Grantee Research Project Results
Final Report: Molecular and Kinetic Characterization of Vinyl Chloride Oxidation and Corresponding Cometabolism of cis- and trans-1,2-Dichloroethylenes
EPA Grant Number: R827612E03Title: Molecular and Kinetic Characterization of Vinyl Chloride Oxidation and Corresponding Cometabolism of cis- and trans-1,2-Dichloroethylenes
Investigators: Freedman, David , Wing, R. , Dean, Ralph
Institution: Clemson University
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2002
Project Amount: $160,000
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (1998) RFA Text | Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)
Objective:
A majority of Superfund sites still have groundwater contaminated with chlorinated ethylenes, including tetrachloroethene (PCE) and trichloroethene (TCE). Anaerobic reductive dechlorination of these and related solvents has typically resulted in the accumulation of daughter products, notably cis-1,2-dichloroethylene (cDCE) and vinyl chloride (VC). One way to treat residual levels of daughter products is to follow anaerobic dechlorination with aerobic treatment. The main objective of this research project was to evaluate aerobic biodegradation of VC by organisms that use it as a sole growth substrate, along with a less well-known phenomenon (i.e., cometabolism of cDCE and, to a lesser extent trans-1,2-dichloroethylene [tDCE] by the VC-grown organisms). The specific objectives and approaches of this research project include:
· Characterization of a culture (strain MF1) that grows on VC and simultaneously cometabolizes DCEs, especially cDCE.
· Evaluation of the pathway by which VC and DCEs are aerobically biodegraded. This involved acetylene inhibition studies of the alkene mono-oxygenase that initiates VC and ethene metabolism, as well as the identification of the epoxides formed when cDCE and tDCE are cometabolized.
· Isolation and identification of two other organisms that also grow on VC. Strain DL1 was isolated on ethene using activated sludge as the inoculum. It transitioned from ethene to VC as a sole source of carbon and energy. Strain AJ was isolated on VC as the sole substrate using soil from a chloroethylene-contaminated Superfund site as the inoculum. In this respect, AJ is the most "environmentally relevant" of the three strains isolated. Molecular characterization of AJ included localization of the gene(s) involved by establishing that the ability to use VC as a growth substrate is associated with a mega-linear plasmid. Portions of the plasmid have been cloned and await sequencing. The ability of AJ to cometabolize DCEs and TCE also was determined.
· Development of a kinetic model that describes cometabolism of cDCE by VC-grown MF1. The model includes terms for cometabolism in the presence or absence of the growth substrate.
Summary/Accomplishments (Outputs/Outcomes):
Pseudomonas aeruginosa strain MF1 was isolated and characterized for its ability to use VC as a growth substrate. Identification included complete sequencing of the isolate's 16S rRNA gene and phenotypic characterization using the BBL Crystal™ system. Due to the slow growth rate of MF1, further molecular characterization was not pursued.
Procedures were developed to identify cDCE- and tDCE-epoxide formation, using the VC-grown enrichment culture from which MF1 was isolated. This included identification by gas chromatography/mass spectrometry and trapping in 4-(4-nitrobenzyl)pyridine. Although the enrichment culture cometabolized both cDCE and tDCE, MF1 showed no activity on tDCE. MF1 cometabolizes 1,1-DCE to a very limited extent, but shows no activity on TCE. The presence of tDCE and TCE enhanced the rate of VC utilization, although the mechanism by which this occurs is not yet known. At high concentrations, 1,1-DCE inhibited VC utilization.
Another P. aeruginosa strain, DL1, was isolated on ethene as a sole source of carbon and energy. When initially provided with VC, DL1 rapidly consumed it via cometabolism. A kinetic model for this process was developed and validated. With additional incubation, DL1 slowly adapted to use of VC as a sole source of carbon and energy. This result suggests that ethene may be a desirable substrate for initial enrichment when seeking cultures that can use VC as a growth substrate.
A culture capable of using VC as a sole substrate was isolated from the Aerojet enrichment culture, derived from a Superfund site in California. It grows considerably faster than MF1 and does not appear to be a P. aeruginosa sp. based on results with the BBL Crystal™ system. More significantly, the ability of AJ to grow on VC and ethene as sole sources of carbon and energy has been definitively linked to the presence of a mega-linear plasmid. A portion of the plasmid has been cloned and awaits sequencing.
A kinetic model was developed to describe cDCE cometabolism and VC metabolism in the presence and absence of one another. The growth substrate (VC) completely inhibits cDCE cometabolism. As soon as VC reaches a low concentration, cDCE is rapidly consumed. This result has practical significance from the standpoint of offering a potential explanation for disappearance of cDCE in the absence of primary substrates other than VC. Two undergraduate and three graduate students, along with two postdoctoral associates, actively participated in this research.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 8 publications | 4 publications in selected types | All 4 journal articles |
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Type | Citation | ||
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Freedman DL, Danko AS, Verce MF. Substrate interactions during aerobic biodegradation of methane, ethene, vinyl chloride and 1,2-dichloroethenes. Water Science and Technology 2001;43(5):333-340. |
R827612E03 (Final) |
not available |
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Verce MF, Ulrich RL, Freedman DL. Characterization of an isolate that uses vinyl chloride as a growth substrate under aerobic conditions. Applied and Environmental Microbiology 2000;66(8):3535-3542. |
R827612E03 (Final) |
not available |
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Verce MF, Ulrich RL, Freedman DL. Transition from cometabolic to growth-linked biodegradation of vinyl chloride by a Pseudomonas sp. isolated on ethene. Environmental Science and Technology 2001;35(21):4242-4251. |
R827612E03 (Final) |
not available |
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Verce MF, Gunsch CG, Danko AS, Freedman DL. Cometabolism of cis-1,2-dichloroethene by aerobic cultures grown on vinyl chloride as the primary substrate. Environmental Science and Technology 2002;36(10):2171-2177. |
R827612E03 (Final) |
not available |
Supplemental Keywords:
biodegradation, bioremediation, cometabolism, chlorinated ethylenes, Pseudomonas aeruginosa, vinyl chloride., Scientific Discipline, Geographic Area, Waste, Water, TREATMENT/CONTROL, Contaminated Sediments, Environmental Chemistry, Remediation, Treatment Technologies, State, Chemistry, Technology, Environmental Engineering, environmental technology, biodegradation, contaminated sediment, remediation technologies, cometabolism, chemical contaminants, catalysts, bioremediation of soils, engineering, halogenated organic contaminants, biotechnology, dehalogenation, kinetic model, South Carolina (SC), assessment methods, phytoremediation, Vinyl chloride, bioremediationProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.