Exploring Mechanisms of Biotic Chlorinated Alkane Reduction: Evidence of Nucleophilic Substitution (SN2) with Vitamin B12.

Chlorinated alkanes are notorious groundwater contaminants. Their natural reductive dechlorination by microorganisms involves reductive dehalogenases (RDases) containing cobamide as a cofactor. However, underlying mechanisms of reductive dehalogenation have remained uncertain. Here, observed products, radical trap experiments, UV-vis, and mass spectra demonstrate that (i) reduction by cobalamin (vitamin B12) involved chloroalkyl-cobalamin complexes (ii) whose formation involved a second-order nucleophilic substitution (SN2). Dual element isotope analysis subsequently linked insights from our model system to microbial reductive dehalogenation. Identical observed isotope effects in reduction of trichloromethane by Dehalobacter CF and cobalamin (Dehalobacter CF, εC = -27.9 ± 1.7‰; εCl = -4.2 ± 0.‰; λ = 6.6 ± 0.1; cobalamin, εC = -26.0 ± 0.9‰; εCl = -4.0 ± 0.2‰; λ = 6.5 ± 0.2) indicated the same underlying mechanism, as did identical isotope effects in the reduction of 1,2-dichloroethane by Dehalococcoides and cobalamin (Dehalococcoides, εC = -33.0 ± 0.4‰; εCl = -5.1 ± 0.1‰; λ = 6.5 ± 0.2; cobalamin, εC = -32.8 ± 1.7‰; εCl = -5.1 ± 0.2‰; λ = 6.4 ± 0.2). In contrast, a different, non-SN2 reaction was evidenced by different isotope effects in reaction of 1,2-dichloroethane with Dehalogenimonas (εC = -23.0 ± 2.0‰; εCl = -12.0 ± 0.8‰; λ = 1.9 ± 0.02) illustrating a diversity of biochemical reaction mechanisms manifested even within the same class of enzymes (RDases). This study resolves open questions in our understanding of bacterial reductive dehalogenation and, thereby, provides important information on the biochemistry of bioremediation.

Linking Increased Isotope Fractionation at Low Concentrations to Enzyme Activity Regulation: 4-Cl Phenol Degradation by Arthrobacter chlorophenolicus A6.

Slow microbial degradation of organic trace chemicals ("micropollutants") has been attributed to either downregulation of enzymatic turnover or rate-limiting substrate supply at low concentrations. In previous biodegradation studies, a drastic decrease in isotope fractionation of atrazine revealed a transition from rate-limiting enzyme turnover to membrane permeation as a bottleneck when concentrations fell below the Monod constant of microbial growth. With degradation of the pollutant 4-chlorophenol (4-CP) by Arthrobacter chlorophenolicus A6, this study targeted a bacterium which adapts its enzyme activity to concentrations. Unlike with atrazine degradation, isotope fractionation of 4-CP increased at lower concentrations, from ε(C) = -1.0 ± 0.5‰ in chemostats (D = 0.090 h-1, 88 mg L-1) and ε(C) = -2.1 ± 0.5‰ in batch (c0 = 220 mg L-1) to ε(C) = -4.1 ± 0.2‰ in chemostats at 90 µg L-1. Surprisingly, fatty acid composition indicated increased cell wall permeability at high concentrations, while proteomics revealed that catabolic enzymes (CphCI and CphCII) were differentially expressed at D = 0.090 h-1. These observations support regulation on the enzyme activity level─through either a metabolic shift between catabolic pathways or decreased enzymatic turnover at low concentrations─and, hence, reveal an alternative end-member scenario for bacterial adaptation at low concentrations. Including more degrader strains into this multidisciplinary analytical approach offers the perspective to build a knowledge base on bottlenecks of bioremediation at low concentrations that considers bacterial adaptation.

Compound-specific chlorine isotope fractionation in biodegradation of atrazine.

