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 an H2-utilizing enrichment culture that reductively dechlorinates tetrachloroethene to vinyl chloride and ethene in the absence of methanogenesis and acetogenesis.

We have been studying an anaerobic enrichment culture which, by using methanol as an electron donor, dechlorinates tetrachloroethene (PCE) to vinyl chloride and ethene. Our previous results indicated that H2 was the direct electron donor for rductive dechlorination of PCE by the methanol-PCE culture. Most-probable-number counts performed on this culture indicated low numbers (< or equal to 10(4)/ml)) of methanogens and PCE dechlorinators using methanol and high numbers (> or equal to 10(6)/ml)) of sulfidogens, methanol-utilizing acetogens, fermentative heterotrophs, and PCE dechlorinators using H2. An anaerobic H2-PCE enrichment culture was derived from a 10(-6) dilution of the methanol-PCE culture. This H2-PCE culture used PCE at increasing rates over time when transferred to fresh medium and could be transferred indefinitely with H2 as the electron donor for the PCE dechlorination, indicating that H2-PCE can serve as an electron donor-acceptor pair for energy conservation and growth. Sustained PCE dechlorination by this culture was supported by supplementation with 0.05 mg of vitamin B12 per liter, 25% (vol/vol) anaerobic digestor sludge supernatant, and 2 mM acetate, which presumably served as a carbon source. Neither methanol nor acetate could serve as an electron donor for dechlorination by the H2-PCE culture, and it did not produce CH4 or acetate from H2-CO2 or methanol, indicating the absence of methanogenic and acetogenic bacteria. Microscopic observatios of the pruified H2-PCE culture showed only two major morphotypes: irregular cocci and small rods.     

Microbial transformation of pentachloronitrobenzene under nitrate reducing conditions

The effect of pentachloronitrobenzene (PCNB) on denitrification was assessed with two denitrifying cultures (PCNB-free control and PCNB-acclimated) developed from a contaminated estuarine sediment. PCNB was transformed to pentachloroaniline (PCA) in the PCNB-acclimated culture repeatedly amended with 0.1 μM PCNB, but further dechlorination or degradation of PCA was not observed for almost 1 year. The effect of PCNB on denitrification was also investigated with the PCNB-free control culture. PCNB at an initial concentration of 13 μM was transformed to PCA simultaneously with nitrate reduction but only after the nitrate concentration was at or below 20 mg N/l. PCNB addition at an initial concentration of 13 μM to the control denitrifying culture developed as PCNB-free culture resulted in a transient accumulation of nitric oxide (NO) and nitrous oxide (N₂O). Similarly to the PCNB-acclimated culture, PCNB transformation to PCA started when the nitrate concentration decreased to about 20 mg N/l. A low degree of nitro group removal resulting in the formation of pentachlorobenzene (PeCB) was also observed in the control culture when amended with 13 μM PCNB. Further transformation or degradation of PCA was not observed in all cultures maintained under active nitrate reducing conditions. Based on the results of this study, the presence of nitrate at low concentrations in anoxic/anaerobic soil and sediments is not expected to negatively affect the biotransformation of PCNB to PCA, but dechlorination or degradation of PCA is not expected under active nitrate reducing conditions.     

Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture

A novel hollow-fiber membrane remediation technology developed in our laboratory for hydrogen delivery to the subsurface was shown to support the dechlorination of perchloroethene (PCE) to cis-dichloroethene. In previous research, the presence of nitrate or sulfate has been observed to inhibit biological reductive dechlorination. In this study hollow-fiber membranes were used to supply hydrogen to a mixed culture to investigate whether adequate hydrogen could be added to support dechlorination in the presence of alternative electron acceptors. By continuously supplying hydrogen through the membrane, the hydrogen concentrations within the reactor were maintained well above the hydrogen thresholds reported to sustain reductive dechlorination. It was hypothesized that by preventing nitrate and sulfate reducers from decreasing hydrogen concentrations to below the dehalorespirer threshold, the inhibition of PCE dechlorination by nitrate and sulfate might be avoided and dechlorination could be stimulated more effectively. Enough membrane-fed hydrogen was supplied to completely degrade the alternative electron acceptors present and initiate dechlorination. Nevertheless, nitrate and sulfate inhibited dechlorinating activity even when hydrogen was not limiting. This suggests that competition for hydrogen was not responsible for the observed inhibition. Subsequent microcosm experiments demonstrated that the denitrification intermediate nitrous oxide was inhibitory at 13 µM.     

