Field Evidence for Intrinsic Aerobic Chlorinated Ethene Cometabolism by Methanotrophs Expressing Soluble Methane Monooxygenase

Idaho National Laboratory's Test Area North is the site of a trichloroethene (TCE) plume resulting from waste injections. Previous investigations revealed that TCE was being attenuated relative to two codisposed internal tracers, tritium and tetrachloroethene, with a half-life of 9 to 21 years. Biological attenuation mechanisms were investigated using a novel suite of assays, including enzyme activity probes designed for the soluble methane monooxygenase (sMMO) enzyme. Samples were analyzed for chlorinated solvents, tritium, redox parameters, primary substrates, degradation products, bacterial community methanotrophic potential, and bacterial DNA. The enzyme probe assays, methanotrophic enrichments and isolations, and DNA analysis documented the presence and activity of indigenous methanotrophs expressing the sMMO enzyme. Three-dimensional groundwater data showed plume-wide aerobic conditions, with low levels of methane and detections of carbon monoxide, a by-product of TCE cometabolism. The TCE half-life attributed to aerobic cometabolism is 13 years relative to tritium, based on the tracer-corrected method. Similarly, a half-life of 8 years was estimated for cis-dichloroethene (DCE). Although these rates are slower than most anaerobic degradation processes, they can be significant for large plumes. This investigation is believed to be the first documentation of intrinsic aerobic TCE and DCE cometabolism in an aquifer by indigenous methanotrophs.     

Does Exposure to Trichloroethene in Low Doses Constitute a Cancer Risk to Humans?

Although chronic exposure to high doses of trichloroethene causes tumors of the lung, liver, and kidney in experimental animals, the epidemiology data in humans exposed to trichloroethene as a whole fail to support a causal association between trichloroethene exposure and cancers of the lungs, liver, or kidneys in humans at environmentally relevant concentrations. Environmentally relevant concentrations of trichloroethene are defined as 50 ppb (50 µg/L) in water or 5 ppb (27 µg/m³) in air. Tumor induction by trichloroethene in rodents exposed to very high doses over their whole lifespan has been observed in the kidney of rats and in the lung and liver of mice. Mechanistic data demonstrate that species-specific processes are involved in the carcinogenicity associated with chronic trichloroethene exposure in rodents. Based on these data and the results of recent well-conducted epidemiology studies, it can be concluded that humans exposed to trichloroethene at environmentally relevant concentrations are not at an increased risk for developing cancer.     

Molecular characterization of microbial populations at two sites with differing reductive dechlorination abilities

This study compares three molecular techniques, including terminal restriction fragment length polymorphism (T-RFLP), RFLP analysis with clone sequencing, and quantitative PCR (Q-PCR) for surveying differences in microbial communities at two contaminated field sites that exhibit dissimilar chlorinated solvent degradation activities. At the Idaho National Engineering and Environmental Laboratory (INEEL), trichloroethene (TCE) was completely converted to ethene during biostimulation with lactate. At Seal Beach, California, perchloroethene (PCE) was degraded only to cis-dichloroethene (cDCE) during biostimulation but was degraded to ethene after bioaugmentation with a dechlorinating culture containing Dehalococcoides strains. T-RFLP analysis showed that microbial community composition differed significantly between the two sites, but was similar within each site among wells that had low or no electron donor exposure. Analysis of INEEL clone libraries by RFLP with clone sequencing revealed a complex microbial population but did not identify any Dehalococcoides strains. Q-PCR targeting the 16S rRNA gene of Dehalococcoides strains – known for their unique capability to dechlorinate solvents completely to ethene – revealed a significant population at INEEL, but no detectable population at Seal Beach prior to bioaugmentation. Detection of Dehalococcoides by Q-PCR correlated with observed dechlorination activity and ethene production at both sites. Q-PCR showed that Dehalococcoides was present in even the pristine well at INEEL, suggesting that the difference in dechlorination ability at the two sites was due to the initial absence of this genus at Seal Beach. Of the techniques tested, Q-PCR quantification of specific dechlorinating species provided the most effective and direct prediction of community dechlorinating potential.     

