources and address the responsible party of the pollution in urban areas.

Carbon and Chlorine Isotope Fractionation During Microbial Degradation of Tetra- and Trichloroethene

Two-dimensional compound-specific isotope analysis (2D-CSIA), combining stable carbon and chlorine isotopes, holds potential for monitoring of natural attenuation of chlorinated ethenes (CEs) in contaminated soil and groundwater. However, interpretation of 2D-CSIA data sets is challenged by a shortage of experimental Cl isotope enrichment factors. Here, isotope enrichments factors for C and Cl (i.e., εC and εCl) were determined for biodegradation of tetrachloroethene (PCE) and trichloroethene (TCE) using microbial enrichment cultures from a heavily CE-contaminated aquifer. The obtained values were εC = -5.6 ± 0.7‰ (95% CI) and εCl = -2.0 ± 0.5‰ for PCE degradation and εC = -8.8 ± 0.2‰ and εCl = -3.5 ± 0.5‰ for TCE degradation. Combining the values for both εC and εCl yielded mechanism-diagnostic εCl/εC ratios of 0.35 ± 0.11 and 0.37 ± 0.11 for the degradation of PCE and TCE, respectively. Application of the obtained εC and εCl values to a previously investigated field site gave similar estimates for the fraction of degraded contaminant as in the previous study, but with a reduced uncertainty in assessment of the natural attenuation. Furthermore, 16S rRNA gene clone library analyses were performed on three samples from the PCE degradation experiments. A species closely related to Desulfitobacterium aromaticivorans UKTL dominated the reductive dechlorination process. This study contributes to the development of 2D-CSIA as a tool for evaluating remediation strategies of CEs at contaminated sites.

Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity

Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 µg l(-1) CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 µg l(-1) CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8-9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 ± 0.1 µg l(-1) extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p-cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides' nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 µM 5',6'-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides' activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.

Molecular Biomarker-Based Biokinetic Modeling of a PCE-Dechlorinating and Methanogenic Mixed Culture

Bioremediation of chlorinated ethenes via anaerobic reductive dechlorination relies upon the activity of specific microbial populations--most notably Dehalococcoides (DHC) strains. In the lab and field Dehalococcoides grow most robustly in mixed communities which usually contain both fermenters and methanogens. Recently, researchers have been developing quantitative molecular biomarkers to aid in field site diagnostics and it is hoped that these biomarkers could aid in the modeling of anaerobic reductive dechlorination. A comprehensive biokinetic model of a community containing Dehalococcoides mccartyi (formerly D. ethenogenes) was updated to describe continuously fed reactors with specific biomass levels based on quantitative PCR (qPCR)-based population data (DNA and RNA). The model was calibrated and validated with subsets of chemical and molecular biological data from various continuous feed experiments (n = 24) with different loading rates of the electron acceptor (1.5 to 482 μeeq/L-h), types of electron acceptor (PCE, TCE, cis-DCE) and electron donor to electron acceptor ratios. The resulting model predicted the sum of dechlorination products vinyl chloride (VC) and ethene (ETH) well. However, VC alone was under-predicted and ETH was over predicted. Consequently, competitive inhibition among chlorinated ethenes was examined and then added to the model. Additionally, as 16S rRNA gene copy numbers did not provide accurate model fits in all cases, we examined whether an improved fit could be obtained if mRNA levels for key functional enzymes could be used to infer respiration rates. The resulting empirically derived mRNA "adjustment factors" were added to the model for both DHC and the main methanogen in the culture (a Methanosaeta species) to provide a more nuanced prediction of activity. Results of this study suggest that at higher feeding rates competitive inhibition is important and mRNA provides a more accurate indicator of a population's instantaneous activity than 16S rRNA gene copies alone as biomass estimates.

Global gene expression of Dehalococcoides within a robust dynamic TCE‐dechlorinating community under conditions of periodic substrate supply

A microarray targeting four sequenced strains in the Dehalococcoides (Dhc) genus was used to analyze gene expression in a robust long-term trichloroethene (TCE)-degrading microbial community (designated ANAS) during feeding cycles that involve conditions of periodic substrate supply. The Dhc transcriptome was examined at three time-points throughout a batch feeding cycle: T1 (27 h) when TCE, dichloroethene (DCE), and vinyl chloride (VC) were present; T2 (54 h) when only VC remained; and T3 (13 days) when Dhc had been starved of substrate for 9 days. Ninety percent of the Dhc open reading frames (ORFs) that were detected in the ANAS DNA were found to be expressed as RNA sometime during the time course, demonstrating extraordinary utilization of the streamlined genome. Ninety-seven percent of these transcripts were differentially expressed during the time course indicating efficiency of transcription through regulation in Dhc. Most Dhc genes were significantly down-regulated at T3 , responding to a lack of substrate as would be expected. The tceA and vcrA genes, which code for proteins with known chlorinated ethene reduction functions, were highly expressed at both T1 and T2 , whereas two other putative reductive dehalogenase genes (DET0173 and DET1545) were most highly expressed at T2 , likely in response to the presence of VC. Hydrogenases were most highly expressed at T1 , reflecting their important role in accumulating electrons used to initiate reductive dechlorination and other biosynthesis pathways. Cobalamin transport genes were preferentially expressed at T2 , reflecting an increase in corrinoid transport as chloroethenes were degraded and a decrease in activity of the transport system after dehalogenation was complete. This is the first application of a microarray targeting a known genus, including both core genomes and identified strain-specific genes, to improve our understanding of transcriptional dynamics within an undefined microbial community.

