Enrichment and Characterization of a Trichloroethene-Dechlorinating Consortium Containing Multiple “Dehalococcoides” Strains

A microbial consortium that reductively dechlorinates trichloroethene, cis-1,2-dichloroethene (cis-DCE), and vinyl chloride (VC) to ethene with methanogenesis was enriched from chloroethene-contaminated soil from Japan. Dechlorination activity was maintained for over 4 years. Using quantitative polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis targeting the “Dehalococcoides” 16S rRNA gene, four strains were detected. Their growth and dechlorination activities were classified into two types: one that grows by converting cis-DCE to ethene and the other that grows by converting cis-DCE to VC. Then, the vcrA and bvcA genes encoding cis-DCE/VC reductive dehalogenases were detected. Inhibitors of methanogenesis (2-bromoethanesulfonate) and sulfidogenesis (molybdate) led to accumulation of cis-DCE and of VC respectively. These results suggest that methanogens and sulfate-reducing bacteria can play a significant role in dechlorination by “Dehalococcoides.”     

Benzoate-driven dehalogenation of chlorinated ethenes in microbial cultures from a contaminated aquifer

Microbial dehalogenation of tetrachloroethene (PCE) and cis-dichloroethene (cis-DCE) was studied in cultures from a continuous stirred tank reactor initially inoculated with aquifer material from a PCE-contaminated site. Cultures amended with hydrogen and acetate readily dechlorinated PCE and cis-DCE; however, this transformation was incomplete and resulted in the accumulation of chlorinated intermediates and only small amounts of ethene within 60 days of incubation. Conversely, microbial PCE and cis-DCE dechlorination in cultures with benzoate and acetate resulted in the complete transformation to ethene within 30 days. Community fingerprinting by denaturing gradient gel electrophoresis (DGGE) revealed the predominance of phylotypes closely affiliated with Desulfitobacterium, Dehalococcoides, and Syntrophus species. The Dehalococcoides culture VZ, obtained from small whitish colonies in cis-DCE dechlorinating agarose cultures, revealed an irregular cell diameter between 200 and 500 nm, and a spherical or biconcave disk-shaped morphology. These organisms were identified as responsible for the dechlorination of cis-DCE to ethene in the PCE-dechlorinating consortia, operating together with the Desulfitobacterium as PCE-to-cis-DCE dehalogenating bacterium and with a Syntrophus species as potential hydrogen-producing partner in cultures with benzoate. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complete reductive dechlorination of tetrachloroethene to ethene by anaerobic microbial enrichment culture developed from sediment

A mixed, anaerobic microbial enrichment culture, AMEC-4P, was developed that uses lactate as the electron donor for the reductive dechlorination of tetrachloroethene (PCE) to ethene. AMEC-4P consistently and completely converted 2 mM PCE to cis-1,2-dichloroethene (cis-DCE) within 13 days, and the intermediate, cis-DCE, was then completely dechlorinated to ethene after 130 days. Dechlorination rates for PCE to cis-DCE, cis-DCE to VC, and VC to ethene were 243, 27, and 41 μmol/l/day, respectively. Geobacter lovleyi and a Dehalococcoides sp. were identified from their 16S rRNA sequences to be the dominant phylotypes in AMEC-4P.     

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas “Dehalococcoides”-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of “Dehalococcoides ethenogenes” strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated.     

Frequent concomitant presence of Desulfitobacterium spp. and "Dehalococcoides" spp. in chloroethene-dechlorinating microbial communities

The presence of chloroethene dechlorination activity as well as several bacterial genera containing mainly organohalide-respiring members was investigated in 34 environmental samples from 18 different sites. Cultures inoculated with these environmental samples on tetrachloroethene and amended weekly with a seven organic electron donor mixture resulted in 11 enrichments with cis-DCE, ten with VC, and 11 with ethene as dechlorination end product, and only two where no dechlorination was observed. “Dehalococcoides” spp. and Desulfitobacterium spp. were detected in the majority of the environmental samples independently of the dechlorination end product formed. The concomitant presence of Dehalococcoides spp. and Desulfitobacterium spp. in the majority of the enrichments suggested that chloroethene dechlorination was probably the result of catalysis by at least two organohalide-respiring genera either in parallel or by stepwise catalysis. A more detailed study of one enrichment on cis-DCE suggested that in this culture Desulfitobacterium spp. as well as Dehalococcoides spp. dechlorinated cis-DCE whereas dechlorination of VC was only catalyzed by the latter.     