Atrazine is a frequently detected groundwater contaminant. It can be microbially degraded by oxidative dealkylation or by hydrolytic dechlorination. Compound-specific isotope analysis is a powerful tool to assess its transformation. In previous work, carbon and nitrogen isotope effects were found to reflect these different transformation pathways. However, chlorine isotope fractionation could be a particularly sensitive indicator of natural transformation since chlorine isotope effects are fully represented in the molecular average while carbon and nitrogen isotope effects are diluted by non-reacting atoms. Therefore, this study explored chlorine isotope effects during atrazine hydrolysis with Arthrobacter aurescens TC1 and oxidative dealkylation with Rhodococcus sp. NI86/21. Dual element isotope slopes of chlorine vs. carbon isotope fractionation (Λ = 1.7 ± 0.9 vs. Λ = 0.6 ± 0.1) and chlorine vs. nitrogen isotope fractionation (Λ = -1.2 ± 0.7 vs. Λ = 0.4 ± 0.2) provided reliable indicators of different pathways. Observed chlorine isotope effects in oxidative dealkylation (εCl = -4.3 ± 1.8‰) were surprisingly large, whereas in hydrolysis (εCl = -1.4 ± 0.6‰) they were small, indicating that C-Cl bond cleavage was not the rate-determining step. This demonstrates the importance of constraining expected isotope effects of new elements before using the approach in the field. Overall, the triple element isotope information brought forward here enables a more reliable identification of atrazine sources and degradation pathways.

Toward Improved Accuracy in Chlorine Isotope Analysis: Synthesis Routes for In-House Standards and Characterization via Complementary Mass Spectrometry Methods.

Increasing applications of compound-specific chlorine isotope analysis (CSIA) emphasize the need for chlorine isotope standards that bracket a wider range of isotope values in order to ensure accurate results. With one exception (USGS38), however, all international chlorine isotope reference materials (chloride and perchlorate salts) fall within the narrow range of one per mille. Furthermore, compound-specific working standards are required for chlorine CSIA but are not available for most organic substances. We took advantage of isotope effects in chemical dehalogenation reactions to generate (i) silver chloride (CT16) depleted in 37Cl/35Cl and (ii) compound-specific standards of the herbicides acetochlor and S-metolachlor (Aceto2, Metola2) enriched in 37Cl/35Cl. Calibration against the international reference standards USGS38 (-87.90 ‰) and ISL-354 (+0.05 ‰) by complementary methods (gas chromatography-isotope ratio mass spectrometry, GC-IRMS, versus gas chromatography-multicollector inductively coupled plasma mass spectrometry, GC-MC-ICPMS) gave a consensus value of δ37ClCT16 = -26.82 ± 0.18 ‰. Preliminary GC-MC-ICPMS characterization of commercial Aceto1 and Metola1 versus Aceto2 and Metola2 resulted in tentative values of δ37ClAceto1 = 0.29 ± 0.29 ‰, δ37ClAceto2 = 18.54 ± 0.20 ‰, δ37ClMetola1 = -4.28 ± 0.17 ‰ and δ37ClMetola2 = 5.12 ± 0.27 ‰. The possibility to generate chlorine isotope in-house standards with pronounced shifts in isotope values offers a much-needed basis for accurate chlorine CSIA.

Reductive Dehalogenation of Trichloromethane by Two Different Dehalobacter restrictus Strains Reveal Opposing Dual Element Isotope Effects.

