Degradation Of cDCE Research Articles

Genomic analysis of Acinetobacter pittii CEP14 reveals its extensive biodegradation capabilities, including cometabolic degradation of cis-1,2-dichloroethene.

Halogenated organic compounds are naturally occurring in subsurface environments; however, accumulation of the degradative intermediate cis-1,2-dichloroethene (cDCE) at soil and groundwater sites contaminated with xenobiotic chlorinated ethenes is a global environmental and public health issue. Identifying microorganisms capable of cDCE degradation in these environments is of interest because of their potential application to bioremediation techniques. In this study, we sequenced, assembled, and analyzed the complete genome of Acinetobacter pittii CEP14, a strain isolated from chloroethene-contaminated groundwater, that has demonstrated the ability for aerobic cometabolic degradation of cDCE in the presence of n-hexane, phenol, and toluene. The A. pittii CEP14 genome consists of a 3.93Mbp-long chromosome (GenBank accession no. CP084921) with a GC content of 38.9% and three plasmids (GenBank accession no. CP084922, CP084923, and CP084924). Gene function was assigned to 83.4% of the 3,930 coding DNA sequences. Functional annotation of the genome revealed that the CEP14 strain possessed all genetic elements to mediate the degradation of a range of aliphatic and aromatic compounds, including n-hexane and phenol. In addition, it harbors gene clusters involved in cytosol detoxification and oxidative stress resistance, which could play a role in the mitigation of toxic chemical intermediates that can arise during the degradation of cDCE. Gene clusters for heavy metal and antibiotic resistance were also identified in the genome of CEP14. These results suggest that CEP14 may be a versatile degrader of xenobiotic compounds and well-adapted to polluted environments, where a combination of heavy metal and organic compound pollution is often found.

Aerobic degradation of cis-dichloroethene by the marine bacterium Marinobacter salsuginis strain 5N-3.

An aerobic bacterium, designated strain 5N-3 (NBRC 113055), that degrades cis-dichloroethene (cDCE) was isolated from a sea sediment in Japan. Strain 5N-3 was able to degrade a certain amount of cDCE in the presence of pyruvate without the action of inducers. In the presence of inducers, such as phenol and benzene, the strain completely removed cDCE. By the application of 16S ribosomal RNA (16S rRNA) gene sequencing and average nucleotide identity analyses, the strain 5N-3 was identified as Marinobacter salsuginis. On the other hand, identified species of Marinobacter are not known to degrade cDCE at all. A draft genome sequence analysis of the strain 5N-3 suggested that the dmp-homologous operon (operon for phenol degradation) may be contributing to the aerobic degradation of cDCE. This is the first report on an aerobic marine bacterium that has been found to degrade cDCE.

Cometabolic degradation of dichloroethenes by Comamonas testosteroni RF2

An environmental isolate Comamonas testosteroni strain RF2, which has been found to cometabolize trichloroethene (TCE) in the presence of phenol and sodium lactate as growth substrates, was tested to investigate its capacity for degrading 1,2-cis-dichloroethene (cDCE), 1,2-trans-dichlorothene (tDCE), and 1,1-dichloroethene (1,1DCE). Degradation assays were performed for single DCEs, as well as for a mixture of DCEs with TCE, which resembled contaminated plume in groundwater. RF2 was capable of efficiently removing all three dichloroethenes (DCEs) at the initial aqueous concentrations of 6.01 mg L−1 for cDCE, 3.80 mg L−1 for tDCE and 0.65 mg L−1 for 1,1DCE, with a removal efficiency of 100% for cDCE, 65.8% for tDCE, and 46.8% for 1,1DCE. Furthermore, complete removal of TCE, cDCE and 1,1DCE (122.5 μg L−1, 84.3 μg L−1 and 51.4 μg L−1, respectively) was observed in a mixture sample that also contained 72.33 μg L−1 of tDCE, which was removed to the amount of 72.3%. Moreover, degradation of cDCE (6.01 mg L−1) led to a 93.8% release of inorganic chloride, and 2,2-dichloroacetaldehyde was determined as the first intermediate of cDCE transformation. The findings of this study suggest that the strain RF2 exhibits the potential to remediate groundwater contaminated with less chlorinated ethenes.

