Quantifying chlorinated ethene degradation during reductive dechlorination at Kelly AFB using stable carbon isotopes

Abstract

Stable isotope analysis of chlorinated ethene contaminants was carried out during a bioaugmentation pilot test at Kelly Air Force Base (AFB) in San Antonio Texas. In this pilot test, cis-1,2-dichloroethene (cDCE) was the primary volatile organic compound. A mixed microbial enrichment culture, KB-1™, shown in laboratory experiments to reduce chlorinated ethenes to non-toxic ethene, was added to the pilot test area. Following bioaugmentation with KB-1™, perchloroethene (PCE), trichloroethene (TCE) and cDCE concentrations declined, while vinyl chloride (VC) concentrations increased and subsequently decreased as ethene became the dominant transformation product. Shifts in carbon isotopic values up to 2.7‰, 6.4‰, 10.9‰ and 10.6‰ were observed for PCE, TCE, cDCE and VC, respectively, after bioaugmentation, consistent with the effects of biodegradation. While a rising trend of VC concentrations and the first appearance of ethene were indicative of biodegradation by 72 days post-bioaugmentation, the most compelling evidence of biodegradation was the substantial carbon isotope enrichment (2.0‰ to 5.0‰) in ä¹³C_cDCE. Fractionation factors obtained in previous laboratory studies were used with isotope field measurements to estimate first-order cDCE degradation rate constants of 0.12 h⁻¹ and 0.17 h⁻¹ at 115 days post-bioaugmentation. These isotope-derived rate constants were clearly lower than, but within a factor of 2–4 of the previously published rate constant calculated in a parallel study at Kelly AFB using chlorinated ethene concentrations. Stable carbon isotopes can provide not only a sensitive means for early identification of the effects of biodegradation, but an additional means to quantify the rates of biodegradation in the field.

Introduction

The chlorinated solvent perchloroethene (PCE) is one of the most frequently detected groundwater contaminants at hazardous waste sites in North America (National Research Council, 1994). PCE is a potential carcinogen and is classified as a priority pollutant by the United States Environmental Protection Agency (EPA) (USEPA, 2001). Under anaerobic conditions, the primary mechanism for the degradation of PCE and other chlorinated ethenes in the environment is reductive dechlorination (Vogel et al., 1987). Reductive dechlorination involves the sequential replacement of chlorine atoms with hydrogen atoms resulting in the transformation of PCE to trichloroethene (TCE), TCE to cis-1,2-dichloroethene (cDCE), cDCE to vinyl chloride (VC) and VC to non-toxic ethene.

Several enrichment cultures capable of mediating reductive dechlorination of chlorinated ethenes have been identified. However, while complete reductive dechlorination of PCE to non-toxic end-products is the ideal outcome, microorganisms that reductively dechlorinate the less chlorinated ethenes are not always present or actively dechlorinating at all contaminated sites (Harkness et al., 1999, Hendrickson et al., 2002, Maymo-Gatell et al., 1997). Incomplete reductive dechlorination has been observed in laboratory (Harkness et al., 1999) and field studies (Ellis et al., 2000), resulting in accumulation of transformation products cDCE, or VC. If the requisite microorganisms are present, then complete dechlorination can be achieved with the addition of electron donors (biostimulation) (Song et al., 2002), or after the addition of a reductively dechlorinating microbial enrichment culture (bioaugmentation) when requisite microorganisms are absent (Major et al., 2002, Ellis et al., 2000, Harkness et al., 1999). Assessing the effectiveness of biodegradation requires monitoring of the primary contaminant and its transformation products, direct microbiological evidence, as well as other geochemical support. In this study, stable carbon isotope analysis was used to verify and quantify the extent of biodegradation at a field site contaminated with chlorinated ethenes during a bioaugmentation pilot study.

Recent laboratory and field studies demonstrated that compound specific isotope analysis (CSIA) can be used to identify biodegradation of chlorinated ethenes (Bloom et al., 2000, Hunkeler et al., 1999, Hunkeler et al., 2002, Kirtland et al., 2003, Sherwood Lollar et al., 1999, Sherwood Lollar et al., 2001, Slater et al., 2001, Vieth et al., 2003). CSIA measures the ratio of two isotopes of an element (e.g. ¹³C and ¹²C) for an individual compound. For carbon, the isotopic ratio is expressed in δ¹³C notation,

$${\delta }^{13}\text{C}=(\frac{R_{\text{sample}}}{R_{\text{std}}}-1)\times 1000$$

where R_sample is the ¹³C/¹²C ratio in a given compound and R_std is the ¹³C/¹²C ratio of the international standard, V-PDB. The δ¹³C value is expressed in units of per mil (‰) (Clark and Fritz, 1997). The analytical error associated with stable carbon isotope analysis by CSIA is ±0.5‰ (Dempster et al., 1997, Mancini et al., 2002). This error incorporates both the internal reproducibility on duplicate measurements, and the accuracy of the measurement with respect to international standards.

