Title: Western Region Hazardous Substance Research Center Project 1-OSU-01
Developing and Optimizing Biotransformation Kinetics for the Bioremediation of Trichloroethylene at NAPL Source Zone Concentrations

Investigators: Lewis Semprini and Mark E. Dolan

Institution: Oregon State University

Research Category:Groundwater, TCE, PCE, Vinyl Chloride, DNAPL, Bioremediation

Project Period:January 2002 – December 2005

Objectives: This project aimed to (1) develop a culture with the ability to reductively dechlorinate TCE to ethylene at very high concentrations (above 1,000 M) and in the presence of DNAPL; (2) characterize microbial growth and measure maximum substrate utilization rates and half velocity coefficients for successive dechlorinations of TCE to ethylene; (3) characterize the microbial consortium by investigating molecular methods to evaluate the diversity of the mixed culture developed in the kinetic studies; (4) provide kinetic information and cultures in support of the Center Projects “Development of the Push-Pull Test to Monitor the Bioaugmentation of Dehalogenating Cultures” and “Development and Evaluation of Field Sensors for Monitoring Bioaugmentation with Anaerobic Dehalogenating Cultures for In-Situ Treatment of TCE.”

Rationale: While TCE reductive dechlorination has been demonstrated under a variety of conditions, most laboratory and field projects have been conducted at TCE concentrations of 100 mg/L or less. However, near NAPL sources concentrations of chlorinated aliphatic hydrocarbons approach their solubilities (>1,000 mg/L for TCE and >150 mg/L for PCE). Studies with different enrichment cultures isolated from contaminated sites have shown good potential for treatment of high concentrations of PCE and TCE. The cultures have different dehalogenation kinetic properties, which indicate that a more effective enrichment culture might be obtained by combining cultures. Research is needed to optimize the transformation kinetics for the consortium that has the ability to reductively dechlorinate high concentrations of TCE and PCE to stoichiometric quantities of ethylene. This project will prove useful for the remediation of chlorinated aliphatic compounds in the NAPL source zone.

Summary of Findings: A culture was developed that can rapidly degrade high concentrations of PCE and TCE to ethylene by mixing two enrichment cultures. The Point Mugu enrichment (PM) rapidly transforms TCE to VC, and slowly transforms VC to ethylene at very high PCE and TCE concentrations. The Evanite enrichment (EV) rapidly transforms PCE to cis-DCE, and vinyl chloride to ethylene. By mixing both cultures rapid transformation of PCE and TCE to ethylene was achieved. We used batch reactor studies to determine transformation kinetics for both cultures, and then when both cultures were combined. Inhibition among the CAHs will also be evaluated. Models were constructed to simulate the results of the sequential transformations over a broad range of concentrations up to the solubility limit of PCE and 50% of the solubility limit of TCE. 

Kinetic studies were conducted with two mixed cultures and a binary culture (a mixture of the two cultures) to describe the reductive dechlorination of chlorinated ethylenes. Inhibition of the CAHs was also studied. The EV culture and the PM obtained from different contaminated sites showed different patterns of reductive dechlorination. The simple batch kinetic method was developed that was easy to implement and produced very reproducible kinetic values. The kmax (based on the total protein content of the culture) for c-DCE of the EV culture was about two times lower than that of the PM culture, reflecting the slower c-DCE biotransformation of the EV culture. The kmax and KS values for VC (2.44 ± 0.36 µmol/mg of protein/day and 602 ± 7.06 µM, respectively) of the PM culture were very different from those of the EV culture (8.08 ± 0.94 µmol/mg of protein/day and 62.6 ± 2.37 µM, respectively). Inhibition studies were performed on the inhibition of the CAH on the transformation of each other. Inhibition studies showed the more chlorinated ethylenes inhibit reductive dechlorination of the less chlorinated. PCE inhibited reductive TCE dechlorination, but not c-DCE dechlorination, while TCE strongly inhibited c-DCE and VC dechlorinations. c-DCE strongly inhibited the transformation of VC. Inhibition constants of each chlorinated ethylene, KI (µmol/L), were comparable to their respective half-velocity coefficients, when a competitive inhibition model was applied.

