Bioaugmentation potential of a vinyl chloride-assimilating Mycobacterium sp., isolated from a chloroethene-contaminated aquifer

An aerobic bacterium, Mycobacterium sp. strain TRW-2 that assimilated vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. The strain TRW-2 also degraded cis-dichloroethene (cis-DCE) in mineral salts medium, but only when VC was present as the primary carbon source. However, no degradation of trans-dichloroethene or trichloroethene occurred in either the presence or absence of added VC. The measured growth yield values were 6.53 and 14.1g protein/mol of VC and ETH utilized, respectively. Inoculation by strain TRW-2 in microcosms prepared with aquifer samples resulted in rapid degradation of VC, whereas native bacteria degraded negligible amounts of VC within the same time period, thus suggesting bioaugmentation potential of the isolate. Phylogenetic analysis of the 16S rDNA sequence of the isolate revealed 98% sequence similarity to the members of the genus Mycobacterium. In summary, the isolate's ability to degrade VC, cis-DCE, and ETH and also its ability to survive and degrade VC in the presence of other microorganisms is relevant to the remediation of VC-impacted aquifers.     

Characterization of a newly isolatedcis-1,2-dichloroethylene and aliphatic compound-degrading bacterium,Clostridium sp. strain KYT-1

Acis-1,2-dichloroethylene (cis-DCE)-degrading anaerobic bacterium,Clostridium sp. strain KYT-1, was isolated from a sediment sample collected from a landfill site in Nanji-do, Seoul, Korea. The KYT-1 strain is a gram-positive, endospore-forming, motile, rod-shaped anaerobic bacterium, of approximately 2.5∼3.0 μm in length. The degradation ofcis-DCE is closely related with the growth of the KYT-1 strain, and it was stopped when the growth of the KYT-1 strain became constant. Although the pathway ofcis-DCE degradation by strain KYT-1 remains to be further elucidated, no accumulation of the harmful intermediate, vinyl chloride (VC), was observed during anaerobiccis-DCE degradation. Strain KYT-1 proved able to degrade a variety of volatile organic compounds, including VC, isomers of DCE (1,1-dichloroethylene,trans-1,2-dichloroethylene, andcis-DCE), trichloroethylene, tetrachloroethylene, 1,2-dichloroethane, 1,1,1-trichloroethane, and 1,1,2-trichloroethane. Strain KYT-1 degradedcis-DCE at a range of temperatures from 15 to 37°C, with an optimum at 30°C, and at a pH range of 5.5 to 8.5, with an optimum at 7.0.     

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.     

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.

     

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.     

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.     

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.”     

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis-dichloroethylene (cis-DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans-dichloroethylene (trans-DCE), and 1,1,1-trichloroethane (1,1,1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans-DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis-DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans-DCE, cis-DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans-DCE oxidation products were also toxic but to a lesser extent. cis-DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity. Received: 17 June 1999 / Accepted: 3 September 1999     

Priority pollutant degradation by the facultative methanotroph, Methylocystis strain SB2

Methylocystis strain SB2, a facultative methanotroph capable of growth on multi-carbon compounds, was screened for its ability to degrade the priority pollutants 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA), and 1,1-dichloroethylene (1,1-DCE), as well as cis-dichloroethylene (cis-DCE) when grown on methane or ethanol. Methylocystis strain SB2 degraded 1,2-DCA and 1,1,2-TCA when grown on either substrate and cis-DCE when grown on methane. Growth of Methylocystis strain SB2 on methane was inhibited in the presence of all compounds, while only 1,1-DCE and cis-DCE inhibited growth on ethanol. No degradation of any chlorinated hydrocarbon was observed in ethanol-grown cultures when particulate methane monooxygenase (pMMO) activity was inhibited with the addition of acetylene, indicating that competition for binding to the pMMO between the chlorinated hydrocarbons and methane limited both methanotrophic growth and pollutant degradation when this strain was grown on methane. Characterization of Methylocystis strain SB2 found no evidence of a high-affinity form of pMMO for methane, nor could this strain utilize 1,2-DCA or its putative oxidative products 2-chloroethanol or chloroactetic acid as sole growth substrates, suggesting that this strain lacks appropriate dehydrogenases for the conversion of 1,2-DCA to glyoxylate. As ethanol: (1) can be used as an alternative growth substrate for promoting pollutant degradation by Methylocystis strain SB2 as the pMMO is not required for its growth on ethanol and (2) has been used to enhance the mobility of chlorinated hydrocarbons in situ, it is proposed that ethanol can be used to enhance both pollutant transport and biodegradation by Methylocystis strain SB2.     

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.     

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.     

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.     

Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes

The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.     