Biodegradation of cis-1,2-dichloroethylene and vinyl chloride in anaerobic cultures enriched from landfill leachate sediment under Fe(III)-reducing conditions

An anaerobic, Fe(III)-reducing enrichment culture, which originatedfrom a sediment sample collected at a landfill in Nanji-do, Seoul, Korea, was capable ofdegrading cis-1,2-dichloroethylene (cis-DCE) and vinylchloride (VC). Although it exhibited the ability under Fe(III)-reducing conditions, the chlorinated ethenes degradationwas not linked to the Fe(III) reduction. During cis-DCE degradation, no VC, ethene, or ethanewas detected through the experimental period. Also, this culture did not accumulate ethene andethane during the VC degradation. It was unlikely that cis-DCE was reductivelydechlorinated to VC and then the VC formed was dechlorinated fast enough. Because the kinetic datashowed that the rate of cis-DCE degradation was 3.5 times higher than that of VC. Whereasglucose supported the culture growth and the degradation, formate, acetate, butyrate, propionate,lactate, pyruvate, and yeast extract did not. The results appeared consistent with the involvement ofoxidative degradation mechanism rather than reductive dechlorination mechanism. The traits of the culturedescribed here are unusual in the anaerobic degradation of chlorinated ethenes and may be usefulfor searching an effective organism and mechanism regarding anaerobic cis-DCE and VC degradation.     

Reductive Dechlorination of Chlorinated Ethenes and 1,2-Dichloroethane by “Dehalococcoides ethenogenes” 195

“Dehalococcoides ethenogenes” 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

Development and characterization of DehaloR^2, a novel anaerobic microbial consortium performing rapid dechlorination of TCE to ethene

A novel anaerobic consortium, named DehaloR^2, that performs rapid and complete reductive dechlorination of trichloroethene (TCE) to ethene is described. DehaloR^2 was developed from estuarine sediment from the Back River of the Chesapeake Bay and has been stably maintained in the laboratory for over 2 years. Initial sediment microcosms showed incomplete reduction of TCE to DCE with a ratio of trans- to cis- isomers of 1.67. However, complete reduction to ethene was achieved within 10 days after transfer of the consortium to sediment-free media and was accompanied by a shift to cis-DCE as the prevailing intermediate metabolite. The microbial community shifted from dominance of the Proteobacterial phylum in the sediment to Firmicutes and Chloroflexi in DehaloR^2, containing the genera Acetobacterium, Clostridium, and the dechlorinators Dehalococcoides. Also present were Spirochaetes, possible acetogens, and Geobacter which encompass previously described dechlorinators. Rates of TCE to ethene reductive dechlorination reached 2.83 mM Cl⁻ d⁻¹ in batch bottles with a Dehalococcoides sp. density of 1.54E+11 gene copies per liter, comparing favorably to other enrichment cultures described in the literature and identifying DehaloR^2 as a promising consortium for use in bioremediation of chlorinated ethene-impacted environments.     

Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter⁻¹ day⁻¹, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Isolation and characterization of tetrachloroethylene- and <Emphasis Type="Italic">cis</Emphasis>-1,2-dichloroethylene-dechlorinating propionibacteria

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹⁴C] PCE and [1,2-¹⁴C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.     

Effects of bioaugmentation on enhanced reductive dechlorination of 1,1,1-trichloroethane in groundwater: a comparison of three sites

Microcosm studies investigated the effects of bioaugmentation with a mixed Dehalococcoides (Dhc)/Dehalobacter (Dhb) culture on biological enhanced reductive dechlorination for treatment of 1,1,1-trichloroethane (TCA) and chloroethenes in groundwater at three Danish sites. Microcosms were amended with lactate as electron donor and monitored over 600 days. Experimental variables included bioaugmentation, TCA concentration, and presence/absence of chloroethenes. Bioaugmented microcosms received a mixture of the Dhc culture KB-1 and Dhb culture ACT-3. To investigate effects of substrate concentration, microcosms were amended with various concentrations of chloroethanes (TCA or monochloroethane [CA]) and/or chloroethenes (tetrachloroethene [PCE], trichloroethene [TCE], or 1,1-dichloroethene [1,1-DCE]). Results showed that combined electron donor addition and bioaugmentation stimulated dechlorination of TCA and 1,1-dichloroethane (1,1-DCA) to CA, and dechlorination of PCE, TCE, 1,1-DCE and cDCE to ethane. Dechlorination of CA was not observed. Bioaugmentation improved the rate and extent of TCA and 1,1-DCA dechlorination at two sites, but did not accelerate dechlorination at a third site where geochemical conditions were reducing and Dhc and Dhb were indigenous. TCA at initial concentrations of 5 mg/L inhibited (i.e., slowed the rate of) TCA dechlorination, TCE dechlorination, donor fermentation, and methanogenesis. 1 mg/L TCA did not inhibit dechlorination of TCA, TCE or cDCE. Moreover, complete dechlorination of PCE to ethene was observed in the presence of 3.2 mg/L TCA. In contrast to some prior reports, these studies indicate that low part-per million levels of TCA (<3 mg/L) in aquifer systems do not inhibit dechlorination of PCE or TCE to ethene. In addition, the results show that co-bioaugmentation with Dhc and Dhb cultures can be an effective strategy for accelerating treatment of chloroethane/chloroethene mixtures in groundwater, with the exception that all currently known Dhc and Dhb cultures cannot treat CA.     