Branched pathways in the degradation of cDCE by cytochrome P450 in Polaromonas sp. JS666

Compound specific isotope analysis (CSIA) is widely used to monitor contaminant remediation in groundwater. CSIA-based approaches that use enrichment (ε) values to assess degradative processes rely on the assumption that the contaminant being investigated will have an ε value that is constant and specific to a catalytic pathway of a microorganism. Distinct ε values have been reported for aerobic degradation of cis-dichloroethene (cDCE), which has led to a number of proposed degradation mechanisms; however, cytochrome P450 catalyzed oxidation is the only biochemical mechanism that has been established in Polaromonas sp. JS666. Using CSIA we measured the ε values for microbial oxidation of cDCE (−18.8‰±1.5‰) and 1,2-dichloroethane (1,2-DCA) (−16.6‰±0.9‰) in wild-type JS666 and the oxidation of cDCE (−13.5‰±2.3‰) from a recombinant E. coli strain expressing the cytochrome P450 enzyme from JS666. This study supports the hypothesis that cytochrome P450 catalyzes the initial step in the degradation pathway of both cDCE and 1,2-DCA and provides evidence that a single enzyme can catalyze multiple pathways with different products and distinct ε values for a single substrate. Therefore, in cases where the products of the reaction cannot, or have not been characterized, caution must be used when employing ε values to interpret mechanisms, pathways, and their applications to environmental contaminant remediation.

Cytochrome P450 Initiates Degradation ofcis-Dichloroethene by Polaromonas sp. Strain JS666

Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes.

Stable Carbon Isotope Fractionation in Chlorinated Ethene Degradation by Bacteria Expressing Three Toluene Oxygenases

One difficulty in using bioremediation at a contaminated site is demonstrating that biodegradation is actually occurring in situ. The stable isotope composition of contaminants may help with this, since they can serve as an indicator of biological activity. To use this approach it is necessary to establish how a particular biodegradation pathway affects the isotopic composition of a contaminant. This study examined bacterial strains expressing three aerobic enzymes for their effect on the 13C/12C ratio when degrading both trichloroethene (TCE) and cis-1,2-dichloroethene (c-DCE): toluene 3-monoxygenase, toluene 4-monooxygenase, and toluene 2,3-dioxygenase. We found no significant differences in fractionation among the three enzymes for either compound. Aerobic degradation of c-DCE occurred with low fractionation producing δ13C enrichment factors of −0.9 ± 0.5 to −1.2 ± 0.5, in contrast to reported anaerobic degradation δ13C enrichment factors of −14.1 to −20.4‰. Aerobic degradation of TCE resulted in δ13C enrichment factors of −11.6 ± 4.1 to −14.7 ± 3.0‰ which overlap reported δ13C enrichment factors for anaerobic TCE degradation of −2.5 to −13.8‰. The data from this study suggest that stable isotopes could serve as a diagnostic for detecting aerobic biodegradation of TCE by toluene oxygenases at contaminated sites.

Sequential Reductive and Oxidative Biodegradation of Chloroethenes Stimulated in a Coupled Bioelectro-Process

This article for the first time demonstrates successful application of electrochemical processes to stimulate sequential reductive/oxidative microbial degradation of perchloroethene (PCE) in mineral medium and in contaminated groundwater. In a flow-through column system, hydrogen generation at the cathode supported reductive dechlorination of PCE to cis-dichloroethene (cDCE), vinyl chloride (VC), and ethene (ETH). Electrolytically generated oxygen at the anode allowed subsequent oxidative degradation of the lower chlorinated metabolites. Aerobic cometabolic degradation of cDCE proved to be the bottleneck for complete metabolite elimination. Total removal of chloroethenes was demonstrated for a PCE load of approximately 1.5 μmol/d. In mineral medium, long-term operation with stainless steel electrodes was demonstrated for more than 300 days. In contaminated groundwater, corrosion of the stainless steel anode occurred, whereas DSA (dimensionally stable anodes) proved to be stable. Precipitation of calcareous deposits was observed at the cathode, resulting in a higher voltage demand and reduced dechlorination activity. With DSA and groundwater from a contaminated site, complete degradation of chloroethenes in groundwater was obtained for two months thus demonstrating the feasibility of the sequential bioelectro-approach for field application.

Aerobic biodegradation of cis-1,2-dichloroethene as sole carbon source: Stable carbon isotope fractionation and growth characteristics

Cis-1,2-dichloroethene (cDCE) is a compound of concern at many chloroethene-contaminated sites, since it tends to accumulate during reductive dechlorination of the higher chlorinated ethenes. Stable carbon isotope fractionation during aerobic cDCE biodegradation was observed in groundwater microcosms under varying incubation conditions (room temperature/groundwater temperature; with/without inorganic nutrients), and resulted in an average stable carbon isotope enrichment factor of −15.2 ± 0.5‰. A new enrichment culture, obtained from groundwater microcosms, degraded cDCE concentrations up to 100 mg L −1, was active at temperatures between 4 and 23 °C, had a pH optimum of ∼7, and could withstand prolonged periods (250 d) of starvation. Microbial growth during degradation of cDCE as sole carbon and energy source was demonstrated by protein formation in mineral medium not containing any known auxiliary substrate. The obtained growth yield was 12.5 ± 1.9 g of protein Mol −1 of cDCE, with a doubling time of 53 ± 2 h at 23 °C. Aerobic degradation of cDCE as sole carbon and energy source appears to be a promising biological process for site remediation.