Trichloromethane (TCM) is a frequently detected and persistent groundwater contaminant. Recent studies have reported that two closely related Dehalobacter strains (UNSWDHB and CF) transform TCM to dichloromethane, with inconsistent carbon isotope effects (ε13CUNSWDHB = -4.3 ± 0.45‰; ε13CCF = -27.5 ± 0.9‰). This study uses dual element compound specific isotope analysis (C; Cl) to explore the underlying differences. TCM transformation experiments using strain CF revealed pronounced normal carbon and chlorine isotope effects (ε13CCF = -27.9 ± 1.7‰; ε37ClCF = -4.2 ± 0.2‰). In contrast, small carbon and unprecedented inverse chlorine isotope effects were observed for strain UNSWDHB (ε13CUNSWDHB = -3.1 ± 0.5‰; ε37ClUNSWDHB = 2.5 ± 0.3‰) leading to opposing dual element isotope slopes (λCF = 6.64 ± 0.14 vs λUNSWDHB = -1.20 ± 0.18). Isotope effects of strain CF were identical to experiments with TCM and Vitamin B12 (ε13CVitamin B12 = -26.0 ± 0.9‰, ε37ClVitamin B12 = -4.0 ± 0.2‰, λVitamin B12 = 6.46 ± 0.20). Comparison to previously reported isotope effects suggests outer-sphere-single-electron transfer or SN2 as possible underlying mechanisms. Cell suspension and cell free extract experiments with strain UNSWDHB were both unable to unmask the intrinsic KIE of the reductive dehalogenase (TmrA) suggesting that enzyme binding and/or mass-transfer into the periplasm were rate-limiting. Nondirected intermolecular interactions of TCM with cellular material were ruled out as reason for the inverse isotope effect by gas/water and gas/hexadecane partitioning experiments indicating specific, yet uncharacterized interactions must be operating prior to catalysis.

Dual element (CCl) isotope approach to distinguish abiotic reactions of chlorinated methanes by Fe(0) and by Fe(II) on iron minerals at neutral and alkaline pH.

A dual element CCl isotopic study was performed for assessing chlorinated methanes (CMs) abiotic transformation reactions mediated by iron minerals and Fe(0) to further distinguish them in natural attenuation monitoring or when applying remediation strategies in polluted sites. Isotope fractionation was investigated during carbon tetrachloride (CT) and chloroform (CF) degradation in anoxic batch experiments with Fe(0), with FeCl2(aq), and with Fe-bearing minerals (magnetite, Mag and pyrite, Py) amended with FeCl2(aq), at two different pH values (7 and 12) representative of field and remediation conditions. At pH 7, only CT batches with Fe(0) and Py underwent degradation and CF accumulation evidenced hydrogenolysis. With Py, thiolytic reduction was revealed by CS2 yield and is a likely reason for different Λ value (Δδ13C/Δδ37Cl) comparing with Fe(0) experiments at pH 7 (2.9 ±â€¯0.5 and 6.1 ±â€¯0.5, respectively). At pH 12, all CT experiments showed degradation to CF, again with significant differences in Λ values between Fe(0) (5.8 ±â€¯0.4) and Fe-bearing minerals (Mag, 2 ±â€¯1, and Py, 3.7 ±â€¯0.9), probably evidencing other parallel pathways (hydrolytic and thiolytic reduction). Variation of pH did not significantly affect the Λ values of CT degradation by Fe(0) nor Py. CF degradation by Fe(0) at pH 12 showed a Λ (8 ±â€¯1) similar to that reported at pH 7 (8 ±â€¯2), suggesting CF hydrogenolysis as the main reaction and that CF alkaline hydrolysis (13.0 ±â€¯0.8) was negligible. Our data establish a base for discerning the predominant or combined pathways of CMs natural attenuation or for assessing the effectiveness of remediation strategies using recycled minerals or Fe(0).

Reductive Outer-Sphere Single Electron Transfer Is an Exception Rather than the Rule in Natural and Engineered Chlorinated Ethene Dehalogenation.

Chlorinated ethenes (CEs) such as perchloroethylene, trichloroethylene and dichloroethylene are notorious groundwater contaminants. Although reductive dehalogenation is key to their environmental and engineered degradation, underlying reaction mechanisms remain elusive. Outer-sphere reductive single electron transfer (OS-SET) has been proposed for such different processes as Vitamin B12-dependent biodegradation and zerovalent metal-mediated dehalogenation. Compound-specific isotope effect (13C/12C, 37Cl/35Cl) analysis offers a new opportunity to test these hypotheses. Defined OS-SET model reactants (CO2 radical anions, S2--doped graphene oxide in water) caused strong carbon (εC = -7.9‰ to -11.9‰), but negligible chlorine isotope effects (εCl = -0.12‰ to 0.04‰) in CEs. Greater chlorine isotope effects were observed in CHCl3 (εC = -7.7‰, εCl = -2.6‰), and in CEs when the exergonicity of C-Cl bond cleavage was reduced in an organic solvent (reaction with arene radical anions in glyme). Together, this points to dissociative OS-SET (SET to a σ* orbital concerted with C-Cl breakage) in alkanes compared to stepwise OS-SET (SET to a π* orbital followed by C-Cl cleavage) in ethenes. The nonexistent chlorine isotope effects of chlorinated ethenes in all aqueous OS-SET experiments contrast strongly with pronounced Cl isotope fractionation in all natural and engineered reductive dehalogenations reported to date suggesting that OS-SET is an exception rather than the rule in environmental transformations of chlorinated ethenes.