Characterization of microbial community structure and population dynamics of tetrachloroethene-dechlorinating tidal mudflat communities

Tetrachloroethene (PCE) and trichloroethene (TCE) are common groundwater contaminants that also impact tidal flats, especially near urban and industrial areas. However, very little is known about dechlorinating microbial communities in tidal flats. Titanium pyrosequencing, 16S rRNA gene clone libraries, and dechlorinator-targeted quantitative real-time PCR (qPCR) characterized reductive dechlorinating activities and populations in tidal flat sediments collected from South Korea’s central west coast near Kangwha. In microcosms established with surface sediments, PCE dechlorination to TCE began within 10 days and 100% of the initial amount of PCE was converted to TCE after 37 days. cis-1,2-Dichloroethene (cis-DCE) was observed as dechlorination end product in microcosms containing sediments collected from deeper zones (i.e., 35–40 cm below ground surface). Pyrosequencing of bacterial 16S rRNA genes and 16S rRNA gene-targeted qPCR results revealed Desulfuromonas michiganensis-like populations predominanted in both TCE and cis-DCE producing microcosms. Other abundant groups included Desulfuromonas thiophila and Pelobacter acidigallici-like populations in the surface sediment microcosms, and Desulfovibrio dechloracetivorans and Fusibacter paucivorans-like populations in the deeper sediment microcosms. Dehalococcoides spp. populations were not detected in these sediments before and after incubation with PCE. The results suggest that tidal flats harbor novel, salt-tolerant dechlorinating populations and that titanium pyrosequencing provides more detailed insight into community structure dynamics of the dechlorinating microcosms than conventional 16S rRNA gene sequencing or fingerprinting methods.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

     

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     

Anaerobic dechlorination of trichloroethene,tetrachloroethene and 1,2-dichloroethane by an acetogenic mixed culture in a fixed-bed reactor

An anaerobic enrichment culture with glucose as the sole source of carbon and energy plus trichloroethene (TCE) as a potential electron acceptor was inoculated with material from a full size anaerobic charcoal reactor that biologically eliminated dichloromethane from contaminated groundwater (Stromeyer et al. 1991). In subcultures of this enrichment complete sequential transformation of 10 µM TCE viacis-dichloroethene and chloroethene to ethene was reproducibly observed. Maintenance of this activity on subcultivation required the presence of TCE in the medium. The enrichment culture was used to inoculate an anaerobic fixed-bed reactor containing sintered glass Raschig elements as support material. The reactor had a total volume of 1780 ml and was operated at 20 °C in an up-flow mode with a flow rate of 50 ml/h. It was fed continuously with 2 mM glucose and 55 µM TCE. Glucose was converted to acetate as the major product and to a minor amount of methane; TCE was quantitatively dehalogenated to ethene. When, in addition to TCE, tetrachloroethene or 1,2-dichloroethane were added to the system, these compounds were also dehalogenated to ethene. In contrast, 1,1,1-trichloroethane was not dehalogenated, but at 40 µM severely inhibited acetogenesis and methanogenesis. When the concentration of TCE in the feed was raised to 220 µM, chloroethene transiently accumulated, but after an adaptation period ethene was again the only volatile product detected in the effluent. The volumetric degradation rate at this stage amounted to 6.2 µmol/l/h. Since complete transformation of TCE occurred in the first sixth of the reactor volume, the degradation capacity of the system is estimated to exceed this value by factor of about ten.Abbreviations CA chloroethane - 1,1-DCA 1,1-dichloroethane - 1,2-DCA 1,2-dichloroethane - 1,1-DCE 1,1-dichloroethene - c-DCE cis-1,2-dichloroethene - t-DCE trans-1,2-dichloroethene - PCE tetrachloroethene, perchloroethene - 1,1,1-TCA 1,1,1-trichloroethane - TCE trichloroethene - VC chloroethene, vinyl chloride     

Biodegradation of vinyl chloride and cis-dichloroethene by a Ralstonia sp. strain TRW-1

An aerobic bacterium, Ralstonia sp. strain TRW-1, that assimilates vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. Phylogenetic analysis of 16S rDNA sequence of the isolate revealed almost 99% sequence similarity to Ralstonia pickettii. To our knowledge, this is the first report describing the isolation of a member of Ralstonia that can degrade VC as the growth substrate. The measured growth yield values for VC and ETH were 11.27 and 18.90 g protein/mole, respectively. The estimated half-velocity constant K _m values for VC and ETH were 9.09±2.97 and 5.73±2.96 μM, respectively. These values are almost three- to tenfold higher than for other VC-assimilating Mycobacterium sp. The strain also degrades cis-dichloroethene (cis-DCE) in mineral salts medium containing yeast-extract, beef-extract, casamino acids, or peptone. This ability of the strain TRW-1 to degrade cis-DCE in the presence of a nontoxic, water-soluble substrate is relevant to in-situ remediation of cis-DCE-contaminated aquifers.     