One-year measurements of chloroethenes in tree cores and groundwater at the SAP Mimoň Site, Northern Bohemia

Chlorinated ethenes (CE) are among the most frequent contaminants of soil and groundwater in the Czech Republic. Because conventional methods of subsurface contamination investigation are costly and technically complicated, attention is directed on alternative and innovative field sampling methods. One promising method is sampling of tree cores (plugs of woody tissue extracted from a host tree). Volatile organic compounds can enter into the trunks and other tissues of trees through their root systems. An analysis of the tree core can thus serve as an indicator of the subsurface contamination. Four areas of interest were chosen at the experimental site with CE groundwater contamination and observed fluctuations in groundwater concentrations. CE concentrations in groundwater and tree cores were observed for a 1-year period. The aim was to determine how the CE concentrations in obtained tree core samples correlate with the level of contamination of groundwater. Other factors which can affect the transfer of contaminants from groundwater to wood were also monitored and evaluated (e.g., tree species and age, level of groundwater table, river flow in the nearby Ploučnice River, seasonal effects, and the effect of the remediation technology operation). Factors that may affect the concentration of CE in wood were identified. The groundwater table level, tree species, and the intensity of transpiration appeared to be the main factors within the framework of the experiment. Obtained values documented that the results of tree core analyses can be used to indicate the presence of CE in the subsurface. The results may also be helpful to identify the best sampling period for tree coring and to learn about the time it takes until tree core concentrations react to changes in groundwater conditions. Interval sampling of tree cores revealed possible preservation of the contaminant in the wood of trees.

Dual Carbon–Chlorine Stable Isotope Investigation of Sources and Fate of Chlorinated Ethenes in Contaminated Groundwater

Chlorinated ethenes (CEs) are ubiquitous groundwater contaminants, yet there remains a need for a method to efficiently monitor their in situ degradation. We report here the first field application of combined stable carbon and chlorine isotope analysis of tetrachloroethene (PCE) and trichloroethene (TCE) to investigate their biodegradation in a heavily contaminated aquifer. The two-dimensional Compound Specific Isotope Analysis (2D-CSIA) approach was facilitated by a recently developed gas chromatography-quadrupole mass spectrometry (GCqMS) method for δ(37)Cl determination. Both C and Cl isotopes showed evidence of ongoing PCE transformation. Applying published C isotope enrichment factors (ε(C)) enabled evaluation of the extent of in situ PCE degradation (11-78%). We interpreted C and Cl isotopes using a numerical reactive transport model along a 60-m flow path. It revealed that combined PCE and TCE mass load was dechlorinated by less than 10%, and that cis-dichloroethene was not further dechlorinated. Furthermore, the 2D-CSIA approach allowed estimation of Cl isotope enrichment factors ε(Cl) (-7.8 to -0.8‰) and characteristic ε(Cl)/ε(C) values (0.42-1.12) for reductive PCE dechlorination at this field site. This investigation demonstrates the benefit of 2D-CSIA to assess in situ degradation of CEs and the applicability of Cl isotope fractionation to evaluate PCE and TCE dechlorination.

Dehalogenation of diverse halogenated substrates by a highly enrichedDehalococcoides-containing culture derived from the contaminated mega-site in Bitterfeld

An enrichment culture dominated by one type of Dehalococcoides sp. (83% of clones) was characterised. This culture, originally derived from contaminated groundwater from the area of Bitterfeld-Wolfen (Saxony-Anhalt, Germany), dehalogenates chlorinated ethenes to ethene. Further, the culture also dehalogenated vinyl bromide (VB) and 1,2-dichloroethane (DCA) to ethene, 1,2,3,4- and 1,2,3,5-tetrachlorobenzene (TeCB), penta- and hexachlorobenzene (PeCB and HCB) to trichlorobenzenes (TCB), lindane to monochlorobenzene (MCB) and pentachlorophenol (PCP) to 2,3,4,6-tetrachlorophenol (TeCP). Growth was proven by quantitative PCR for all active cultures, except for those with TeCB, lindane and PCP. The growth yields obtained ranged from (2.9 ± 0.7) × 10(7) cells μmol(-1) Br(-) released on VB to (34.8 ± 5.4) × 10(7) cells μmol(-1) Cl(-) released on VC. Genes coding for nine putative reductive dehalogenases, the enzymes that mediate the respiratory process of dehalogenation, were identified. Phylogenetic analysis revealed eight reductive dehalogenases with similar sequences in other Dehalococcoides strains and one unique sequence.