Mechanistic Investigation of Chlorinated Ethylene Degradation using Chlorine and Carbon Isotope Fractionation

Chloroethenes are large-scale industrial products, and detected as toxic contaminants in the environment. Their reductive dechlorination is a clear remediation approach, which has been the focus of several studies, using biotic and abiotic model systems. Despite the progress toward understanding the underlying process the formation of toxic and harmless products remains incompletely understood, and some of the proposed mechanistic hypotheses have led to inconsistencies. A recently developed analytical method of continuous flow compound specific chlorine isotope analysis was used in this study to further uncover the underlying mechanisms of reductive dechlorination. In the first instance, the newly created chlorine isotope data was analyzed towards the basic question how isotope effects of chlorine are manifested in the respective products during biodegradation. The developed mathematical framework gave first insights into position specific chlorine isotope effects during biodegradation of chloroethenes. From their interpretation, the structural selectivity in the biotic reductive dechlorination of TCE could be allocated to two chlorine substituents. This information allowed a systematic discussion with respect to the hypothesized mechanisms. Further degradation experiments with two different microbial strains were investigated by combined analysis of carbon and chlorine isotope effects in dual isotope plots in comparison to model reactions that are commonly used to mimic microbial dechlorination. Similar mechanisms were indicated for biodegradation and reactions with cobalamin (Vitamin B12), the enzymatic cofactor of dehalogenase enzymes. In contrast a different mechanism was indicated for reactions with cobaloxime, a commonly used mimicking reagent for cobalamin. The results demonstrate the strength of dual isotope plots as an indicator of the authenticity of a model reaction for the actual system with respect to the underlying mechanisms. The method of two dimensional isotope analysis was further investigated for its application towards the environmental clean-up technology of permeable reactive barriers (PRB) with zero-valent iron (ZVI). Dual isotope plots and product related carbon isotope fractionation were explored here as two discrete approaches to distinguish the effectiveness of transformation by ZVI as opposed to natural biodegradation. The results of this work exemplify the potential of chlorine and carbon isotope analysis to assess the sustainable removal of contaminants and their degradation pathways directly in real-world transformations. Moreover, it opens the perspective for future work to pinpoint mechanisms of the important environmental dehalogenation reactions by applying the approach on further model reactions with distinct mechanisms

Contrasting dual (C, Cl) isotope fractionation offers potential to distinguish reductive chloroethene transformation from breakdown by permanganate

cis-1,2-Dichloroethene (cis-DCE) and trichloroethene (TCE) are persistent, toxic and mobile pollutants in groundwater systems. They are both conducive to reductive dehalogenation and to oxidation by permanganate. In this study, the potential of dual element (C, Cl) compound specific isotope analyses (CSIA) for distinguishing between chemical oxidation and anaerobic reductive dechlorination of cis-DCE and TCE was investigated. Well-controlled cis-DCE degradation batch tests gave similar carbon isotope enrichment factors εC (‰), but starkly contrasting dual element isotope slopes Δδ(13)C/Δδ(37)Cl for permanganate oxidation (εC=-26‰±6‰, Δδ(13)C/Δδ(37)Cl≈-125±47) compared to reductive dechlorination (εC=-18‰±4‰, Δδ(13)C/Δδ(37)Cl≈4.5±3.4). The difference can be tracked down to distinctly different chlorine isotope fractionation: an inverse isotope effect during chemical oxidation (εCl=+0.2‰±0.1‰) compared to a large normal isotope effect in reductive dechlorination (εCl=-3.3‰±0.9‰) (p≪0.05). A similar trend was observed for TCE. The dual isotope approach was evaluated in the field before and up to 443days after a pilot scale permanganate injection in the subsurface. Our study indicates, for the first time, the potential of the dual element isotope approach for distinguishing cis-DCE (and TCE) concentration drops caused by dilution, oxidation by permanganate and reductive dechlorination both at laboratory and field scale