Factors controlling the carbon isotope fractionation of tetra- and trichloroethene during reductive dechlorination by Sulfurospirillum ssp. and Desulfitobacterium sp. strain PCE-S

Carbon stable isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE) was investigated during reductive dechlorination. Growing cells of Sulfurospirillum multivorans, Sulfurospirillum halorespirans, or Desulfitobacterium sp. strain PCE-S, the respective crude extracts and the abiotic reaction with cyanocobalamin (vitamin B(12)) were used. Fractionation of TCE (alphaC=1.0132-1.0187) by S. multivorans was more than one order of magnitude higher than values previously observed for tetrachloroethene (PCE) (alphaC=1.00042-1.0017). Similar differences in fractionation were observed during reductive dehalogenation by the close relative S. halorespirans with alphaC=1.0046-1.032 and alphaC=1.0187-1.0229 for PCE and TCE respectively. TCE carbon isotope fractionation (alphaC=1.0150) by the purified PCE-reductive dehalogenase from S. multivorans was more than one order of magnitude higher than fractionation of PCE (alphaC=1.0017). Carbon isotope fractionation of TCE by Desulfitobacterium sp. strain PCE-S (alphaC=1.0109-1.0122) as well as during the abiotic reaction with cyanocobalamin (alphaC=1.0154) was in a similar range to previously reported values for fractionation by mixed microbial cultures. In contrast with previous results with PCE, no effects due to rate limitations, uptake or transport of the substrate to the reactive site could be observed during TCE dechlorination. Our results show that prior to a mechanistic interpretation of stable isotope fractionation factors it has to be carefully verified how other factors such as uptake or transport affect the isotope fractionation during degradation experiments with microbial cultures.     

Phreatophyte influence on reductive dechlorination in a shallow aquifer contaminated with trichloroethene (TCE)

Phytoremediation uses the natural ability of plants to degrade contaminants in groundwater. A field demonstration designed to remediate aerobic shallow ground‐water contaminated with trichloroethene began in April 1996 with the planting of cottonwood trees, a short‐rotation woody crop, over an approximately 0.2‐ha area at the Naval Air Station, Fort Worth, Texas. The project was developed to demonstrate capture of contaminated groundwater and degradation of contaminants by phreatophytes. Analyses from samples of groundwater collected from July 1997 to June 1998 indicate that tree roots have the potential to create anaerobic conditions in the groundwater that will facilitate degradation of trichloroethene by microbially mediated reductive dechlorination. Organic matter from root exudates and decay of tree roots probably stimulate microbial activity, consuming dissolved oxygen. Dissolved oxygen concentrations, which varied across the site, were smallest near a mature cottonwood tree (about 20 years of age and 60 meters southwest of the cottonwood plantings) where degradation products of trichloroethene were measured. Oxidation     

Anaerobic Biodegradation and Hydrogeochemical Controls on Natural Attenuation of Trichloroethene in an Inland Forested Wetland

A field and laboratory investigation of natural attenuation, focusing on anaerobic biodegradation, was conducted in a forested wetland where a plume of trichloroethene discharges from a sand aquifer through organic-rich wetland and stream-bottom sediments. The rapid response of the wetland hydrology to precipitation events altered groundwater flow and geochemistry during wet conditions in the spring compared to the drier conditions in the summer and fall. During dry conditions, partial reductive dechlorination of trichloroethene to cis-1,2-dichloroethene occurred in methanogenic wetland porewater. Influx of oxygenated recharge during wet conditions resulted in a change from methanogenic to iron-reducing conditions and a lack of 1,2-dichloroethene production in the wet spring conditions. During these wet conditions, dilution was the primary attenuation mechanism evident for trichloroethene in the wetland porewater. Trichloroethene degradation was insignificant in anaerobic microcosms constructed with the shallow wetland sediment, and microbiological analyses showed a low microbial biomass and absence of known dehalorespiring microorganisms. Despite the typically organic-rich characteristic of wetland sediments, natural attenuation by anaerobic degradation may not be an effective groundwater remediation for chlorinated solvents at all sites.     

A Long-Term Field Study of In Situ Bioremediation in a Fractured Conglomerate Trichloroethene Source Zone

ABSTRACT An 8-year bioremediation field study was conducted in a trichloroethene (TCE)-contaminated, highly indurated (i.e., hard), recharge-limited (i.e., contains little water) conglomerate where common remediation strategies, such as groundwater recirculation and direct push installation of a large well network, could not be used. A tracer test using isotopically distinct water from the Hetch Hetchy Reservoir indicated that remediation fluids mainly flowed through fractures and sand lenses in the conglomerate. This was confirmed during in situ bioremediation of the site, in which Dehalococcoides (from a bioaugmentation culture) and volatile fatty acids (from injection of lactate) were the most accurate indicators of transport between wells. Some contaminants were also displaced out of the area due to injection of tracer water. Despite these difficulties, dissolved contaminant mass decreased by an estimated 80% by the end of the test, reaching the lowest values ever recorded at this site. Furthermore, the persistence of ethene 4 years after bioaugmentation suggests that the dechlorinating capacity of the remaining microbial community is comparable to the matrix diffusion of TCE into the dissolved phase.     

