Julian Renpenning

Combined C and Cl isotope effects indicate differences between corrinoids and enzyme (Sulfurospirillum multivorans 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 Sulfurospirillum multivorans was investigated using isotope analysis of carbon and chlorine. Crude extracts containing PceA--harboring either a native norpseudo-B12 or the alternative nor-B12 cofactor--were applied for dehalogenation of tetrachloroethene (PCE) or trichloroethene (TCE), and compared to abiotic dehalogenation with the respective purified corrinoids (norpseudovitamin B12 and norvitamin 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 (εC/εCl) 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, εC/εCl of PCE depended in addition on the corrinoid type: εC/εCl values of 4.6 and 5.0 for vitamin B12 and norvitamin B12 were significantly different compared to values of 6.9 and 7.0 for norpseudovitamin 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.

On-Line Hydrogen-Isotope Measurements of Organic Samples Using Elemental Chromium: An Extension for High Temperature Elemental-Analyzer Techniques

The high temperature conversion (HTC) technique using an elemental analyzer with a glassy carbon tube and filling (temperature conversion/elemental analysis, TC/EA) is a widely used method for hydrogen isotopic analysis of water and many solid and liquid organic samples with analysis by isotope-ratio mass spectrometry (IRMS). However, the TC/EA IRMS method may produce inaccurate δ(2)H results, with values deviating by more than 20 mUr (milliurey = 0.001 = 1‰) from the true value for some materials. We show that a single-oven, chromium-filled elemental analyzer coupled to an IRMS substantially improves the measurement quality and reliability for hydrogen isotopic compositions of organic substances (Cr-EA method). Hot chromium maximizes the yield of molecular hydrogen in a helium carrier gas by irreversibly and quantitatively scavenging all reactive elements except hydrogen. In contrast, under TC/EA conditions, heteroelements like nitrogen or chlorine (and other halogens) can form hydrogen cyanide (HCN) or hydrogen chloride (HCl) and this can cause isotopic fractionation. The Cr-EA technique thus expands the analytical possibilities for on-line hydrogen-isotope measurements of organic samples significantly. This method yielded reproducibility values (1-sigma) for δ(2)H measurements on water and caffeine samples of better than 1.0 and 0.5 mUr, respectively. To overcome handling problems with water as the principal calibration anchor for hydrogen isotopic measurements, we have employed an effective and simple strategy using reference waters or other liquids sealed in silver-tube segments. These crimped silver tubes can be employed in both the Cr-EA and TC/EA techniques. They simplify considerably the normalization of hydrogen-isotope measurement data to the VSMOW-SLAP (Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation) scale, and their use improves accuracy of the data by eliminating evaporative loss and associated isotopic fractionation while handling water as a bulk sample. The calibration of organic samples, commonly having high δ(2)H values, will benefit from the availability of suitably (2)H-enriched reference waters, extending the VSMOW-SLAP scale above zero.

Compound-Specific Hydrogen Isotope Analysis of Heteroatom-Bearing Compounds via Gas Chromatography–Chromium-Based High-Temperature Conversion (Cr/HTC)–Isotope Ratio Mass Spectrometry

The traditional high-temperature conversion (HTC) approach toward compound-specific stable isotope analysis (CSIA) of hydrogen for heteroatom-bearing (i.e., N, Cl, S) compounds has been afflicted by fractionation bias due to formation of byproducts HCN, HCl, and H2S. This study presents a chromium-based high-temperature conversion (Cr/HTC) approach for organic compounds containing nitrogen, chlorine, and sulfur. Following peak separation along a gas chromatographic (GC) column, the use of thermally stable ceramic Cr/HTC reactors at 1100-1500 °C and chemical sequestration of N, Cl, and S by chromium result in quantitative conversion of compound-specific organic hydrogen to H2 analyte gas. The overall hydrogen isotope analysis via GC-Cr/HTC-isotope ratio mass spectrometry (IRMS) achieved a precision of better than ± 5 mUr along the VSMOW-SLAP scale. The accuracy of GC-Cr/HTC-IRMS was validated with organic reference materials (RM) in comparison with online EA-Cr/HTC-IRMS and offline dual-inlet IRMS. The utility and reliability of the GC-Cr/HTC-IRMS system were documented during the routine measurement of more than 500 heteroatom-bearing organic samples spanning a δ(2)H range of -181 mUr to 629 mUr.

