Use of isotopic (C, Cl) and molecular biology tools to assess biodegradation in a source area of chlorinated ethenes after biostimulation with Emulsified Vegetable Oil (EVO).

Enhanced In Situ Bioremediation (EISB) using Emulsified Vegetable Oil (EVO) as a long-term electron donor has gained prominence for the treatment of groundwater contaminated with chlorinated ethenes (CEs). This study explores the potential of isotopic and molecular biology tools (MBT) to investigate the CEs (PCE, TCE and cis-DCE) bioremediation using EVO in a contaminated site. A multiple approach using C and Cl-CSIA, quantification of Dehalococcoides (Dhc) and specific reductive dechlorination (RD) gene population, and hydrochemical data in microcosm experiments and field samples was applied. Despite the high partitioning of CEs into the EVO phase, the carbon isotopic values of the remaining CEs fraction in the aqueous phase did not exhibit significant changes caused by phase partitioning in laboratory experiments. Both microcosm experiments and field data revealed a rapid RD of PCE and TCE, resulting in the transient accumulation of cis-DCE, which was slowly degraded to vinyl chloride (VC). These results agreed with the presence of Dhc populations and a shift to stronger reducing conditions in the field: i) RD functional genes (tceA, vcrA and bvcA) exhibited a trend to higher values and ii) a substantial increase in Dhc populations (up to 30% of the total bacterial populations) was observed over time. The dual-element isotope slope ΛC-Cl for RD of cis-DCE obtained from field data (ΛC - Cl = 5 ± 3) was similar to the one determined from the microcosm experiments under controlled anoxic conditions (ΛC - Cl = 4.9 ± 0.8). However, ΛC-Cl values differ from those reported so far for laboratory studies with Dhc strains and mixed cultures containing Dhc, i.e., between 8.3 and 17.8. This observation underscores the potential variety of reductive dehalogenases involved during cis-DCE RD and the importance of determining site-specific Λ and ɛ values in order to improve the identification and quantification of transformation processes in the field.

Dual Carbon-Chlorine Isotope Analysis Indicates Distinct Anaerobic Dichloromethane Degradation Pathways in Two Members of Peptococcaceae.

Dichloromethane (DCM) is a probable human carcinogen and frequent groundwater contaminant and contributes to stratospheric ozone layer depletion. DCM is degraded by aerobes harboring glutathione-dependent DCM dehalogenases; however, DCM contamination occurs in oxygen-deprived environments, and much less is known about anaerobic DCM metabolism. Some members of the Peptococcaceae family convert DCM to environmentally benign products including acetate, formate, hydrogen (H2), and inorganic chloride under strictly anoxic conditions. The current study applied stable carbon and chlorine isotope fractionation measurements to the axenic culture Dehalobacterium formicoaceticum and to the consortium RM comprising DCM degrader Candidatus Dichloromethanomonas elyunquensis. Degradation-associated carbon and chlorine isotope enrichment factors (εC and εCl) of -42.4 ± 0.7‰ and -5.3 ± 0.1‰, respectively, were measured in D. formicoaceticum cultures. A similar εCl of -5.2 ± 0.1‰, but a substantially lower εC of -18.3 ± 0.2‰, were determined for Ca. Dichloromethanomonas elyunquensis. The εC and εCl values resulted in distinctly different dual element C-Cl isotope correlations (ΛC/Cl = Δδ13C/Δδ37Cl) of 7.89 ± 0.12 and 3.40 ± 0.03 for D. formicoaceticum and Ca. Dichloromethanomonas elyunquensis, respectively. The distinct ΛC/Cl values obtained for the two cultures imply mechanistically distinct C-Cl bond cleavage reactions, suggesting that members of Peptococcaceae employ different pathways to metabolize DCM. These findings emphasize the utility of dual carbon-chlorine isotope analysis to pinpoint DCM degradation mechanisms and to provide an additional line of evidence that detoxification is occurring at DCM-contaminated sites.

Stable chlorine isotope analysis of chlorinated acetic acids using gas chromatography/quadrupole mass spectrometry.

RATIONALE: The environmental occurrence of chlorinated acetic acids (CAAs) has been extensively studied, but the sources and transport are still not yet fully understood. A promising approach for source apportionment and process studies is the isotopic characterization of target compounds. We present the first on-line stable chlorine isotope analysis of CAAs by use of gas chromatography/quadrupole mass spectrometry (GC/qMS). METHODS: Following approved procedures for concentration analysis, CAAs extracted into MTBE were methylated to GC-amenable methyl esters (mCAAs). These mCAAs were then analyzed by GC/qMS for their stable chlorine isotope composition using a sample/standard-bracketing approach (CAA standards in the range δ(37) Cl -6.3 to -0.2 ‰, Standard Mean Ocean Chloride). RESULTS: Cross-calibration of the herein presented method with off-line reference methods (thermal ionization and continuous-flow GC isotope ratio mass spectrometry; TI-MS and CF-GC/IRMS, respectively) shows good agreement between the methods (regression slope for GC/qMS vs reference method data sets: 0.92 ± 0.29). Sample amounts as small as 10 pmol Cl can herewith be analyzed with a precision of 0.1 to 0.4 ‰. CONCLUSIONS: This method should be useful for environmental studies of CAAs at ambient concentrations in precipitations (<0.06 to 100 nmol L(-1) ), surface waters (<0.2 to 5 nmol L(-1) ) and soil (<0.6 to 2000 nmol kg(-1) dry soil) where conventional off-line methods cannot be applied.

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 δ(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 (1σ ≈ 0.1‰), to GC/qMS (1σ ≈ 0.2-0.5‰ for Agilent GC/qMS and 1σ ≈ 0.2-0.9‰ for Thermo GC/qMS). Nonetheless, δ(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.