C and Cl Isotope Fractionation of 1,2-Dichloroethane Displays Unique δ¹³C/δ³⁷Cl Patterns for Pathway Identification and Reveals Surprising C–Cl Bond Involvement in Microbial Oxidation

This study investigates dual element isotope fractionation during aerobic biodegradation of 1,2-dichloroethane (1,2-DCA) via oxidative cleavage of a C–H bond (<i>Pseudomonas</i> sp. strain DCA1) versus C–Cl bond cleavage by S_N2 reaction (<i>Xanthobacter autotrophicus</i> GJ10 and <i>Ancylobacter aquaticus</i> AD20). Compound-specific chlorine isotope analysis of 1,2-DCA was performed for the first time, and isotope fractionation (ε_bulk^Cl) was determined by measurements of the same samples in three different laboratories using two gas chromatography–isotope ratio mass spectrometry systems and one gas chromatography–quadrupole mass spectrometry system. Strongly pathway-dependent slopes (Δδ¹³C/Δδ³⁷Cl), 0.78 ± 0.03 (oxidation) and 7.7 ± 0.2 (S_N2), delineate the potential of the dual isotope approach to identify 1,2-DCA degradation pathways in the field. In contrast to different ε_bulk^C values [−3.5 ± 0.1‰ (oxidation) and −31.9 ± 0.7 and −32.0 ± 0.9‰ (S_N2)], the obtained ε_bulk^Cl values were surprisingly similar for the two pathways: −3.8 ± 0.2‰ (oxidation) and −4.2 ± 0.1 and −4.4 ± 0.2‰ (S_N2). Apparent kinetic isotope effects (AKIEs) of 1.0070 ± 0.0002 (¹³C-AKIE, oxidation), 1.068 ± 0.001 (¹³C-AKIE, S_N2), and 1.0087 ± 0.0002 (³⁷Cl-AKIE, S_N2) fell within expected ranges. In contrast, an unexpectedly large secondary ³⁷Cl-AKIE of 1.0038 ± 0.0002 reveals a hitherto unrecognized involvement of C–Cl bonds in microbial C–H bond oxidation. Our two-dimensional isotope fractionation patterns allow for the first time reliable 1,2-DCA degradation pathway identification in the field, which unlocks the full potential of isotope applications for this important groundwater contaminant

Combined C and Cl Isotope Effects Indicate Differences between Corrinoids and Enzyme (<i>Sulfurospirillum multivorans</i> PceA) in Reductive Dehalogenation of Tetrachloroethene, But Not Trichloroethene

The role of the corrinoid cofactor in reductive dehalogenation catalysis by tetrachloroethene reductive dehalogenase (PceA) of <i>Sulfurospirillum multivorans</i> was investigated using isotope analysis of carbon and chlorine. Crude extracts containing PceAharboring either a native <i>norpseudo</i>-B₁₂ or the alternative <i>nor</i>-B₁₂ cofactorwere applied for dehalogenation of tetrachloroethene (PCE) or trichloroethene (TCE), and compared to abiotic dehalogenation with the respective purified corrinoids (<i>norpseudo</i>vitamin B₁₂ and <i>nor</i>vitamin B₁₂), as well as several commercially available cobalamins and cobinamide. Dehalogenation of TCE resulted in a similar extent of C and Cl isotope fractionation, and in similar dual-element isotope slopes (ε_C/ε_Cl) of 5.0–5.3 for PceA enzyme and 3.7–4.5 for the corrinoids. Both observations support an identical reaction mechanism. For PCE, in contrast, observed C and Cl isotope fractionation was smaller in enzymatic dehalogenation, and dual-element isotope slopes (2.2–2.8) were distinctly different compared to dehalogenation mediated by corrinoids (4.6−7.0). Remarkably, ε_C/ε_Cl of PCE depended in addition on the corrinoid type: ε_C/ε_Cl values of 4.6 and 5.0 for vitamin B₁₂ and <i>nor</i>vitamin B₁₂ were significantly different compared to values of 6.9 and 7.0 for <i>norpseudo</i>vitamin B₁₂ and dicyanocobinamide. Our results therefore suggest mechanistic and/or kinetic differences in catalytic PCE dehalogenation by enzymes and different corrinoids, whereas such differences were not observed for TCE

