Kinetics and Modeling of Reductive Dechlorination at High PCE and TCE Concentrations Seungho Yu, Lewis Semprini Department of Civil, Construction, and Environmental Engineering, Oregon State University, Corvallis, Oregon 97331; telephone: 541-737-6895; fax: 541-737-3099; e-mail: [email protected] Received 26 March 2004; accepted 8 July 2004 Published online 21 September 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20260 Abstract: Two biokinetic models employing the Michaelis- INTRODUCTION Menten equation for anaerobic reductive dechlorination of tetrachloroethylene (PCE) and trichloroethylene (TCE) Chlorinated ethylenes are ubiquitous soil and groundwater were developed. The models were compared with results contaminants (Westrick et al., 1984), for which bioreme- from batch kinetic tests conducted over a wide range of diation is a promising technology. Anaerobic reductive de- PCE and TCE concentrations with two different dechlorin- chlorination is an important process in the subsurface, ating cultures. One model applies Michaelis-Menten ki- netics with competitive inhibition among chlorinated especially for the dechlorination of highly chlorinated com- aliphatic hydrocarbons (CAHs), while the other model in- pounds such as PCE and TCE, which can be present at high cludes both competitive inhibition and Haldane inhibition concentrations due to the presence of dense non-aqueous at high CAH concentrations. Model simulations with com- phase liquid (DNAPL; Rittmann et al., 1994). petitive inhibition simulated the experimental results well Although intrinsic biotransformation of PCE and TCE for PCE concentrations lower than 300 AM. However, sim- ulations deviated from the experimental observations for occurs, observations at many contaminated sites show trans- PCE or TCE concentrations greater than 300–400 AM. formation often stalls at c-DCE and VC (Major et al., 2002). The kinetic model that incorporated both competitive and This may be due to an absence of appropriate dechlorinating Haldane inhibitions better simulated experimental data for bacteria. A study of 24 chlorinated ethylene-contaminated PCE concentrations near the solubility limit (1000 AM), and sites reported that Dehalococcoides organisms were not de- TCE concentrations at half its solubility limit (4000 AM). Based on the modeling analysis of the experimental re- tected at sites where incomplete dechlorination was ob- sults, the PM culture (Point Mugu, CA) had very high Hal- served (Hendrickson et al., 2002). Bioaugmentation with dane inhibition constants for cis-1,2-dichlororethylene dechlorinating cultures that can completely transform PCE (c-DCE) and vinyl chloride (VC) (6000 and 7000 AM, re- and TCE to ethylene (ETH) can be an appropriate strategy spectively), indicating very weak Haldane inhibition, while to enhance reductive dechlorination (Ellis et al., 2000; the EV culture (the Evanite site in Corvallis, OR) had lower Haldane inhibition constants for TCE, c-DCE, and VC of Harkness et al., 1999). 900, 750, and 750 AM, respectively. The BM culture (a bi- The use of a binary mixed culture consisting of two nary mixed culture of the PM and EV cultures) had trans- different aerobic pure cultures has been reported to show formation abilities that represented the mixture of the EV interspecies interactions and changes in the rate and extent and PM cultures. Model simulations of the BM culture of biodegradation (Fairlee et al., 1997; Rogers et al., 2000). transformation abilities were well represented by separate rate equations and model parameters for the two indepen- The studies revealed that the utilization of a binary mixed dent cultures that were simultaneously solved. Modeling culture could enhance the biodegradation of mixed pollu- results indicated that a combination of competitive and tants such as toluene, phenol, and benzene. Previously, we Haldane inhibition kinetics is required to simulate dechlo- reported (Yu and Semprini, 2002a) that a binary anaerobic rination over a broad range of concentrations up to the mixed culture isolated from two different contaminated solubility limit of PCE and half the solubility limit of TCE. B 2004 Wiley Periodicals, Inc. groundwaters was more effective in reductively dechlorin- Keywords: reductive dechlorination; PCE; TCE; compet- ating PCE to ETH than each individual culture. itive inhibition; Haldane inhibition Various models have been developed that describe re- ductive dechlorination of chlorinated ethylenes. For com- plete reductive dechlorination of PCE to ETH, competitive inhibition of VC dechlorination by the other chlorinated eth- Correspondence to: Lewis Semprini Contract grant sponsor: U.S. Environmental Protection Agency- ylenes was assumed, and model simulations were