Stable Carbon Isotope Enrichment Factors for - American Chemical

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Stable Carbon Isotope Enrichment Factors for cis-1,2-Dichloroethene and Vinyl Chloride Reductive Dechlorination by Dehalococcoides Kelly E. Fletcher,† Ivonne Nijenhuis,§ Hans-Hermann Richnow,§ and Frank E. L€offler*,†,‡,^,||,# School of Civil and Environmental Engineering and ‡School of Biology, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, Georgia 30332, United States § Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, D-04318 Leipzig, Germany ^ Department of Microbiology and Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee 37996, United States # Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States )

†

bS Supporting Information ABSTRACT: Compound-specific stable isotope analysis (CSIA) is a promising tool for monitoring in situ microbial activity, and enrichment factors (ε values) determined using CSIA can be employed to estimate compound transformation. Although ε values for some dechlorination reactions catalyzed by Dehalococcoides (Dhc) have been reported, reproducibility between independent experiments, variability between different Dhc strains, and congruency between pure and mixed cultures are unknown. In experiments conducted with pure cultures of Dhc sp. strain BAV1, ε values for 1,1-DCE, cis-DCE, trans-DCE, and VC were -5.1, -14.9, -20.8, and -23.2%, respectively. The ε value for 1,1-DCE dechlorination was 48.9% higher than the value reported in a previous study, but ε values for other chlorinated ethenes were equal between independent experiments. For the dechlorination of cis-DCE and VC by Dhc strains BAV1, FL2, GT, and VS, average ε values were -18.4 and -23.2%, respectively. cis-DCE and VC ε values determined in pure Dhc cultures with different reductive dehalogenase genes (e.g., vcrA vs bvcA) varied by less than 36.8 and 8.3%, respectively. In the BDI consortium, ε values for cis-DCE and VC dechlorination were -25.3% and -19.9%, 31.6% higher and 15.3% lower, respectively, compared to the average ε value for Dhc pure cultures. As cis-DCE and VC ε values are all within the same order-ofmagnitude and fractionation is always measured during Dhc dechlorination, CSIA may be a valuable approach for monitoring in situ cis-DCE and VC reductive dechlorination.

’ INTRODUCTION Chlorinated solvents such as tetrachloroethene (PCE) and trichloroethene (TCE) are widespread groundwater contaminants due to extensive use and inappropriate disposal practices.1 One approach for the cleanup of chlorinated ethene-contaminated sites is bioremediation.2-4 Anoxic bioremediation of chlorinated ethenes relies on stepwise reductive dechlorination wherein carbon-chlorine bonds are cleaved and each chlorine atom is replaced with a hydrogen atom.5 In this manner, PCE is dechlorinated to TCE, TCE is dechlorinated to a dichloroethene (DCE) (usually cis-1,2-DCE [cis-DCE]), DCE is dechlorinated to vinyl chloride (VC), and VC is dechlorinated to nontoxic ethene.5 Phylogenetically diverse bacteria dechlorinate PCE and TCE to cis-DCE, but so far, only Dehalococcoides (Dhc) strains have been implicated in DCE and VC dechlorination.6 Dhc are not present at r 2011 American Chemical Society

all contaminated sites 5 and only some Dhc strains respire VC;7-9 therefore, dechlorination may be incomplete, causing the so-called cis-DCE 10-12 or VC “stall”.10,13 Because abiotic processes such as dilution, sorption, and volatilization contribute to decreasing contaminant concentrations,14,15 multiple lines-of-evidence are required to demonstrate that in situ microbial reductive dechlorination is occurring.16 Compound-specific stable isotope analysis (CSIA) relies on the faster rate of biotransformation of isotopically light 12C-containing compounds than the heavier 13C-containing compounds [ref 15 Received: November 5, 2010 Accepted: February 21, 2011 Revised: February 16, 2011 Published: March 10, 2011 2951

