Article pubs.acs.org/est
3D-CSIA: Carbon, Chlorine, and Hydrogen Isotope Fractionation in Transformation of TCE to Ethene by a Dehalococcoides Culture Tomasz Kuder,†,* Boris M. van Breukelen,‡ Mindy Vanderford,§ and Paul Philp† †
School of Geology and Geophysics, University of Oklahoma, 100 E. Boyd Street, SEC 710, Norman, Oklahoma 73019, United States Department of Earth Sciences, VU University Amsterdam, De Boelelaan 1085, NL-1081 HV Amsterdam, The Netherlands § GSI Environmental Inc., 2211 Norfolk Street, Suite 1000, Houston, Texas 77098, United States ‡
S Supporting Information *
ABSTRACT: Carbon (C), chlorine (Cl), and hydrogen (H) isotope effects were determined during dechlorination of TCE to ethene by a mixed Dehalococcoides (Dhc) culture. The C isotope effects for the dechlorination steps were consistent with data published in the past for reductive dechlorination (RD) by Dhc. The Cl effects (combined with an inverse H effect in TCE) suggested that dechlorination proceeded through nucleophilic reactions with cobalamin rather than by an electron transfer mechanism. Depletions of 37Cl in daughter compounds, resulting from fractionation at positions away from the dechlorination center (secondary isotope effects), further support the nucleophilic dechlorination mechanism. Determination of C and Cl isotope ratios of the reactants and products in the reductive dechlorination chain offers a potential tool for differentiation of Dhc activity from alternative transformation mechanisms (e.g., aerobic degradation and reductive dechlorination proceeding via outer sphere mechanisms), in studies of in situ attenuation of chlorinated ethenes. Hydrogenation of the reaction products (DCE, VC, and ethene) showed a major preference for the 1H isotope. Detection of depleted dechlorination products could provide a line of evidence in discrimination between alternative sources of TCE (e.g., evolution from DNAPL sources or from conversion of PCE).
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INTRODUCTION Chlorinated ethenes (CEs) are among the most ubiquitous groundwater contaminants.1 Under reducing conditions, tetrachloroethene (PCE) or trichloroethene (TCE), undergo biological reductive dechlorination (RD), potentially all the way to nontoxic ethene (Figure 1).2 RD may be an element of natural attenuation or may be stimulated by addition of electron donor and/or dechlorinating culture.3 While certain microbial cultures are capable of complete transformation of PCE to nontoxic ethene, the common concern is accumulation of toxic reaction intermediates, cis-dichloroethene (cDCE), and/or vinyl chloride (VC).2 Successful remediation of CEs requires that the parent contaminant is not only destroyed, but also accumulations of toxic degradation products be avoided. Compound-specific isotope analysis (CSIA) may potentially provide direct evidence of ongoing degradation of the parent contaminants and the intermediates, through detection of characteristic changes of isotope ratios (isotope fractionation) in the compounds undergoing degradation and degradation products. CSIA permits detection and quantification of such isotope effects, as contaminant-specific evidence of degradation.4 CSIA applications are most numerous in studies of CEs, benzene and MtBE,4−9 including applications to discriminate between biotic and abiotic RD and aerobic biodegradation of CEs.10−14 While most applications center on carbon isotope ratios, carbon isotope fractionation may be similar among © 2013 American Chemical Society
different transformation pathways inhibiting their distinction, and/or the fractionation may be poorly constrained for a given pathway, resulting in large uncertainties of calculated contaminant transformation.4 These limitations could be overcome by combining CSIA of two (or more) elements (so-called 2D-CSIA). The proportional isotope enrichment for one element versus enrichment for another element is often pathway-specific, strengthening interpretation of CSIA results.8,15−17 CSIA of chlorine and carbon may be particularly informative in identification of degradation pathways of CEs.11,18−22 In the present study, isotope effects were determined for dechlorination of TCE to ethene, by a mixed culture containing Dehalococcoides (Dhc) species.23,24 Dhc is one of the key groups of RD organisms, and their activity may be particularly significant at sites where RD proceeds all the way to nontoxic ethene.25 While there is an extensive body of work available on carbon isotope fractionation during RD in general,4 and on Dhc-mediated RD in particular,11,12,26−28 a limited amount of data are available for other isotopes. A few examples of utilization of chlorine isotope effects in CEs degradation studies Received: Revised: Accepted: Published: 9668