Enhanced biodegradation of hexachlorocyclohexane in upflow anaerobic sludge blanket reactor using methanol as an electron donor

Anaerobic dechlorination of technical grade hexachlorocyclohexane (THCH) was studied in a continuous upflow anaerobic sludge blanket (UASB) reactor with methanol as a supplementary substrate and electron donor. A reactor without methanol served as the experimental control. The inlet feed concentration of THCH in both the experimental and the control UASB reactor was 100 mg l(-1). After 60 days of continuous operation, the removal of THCH was >99% in the methanol-supplemented reactor as compared to 20-35% in the control reactor. THCH was completely dechlorinated in the methanol fed reactor at 48 h HRT after 2 months of continuous operation. This period was also accompanied by increase in biomass in the reactor, which was not observed in the experimental control. Batch studies using other supplementary substrates as well as electron donors namely acetate, butyrate, formate and ethanol showed lower % dechlorination (<85%) and dechlorination rates (<3 mg g(-1)d(-1)) as compared to methanol (98%, 5 mg g(-1)d(-1)). The optimum concentration of methanol required, for stable dechlorination of THCH (100 mg l(-1)) in the UASB reactor, was found to be 500 mg l(-1). Results indicate that addition of methanol as electron donor enhances dechlorination of THCH at high inlet concentration, and is also required for stable UASB reactor performance.     

Transformation of carbon tetrachloride under sulfate reducing conditions

The removal of carbon tetrachloride under sulfate reducing conditions was studied in an an aerobic packed-bed reactor. Carbon tetrachloride, up to a concentration of 30 μM, was completely converted. Chloroform and dichloromethane were the main transformation products, but part of the carbon tetrachloride was also completely dechlorinated to unknown products. Gram-positive sulfate-reducing bacteria were involved in the reductive dechlorination of carbon tetrachloride to chloroform and dichloromethane since both molybdate, an inhibitor of sulfate reduction, and vancomycin, an inhibitor of gram-positive bacteria completely inhibited carbon tetrachloride transformation. Carbon tetrachloride transformation by these bacteria was a cometabolic process and depended on the input of an electron donor and electron acceptor (sulfate). The rate of carbon tetrachloride transformation by sulfate reducing bacteria depended on the type of electron donor present. A transformation rate of 5.1 nmol·ml^-1·h^-1 was found with ethanol as electron donor. At carbon tetrachloride concentrations higher than18 μM, sulfate reduction and reductive dechlorination of carbon tetrachloride decreased and complete inhibition was observed at a carbon tetrachloride concentration of 56.6 μM. It is not clear what type of microorganisms were involved in the observed partial complete dechlorination of carbon tetrachloride. Sulfate reducing bacteria probably did not play a role since inhibition of these bacteria with molybdate had no effect on the complete dechlorination of carbon tetrachloride. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of enrichments of sulfate reducing bacteria from pristine hydrothermal vents sediments as potential inoculum for reducing trichloroethylene