Substrate Hydrophobicity and Cell Composition Influence the Extent of Rate Limitation and Masking of Isotope Fractionation during Microbial Reductive Dehalogenation of Chlorinated Ethenes

This study investigated the effect of intracellular microscale mass transfer on microbial carbon isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE). Significantly stronger isotope fractionation was observed for crude extracts vs intact cells of Sulfurospirillum multivorans, Geobacter lovleyi, Desulfuromonas michiganensis, Desulfitobacterium hafniense strain PCE-S, and Dehalobacter restrictus. Furthermore, carbon stable isotope fractionation was stronger for microorganisms with a Gram-positive cell envelope compared to those with a Gram-negative cell envelope. Significant differences were observed between model organisms in cellular sorption capacity for PCE (S. multivorans-K(d-PCE) = 0.42-0.51 L g(-1); D. hafniense-K(d-PCE) = 0.13 L g(-1)), as well as in envelope hydrophobicity (S. multivorans 33.0° to 72.2°; D. hafniense 59.1° to 60.8°) when previously cultivated with fumarate or PCE as electron acceptor, but not for TCE. Cell envelope properties and the tetrachloroethene reductive dehalogenase (PceA-RDase) localization did not result in significant effects on observed isotope fractionation of TCE. For PCE, however, systematic masking of isotope effects as a result of microscale mass transfer limitation at microbial membranes was observed, with carbon isotope enrichment factors of -2.2‰, -1.5 to -1.6‰, and -1.0‰ (CI95% < ± 0.2‰) for no membrane, hydrophilic outer membrane, and outer + cytoplasmic membrane, respectively. Conclusively, rate-limiting mass transfer barriers were (a) the outer membrane or cell wall and (b) the cytoplasmic membrane in case of a cytoplasmic location of the RDase enzyme. Overall, our results indicate that masking of isotope fractionation is determined by (1) hydrophobicity of the degraded compound, (2) properties of the cell envelope, and (3) the localization of the reacting enzyme.

Development and Validation of an Universal Interface for Compound-Specific Stable Isotope Analysis of Chlorine (37Cl/35Cl) by GC-High-Temperature Conversion (HTC)-MS/IRMS

A universal application of compound-specific isotope analysis of chlorine was thus far limited by the availability of suitable analysis techniques. In this study, gas chromatography in combination with a high-temperature conversion interface (GC-HTC), converting organic chlorine in the presence of H2 to gaseous HCl, was coupled to a dual-detection system, combining an ion trap mass spectrometer (MS) and isotope-ratio mass spectrometer (IRMS). The combination of the MS/IRMS detection enabled a detailed characterization, optimization, and online monitoring of the high-temperature conversion process via ion trap MS as well as a simultaneous chlorine isotope analysis by the IRMS. Using GC-HTC-MS/IRMS, chlorine isotope analysis at optimized conversion conditions resulted in very accurate isotope values (δ(37)Cl(SMOC)) for measured reference material with known isotope composition, including chlorinated ethylene, chloromethane, hexachlorocyclohexane, and trichloroacetic acids methyl ester. Respective detection limits were determined to be <15 nmol Cl on column with achieved precision of <0.3‰.