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Environmental Science & Technology and references therein], and has been used to demonstrate and quantify in situ transformation of chlorinated ethenes.17-23 Using isotope analysis, compound-specific enrichment factors (ε values) are calculated with the Rayleigh equation.24-26 At a contaminated site, in situ biotransformation can be quantified if the initial isotope composition of the compound(s) of interest, the shift in the isotope composition, and the ε value are known. The isotope effects for PCE and TCE reductive dechlorination are highly variable.14 During dechlorination of PCE by Desulfuromonas michiganensis and Geobacter lovleyi strain SZ, no significant fractionation occurred and therefore, the ε values for PCE dechlorination were below -0.4%.14 Conversely, the ε for PCE dechlorination by Desulfitobacterium sp. strain Viet1 was -16.7%.14 Similarly, ε values for dechlorination of TCE range from -2.5 to -18.9%.14,27 Because biotransformation is quantified based on ε values, the larger the percentage difference of ε values, the larger the uncertainty associated with the quantity of a contaminant that has been biotransformed. The large percent variability of ε for PCE and TCE dechlorination indicates that PCE and TCE biotransformation can be overestimated by as much as 3.8 and 3.7 fold, respectively, following isotope shifts of 5%, rendering estimates of in situ biotransformation challenging.14,28 Estimating biotransformation of dechlorination intermediates (i.e., DCEs and VC) with CSIA is complicated by the challenge of determining the initial isotope composition of the dechlorination intermediate due to the simultaneous formation of intermediates from higher chlorinated ethenes and the removal of intermediates due to reductive dechlorination.12,29,30 Therefore, the Rayleigh equation cannot be applied directly for determining the amount of a dechlorination intermediate that has undergone biotransformation unless the parent compound (e.g., PCE, TCE) has been completely transformed to an intermediate (e.g., cis-DCE, VC) that has not been degraded. Such a scenario often occurs at “stalled” sites.30 Previously determined ε values for cis-DCE and VC dechlorination in defined pure and mixed cultures ranged from -14.1 to -29.7% and from -21.5 to -26.6%, respectively.27,28,31 To apply CSIA as a reliable tool for monitoring contaminant fate in situ, the causes for the observed variability (i.e., the range of measured ε values) must be understood. Interestingly, in only a single case has the reproducibility of ε values for reductive dechlorination reactions catalyzed by pure cultures been determined in independent experiments. Cichocka et al.14 and Lee et al. 28 independently determined ε values of -13.7 ( 1.8% and -9.6 ( 0.4%, respectively, for the dechlorination of TCE by Dhc ethenogenes strain 195, a difference of 35.2%. A number of different reductive dehalogenase (RDase) enzyme systems have been implicated in chlorinated ethene dechlorination. TceA of Dhc strains 195 and FL2 catalyzes dechlorination of TCE and cis-DCE to VC7,32,33 and VcrA of strains VS and GT catalyzes dechlorination of DCEs and VC to ethene.9,34 BvcA of Dhc strain BAV1 has been implicated in the dechlorination of VC to ethene.35 Studies conducted with Dhc pure cultures have only investigated fractionation in cultures containing the bvcA and tceA genes28 and it is unclear if Dhc strains possessing the distinct VcrA RDase produce different ε values for the DCE and/or VC transformation steps. Additionally, few studies have attempted to correlate DCE and VC fractionation between pure and mixed cultures.28 This study addresses these key knowledge gaps in isotope effects for Dhccatalyzed reductive dechlorination of cis-DCE and VC and determines (i) if ε values measured in Dhc pure cultures are

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reproducible in independent experiments, (ii) if compoundspecific ε values vary between different Dhc strains possessing unique RDases, and (iii) if the isotope effects determined in Dhc pure cultures for cis-DCE and VC dechlorination correlate with those measured in a Dhc-containing consortium.