The evaluation of enrichments from pristine hydrothermal vents sediments on its capability of reducing trichloroethylene (TCE) under sulfate reducing conditions with lactate and volatile fatty acids (VFAs) as substrates was performed. Effect of the possible TCE biodegradation intermediates cis and trans 1,2 dichloroethenes on sulfate reduction (SR) was also evaluated. The influence of cyanocobalamin (CNB₁₂) and riboflavin (RF) on the SR and biodegradation of TCE was also determined. Sediments from the vents were incubated at 37°C and supplemented with 4 g l⁻¹ SO₄ ²⁻, lactate or VFAs and amended in the corresponding treatments with either CNB₁₂ or RF in separated experiments. A percentage of TCE removal of 86 (150 μmol l⁻¹ initial concentration) was attained coupled to 48% sulfate depletion with lactate as substrate. Up to 93% removal of TCE (300 μmol l⁻¹ initial concentration) and 40% of sulfate was reached for VFAs as electron donor. A combination of lactate and CNB₁₂ yielded the best SR. The overall results suggest a syntrophic association in this microbial community in which sulfate reducers, dehalogenating, and probably halorespiring bacteria may be interacting and taking advantage of the fermentation of substrates differently, but without interruption of SR in spite of the fact that TCE was always present. It was also clear that sulfate reduction must be established in the cultures before any degradation can occur. The microbial community present in these hydrothermal vents sediments could be a new source of inoculum for bioreactors designed for dechlorination purposes.     

Transformation of carbon tetrachloride in an anaerobic packed-bed reactor without addition of another electron donor

Carbon tetrachloride (52 M) was biodegraded for more than 72% in an anaerobic packed-bed reactor without addition of an external electron donor. The chloride mass balance demonstrated that all carbon tetrachloride transformed was completely dechlorinated. Chloroform and dichloromethane were sometimes also found as transformation products, but neither accumulated to significant levels in comparison to the amount of carbon tetrachloride transformed. Transformation of carbon tetrachloride in the absence of an added electron donor suggests that carbon tetrachloride itself is the source of energy for the biological reaction observed, and possibly the source of carbon for cell growth. No such mechanism is yet known. The pathway of carbon tetrachloride transformation is not clear; it may be dehalogenated by hydrolytic reduction to carbon monoxide or formic acid which are electron demanding transformations. Carbon monoxide or formic acid may be further utilized and serve as electron donor. Complete dechlorination of carbon tetrachloride according to this pathway is independent of a second electron donor or electron acceptor, as with a fermentation process. Vancomycin, an inhibitor of gram positive eubacteria, severely inhibited carbon tetrachloride transformation in batch incubations with an enrichment culture from the reactor, indicating that gram positive eubacteria were involved in carbon tetrachloride transformation. Batch experiments with bromoethanesulfonic acid, used to inhibit methanogens, and molybdate, an inhibitor of sulfate reduction in sulfate reducing bacteria, demonstrated that neither methanogens nor sulfate reducers were involved in the complete dechlorination of carbon tetrachloride.     

Effects of electron donor and microbial concentration on the enhanced dechlorination of carbon tetrachloride by anaerobic consortia

 Reductive dechlorination of carbon tetra-chloride (CCl₄) by anaerobic bacterial communities from anaerobic digester sludge with the amendment of low concentrations of electron donors and microorganisms was undertaken to evaluate the influence of electron donors and microbial concentration on the rate of dechlorination of CCl₄. Humic acid, acetate, and glucose were selected to examine the feasibility of the electron donor with respect to the remediation of a contaminated subsurface. The addition of an electron donor and microorganisms significantly enhanced the dechlorination rate of carbon tetrachloride. The addition of an electron donor increased the cell numbers of anaerobic consortia, thereby increasing the rate of dechlorination. Glucose was a better electron donor than acetate and humic acid under reducing environments. The pseudo-first-order degradation rate constants of CCl₄ ranged from 0.0057 day^-1 to 0.135 day^-1, depending on the conditions of the electron donor and biomass supplemented. Furthermore, the addition of the electron donor in the batches amended with 0.56 mg volatile suspended solids (VSS)/l biomass had a higher enhanced efficiency than those with 1.7 mg VSS/l biomass. These results suggest that there is a potential for stimulating the dechlorinating capability of anaerobic consortia to remedy the chlorinated hydrocarbons in the oligotrophic environment if the conditions of the supplementing electron donor are properly selected. Received: 14 August 1995/Received last revision: 15 March 1996/Accepted: 15 April 1996     