January 29, 2013 July 15, 2013 July 29, 2013 July 29, 2013 dx.doi.org/10.1021/es400463p | Environ. Sci. Technol. 2013, 47, 9668−9677
Environmental Science & Technology
Article
using liquid medium, prepared as described elsewhere.36 The H isotope composition (δ2H) of the medium was −42 ‰. The microcosms were spiked with lactate electron donor (5 mM) and TCE (165 μM). The microcosms were incubated upside down, in the dark, with periodic monitoring for TCE concentration decreases and the presence of TCE degradation products. The bottles were filled completely with the liquid medium to eliminate partitioning between the liquid and gas phase. During incubation, the headspace was limited to a peasized gas bubble. Dechlorination activity was initially established after a lag time of 4 months. After stoichiometric transformation of TCE to ethene, the medium was sparged with nitrogen to remove the volatile degradation products (confirmed by gas chromatography/mass spectrometry (GC/MS) analysis) prior to fresh TCE and lactate amendment. In the follow up amendments, TCE degradation commenced without lag. The data for this study (TCE and daughter compounds concentrations and C, Cl, and H isotope ratios) were collected from the third TCE amendment, from a single microcosm bottle (a backup set of samples was collected from a duplicate microcosm bottle; the results of VOCs concentrations and C isotope ratio analysis from those samples are shown in Supporting Information, SI, Figure S1). Collection of Samples. Samples were collected for analysis of the CEs and ethene with simultaneous replenishment of the volume withdrawn with fresh medium to maintain headspacefree conditions. The resulting dilutions did not change molar fractions of the individual compounds present as no differential headspace-water partitioning occurred. The analyte concentrations determined by GC/MS were corrected by dilution factors, to permit Rayleigh-type data assessment (per eq 1). Aliquots of 50 μL were withdrawn for CEs and ethene concentration analysis, initially at least twice a day, followed by less frequent sampling in the later stages of incubation. The samples were analyzed by GC/MS immediately after sample collection. The need for collection of larger volumes for CSIA was determined by extrapolation of the trends of the target analyte concentrations, to ideally obtain a resolution of ∼10 μM along the degradation profiles. The CSIA sample volumes varied from 200 μL (initial phase of incubation with high concentration TCE present) to 4 mL for the samples where some of the analytes were present at low concentration. The CSIA samples were stored at 4 °C, in 20 mL vials with Teflonlined closures, filled completely with reagent-grade water that was previously acidified with H2SO4 to pH = 1. Given that the CSIA samples’ collection time was known (immediately after GC/MS analysis), the concentrations of CEs in the CSIA samples were read from the concentration profiles determined previously by GC/MS. Analytical Techniques. The concentrations and isotope ratios of CEs and ethene were performed using gas chromatography-quadrupole mass spectrometry (concentrations and Cl isotope ratios) or gas chromatography-isotope ratio mass spectrometry (C and H isotope ratios). H CSIA was performed using a custom chromium metal reactor35 for conversion of the CEs and/or ethene to H2. Further details of the analytical methods, including a discussion of analytical bias in H CSIA are included in the SI. Calculation of Enrichment Factors and KIEs. The isotope ratios of an element E are expressed using delta (δ) notation, where δheavyE = Rsample/Rstandard − 1 (expressed in ‰). R is heavyE/lightE, e.g., 13C/12C. The 13C/12C, 2H/1H and
Figure 1. Definition of the Cl positions in TCE to ethene transformation chain and the alternative reaction pathways in reductive dechlorination of chlorinated ethenes by cobalamin (LCo).45,46 Position-specific dechlorination of TCE at ClC results in formation of the cis-DCE isomer, while the two chlorine atoms of cDCE (ClA and ClB) are equivalent in reactivity in transformation to VC. The position-specific Cl isotope effects (ε) will be referred to as εCl‑A,B,C (TCE transformation to cDCE), εCl,VC (Cl atom of cDCE retained by VC), and εCl,Cl− (Cl atom of cDCE released as Cl−). Scenario A represents the nucleophilic addition pathway and Scenario B represents the nucleophilic substitution or single electron transfer SET pathways (simplified by omission of alternative reaction of the chlorovinyl radical). Positions indicated by “X” can be taken by Cl or H atoms, depending on the CE substrate.