Compound-Specific Chlorine Isotope Analysis: A Comparison of Gas Chromatography/Isotope Ratio Mass Spectrometry and Gas Chromatography/Quadrupole Mass Spectrometry Methods in an Interlaboratory Study

Chlorine isotope analysis of chlorinated hydrocarbons like trichloroethylene (TCE) is of emerging demand because these species are important environmental pollutants. Continuous flow analysis of noncombusted TCE molecules, either by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) or by GC/quadrupole mass spectrometry (GC/qMS), was recently brought forward as innovative analytical solution. Despite early implementations, a benchmark for routine applications has been missing. This study systematically compared the performance of GC/qMS versus GC/IRMS in six laboratories involving eight different instruments (GC/IRMS, Isoprime and Thermo MAT-253; GC/qMS, Agilent 5973N, two Agilent 5975C, two Thermo DSQII, and one Thermo DSQI). Calibrations of (37)Cl/(35)Cl instrument data against the international SMOC scale (Standard Mean Ocean Chloride) deviated between instruments and over time. Therefore, at least two calibration standards are required to obtain true differences between samples. Amount dependency of &delta;(37)Cl was pronounced for some instruments, but could be eliminated by corrections, or by adjusting amplitudes of standards and samples. Precision decreased in the order GC/IRMS (1&sigma; &asymp; 0.1&permil;), to GC/qMS (1&sigma; &asymp; 0.2-0.5&permil; for Agilent GC/qMS and 1&sigma; &asymp; 0.2-0.9&permil; for Thermo GC/qMS). Nonetheless, &delta;(37)Cl values between laboratories showed good agreement when the same external standards were used. These results lend confidence to the methods and may serve as a benchmark for future applications

Reductive Dechlorination of TCE by Chemical Model Systems in Comparison to Dehalogenating Bacteria: Insights from Dual Element Isotope Analysis (¹³C/¹²C, ³⁷Cl/³⁵Cl)

Chloroethenes like trichloroethene (TCE) are prevalent environmental contaminants, which may be degraded through reductive dechlorination. Chemical models such as cobalamine (vitamin B₁₂) and its simplified analogue cobaloxime have served to mimic microbial reductive dechlorination. To test whether in vitro and in vivo mechanisms agree, we combined carbon and chlorine isotope measurements of TCE. Degradation-associated enrichment factors ε_carbon and ε_chlorine (i.e., molecular-average isotope effects) were −12.2‰ ± 0.5‰ and −3.6‰ ± 0.1‰ with <i>Geobacter lovleyi</i> strain SZ; −9.1‰ ± 0.6‰ and −2.7‰ ± 0.6‰ with <i>Desulfitobacterium hafniense</i> Y51; −16.1‰ ± 0.9‰ and −4.0‰ ± 0.2‰ with the enzymatic cofactor cobalamin; −21.3‰ ± 0.5‰ and −3.5‰ ± 0.1‰ with cobaloxime. Dual element isotope slopes m = Δδ¹³C/ Δδ³⁷Cl ≈ ε_carbon/ε_chlorine of TCE showed strong agreement between biotransformations (3.4 to 3.8) and cobalamin (3.9), but differed markedly for cobaloxime (6.1). These results (i) suggest a similar biodegradation mechanism despite different microbial strains, (ii) indicate that transformation with isolated cobalamin resembles in vivo transformation and (iii) suggest a different mechanism with cobaloxime. This model reactant should therefore be used with caution. Our results demonstrate the power of two-dimensional isotope analyses to characterize and distinguish between reaction mechanisms in whole cell experiments and in vitro model systems