’ MATERIALS AND METHODS Cultures and Growth Conditions. The following pure Dhc cultures were used in this study: strain BAV1,6 strain FL2,7 strain GT,34 and strain VS.36 Strain BAV1 respires all DCE isomers and VC,6 strain FL2 respires TCE, cis-DCE, and trans-1,2-DCE (transDCE),7 strain GT and strain VS respire TCE, 1,1-DCE, cis-DCE, and VC.34,36 Isotope fractionation was also measured in the PCE-toethene dechlorinating Bio-Dechlor INOCULUM (BDI) consortium, which contains strain BAV1, strain FL2, and strain GT.37 Fractionation was determined only for the parent compound initially provided (e.g., to determine cis-DCE and VC ε values for dechlorination by Dhc strain BAV1, two independent experiments were conducted with cis-DCE or VC provided as electron acceptor). Duplicate or triplicate cultures were constructed via inoculation (3%, vol/vol) of 160-mL (nominal volume) glass serum bottles containing approximately 100 mL of anoxic, bicarbonate buffered (30 mM) mineral salts medium38 amended with vitamins39 and reduced with 1.4 mM Ti(III) citrate.40 TCE and DCEs were amended to cultures dissolved in 0.1 mL of methanol and VC was amended to cultures in gaseous form.41 Final aqueous chlorinated ethene concentrations ranged from 12 to 59 mg/L. Dhc pure cultures received 4 mL of hydrogen and 5 mM acetate whereas BDI cultures were amended with 5 mM of lactate. Cultures were incubated at 25 C in the dark. Analytical Techniques and Sample Collection. Chlorinated ethene and ethene concentrations were quantified by gas chromatography (GC) as described.38 To determine isotope compositions, aqueous samples (7 mL) were collected and placed in 10mL vials with 1 mL of 1 M NaOH. Immediately after sample addition, vials were closed with Teflon-lined butyl rubber septa. Headspace samples (0.05-1.0 mL) were removed from sample vials incubated at 25 to 60 C and analyzed using a GCcombustion-isotope ratio monitoring mass spectrometer (GCC-IRM-MS).14,38 Based on the repeated analysis of select sample vials, heating did not affect the isotope compositions. Each sample vial was analyzed at least twice and the standard deviation between measurements was typically less than 0.5%. Chlorinated ethene concentrations were generally quantified twice as often as isotope compositions. Isotope Fractionation Calculations. Stable carbon isotope ε values for dechlorination reactions were calculated according to the Rayleigh model:

ðε=1000ÞlnðCt =C0 Þ ¼ lnðR t =R 0 Þ

ð1Þ

where Ct is the number of moles per bottle of the parent compound at time t and C0 is the initial number of moles of the parent compound per bottle [ref 15 and references therein]. The Rt/R0 term was calculated according to the equation:     1000 þ δ13 C0 þ Δδ13 C = 1000 þ δ13 C0 R t =R 0 ¼ ð2Þ where δ13C0 is the carbon isotope composition of the parent compound at time zero and Δδ13C is the shift in the carbon isotope composition from time zero to time t [ref 15 and 2952

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references therein]. Initially, ε values were calculated for each individual culture. In all cases, ε values generated by duplicate or triplicate cultures (e.g., two independent BAV1 cultures amended with cis-DCE), were equivalent; therefore, linear regressions combining data from replicate cultures were used to generate final ε values. The statistical error of ε was determined based on the standard error of the slope of the linear regression. The percent difference between ε values was calculated according to: PD ¼ ðabsðεA - εB Þ=ððεA þ εB Þ=2ÞÞ100

ð3Þ

where PD is the percent difference between unique ε values εA and εB. Parent compound losses in the culture vessels were due to biotransformation as well as sample removal. The total number of moles of parent compound withdrawn over the course of the experiment as a percentage of the moles of parent compound initially present was dependent on the number of samples collected, the amount of the parent compound that had been transformed at each sampling point, and the partitioning properties of the specific compound. The percentage of the parent compound that was withdrawn from the cultures ranged from 3.2 to 25.9%. To account for the effect of sample removal, Ct was corrected as follows: Ct , correct ¼ Ct þ Cr, t-1

ð4Þ

where Ct,correct is the corrected number of moles of the parent compound present in each bottle at time t and Cr,t-1 is the cumulative number of moles of parent compound removed per bottle due to sample collection at time t - 1. Similarly, C0 was also corrected according to the equation C0, correct ¼ C0 þ Cr, t-1