Complete Biological Dehalogenation of Chlorinated Ethylenes in Sulfate Containing Groundwater

The ability of dehalogenating bacteria to compete with sulfate reducing bacteria for electron donor was studied in microcosms that simulated groundwater contaminated with both chlorinated ethylenes and fuel hydrocarbon compounds. Results demonstrate that reductive dehalogenation of perchloroethylene to ethylene can proceed in the presence of > 100 mg l(-1) sulfate. The hydrogen concentration, which was 2.5 nM in the presence of approximately 150 mg l(-1) sulfate and in the absence of chlorinated compounds, decreased to 0.7 nM during the dechlorination of trichloroethylene and increased to 1.6 nM during the dechlorination of cis-dichloroethylene and vinyl chloride. With only sediment associated donor ("historical" donor) present, dechlorination of trichloroethylene proceeded slowly to ethylene (on a time scale of several years). Addition of toluene, a model hydrocarbon compound, stimulated dechlorination indirectly. Toluene degradation was rapid and linked to sulfate utilization, and presumably formed fermentable substrates that served as hydrogen donors. Dehalogenation was inhibited in soil free microcosms containing 5 mM sulfide, but inhibition was not observed when either aquifer sediment or 5 mM ferrous chloride was added.     

Population Dynamics of Polychlorinated Biphenyl-Dechlorinating Microorganisms in Contaminated Sediments

The growth dynamics of polychlorinated biphenyl (PCB)-dechlorinating microorganisms were determined for the first time, along with those of sulfate reducers and methanogens, by using the most-probable-number technique. The time course of Aroclor 1248 dechlorination mirrored the growth of dechlorinators; dechlorination ensued when the dechlorinating population increased by 2 orders of magnitude from 2.5 x 10(sup5) to 4.6 x 10(sup7) cells g of sediment(sup-1), at a specific growth rate of 6.7 day(sup-1) between 2 and 6 weeks. During this period, PCB-dechlorinating microorganisms dechlorinated Aroclor 1248 at a rate of 3.9 x 10(sup-8) mol of Cl g of sediment(sup-1) day(sup-1), reducing the average number of Cl molecules per biphenyl from 3.9 to 2.8. The growth yield was 4.2 x 10(sup13) cells mol of Cl dechlorinated(sup-1). Once dechlorination reached a plateau, after 6 weeks, the number of dechlorinators began to decrease. On the other hand, dechlorinators inoculated into PCB-free sediments decreased over time from their initial level, suggesting that PCBs are required for their selective enrichment. The numbers of sulfate reducers and methanogens increased in both PCB-free and contaminated sediments, showing little difference between them. The maximum population size of sulfate reducers was about an order of magnitude higher than that of dechlorinators, whereas that of methanogens was slightly less. Unlike those of dechlorinators, however, numbers of both sulfate reducers and methanogens remained high even when dechlorination ceased. The results of this study imply that PCB concentrations may have to exceed a certain threshold to maintain the growth of PCB dechlorinators.     

Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria

Abstract Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE). Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor. Resting cells of S. ovata reductively dechlorinated PCE at a rate of 9.8 nmol h⁻¹ (mg protein)⁻¹ to trichloroethylene (TCE) as the sole product. The dechlorination activity depended on concomitant acetogenesis from methanol and CO₂. Cell-free extracts of S. ovata, Clostridium formicoaceticum, Acetobacterium woodii , and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors. Corrinoids were shown in S. ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide. Rates of dechlorination followed a pseudo-first-order kinetic.     