Batch tests to study the sequential transformation of PCE to ETH were also performed over a factor of 30 change in concentration of PCE, up to it solubility limit in water (1128 M), with the EV, PM, and a 50/50 mixture of both cultures to yield a binary culture (BM). Additional studies were performed with TCE up to a concentration of 4173 M (550 mg/L), which represents 50% of its solubility limit in water. Simulations of the successive transformations of PCE to ETH, and TCE to ETH using the independently derived kinetic parameters matched well the results of batch kinetic tests for initial PCE concentration up to around 317 M. The simulations included the growth on the chlorinated solvents, and Monod kinetics including competitive inhibition. Above this concentration simulations deviated from the experimental observations, and predicted more rapid transformation of VC than was observed. The results suggest potential toxicity or inhibition at the higher concentrations of PCE and TCE. Simulations were performed with Halden kinetics incorporated into the transformation models, where high concentrations of a contaminant inhibit its transformation. In order to explain the experimental observations Halden kinetics for TCE transformation were required for both the EV and the PM cultures and for c-DCE and VC transformation by the EV culture. The EV culture appeared to be more inhibited at higher CAH concentrations. TCE concentrations, up to 4173 M (550 mg/L) were transformed by both the EV and PM culture, with the PM culture more rapid transforming the TCE to VC and ethene. The results indicate less inhibition of the PM culture at higher concentrations. Batch experimental results indicate that the BM culture, which represent a mixture of both cultures, has better transformation abilities that either of the single cultures. Simulations for the BM culture, using individual transformation abilities of each culture, support the experimental observations of more diverse dechlorination ability than either of the single mixed cultures.

Molecular methods analysis using PCR reactions with Dehalococcoides-specific primers and Desulfuromonas-specific primers found Dehalococcoides-like microorganisms in both the cultures, but not Desulfuromonas-like microorganisms. The molecular methods could not distinguish between the Dehalococcoides species of the EV and the PM cultures.



Journal Articles

Pon G. M.R. Hyman, and L. Semprini (2003). Acetylene Inhibition of Trichloroethene and Vinyl Chloride Reductive Dechlorination. Environmental Science and Technology, 37 3181-3188.

Pon, G. and L. Semprini (2004). Anaerobic Reductive Dechlorination of 1-chloro-1-fluoroethene to Track the Transformation of Vinyl Chloride. Environmental Science and Technology, 38 6803-6808.

Yu, S., and L. Semprini (2002). Comparison of Trichloroethylene Reductive Dehalogenation by Microbial Communities Stimulated on Silicon-based Organic Compounds as Slow-release Anaerobic Substrates. Water Research 36(20): 4985-4996.

Yu S. and L.Semprini (2004). Kinetics and Modeling of Reductive Dechlorination at High PCE and TCE Concentrations. Biotechnology and Bioengineering, 88 451-464.

Yu, S., M.E. Dolan, and L. Semprini (2005). Kinetics and Inhibition of Reductive Dechlorination of Chlorinated Ethylenes by Two Different Mixed Cultures. Environmental Science and Technology,39 195-205.


Conferences Proceedings and Presentations

Yu S. and L. Semprini (2002). Dechlorination of PCE DNAPL with TBOS Using a Binary Mixed Culture. The 3rd International Conference on Remediation of Chlorinated and Recalcitrant Compounds, Monterey, CA (2B-49) (May 20-23).

Yu, S and L. Semprini (2003). Kinetic Studies and Comparison for Reductive Dechlorination by a Binary Mixed Culture. The Seventh International Symposium on In Situ and On-Site Bioremediation, Orlando FL (June, 2-5).



Yu, S. (2004). Kinetic and Modeling Investigations of Anaerobic Reductive Dechlorination of Chlorinated Ethylenes Using Single and Binary Mixed Cultures and Silcon-Based Organic Compounds as Slow-release Substrates. Ph.D., Oregon State University.

Pon. G. (2004). Inhibition, Kinetic and Modeling Studies of Acetylene and 1-chlorofluoroethene on Reductive Dechlorination of TCE and Vinyl Chloride. Ph.D, Oregon State University.


Supplemental Keywords: biotransformation; groundwater; NAPL; VOCs; bioremediation; chlorinated solvent; remediation technologies; in-situ