ð5Þ

where C0,correct is the corrected number of moles of the parent compound initially present in each bottle. Note that at time zero, C0,correct is equal to C0. By replacing Ct with Ct,correct and C0 with C0,correct in the Rayleigh model, calculated ε values only take into account the amount of the parent compound that was removed due to biotransformation. Quantifying Biotransformation Based on ε Values. To quantify biotransformation of the parent compound based on a known ε, the following equation was derived based on eqs 1 and 2:     1000=ε ln 1000 þ δ13 C0 þ Δδ13 C Ct =C0 ¼ e ∧   = 1000 þ δ13 C0 ð6Þ The percent of a compound that has undergone biotransformation, B, was defined as14 B ¼ ð1 - Ct =C0 Þ100 ð7Þ B was calculated for varying shifts in isotope composition (i.e., Δδ13C) based on known ε values. Shifts in isotope composition are determined based on the initial isotope composition of the parent compound, δ13C0, and the initial isotope composition was assumed to be -28% as described previously.14 B values were calculated using ε values determined experimentally and were compared to experimentally measured B values (Figure S1 in the Supporting Information). The average difference between experimental measurements and B values determined using eqs 6 and 7 across all experiments was 5.8%.

Figure 1. Rayleigh plot showing 1,1-DCE (b), cis-DCE (O), transDCE (0), and VC (9) transformation by Dhc sp. strain BAV1. Error bars depict one standard deviation (2σ) for duplicate or triplicate isotope measurements. In most cases, error bars are not visible because standard deviations are covered by the symbols.

’ RESULTS AND DISCUSSION Reproducibility of ε Values for Reactions Catalyzed by Dhc sp. Strain BAV1. Fractionation during dechlorination by strain

BAV1 was investigated to assess if independently determined ε values for dechlorination by a pure culture are equal. Fractionation of all DCE isomers and VC by strain BAV1 fit the Rayleigh model (Figure 1). The ε values for trans-DCE and VC dechlorination were -20.8 ( 2.8 and -23.2 ( 1.8%, respectively, and are within standard errors of ε measured in BAV1 cultures by Lee et al. (Table 1).28 The ε value for cis-DCE dechlorination was -14.9 ( 0.5% and is not discernibly different from the value measured by Lee et al. (Table 1). Conversely, ε values determined for 1,1-DCE dechlorination varied. In the current study, the ε value was -5.1 ( 0.3%, but the value determined by Lee et al. for 1,1-DCE dechlorination was -8.4 ( 0.3%,28 48.9% greater than the value measured in the current study (Table 1). These results demonstrate that ε values determined in independent experiments with the same Dhc strain and compound may vary by as much as 48.9%. Similarly, TCE fractionation during dechlorination by strain 195, measured previously in two independent experiments14,28 differed by 35.2% (Table 1). Variability in ε for Dechlorination Reactions Catalyzed by Distinct Dhc Strains. Fractionation in Dhc strain FL2, strain GT, and strain VS cultures was measured and compared to fractionation in strain BAV1 and strain 195 cultures to determine how isotope effects varied among Dhc strains with distinct RDases. TCE and cis-DCE fractionation in cultures of strain FL2 were fit to the Rayleigh model (Figure S2) and yielded ε values of -8.0 ( 0.4 and -15.8 ( 1.1%, respectively. Similarly, in strain GT and strain VS cultures, fractionation of cis-DCE and VC fit the Rayleigh model (Figures S3 and S4), and ε values of -21.6 ( 1.3 to -17.6 ( 2.7% and -23.8 ( 1.1 to -22.1 ( 1.3%, respectively, were determined (Table 1). Both Dhc strain FL2 and strain 195 possess the tceA gene,7,33 but ε values for TCE and cis-DCE fractionation were smaller for dechlorination reactions catalyzed by strain FL2 than for dechlorination reactions reported for strain 19514 (Table 1). TCE 2953