Complete dechlorination of tetrachloroethene to ethene in presence of methanogenesis and acetogenesis by an anaerobic sediment microcosm

An anaerobic consortium taken from brackish sediments, enriched byPCE/CH₃OH sequential feeding, was capable of completely dechlorinating tetrachloroethene(PCE) to ethene (ETH). In batch experiments, PCE (0.5 mM) was dechlorinated to ethene (ETH) in approximately 75 h with either CH₃OH or H₂ as the electron donor. When VC (0.5 mM) was added instead of PCE it was dechlorinated without any initial lag by the PCE/CH₃OHenriched consortium, although at a lower dechlorination rate. In batch tests H₂ could readilyreplace CH₃OH for supporting PCE dechlorination, with a similar PCE dechlorination rate andproduct distribution with respect to those observed with methanol. This indicates that H₂ productionduring CH₃OH fermentation was not the rate-limiting step of PCE or VC dechlorination.Acetogenesis was the predominant activity when methanol was present. A remarkable homoacetogenicactivity was also observed when hydrogen was supplied instead of methanol.     

Interaction between methanogenic and sulfate-reducing microorganisms during dechlorination of a high concentration of tetrachloroethylene

A methanogenic and sulfate-reducing consortium, which was enriched on medium containing tetrachloroethylene (PCE), had the ability to dechlorinate high concentrations of PCE. Dehalogenation was due to the direct activity of methanogens. However, interactions between methanogenic and sulfate-reducing bacteria involved modification of the dechlorination process according to culture conditions. In the absence of sulfate, the relative percentage of electrons used in PCE dehalogenation increased after an addition of lactate in batch conditions. The sulfate reducers would produce further reductant from lactate catabolism. This reductant might be used by methanogenic bacteria in PCE dechlorination. A mutualistic interaction was observed in the absence of sulfate. However in the presence of sulfate, methanogenesis and dechlorination decreased because of interspecific competition, probably between the H(2)-oxydizing methanogenic and sulfate-reducing bacteria in batch conditions. In the semicontinuous fixed-bed reactor, the presence of sulfate did not affect dechlorination and methanogenesis. The sulfate-reducing bacteria may not be competitors of H(2)-consuming methanogens in the reactor because of the existence of microbial biofilm. The presence of the fixed film may be an advantage for bioremediation and industrial treatment of effluent charged in sulfate and PCE. This is the first report on the microbial ecology of a methanogenic and sulfate-reducing PCE-enrichment consortium.     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

Reductive dechlorination of 2-chlorophenol in a hydrogenotrophic, gas-permeable, silicone membrane bioreactor

A gas-permeable silicone membrane bioreactor was used to cultivate the biofilm under hydrogenotrophic condition for reductive dechlorination of 2-chlorophenol (2-CP). The anaerobic sludge obtained from a swine wastewater treatment plant was immobilized by polyvinyl alcohol (PVA) so as to form a biofilm on the surface of the silicone tube. After acclimating for about 4 months, the bioreactor showed a high dechlorinating performance. Under the condition of continuous feeding with 2-CP at 25 mg/l and the hydraulic retention time of 15 h, the 2-CP removal efficiency reached 92.8% (2-CP decay rate: 0.67 g/m² d of surface area of silicone tube). H₂ was used as electron donor for dechlorinating 2-CP, and produced the dechlorinating intermediate, phenol. Both nitrate and sulfate played important roles in inhibiting 2-CP dechlorination through different biological mechanisms. Nitrate can be easily utilized as an electron acceptor by the biofilm, while sulfate cannot. Results of this study demonstrated that nitrate competed with 2-CP as the electron acceptor, while sulfate retarded the activity of hydrogen-dechlorinating bacteria and thus inhibited the 2-CP dechlorination.     