Optimization of on-line hydrogen stable isotope ratio measurements of halogen- and sulfur-bearing organic compounds using elemental analyzer–chromium/high-temperature conversion isotope ratio mass spectrometry (EA-Cr/HTC-IRMS)

RATIONALE Accurate hydrogen isotopic analysis of halogen- and sulfur-bearing organics has not been possible with traditional high-temperature conversion (HTC) because the formation of hydrogen-bearing reaction products other than molecular hydrogen (H2 ) is responsible for non-quantitative H2 yields and possible hydrogen isotopic fractionation. Our previously introduced, new chromium-based EA-Cr/HTC-IRMS (Elemental Analyzer-Chromium/High-Temperature Conversion Isotope Ratio Mass Spectrometry) technique focused primarily on nitrogen-bearing compounds. Several technical and analytical issues concerning halogen- and sulfur-bearing samples, however, remained unresolved and required further refinement of the reactor systems. METHODS The EA-Cr/HTC reactor was substantially modified for the conversion of halogen- and sulfur-bearing samples. The performance of the novel conversion setup for solid and liquid samples was monitored and optimized using a simultaneously operating dual-detection system of IRMS and ion trap MS. The method with several variants in the reactor, including the addition of manganese metal chips, was evaluated in three laboratories using EA-Cr/HTC-IRMS (on-line method) and compared with traditional uranium-reduction-based conversion combined with manual dual-inlet IRMS analysis (off-line method) in one laboratory. RESULTS The modified EA-Cr/HTC reactor setup showed an overall H2 -recovery of more than 96% for all halogen- and sulfur-bearing organic compounds. All results were successfully normalized via two-point calibration with VSMOW-SLAP reference waters. Precise and accurate hydrogen isotopic analysis was achieved for a variety of organics containing F-, Cl-, Br-, I-, and S-bearing heteroelements. The robust nature of the on-line EA-Cr/HTC technique was demonstrated by a series of 196 consecutive measurements with a single reactor filling. CONCLUSIONS The optimized EA-Cr/HTC reactor design can be implemented in existing analytical equipment using commercially available material and is universally applicable for both heteroelement-bearing and heteroelement-free organic-compound classes. The sensitivity and simplicity of the on-line EA-Cr/HTC-IRMS technique provide a much needed tool for routine hydrogen-isotope source tracing of organic contaminants in the environment. Copyright

Online isotope analysis of 37Cl/35Cl universally applied for semi-volatile organic compounds using GC-MC-ICPMS

Stable chlorine isotope analysis of organic compounds is potentially applicable in various fields in forensics and environmental analytics to investigate the fate of these substances in the environment, but a wider use of this technique is still hampered by the limited applicability of available offline and online techniques. In a previous study we presented a method for compound-specific chlorine isotope analysis of volatile organics including chlorinated methanes, ethanes and ethenes using gas chromatography interfaced with multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS). In the current study we modified further the setup in order to extend the range of analytes towards semi-volatile organic substances with boiling points of up to 350 °C. The modified method was evaluated by using offline characterized in-house reference materials, such as chloroethenes, chloroacetic acid and hexachlorocyclohexenes. Additionally, analysis of various chlorinated benzenes, chlorinated phenols, chlordecone, dichlorodiphenyltrichloroethane (DDT) and related derivatives was demonstrated. The analytical precision (1σ) was usually better than ±0.2 mUr for single compound and ±0.3 mUr for compound-specific analysis of mixtures. Achieved accuracy was within ±0.2 mUr compared to available offline values. The isotopic detection limit could be significantly improved by one order of magnitude (250 pmol Cl on column, corresponding to ∼10 ng Cl) and is superior to other online state of the art approaches. The demonstrated method allows for the compound-specific stable chlorine isotope analysis of virtually all GC-compatible organics with versatility, high accuracy, and sensitivity.