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Table 1. Compound-Specific ε Values for the Reductive Dechlorination of Chlorinated Ethenes by Dhc Isolates and DhcContaining Consortia (Values in Bold Font Were Generated in the Current Study and Have Been Corrected for the Effects of Sample Removal) culture

TCE

cis-DCE

1,1-DCE ε (%)

ε (%)

trans-DCE ε (%)

vinyl chloride ε (%)

ε (%)

R

-13.7 ( 1.8a

0.95a

NTa,d

-9.6 ( 0.4b

0.99b

-5.8 ( 0.5b

0.97b

-21.1 ( 1.8b

0.97b

-8.4 ( 0.3b

0.99b

-16.9 ( 1.4b

0.98b

-21.4 ( 0.9b

0.99b

-24.0 ( 2.0b

0.97b

-5.1 ( 0.3

0.97

-14.9 ( 0.5

0.99

-20.8 ( 2.8

0.92

-23.2 ( 1.8

0.96

NA NT

-15.8 ( 1.1 -21.6 ( 1.3

0.97 0.97

NT NA

NA -23.8 ( 1.1

0.99

0.89

NA

-22.1 ( 1.2

0.97

-21.1 ( 1.5

-23.2 ( 1.1

2

R

2

2

R

2

R

R2

Dhc pure cultures 195 (tceA) BAV1 (bvcA)

NA

NTa

FL2 (tceA) GT (vcrA)

-8.0 ( 0.4 NT

VS (vcrA)

NT

NT

-17.6 ( 2.7

-9.8 ( 2.6

-6.3 ( 1.2

-18.4 ( 2.8

Average

0.98

NAe

NA

Dhc-containing consortia ANASb

-16.0 ( 0.6

0.99

-23.9 ( 1.2

-29.7 ( 1.6

0.99

-28.3 ( 1.4

-22.7 ( 0.8

0.99

BDI

-15.3 ( 0.8f

NPg

NT

-25.3 ( 1.0

0.99

NT

-19.9 ( 1.6

0.96

-2.5 to -13.8

0.91

NT

-14.1 to -20.4

0.92

NA

-21.5 to -26.6

0.96

KB-1c

0.98

0.99

a

Data from Chichocka et al.14 b Data from Lee et al.28 c Data from Bloom et al.,27 Duhamel et al.,42 and Slater et al.31 d NT indicates that fractionation was not quantified although the chlorinated ethene supports organohalide respiration and growth. e NA indicates that fractionation was not quantified because the chlorinated ethene does not support organohalide respiration. f Data from Liang et al.47 g NP indicates that an R2 value was not reported in Liang et al.47

fractionation during dechlorination by strain 195, measured previously in two independent experiments, differed by 4.1% with an average ε value of -11.7 ( 2.9%,14,28 a difference of 3.7% compared to the dechlorination of TCE by strain FL2 (Table 1). Therefore, the difference in ε for TCE reductive dechlorination measured for the same Dhc strain in independent experiments was greater than the difference measured between different Dhc strains harboring tceA. For the dechlorination of cisDCE, ε values for strain FL2 and strain 195 differed by 28.7%. Interestingly, the ε value for cis-DCE dechlorination by strain FL2 was statistically identical to the value measured for strain BAV1 (Table 1). Strain BAV1 does not possess tceA35 and a protein BLAST analysis revealed that none of the strain BAV1 RDases share greater than 41% amino acid identity with TceA. Similarly, the ε for dechlorination of cis-DCE by strain 195 was statistically identical to that of strain GT (Table 1), even though strain GT does not possess tceA34 and no strain GT RDase shares greater than 37% amino acid identity with TceA. Dhc strain GT and strain VS both possess the vcrA gene encoding VcrA, which catalyzes the dechlorination of 1,1-DCE, cis-DCE, and VC,9,34 and the ε values for the same dechlorination reactions performed by these Dhc strains were within statistical error (Table 1). ε values for cis-DCE dechlorination by strain VS were also within the statistical error of values measured for strains BAV1 and FL2 as well as those reported previously for strain 195.14,28 Further, the ε values for VC dechlorination by strain BAV1, strain GT, and strain VS are all within statistical error (Table 1). The percent differences between ε values for reactions catalyzed by all Dhc pure cultures were within the variability reported between ε determined in independent experiments with the same strain. For example, ε for dechlorination of cis-DCE in Dhc pure cultures ranged from -14.9 to -21.6%, which corresponds to a percentage difference of 36.7%, less than the difference of 48.9% measured for the dechlorination of 1,1-DCE by strain