are available to date11,21,22,29−32 including one on biodegradation mediated by Dhc.11 Only one study to date provided limited data on hydrogen fractionation during biotic RD.33 The main goal of this work is the detailed assessment of mechanisms of degradation and attendant isotope fractionation mechanisms during the RD sequence from TCE to ethene. Such information will be a benchmark in assessing CEs degradation at sites with potential Dhc activity. Until recently, the feasibility for 2D- or 3D- (including C, Cl, and H isotopes) CSIA was limited by the low availability of highly specialized Cl CSIA instrumentation34 and lack of a robust methodology for hydrogen CSIA in chlorinated compounds. Herein we present C, Cl, and H CSIA results for TCE in microcosm studies using Dhc culture. The H CSIA data were obtained by using a novel pyrolysis reactor type, compatible with analysis of chlorinated species.35
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MATERIALS AND METHODS Microcosm Experiment. Microcosms were set up with Bio-Dechlor Inoculum (BDI) culture.24 BDI is a consortium of Dehalococcoides (Dhc) species that is capable of complete dechlorination of PCE to ethene. The microcosms were set up in 72 mL (actual volume) serum bottles with crimp closures, 9669
dx.doi.org/10.1021/es400463p | Environ. Sci. Technol. 2013, 47, 9668−9677
Environmental Science & Technology
Article
Figure 2. Evolution of concentrations and C, Cl and H isotope ratios in transformation of TCE to ethene in the BDI microcosm (see SI Figure S1 for the results from a duplicate BDI culture). (A) Trends of CEs and ethene concentrations over time of BDI incubation (the inset shows the trends for the three dichloroethene isomers); (B−D) δ13C, δ37Cl, and δ2H data. TCE (⧫), cDCE (Δ), VC (◊), ethene (●), 1,1DCE (○) tDCE (×) and net of the four major compounds: TCE, cDCE, VC, and ethene (+). No data are shown for the lag period prior to the onset of VC degradation. 37
Cl/35Cl standards are Vienna Pee Dee Belemnite (VPDB), Vienna Standard Mean Ocean Water (VSMOW), and Standard Mean Ocean Chloride (SMOC), respectively. Note that the notation used omits the commonly used 103 factor.37 Isotope fractionation in degradation of a parent compound (here: TCE) is calculated by eq 1, where ε is the kinetic isotope enrichment factor, Ct and C0 are substrate concentrations at time t and the initial time, respectively; Rt and R0 are isotope ratios for given element, at time t and the initial time, respectively. Typically, measurable isotope fractionation is most strongly expressed for the atoms at the reaction center (primary isotope effects). Measurable effects may also occur at remote atoms not directly involved in the reaction (secondary isotope effects).
ln(R t /R 0) = ln(Ct /C0) × ε
(1)
The enrichment factor defines the magnitude of isotope fractionation: ε = heavyk/lightk − 1 (expressed in ‰),37 where k is the kinetic rate constant, for the molecules with and without the heavy isotope species present, respectively. For cDCE, the observed isotope ratios reflected concurrent production and degradation of the compound. Therefore, calculation of ε of cDCE degradation by eq 1 was not possible, however, eq 1 could be applied to VC (VC degradation commenced when TCE and cDCE were no longer present). ε ≈ δdaughter,t ≈ 0 − δparent,t ≈ 0 9670
(2)
dx.doi.org/10.1021/es400463p | Environ. Sci. Technol. 2013, 47, 9668−9677
Environmental Science & Technology
Article
Table 1. Bulk and Position-Specific Isotope Effects for Individual RD Transformations eq 3 parameters RD step
ε (‰)
εCl/εC
scenario
TCE to cDCE
εC = −16.4 ± 0.4a εC = −15.3b εCl = −3.6 ± 0.3a εCl‑A,B = −3.3b εH = +34 ± 11a
0.21 ± 0.2a
Ae
εC = −26.8b |εCl|< |−3.2|c εCl,VC = −1.7b