Evidence for modified mechanisms of chloroethene oxidation in Pseudomonas butanovora mutants containing single amino acid substitutions in the hydroxylase alpha-subunit of butane monooxygenase

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O₂ uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-α mutants might allow insights into the catalytic mechanism of BMO to be obtained.     

Evaluation of different culture conditions ofClostridium bifermentans DPH-1 for cost effective PCE degradation

Clostridium bifermentans strain DPH-1 has already been found to dechlorinate perchloroethylene (PCE) tocis-dichloroethylene (cis-DCE)via trichloroethylene (TCE). In this study, our investigation on different culture conditions of this DPH-1 strain was extended to find a more efficient and cost effective growth medium composition for this DPH-1 strain in bioremediation practices. Temperature dependency of strain DPH-1 showed that the growth starting time and PCE degradation at 15°C was very slow compared to that of 30°C, but complete PCE degradation occurred in both cases. For the proper utilization of strain DPH-1 in more cost effective bioremediation practices, a simpler composition of an effective media was studied. One component of the culture medium, yeast extract, had been substituted by molasses, which served as a good source of electron donor. The DPH-1 strain in the medium containing molasses, in the presence of K₂HPO₄ and KH₂PO₄, showed identical bacterial multiplication (0.135 mg protein mL⁻¹h⁻¹) and PCE degradation rates (0.38 μM/h) to those of the yeast extract containing medium.     

3-Nitrotoluene dioxygenase from Diaphorobacter sp. strains: cloning,sequencing and evolutionary studies

The first step in the degradation of 3-nitrotoluene by Diaphorobacter sp. strain DS2 is the dihydroxylation of the benzene ring with the concomitant removal of nitro group. This is catalyzed by a dioxygenase enzyme system. We report here the cloning and sequencing of the complete dioxygenase gene with its putative regulatory sequence from the genomic DNA of Diaphorobacter sp. strains DS1, DS2 and DS3. Analysis of the 5 kb DNA stretch that was cloned, revealed five complete open reading frames (ORFs) encoding for a reductase, a ferredoxin and two dioxygenase subunits with predicted molecular weights (MW) of 35, 12, 50 and 23 kDa respectively. A regulatory protein was also divergently transcribed from the reductase subunit and has a predicated MW of 34 kDa. Presence of parts of two functional ORFs in between the reductase and the ferredoxin subunits reveals an evolutionary route from a naphthalene dioxygenase like system of Ralstonia sp. strain U2. Further a 100 % identity of its ferredoxin subunit reveals its evolution via dinitrotoluene dioxygenase like system present in Burkholderia cepacia strain R34. A modeled structure of oxygenase_3NT from strain DS2 was generated using nitrobenzene dioxygenase as a template. The modeled structure only showed minor changes at its active site. Comparison of growth patterns of strains DS1, DS2 and DS3 revealed that Diaphorobacter sp. strain DS1 has been evolved to degrade 4-nitrotoluene better by an oxidative route amongst all three strains.     

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.     

Kinetics of the aerobic co-metabolism of 1,1-dichloroethylene by Achromobacter sp.: a novel benzene-grown culture

Batch experiments were performed for the aerobic co-metabolism of 1,1-dichloroethylene (1,1-DCE) by Achromobacter sp., identified by gene sequencing of 16S rRNA and grown on benzene. Kinetic models were employed to simulate the co-metabolic degradation of 1,1-DCE, and relevant parameters were obtained by non-linear least squares regression. Benzene at 90 mg L^?1 non-competitively inhibited degradation of 1,1-DCE (from 125 to 1,200 μg L^?1). The maximum specific utilization (k_c) rate and the half-saturation constant (K_c) for 1,1-DCE were 54 ± 0.85 μg h^?1 and 220 ± 6.8 μg L^?1, respectively; the k_b and K_b for benzene were 13 ± 0.18 mg h^?1 and 28 ± 0.42 mg L^?1, respectively. This study provides a theoretical basis to predict the natural attenuation when benzene and 1,1-DCE occur as co-contaminants.     

Evaluation of transformation capacity for degradation of ethylene chlorides by<Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

The transformation capacity (T_c) ofMethylosinus trichosporium OB3b in the degradation of ethylene chlorides was determined by measuring the decrease of soluble methane monooxygenase (sMMO) activity of resting cells in batch experiments. All measurements of sMMO activity were taken in the presence of 20 mM formate to avoid the deficiency of reducing power, and within 2 hrs to avoid the effect of natural inactivation from instability of the resting cells. The constant T_c values of 0.58±0.132 and 0.80±0.17 μmol/mg cell were obtained for trichloroethylene (TCE) and 1,2-dichloroethylene (cis andtrans-1,2-DCE), respectively, regardless of their concentrations. The transformation capacity measured by this method can be used to predict the amount of cells that should be stimulated inin-situ bioremediation.     

Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k _cat/K _m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate. Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998