BAV1 in independent experiments. Therefore, the differences between ε values measured in cultures of unique Dhc strains cannot be considered strain-specific. Based on this finding, ε values from Dhc pure cultures were averaged to generate values that are applicable to Dhc pure cultures in general (Table 1). Because reductive dechlorination is the mechanism of chlorinated ethene transformation used by all Dhc isolates, the similarity of isotope effects between Dhc isolates supports the fundamental fractionation principle that fractionation extent is determined by the mechanism of the chemical bond cleavage [ref 15 and references therein]. Similarly, in a previous study, ε values determined for the dichloroelimination of 1,2-dichloropropane by two distinct Dhc strains were statistically identical.38 Variation of ε between Dhc Pure and Dhc-Containing Mixed Cultures. To determine if fractionation in pure and mixed cultures differs, ε values for dechlorination of cis-DCE and VC were measured in the BDI consortium and compared to the average ε values determined for Dhc pure cultures. Fractionation of cis-DCE and VC during dechlorination by the BDI consortium fit the Rayleigh model (Figure S5) and ε values were -25.3 ( 1.0 and -19.9 ( 1.6% for cis-DCE and VC dechlorination, respectively (Table 1). The ε values for dechlorination of cis-DCE and VC in the BDI consortium were 31.6% greater and 15.3% lower, respectively, than the average ε values for cis-DCE and VC dechlorination in all Dhc pure cultures. In both cases, the percent differences between BDI and average Dhc pure culture ε values were less than those determined by different laboratories using the same Dhc strains; therefore, differences between ε measured in the BDI consortium and in Dhc pure cultures are not a function of culture composition. Previous studies have investigated isotope fractionation of cisDCE and VC in the ANAS and KB-1 consortia,27,28,31 which dechlorinate TCE and PCE, respectively, to ethene and contain multiple Dhc strains.42,43 The ε for dechlorination of cis-DCE in 2954