Complete transformation of 1,1,1-trichloroethane to chloroethane by a methanogenic mixed population

A methanogenic mixed population in a packed-bed reactor completely transformed 1,1,1-trichloroethane (10 μM) to chloroethane by a cometabolic process. Chloroethane was not further transformed. Acetate and methanol served as electron donors. Complete transformation of 1,1,1-trichloroethane to chloroethane only occurred when sufficient electron donor was fed into the reactor. Otherwise, besides chloroethane, 1,1-dichloroethane was also found as a product. The products of 1,1,1-trichloroethane transformation also depended on the type of electron donor present. With acetate, the degree of dechlorination was higher, i.e. more 1,1,1-trichloroethane was transformed to chloroethane than with methanol. In an enrichment culture obtained from the reactor contents, 1,1,1-trichloroethane was only transformed to 1,1-dichloroethane and was not further metabolized. Methanol, acetate, formate, ethanol, 2-propanol, trimethylamine and H₂, but not dimethylamine and methylamine, served as electron donors for 1,1,1-trichloroethane transformation by this enrichment culture. Both nitrate and nitrite inhibited 1,1,1-trichloroethane transformation; while nitrate completely inhibited 1,1,1-trichloroethane dechlorination, some conversion did occur in the presence of nitrite. The product(s) of this conversion remain unknown, since no chlorinated hydrocarbons were detected. Received: 19 June 1998 / Received revision: 14 September 1998 / Accepted: 17 September 1998     

Initial Characterization of a Reductive Dehalogenase from Desulfitobacterium chlororespirans Co23

Desulfitobacterium chlororespirans Co23 is capable of using 3-chloro-4-hydroxybenzoate as terminal electron acceptor for growth. Membrane preparations from cells grown fermentatively on pyruvate in the presence of 3-chloro-4-hydroxybenzoate dechlorinated this compound at a rate of 3.9 nmol min(sup-1) mg of protein(sup-1). Fivefold-greater dechlorination rates were measured with reduced methyl viologen as the artificial electron donor. Reduced benzyl viologen, NADH, NADPH, reduced flavin adenine dinucleotide, and reduced flavin mononucleotide could not substitute for reduced methyl viologen. The maximal initial rate of catalysis was achieved at pH 6.5 and 60(deg)C. The membrane-bound dechlorinating enzyme system was not oxygen sensitive and was stable at 57(deg)C for at least 2 h. Sulfite inhibited dechlorination in cell-free assays, whereas sulfate did not. Several chlorophenols were dehalogenated exclusively in the ortho position by cell extracts.     

Reductive dechlorination of high concentrations of tetrachloroethene to ethene by an anaerobic enrichment culture in the absence of methanogenesis.

Tetrachloroethene, also known as perchloroethylene (PCE), is a common groundwater contaminant throughout the United States. The incomplete reductive dechlorination of PCE--resulting in accumulations of trichloroethene, dichloroethene isomers, and/or vinyl chloride--has been observed by many investigators in a wide variety of methanogenic environments. Previous mixed-culture studies have demonstrated that complete dechlorination to ethene is possible, although the final dechlorination step from vinyl chloride to ethene is rate limiting, with significant levels of vinyl chloride typically persisting. In this study, anaerobic methanol-PCE enrichment cultures which proved capable of dechlorinating high concentrations PCE to ethene were developed. Added concentrations of PCE as high as 550 microM (91-mg/liter nominal concentration; approximately 55-mg/liter actual aqueous concentration) were routinely dechlorinated to 80% ethene and 20% vinyl chloride within 2 days at 35 degrees C. The methanol level used was approximately twice that needed for complete dechlorination of PCE to ethene. The observed transformations occurred in the absence of methanogenesis, which was apparently inhibited by the high concentrations of PCE. When incubation was allowed to proceed for as long as 4 days, virtually complete conversion of PCE to ethene resulted, with less than 1% persisting as vinyl chloride. An electron balance demonstrated that methanol consumption was completely accounted for by dechlorination (31%) and acetate production (69%). The high volumetric rates of PCE dechlorination (up to 275 mumol/liter/day) and the relatively large fraction (ca. one-third) of the supplied electron donor used for dechlorination suggest that reductive dechlorination could be exploited for bioremediation of PCE-contaminated sites.