Tetrachloroethene and 3-chlorobenzoate dechlorination activities are co-induced inDesulfomonile tiedjei DCB-1

Desulfomonile tiedjei, a strict anaerobe capable of reductively dechlorinating 3-chlorobenzoate, also dechlorinates tetrachloroethene and trichloroethene. It is not known, however, if the aryl and aliphatic dechlorination activities are catalyzed by the same enzymatic system. Cultures induced for 3-chlorobenzoate activity dechlorinated tetrachloroethene and trichloroethene to lower chlorinated products while uninduced parallel cultures did not dechlorinate either substrate. The observed rate of PCE dechlorination in induced cultures was 22 µmol h^–1 g protein^–1, which is considerably faster than previous rates obtained with defined cultures of this organism. These results show that both dechlorination activities are co-induced and therefore, that the dechlorination mechanisms may share at least some components.Abbreviations PCE tetrachloroethene - TCE trichloroethene - cis-DCE cis-dichloroethene - trans-DCE trans-dichloroethene - 3FBz 3-fluorobenzoate - 3ClBz 3-chlorobenzoate     

Comparison of the microbial population dynamics and phylogenetic characterization of a CANOXIS reactor and a UASB reactor degrading trichloroethene

AIMS: To understand the microbial ecology underlying trichloethene (TCE) degradation in a coupled anaerobic/aerobic single stage (CANOXIS) reactor oxygenated with hydrogen peroxide (H2O2) and in an upflow anaerobic sludge bed (UASB) reactor. METHODS AND RESULTS: The molecular study of the microbial population dynamics and a phylogenetic characterization were conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). In both reactors, TCE had a toxic effect on two uncultured bacterial populations whereas oxygen favoured the growth of aerobic species belonging to Rhizobiaceae and Dechloromonas. No methanotrophic bacteria were detected when targeting 16S rRNA gene with universal primers. Alternatively, pmo gene encoding the particulate methane monooxygenase of Methylomonas sp. LW21 could be detected in the coupled reactor when H2O2 was supplied at 0.7 g O2 l day(-1). CONCLUSIONS: Methylomonas sp. LW21 that could be responsible for the aerobic degradation of the TCE by-products is not among the predominant bacterial populations in the coupled reactor. It seems to have been outcompeted by heterotrophic bacteria (Rhizobiaceae and Dechloromonas sp.) for oxygen. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained show the limitations of the coupled reactor examined in this study. Further investigations should focus on the operating conditions of this reactor in order to favour the growth of the methanotrophs.     

Transpiration and metabolisation of TCE by willow plants – a pot experiment

Willows were grown in glass cylinders filled with compost above water-saturated quartz sand, to trace the fate of TCE in water and plant biomass. The experiment was repeated once with the same plants in two consecutive years. TCE was added in nominal concentrations of 0, 144, 288, and 721 mg l^?1. Unplanted cylinders were set-up and spiked with nominal concentrations of 721 mg l^?1 TCE in the second year. Additionally, ¹³C-enriched TCE solution (δ¹³C = 110.3 ‰) was used. Periodically, TCE content and metabolites were analyzed in water and plant biomass. The presence of TCE-degrading microorganisms was monitored via the measurement of the isotopic ratio of carbon (¹³C/¹²C) in TCE, and the abundance of ¹³C-labeled microbial PLFAs (phospholipid fatty acids). More than 98% of TCE was lost via evapotranspiration from the planted pots within one month after adding TCE. Transpiration accounted to 94 to 78% of the total evapotranspiration loss. Almost 1% of TCE was metabolized in the shoots, whereby trichloroacetic acid (TCAA) and dichloroacetic acid (DCAA) were dominant metabolites; less trichloroethanol (TCOH) and TCE accumulated in plant tissues. Microbial degradation was ruled out by δ¹³C measurements of water and PLFAs. TCE had no detected influence on plant stress status as determined by chlorophyll-fluorescence and gas exchange.