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Environmental Science & Technology the ANAS consortium was -29.7 ( 1.6%,28 which is 47.0% greater than the average ε for cis-DCE reductive dechlorination for all measured Dhc pure cultures (Table 1). The ε for VC dechlorination in the ANAS consortium was -22.7 ( 0.8%,28 which is within the standard error of the average ε of -23.2 ( 1.1% for VC dechlorination by Dhc pure cultures (Table 1). Several studies have examined isotope fractionation in the KB-1 consortium and reported ε values for cis-DCE and VC dechlorination range from -14.1 to -20.4% and from -21.5 to -26.6%, respectively.27,31 Therefore, ε values for cis-DCE dechlorination in the KB-1 consortium range from 26.5% less than to within the standard error of the average ε value for Dhc pure cultures (Table 1). Similarly, ε values for VC dechlorination in the KB-1 consortium range from 7.6% less than to 13.7% greater than the average ε value for Dhc pure cultures. In the case of both the ANAS and KB-1 consortia, the percent differences between ε values were within the variability reported between ε values determined in independent experiments with the same Dhc strain. Therefore, similar to consortium BDI, differences between ε generated in the ANAS and KB-1 consortia and by Dhc pure cultures cannot be considered a function of culture composition. The fractionation of chlorinated ethenes has also been investigated in microcosms23 and a wetland model system.19 The ε values determined for cis-DCE and VC dechlorination in microcosms established with sediments and groundwater from a contaminated site in Louisiana, USA, were -19.9 ( 1.5 and -31.1 ( 0.4%, respectively.23 While the ε value for cis-DCE dechlorination in microcosms is within the error of the average ε value for cisDCE dechlorination determined in Dhc pure cultures (Table 1), the ε value determined in microcosms for VC dechlorination is 29.1% greater than the average ε value for VC dechlorination by Dhc pure cultures. Even so, 29.1% is less than the percent difference between ε values measured in independent experiments conducted with the same Dhc strain; therefore, differences between ε generated in microcosms and by Dhc pure cultures cannot be considered a function of culture composition. In a model wetland system, the ε values determined for reductive cis-DCE dechlorination varied between approximately -15 and -33%.19 ε values ranging from -15.6 to -21.2% are within the standard error of the average ε value for cis-DCE dechlorination by Dhc pure cultures (Table 1) and ε values of up to -30.3% fall within the variability determined for Dhc pure cultures measured in independent experiments. However, the highest ε value measured in the model wetland, -33%, is outside the range of values determined in defined laboratory systems. Implications for Estimates of in Situ Biotransformation. CSIA is used to quantify in situ biotransformation based on the shift in isotope composition and ε value. To assess the impact of the variability of ε values on the in situ quantification of cis-DCE and VC dechlorination, the percentages of cis-DCE and VC biotransformed were calculated based on both the minimum and maximum ε values for shifts in isotope composition from 0% to 80% (Figure 2). These estimates of biotransformation are reliant upon the assumption that the initial isotope composition of the intermediate (i.e., cis-DCE or VC) is known and therefore, that the intermediate is no longer being formed and has not yet been degraded. Estimates are also accurate when cis-DCE or VC is the original, parent compound and has not yet undergone degradation. For cis-DCE dechlorination, ε varied from -14.1 to -33.0%. The percent of cis-DCE that has undergone biotransformation based on an isotope shift of 5% and an ε of -14.1% is 30.5%.

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Figure 2. Biotransformation extent of cis-DCE (A) and of VC (B) versus shifts in isotope composition. Solid lines are calculated using minimum ε and dashed lines are calculated based on maximum ε values.

Similarly, the percent of cis-DCE that has undergone biotransformation following an isotope shift of 5% based on the highest ε value measured (i.e., -33.0%) is 14.4% (Figure 2). Therefore, at most, cis-DCE biotransformation can be overestimated by 2.1 times for an isotope shift of 5%. For isotope shifts of 15% and greater and of 30% and greater, cis-DCE biotransformation can be overestimated by a maximum of 1.8 and 1.5 times, respectively. The ε values for VC dechlorination varied over 11.2%, from -19.9 to -31.1%, and therefore, VC biotransformation can be overestimated by 1.5 times for an isotope shift of 5%. VC biotransformation can be overestimated by no more than 1.4 times for isotope shifts of at least 20%. Overestimates of biodegradation are associated only with estimates of biodegradation at “stalled” sites or sites where cis-DCE or VC is the parent compound. Further, overestimate ranges are applicable only at field sites where in situ ε values fall within previously reported ε values. Interestingly, in a previous study using an optimization model,44 Morrill et al. determined that it is justifiable to apply experimentally derived cis-DCE ε values to field sites where cisDCE is simultaneously being formed and degraded because ε values for cis-DCE dechlorination are consistent whether or not cis-DCE is introduced as the parent compound. Therefore, the overestimation ranges determined for “stalled” sites may also apply to sites where cis-DCE is being simultaneously produced and transformed. Conversely, in the same study, VC ε values varied by as much as 10.1% based on if VC was the original parent compound or was formed as an intermediate. Although ε values for dechlorination of cis-DCE and VC vary by as much as 80.3%, ε values were all the same order-ofmagnitude and fractionation always occurred during dechlorination. Conversely, the percent differences between ε values determined for PCE and TCE dechlorination are up to 190.6 and 152.3%, respectively. Even so, in general, ε values determined for PCE and TCE dechlorination are less variable between phylogenetically related bacteria than between more distantly related bacteria.14 Isotope fractionation is mainly determined by the bond cleavage mechanism but is also affected by the slow, nonfractionating steps that precede bond cleavage and decrease fractionation extent such as transport and binding of the substrate to the RDase;45,46 therefore, the larger variability in ε for reductive dechlorination reactions catalyzed by phylogenetically distant organisms may be due to unique structural features of the bacterial cell or distinct mechanisms of catalytic C-Cl bond cleavage. Although ε values determined in situ may differ from those determined under defined, laboratory conditions, the limited number of phylogenetically related isolates currently 2955