Treatment of chlorinated volatile organic compounds in upflow wetland mesocosms

Sorption, biodegradation and hydraulic parameters were determined in the laboratory for two candidate soil substrate mixtures for construction of an upflow treatment wetland for volatile organic compounds (VOCs) at a Superfund site. The major parent contaminants in the groundwater at the Superfund site were cis-1,2-dichloroethene (cis-1,2-DCE) and 1,1,1-trichloroethane (1,1,1-TCA). The two mixtures; one a mixture of sand and peat, the other a mixture of sand, peat and Bion Soil, a product derived from agricultural wastes; were selected from ten possible mixtures based on the results of hydraulic and geotechnical testing. The sand and peat mixture had an average hydraulic conductivity of 4.95×10⁻⁴ cm/s with a critical flow of 39.5 gpm/acre (368 l/min/ha) without fluidization of the bed. The sand, peat and Bion Soil mixture had an average hydraulic conductivity of 3.02×10⁻⁴ cm/s with a critical flow of 36.8 gpm/acre (344 l/min/ha) without fluidization of the bed. Retardation coefficients ranged from 1 to 7.3 for target VOCs with higher coefficients observed in the mixture containing the Bion Soil. Consistently higher spatial and temporal first-order removal rate constants were observed in the sand, peat and Bion Soil mixture (cis-1,2-DCE, 0.84±0.36/day; 1,1,1-TCA, 6.52±3.12/day) than in the sand and peat mixture (cis-1,2-DCE, 0.37±0.13/day; 1,1,1-TCA, 1.48±0.42/day). Results from anaerobic microcosm studies confirmed that biodegradation was occurring in the columns and that the sand, peat and Bion Soil mixture had higher degradation rate than the sand and peat mixture. Vinyl chloride (VC) was identified as a ‘design’ contaminant since it is a proven carcinogen and had the lowest removal rate constant for both substrate mixtures. Effective wetland bed depths for VC removal of 900 and 210 cm will be required for peat and sand alone and sand, peat and Bion Soil mixtures, respectively.     

Microcosm evaluation of bioaugmentation after field-scale thermal treatment of a TCE-contaminated aquifer

This paper investigates effects of combining thermal and biological remediation, based on laboratory studies of trichloroethene (TCE) degradation. Aquifer material was collected 6 months after terminating a full-scale Electrical Resistance Heating (ERH), when the site had cooled from approximately 100°C to 40°C. The aquifer material was used to construct bioaugmented microcosms amended with the mixed anaerobic dechlorinating culture, KB-1^TM, and an electron donor (5 mM lactate). Microcosms were bioaugmented during cooling at 40, 30, 20, and 10°C, as temperatures continually decreased during laboratory incubation. Redox conditions were generally methanogenic, and electron donors were present to support dechlorination. For microcosms bioaugmented at 10°C and 20°C, dechlorination stalled at cis-dichloroethene (cDCE) and vinyl chloride (VC) 150 days after bioaugmentation. However, within 300 days of incubation ethene was produced in the majority of these microcosms. In contrast, dechlorination was rapid and complete in microcosms bioaugmented at 30°C. Microcosms bioaugmented at 40°C also showed rapid dechlorination, but stalled at cDCE with partial VC and ethene production, even after 150 days of incubation when the temperature had decreased to 10°C. These results suggest that sequential bioremediation of TCE is possible in field-scale thermal treatments after donor addition and bioaugmentation and that the optimal bioaugmentation temperature is approximately 30°C. When biological and thermal remediations are to be applied at the same location, three bioremediation approaches could be considered: (a) treating TCE in perimeter areas outside the source zone at temperatures of approximately 30°C; (b) polishing TCE concentrations in the original source zone during cooling from approximately 30°C to ambient groundwater temperatures; and (c) using bioremediation in downgradient areas taking advantages of the higher temperature and potential release of organic matter.     

Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a Dehalococcoides Species

A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 μmol liter⁻¹day⁻¹, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (K_S) for VC was 5.8 μM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30°C, and negligible dechlorination occurred at 4 and 35°C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H₂ as electron donor. VC-dechlorinating cultures consumed H₂ to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.     

Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.