A shift in the substrate's ratio of heavy to light isotopes is known as isotopic fractionation and is the result of different reaction rates for each isotopic species (e.g. ¹³C and ¹²C). Molecules containing the lighter isotope have a tendency to react at a faster rate than molecules containing the heavier isotope (Faure, 1986). As dissolved TCE degrades via reductive dechlorination to cDCE, the ratio of ¹³C/¹²C of the remaining TCE will increase due to the greater rate of incorporation of the isotopically light (¹²C) molecules into the breakdown product (Sherwood Lollar et al., 1999). For example, during anaerobic biodegradation of TCE using a mixed consortium of bacteria cultured from contaminated soil from the Department of Energy's Pinellas site Largo, FL (the Pinellas consortium, Ellis et al., 2000), Sherwood Lollar et al. (1999) showed that δ¹³C values for TCE shifted from −30.4‰ to values as enriched as −16.0‰. Other studies of dechlorination of highly chlorinated ethenes have documented significant isotopic shifts in the breakdown products of reductive dechlorination as well (Bloom et al., 2000, Hunkeler et al., 1999, Slater et al., 2001). The less chlorinated biodegradation products are initially more depleted in ¹³C with respect to the initial substrate from which they are derived, due to the preferential incorporation of ¹²C into the transformation products. Subsequently, each biodegradation product shows a trend of increasing isotopic enrichment as it is itself degraded.

Laboratory studies have demonstrated that carbon isotopic fractionation trends measured during the reduction of TCE, cDCE and VC all fit a Rayleigh model for closed systems (Mariotti et al., 1981), indicating that carbon stable isotopes may provide both a quantitative and qualitative indicator of biodegradation (Bloom et al., 2000, Hunkeler et al., 1999, Hunkeler et al., 2002, Sherwood Lollar et al., 1999, Slater et al., 2001). In contrast, non-degradative processes such as sorption, dissolution, and volatilization involve only relatively small isotopic shifts at equilibrium, which in many cases are less than analytical uncertainty (Dempster et al., 1997, Harrington et al., 1999, Huang et al., 1999, Slater et al., 1999, Slater et al., 2000). Therefore, isotopic measurements have the potential to differentiate between concentration decreases due to degradative and non-degradative processes, and to verify biodegradation.

To date, several field studies have used CSIA to verify natural attenuation of chlorinated ethenes via reductive dechlorination (Hunkeler et al., 1999, Kirtland et al., 2003, Sherwood Lollar et al., 2001, Song et al., 2002, Vieth et al., 2003). Hunkeler et al. (1999) concluded that the enriched isotopic values of transformation products cDCE and VC could be used to monitor the last two steps of reductive dechlorination. Sherwood Lollar et al. (2001) demonstrated that enrichment in the isotopic composition of the primary contaminants PCE and TCE could verify the initial steps of reductive dechlorination. Sherwood Lollar et al. (2001) also used δ¹³C values of TCE to estimate the extent of in situ degradation. While investigating reductive dechlorination of TCE, Song et al. (2002) observed large isotopic fractionation for transformation products cDCE, VC and ethene, but not for the original TCE. The lack of isotopic shift in TCE was attributed to proximity of the sampling wells to the source zone, which provided a continual source of TCE to the plume and diluted the isotopic shifts in dissolved TCE resulting from biodegradation (Song et al., 2002).

In this study, stable carbon isotopic values were measured for PCE and its transformation products during enhanced microbial degradation of PCE in a controlled recirculation system at Kelly AFB. The first objective of this study was to determine if isotopic values of PCE and its transformation products measured during a field pilot test could provide an additional line of evidence for verification of biodegradation. The second objective of this study was to calculate the biodegradation rate constant for cDCE using the isotope data and to compare this estimate to a rate constant calculated using concentration data only in a parallel study at Kelly AFB (Major et al., 2002).