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Environmental Science & Technology known to be capable of catalyzing reductive dechlorination of cis-DCE and VC gives credence to the application of CSIA for quantification of in situ cis-DCE and VC reductive dechlorination.

’ ASSOCIATED CONTENT

bS

Supporting Information. Rayleigh plots for dechlorination catalyzed by Dhc strain FL2, strain GT, strain VS, and the BDI consortium. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: frank.loeffl[email protected].

’ ACKNOWLEDGMENT We thank Matthias Gehre, Ursula G€unther, and Erik Miyoshi for support performing the isotope analyses, M. Alan Nevins for assistance with sample collection, and Alfred Spormann for providing culture VS. This research was supported by the Strategic Environmental Research and Development Program (SERDP) under Contract W912HQ-07-C-0036 (project ER1586). Additional financial support was provided by the European Union Early Stage Training Marie Curie AXIOM Program under contract MEST-CT-2004-8332. K.E.F. acknowledges financial support through a NSF graduate research fellowship. ’ REFERENCES (1) National Research Council. Contaminants in the Subsurface: Source Zone Assessment and Remediation; National Academy Press: Washington, DC, 2004. (2) Ritalahti, K. M.; L€offler, F. E.; Rasch, E. E.; Koenigsberg, S. S. Bioaugmentation for chlorinated ethene detoxification: Bioaugmentation and molecular diagnostics in the bioremediation of chlorinated ethene-contaminated sites. Ind. Biotechnol. 2005, 1, 114–118. (3) Lendvay, J. M.; L€offler, F. E.; Dollhopf, M.; Aiello, M. R.; Daniels, G.; Fathepure, B. Z.; Gebhard, M.; Heine, R.; Helton, R.; Shi, J.; Krajmalnik-Brown, R.; Major, C. L.; Barcelona, M. J.; Petrovskis, E.; Hickey, R.; Tiedje, J. M.; Adriaens, P. Bioreactive barriers: A comparison of bioaugmentation and biostimulation for chlorinated solvent remediation. Environ. Sci. Technol. 2003, 37 (7), 1422–1431. (4) Major, D. W.; McMaster, M. L.; Cox, E. E.; Edwards, E. A.; Dworatzek, S. M.; Hendrickson, E. R.; Starr, M. G.; Payne, J. A.; Buonamici, L. W. Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene. Environ. Sci. Technol. 2002, 36 (23), 5106–5116. (5) L€offler, F. E.; Edwards, E. A. Harnessing microbial activities for environmental cleanup. Curr. Opin. Biotechnol. 2006, 17 (3), 274–284. (6) He, J. Z.; Ritalahti, K. M.; Yang, K. L.; Koenigsberg, S. S.; L€offler, F. E. Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium. Nature 2003, 424 (6944), 62–65. (7) He, J. Z.; Sung, Y.; Krajmalnik-Brown, R.; Ritalahti, K. M.; L€offler, F. E. Isolation and characterization of Dehalococcoides sp. strain FL2, a trichloroethene (TCE)- and 1,2-dichloroethene-respiring anaerobe. Environ. Microbiol. 2005, 7 (9), 1442–1450. (8) Maymo-Gatell, X.; Anguish, T.; Zinder, S. H. Reductive dechlorination of chlorinated ethenes and 1,2-dichloroethane by “Dehalococcoides ethenogenes” 195. Appl. Environ. Microbiol. 1999, 65 (7), 3108–3113. (9) M€uller, J. A.; Rosner, B. M.; von Abendroth, G.; MeshulamSimon, G.; McCarty, P. L.; Spormann, A. M. Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. strain VS

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