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 <i>Sulfurospirillum multivorans</i>, <i>Geobacter lovleyi</i>, <i>Desulfuromonas michiganensis</i>, <i>Desulfitobacterium hafniense</i> strain PCE-S, and <i>Dehalobacter restrictus</i>. 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 (<i>S. multivorans</i>-K_d‑PCE = 0.42–0.51 L g^–1; <i>D. hafniense</i>-K_d‑PCE = 0.13 L g^–1), as well as in envelope hydrophobicity (<i>S. multivorans</i> 33.0° to 72.2°; <i>D. hafniense</i> 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‰ (CI_95% < ± 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

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

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 δ²H range of −181 mUr to 629 mUr

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