Triple-element compound-specific stable isotope analysis of 1,2-dichloroethane for characterization of the underlying dehalogenation reaction in two Dehalococcoides mccartyi strains

ABSTRACT Chlorinated ethanes belong to the most common groundwater and soil contaminants. Of these, 1,2‐dichloroethane (1,2‐DCA) is a man‐made, persistent and toxic contaminant, released due to improper waste treatment at versatile production sites. This study investigated the anaerobic transformation of 1,2‐DCA by Dehalococcoides mccartyi strain 195 and strain BTF08 using triple‐element compound‐specific stable isotope analysis of carbon, chlorine and hydrogen for the first time. Isotope fractionation patterns for carbon (&egr;CBTF08 = ‐28.4 ± 3.7‰; &egr;C195 = ‐30.9 ± 3.6‰) and chlorine (&egr;ClBTF08 = ‐4.6 ± 0.7‰; &egr;Cl195 = ‐4.2 ± 0.5‰) within both investigated D. mccartyi strains, as well as the dual‐element analysis (&Lgr;BTF08 = 6.9 ± 1.2; &Lgr;195 = 7.1 ± 0.2), supported identical reaction mechanisms for dehalogenation of 1,2‐DCA. Hydrogen isotope fractionation analysis revealed dihaloelimination as prevalent reaction mechanism. Vinyl chloride as major intermediate could be excluded by performing the experiment in deuterated aqueous media. Furthermore, evaluation of the derived apparent kinetic isotope effects (AKIECBTF08 = 1.029/AKIEC195 = 1.031; AKIEClBTF08 = 1.005/AKIECl195 = 1.004) pointed towards simultaneous abstraction of both involved chlorine‐substituents in a concerted matter. It was shown that D. mccartyi strain BTF08 and strain 195 are capable of complete, direct dihaloelimination of 1,2‐DCA to ethene. &NA; Graphical Abstract Figure. Triple‐element compound‐specific stable isotope analysis provides evidence that 1,2‐dichloroethane dehalogenation involves a dihaloelimination mechanism with concerted chlorine removal that avoids the formation of toxic vinyl chloride.

Compound Specific Stable Chlorine Isotopic Analysis of Volatile Aliphatic Compounds Using Gas Chromatography Hyphenated with Multiple Collector Inductively Coupled Plasma Mass Spectrometry

Stable chlorine isotope analysis is increasingly used to characterize sources, transformation pathways, and sinks of organic aliphatic compounds, many of them being priority pollutants in groundwater and the atmosphere. A wider use of chlorine isotopes in environmental studies is still inhibited by limitations of the different analytical techniques such as high sample needs, offline preparation, confinement to few compounds and mediocre precision, respectively. Here we present a method for the δ37Cl determination in volatile aliphatic compounds using gas chromatography coupled with multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS), which overcomes these limitations. The method was evaluated by using a suite of five previously offline characterized in-house standards and eight chlorinated methanes, ethanes, and ethenes. Other than in previous approaches using ICP methods for chlorine isotopes, isobaric interference of the 36ArH dimer with 37Cl was minimized by employing dry plasma conditions. Samples containing 2-3 nmol Cl injected on-column were sufficient to achieve a precision (σ) of 0.1 mUr (1 milliurey = 0.001 = 1‰) or better. Long-term reproducibility and accuracy was always better than 0.3 mUr if organics were analyzed in compound mixtures. Standardization is carried out by using a two-point calibration approach. Drift, even though very small in this study, is corrected by referencing versus an internal standard. The presented method offers a direct, universal, and compound-specific procedure to measure the δ37Cl of a wide array of organic compounds overcoming limitations of previous techniques with the benefits of high sensitivity and accuracy comparable to the best existing approaches.

Evaluation of the Microbial Reductive Dehalogenation Reaction Using Compound-Specific Stable Isotope Analysis (CSIA)

In recent decades, concepts involving compound-specific stable isotope analysis have evolved allowing the assessment of organohalide biotransformation in situ as well as evaluating complex (bio)chemical reactions. The stable isotope composition can provide information on the source of a specific chemical and changes over time or space additionally allow to assess degradation pathways and reaction mechanisms involved during (bio)transformation. This chapter provides a basic introduction into stable isotope analysis, an overview of the application of compound-specific stable isotope analysis (CSIA ) for investigation of the microbial reductive dehalogenation reaction of mainly chlorinated ethenes and summarizes recent advances and results.