Inhibition of aerobic metabolic cis-1,2-di-chloroethene biodegradation by other chloroethenes

The presence of other chloroethenes influences aerobic metabolic biodegradation of cis-1,2-dichloroethene ( cDCE). A new metabolically cDCE degrading enrichment culture was identified as also being capable of degrading vinyl chloride (VC), but not 1,1 -dichloroethene (1,1DCE), trans-1,2-dichloroethene ( tDCE), trichloroethene (TCE), or tetrachloroethene (PCE). The fastest degradation of cDCE was observed in the absence of any other chloroethene. In the presence of a second chloroethene (40–90 μM), the rate of cDCE (60 μM) degradation decreased in the following order: cDCE (+PCE) > cDCE (+ tDCE) > cDCE (+VC)> cDCE (+1,1DCE) ≈ cDCE (+TCE). With increasing concentrations of VC, ranging from 10 to 110 μM, the rate of cDCE (60 μM) degradation decreased. This study demonstrates that the inhibiting effects of chloroethene mixtures have to be considered during laboratory studies and bioremediation approaches based on metabolic cDCE degradation.

Growth kinetics and stable carbon isotope fractionation during aerobic degradation of cis-1,2-dichloroethene and vinyl chloride

Assessing changes in the isotopic signature of contaminants is a promising new tool to monitor microbial degradation processes. In this study, chloroethene degradation was proven by depletion of chloroethenes, formation of chloride, increase in protein content and stable carbon isotope fractionation. Aerobic degradation of vinyl chloride (VC) was found to proceed metabolically, with degradation rates of 0.48 and 0.29d−1; and growth yields of 9.7 and 6.4g of protein/mol of VC at room and groundwater temperature, respectively. Cis-1,2-dichloroethene (cDCE) was degraded cometabolically under aerobic conditions when VC was provided as growth substrate. Aerobic degradation was associated with significant stable carbon isotope fractionation, with enrichment factors ranging from −5.4±0.4‰ for metabolic degradation of VC to −9.8±1.7‰ for cometabolic degradation of cDCE. Thus, it was demonstrated that stable carbon isotope fractionation is suitable for assessing aerobic chloroethene degradation, which can contribute significantly to site remediation.

A 1,1,1-Trichloroethane-Degrading Anaerobic Mixed Microbial Culture Enhances Biotransformation of Mixtures of Chlorinated Ethenes and Ethanes

1,1,1-trichloroethane (1,1,1-TCA) is a common groundwater pollutant as a result of improper disposal and accidental spills. It is often found as a cocontaminant with trichloroethene (TCE) and inhibits some TCE-degrading microorganisms. 1,1,1-TCA removal is therefore required for effective bioremediation of sites contaminated with mixed chlorinated organics. This study characterized MS, a 1,1,1-TCA-degrading, anaerobic, mixed microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States. MS reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) but not further. Cloning of bacterial 16S rRNA genes revealed among other organisms the presence of a Dehalobacter sp. and a Desulfovibrio sp., which are both phylogenetically related to known dehalorespiring strains. Monitoring of these populations with species-specific quantitative PCR during degradation of 1,1,1-TCA and 1,1-DCA showed that Dehalobacter proliferated during dechlorination. Dehalobacter growth was dechlorination dependent, whereas Desulfovibrio growth was dechlorination independent. Experiments were also performed to test whether MS could enhance TCE degradation in the presence of inhibiting levels of 1,1,1-TCA. Dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) in KB-1, a chloroethene-degrading culture used for bioaugmentation, was inhibited with 1,1,1-TCA present. When KB-1 and MS were coinoculated, degradation of cDCE and VC to ethene proceeded as soon as the 1,1,1-TCA was dechlorinated to 1,1-DCA by MS. This demonstrated the potential application of the MS and KB-1 cultures for cobioaugmentation of sites cocontaminated with 1,1,1-TCA and TCE.

In situ bioremediation of a cis-dichloroethylene-contaminated aquifer utilizing methane-rich groundwater from an uncontaminated aquifer

At a trichloroethylene (TCE)-contaminated site in Chikura, Chiba, Japan, TCE had spread over to the first and second aquifers over years. After 8 years of pumping and treatment, finally derivative of TCE, cis-dichloroethylene (c-DCE) remained only in the second aquifer. In this study, feasibility of a low cost in situ bioremediation utilizing groundwater of the third aquifer, which contained natural dissolved methane possibly derived from natural gas field nearby, to stimulate methane-oxidizing bacteria was examined. In vitro experiment showed that a mixture of the groundwater from the second and third aquifers stimulated a growth of methane oxidizing bacteria and enhanced c-DCE degradation. The groundwater of the third aquifer was introduced into the second aquifer in situ. The population of methanotrophs with high V max and K m for methane uptake increased, resulting in successful degradation of c-DCE at a monitoring well 2 m downgradient of the injection well.