compared sponsored Western Region Hazardous Substance Research Center with the experimental data (Tandoi et al., 1994). Fennell Contract grant number: R-828772 and Gossett (1998) simulated both fermentation of electron B 2004 Wiley Periodicals, Inc. donors and competition for the evolved H2 between hy- samples (200 AL) was accomplished using an HP-5890 GC drogenotrophic PCE dechlorinators and methanogens, but with a thermal conductivity detector (TCD), operated iso- did not consider inhibition between the chlorinated ethyl- thermally at 220jC. The gas samples were chromato- enes. Tonnaer et al. (1997) also incorporated competitive graphically separated with a Carboxen 1000 column (15 ft dechlorination between PCE and TCE in a model to predict  1/8 in; Supelco, Bellefonte, PA), using argon as carrier gas PCE reductive dechlorination. Garant and Lynd (1998) at a flow rate of 15 mL/min. The hydrogen detection limit showed that competitive kinetics for complete reductive was 4 nM (aqueous concentration). dechlorination of PCE achieved better model fits of the experimental data than noncompetitive inhibition. Other Enrichment Cultures and Kinetic Batch Reactors modeling studies included competitive inhibition between c-DCE and VC with a dechlorinating culture using c-DCE The two anaerobic mixed cultures used in this study were and VC as electron acceptors for energy (Cupples et al., isolated from subsurface samples from Point Mugu Naval 2004; Haston 1999). Weapon Facility, California (PM) and the Evanite site in Recently, we experimentally determined maximum rates Corvallis, Oregon (EV). The third culture used was a binary (kmax) and half-velocity coefficients (KS) for each step of mixed culture (BM), representing a mixture of the PM and the dechlorination of PCE to ETH for two different mixed EV cultures (1:1 volume ratio). Details of the enrichment cultures (Yu, 2003) and evaluated inhibition between chlo- process are provided by Yu (2003). PCR assay targeting rinated ethylenes. The study presented here aims to sim- genes encoding for 16S ribosomal RNA indicated that ulate with independently measured kinetic parameters the the PM and EV cultures had Dehalococcoides-like micro- sequential transformation of PCE and TCE to ETH over a organisms present with the ability of complete reduc- broad range of concentrations up to the solubility limit of tive dechlorination to ETH (Yu, 2003). The two cultures PCE and half the solubility limit of TCE in water. The ki- were grown in separate batch mother reactors (total volume netic models developed for each culture were also com- 1.2 L with liquid volume of 1 L) as described in Yu and bined to simulate the transformation ability of a binary Semprini (2002b) and Yu (2003). The mother reactors were mixed culture composed of each dechlorinating culture. amended with an aqueous TCE or PCE concentration of 100 mg/L for the PM and EV cultures, respectively. 1- Butanol (40–60 mg/L) was added as a fermenting substrate MATERIALS AND METHODS and hydrogen (10% in the headspace) was added as an electron donor. The liquid cultures for kinetic studies were obtained from the batch mother reactor after 50–100% of Chemicals the PCE or TCE added was converted to ETH. Chlorinated ethylenes including PCE (99.9%, spectropho- Batch kinetic reactors [serum bottles—156 mL total tometric grade), TCE (99.9%), and c-1,2-dichloroethylene volume with 125 mL liquid—fitted with gray chlorobutyl (c-DCE) (97%) used for preparing stock feed solutions and rubber septa (Wheaton Industries, Millville, NJ)] were con- analytical standards were obtained from Acros Organics structed in an anaerobic glove box. The reactors contained (Pittsburgh, PA). Gaseous VC and ETH (both 99.5%, Al- 125 mL enrichment liquid culture and 31 mL anaerobic gas drich Chemical, Milwaukee, WI) were used to prepare ana- headspace. After construction in the glove box, the reactors lytical standards. Hydrogen gas (99%, Airco, Inc., Albany, were purged with a mixed gas mixture of N2 (70%), CO2 OR) was used as an electron donor for dechlorination and (20%), and H2 (10%) that was treated in a tube furnace to 1-butanol (99.8%, HPLC grade, Aldrich Chemical, Mil- remove trace oxygen. After purging, neat PCE or TCE and waukee, WI) was used as a fermenting substrate. butanol were added to each reactor to achieve the desired initial concentration. To evaluate different PCE concen- trations (Table I), the kinetic reactors were purged after Analytical Methods each experimental run, and higher PCE concentrations were The sample gas concentrations of all chlorinated ethylenes, introduced along with butanol and hydrogen. The batch ETH, and hydrogen were determined using gas chromatog- reactors were shaken at 200 rpm and incubated at 20jC. raphy.