Carbon and Chlorine Isotope Fractionation Patterns Associated with Different Engineered Chloroform Transformation Reactions.

To use compound-specific isotope analysis for confidently assessing organic contaminant attenuation in the environment, isotope fractionation patterns associated with different transformation mechanisms must first be explored in laboratory experiments. To deliver this information for the common groundwater contaminant chloroform (CF), this study investigated for the first time both carbon and chlorine isotope fractionation for three different engineered reactions: oxidative C-H bond cleavage using heat-activated persulfate, transformation under alkaline conditions (pH ∼ 12) and reductive C-Cl bond cleavage by cast zerovalent iron, Fe(0). Carbon and chlorine isotope fractionation values were -8 ± 1‰ and -0.44 ± 0.06‰ for oxidation, -57 ± 5‰ and -4.4 ± 0.4‰ for alkaline hydrolysis (pH 11.84 ± 0.03), and -33 ± 11‰ and -3 ± 1‰ for dechlorination, respectively. Carbon and chlorine apparent kinetic isotope effects (AKIEs) were in general agreement with expected mechanisms (C-H bond cleavage in oxidation by persulfate, C-Cl bond cleavage in Fe(0)-mediated reductive dechlorination and E1CB elimination mechanism during alkaline hydrolysis) where a secondary AKIECl (1.00045 ± 0.00004) was observed for oxidation. The different dual carbon-chlorine (Δδ13C vs Δδ37Cl) isotope patterns for oxidation by thermally activated persulfate and alkaline hydrolysis (17 ± 2 and 13.0 ± 0.8, respectively) vs reductive dechlorination by Fe(0) (8 ± 2) establish a base to identify and quantify these CF degradation mechanisms in the field.

Compound-Specific Chlorine Isotope Analysis of Tetrachloromethane and Trichloromethane by Gas Chromatography-Isotope Ratio Mass Spectrometry vs Gas Chromatography-Quadrupole Mass Spectrometry: Method Development and Evaluation of Precision and Trueness.

Compound-specific chlorine isotope analysis of tetrachloromethane (CCl4) and trichloromethane (CHCl3) was explored by both, gas chromatography-isotope ratio mass spectrometry (GC-IRMS) and GC-quadrupole MS (GC-qMS), where GC-qMS was validated in an interlaboratory comparison between Munich and Neuchâtel with the same type of commercial GC-qMS instrument. GC-IRMS measurements analyzed CCl isotopologue ions, whereas GC-qMS analyzed the isotopologue ions CCl3, CCl2, CCl (of CCl4) and CHCl3, CHCl2, CHCl (of CHCl3), respectively. Lowest amount dependence (good linearity) was obtained (i) in H-containing fragment ions where interference of 35Cl- to 37Cl-containing ions was avoided; (ii) with tuning parameters favoring one predominant rather than multiple fragment ions in the mass spectra. Optimized GC-qMS parameters (dwell time 70 ms, 2 most abundant ions) resulted in standard deviations of 0.2‰ (CHCl3) and 0.4‰ (CCl4), which are only about twice as large as 0.1‰ and 0.2‰ for GC-IRMS. To compare also the trueness of both methods and laboratories, samples from CCl4 and CHCl3 degradation experiments were analyzed and calibrated against isotopically different reference standards for both CCl4 and CHCl3 (two of each). Excellent agreement confirms that true results can be obtained by both methods provided that a consistent set of isotopically characterized reference materials is used.