Stable carbon isotope fractionation during aerobic biodegradation of chlorinated ethenes.

Stable isotope analysis is recognized as a powerful tool for monitoring, assessing, and validating in-situ bioremediation processes. In this study, kinetic carbon isotope fractionation factors (epsilon) associated with the aerobic biodegradation of vinyl chloride (VC), cis-1,2-dichloroethylene (cDCE), and trichloroethylene (TCE) were examined. Of the three solvents, the largest fractionation effects were observed for biodegradation of VC. Both metabolic and cometabolic VC degradation were studied using Mycobacterium aurum L1 (grown on VC), Methylosinus trichosporium OB3b (grown on methane), Mycobacterium vaccae JOB5 (grown on propane), and two VC enrichment cultures seeded from contaminated soils of Alameda Point and Travis Air Force Base, CA. M. aurum L1 caused the greatest fractionation (epsilon = -5.7) while for the cometabolic cultures, epsilon values ranged from -3.2 to -4.8. VC fractionation patterns for the enrichment cultures were within the range of those observed for the metabolic and cometabolic cultures (epsilon = -4.5 to -5.5). The fractionation for cometabolic degradation of TCE by Me. trichosporium OB3b was low (epsilon = -1.1), while no quantifiable carbon isotopic fractionation was observed during the cometabolic degradation of cDCE. For all three of the tested chlorinated ethenes, isotopic fractionation measured during aerobic degradation was significantly smaller than that reported for anaerobic reductive dechlorination. This study suggests that analysis of compound-specific isotopic fractionation could assist in determining whether aerobic or anaerobic degradation of VC and cDCE predominates in field applications of in-situ bioremediation. In contrast, isotopic fractionation effects associated with metabolic and cometabolic reactions are not sufficiently dissimilar to distinguish these processes in the field.

Ethene as an auxiliary substrate for the cooxidation of cis-1, 2-dichloroethene and vinyl chloride

Cultures able to dechlorinate cis-1,2-dichloroethene (cDCE) were selected with ethene (3-20%, v/v) as the sole source of carbon and energy. One mixed culture (K20) could degrade cDCE (400 &mgr;mol l(-1)) or vinyl chloride (100 &mgr;mol l(-1)) in the presence of ethene (</= 80 &mgr;mol l(-1) and </= 210 &mgr;mol l(-1), respectively). This culture consists of at least five bacterial strains. All five strains were able to degrade cDCE cometabolically in pure culture. The mixed culture K20 was highly tolerant against cDCE (up to 6 mmol l(-1) in the liquid phase). Degradation of cDCE (200 &mgr;mol l(-1)) was not affected by the presence of trichloroethene (100 &mgr;mol l(-1)) or tetrachloroethene (100 &mgr;mol l(-1)). Transformation yields (T(y), defined as unit mass of chloroethene degraded per unit mass of ethene consumed) of the mixed culture K20 were relatively high (0.51 and 0.61 for cDCE and vinyl chloride, respectively). The yield for cDCE with ethene as auxiliary substrate was ninefold higher than any values reported with methane or methane/formate as auxiliary substrate. The viability of the cells of the mixed culture K20 (0.3 mg of cells ml(-1)) was unaffected by the transformation of </= 200 &mgr;mol l(-1) cDCE in 300 min.

嫌気・好気直列バイオリアクターを用いたテトラクロロエチレンの連続分解

Degradation of tetrachloroethene (PCE) by using a two-stage anaerobic/aerobic bioreactor system was investigated. A PCE-dechlorinating anaerobic consortium was immobilized on the surface of crushed scallop shells. The maximum rate of PCE dechlorination to cis-1, 2-dichloroethene (cDCE) with an anaerobic fixed-bed upflow reactor containing the biofilm on the shell was 30μmol PCE·l-1 reactor volume·h-1. A phenol-oxidizing bacterium, capable of co-metabolic degradation of cDCE was immobilized in κ-carrageenan gel beads. Gaseous cDCE purged from effluent of the anaerobic reactor was degraded with an air-lift aerobic reactor containing the immobilized cells. To degrade PCE continuously, yeast extract was added to the anaerobic reactor feed, and phenol was added to the aerobic reactor feed. The life of the two-stage anaerobic/aerobic bioreactor had been tested for one month at a PCE supply rate of 17μmol PCE·l-1 reactor volume·h-1. The activity of the aerobic reactor decreased gradually, although the activity of the anaerobic reactor was stable during the experimental period.