Molecular and Kinetic Characterization of Vinyl Chloride Oxidation and Corresponding Cometabolism of cis- and trans-1,2-DichloroethylenesEPA Grant Number: R827612E03
Title: Molecular and Kinetic Characterization of Vinyl Chloride Oxidation and Corresponding Cometabolism of cis- and trans-1,2-Dichloroethylenes
Investigators: Freedman, David , Dean, Ralph , Wing, R.
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)
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 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 project is to evaluate aerobic biodegradation of VC by organisms that use it as a sole growth substrate, along with a newly discovered 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 that will be used to achieve them are:
1) molecular characterization of a culture we have isolated that grows on VC and simultaneously cometabolizes DCEs, especially cDCE. This will include cloning and sequencing of the gene(s) involved. A bacterial artificial chromosome library will be created and ligated DNA will be transformed into E. coli. Pools of BAC clones will be screened for their ability to grow on VC. BAC inserts will be sequenced and evaluated in terms of candidate VC-catabolic genes. The identity of the culture will be determined by analysis of its 16S rDNA sequence;
2) determination of the pathway by which VC and DCEs are biodegraded. Preliminary evidence indicates the formation of epoxides. This will be confirmed by GC/MS analysis of volatile unknowns, using chemically synthesized and biologically produced (using M. trichosporium Ob3b) epoxides as standards. Additional evidence of epoxide formation will be obtained using a colorometric test involving reaction with 4-(4-nitrobenzyl)pyridine;
3) isolation and identification of a second organism that also grows on VC, using soil inoculum from a chloroethylene-contaminated Superfund site. An enrichment culture that uses VC as a sole substrate has already been developed with this inoculum. An isolate will be obtained and identified by analysis of its 16S rDNA sequence. The ability of this organism to cometabolize DCEs and TCE will also be determined; and
4) evaluation of a kinetic model that describes cometabolism of DCEs by the VC-grown isolates. The model includes terms for the loss of microbial biomass or enzyme activity caused by auto-oxidation, proteolysis, depletion of reducing power, product toxicity, and suicide inactivation. It also allows for evaluation of cometabolism in the presence of the growth substrate, i.e., VC. The effect of long-term exposure of the culture to VC and cDCE on the rate at which each is biodegraded will be determined.
The results of this project will help to improve our understanding of in situ biodegradation of chlorinated aliphatic compounds, particularly when an aerobic zone follows incomplete anaerobic dechlorination. Understanding the capabilities of organisms that use VC as a sole growth substrate and simultaneously cometabolize DCEs will advance the prospects for successfully applying natural attenuation.