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

Enantioselective Carbon Stable Isotope Fractionation of Hexachlorocyclohexane during Aerobic Biodegradation by <i>Sphingobium</i> spp.

Carbon isotope fractionation was investigated for the biotransformation of γ- and α- hexachlorocyclohexane (HCH) as well as enantiomers of α-HCH using two aerobic bacterial strains: <i>Sphingobium indicum</i> strain B90A and <i>Sphingobium japonicum</i> strain UT26. Carbon isotope enrichment factors (ε_c) for γ-HCH (ε_c = −1.5 ± 0.1‰ and −1.7 ± 0.2‰) and α-HCH (ε_c = −1.0 ± 0.2‰ and −1.6 ± 0.3‰) were similar for both aerobic strains, but lower in comparison with previously reported values for anaerobic γ- and α-HCH degradation. Isotope fractionation of α-HCH enantiomers was higher for (+) α-HCH (ε_c = −2.4 ± 0.8 ‰ and −3.3 ± 0.8 ‰) in comparison to (−) α-HCH (ε_c = −0.7 ± 0.2‰ and −1.0 ± 0.6‰). The microbial fractionation between the α-HCH enantiomers was quantified by the Rayleigh equation and enantiomeric fractionation factors (ε_e) for <i>S. indicum</i> strain B90A and <i>S. japonicum</i> strain UT26 were −42 ± 16% and −22 ± 6%, respectively. The extent and range of isomer and enantiomeric carbon isotope fractionation of HCHs with <i>Sphingobium</i> spp. suggests that aerobic biodegradation of HCHs can be monitored in situ by compound-specific stable isotope analysis (CSIA) and enantiomer-specific isotope analysis (ESIA). In addition, enantiomeric fractionation has the potential as a complementary approach to CSIA and ESIA for assessing the biodegradation of α-HCH at contaminated field sites

Coupling of a Headspace Autosampler with a Programmed Temperature Vaporizer for Stable Carbon and Hydrogen Isotope Analysis of Volatile Organic Compounds at Microgram per Liter Concentrations

One major challenge for the environmental application of compound-specific stable isotope analysis (CSIA) is the necessity of efficient sample treatment methods, allowing isolation of a sufficient mass of organic contaminants needed for accurate measurement of the isotope ratios. Here, we present a novel preconcentration techniquethe coupling of a headspace (HS) autosampler with a programmed temperature vaporizer (PTV)for carbon (δ¹³C) and hydrogen (δ²H) isotope analysis of volatile organic compounds in water at concentrations of tens of micrograms per liter. The technique permits large-volume injection of headspace samples, maintaining the principle of simple static HS extraction. We developed the method for multielement isotope analysis (δ¹³C and δ²H) of methyl <i>tert</i>-butyl ether (MTBE), benzene, toluene, ethylbenzene, and <i>o</i>-xylene (BTEX), and analysis of δ¹³C for chlorinated benzenes and ethenes. Extraction and injection conditions were optimized for maximum sensitivity and minimum isotope effects. Injection of up to 5 mL of headspace sample from a 20 mL vial containing 13 mL of aqueous solution and 5 g of NaCl (10 min of incubation at 90 °C) resulted in accurate δ¹³C and δ²H values. The method detection limits (MDLs) for δ¹³C were from 2 to 60 μg/L (MTBE, BTEX, chlorinated ethenes, and benzenes) and 60–97 μg/L for δ²H (MTBE and BTEX). Overall, the HS–PTV technique is faster, simpler, isotope effect-free, and requires fewer treatment steps and less sample volume than other extraction techniques used for CSIA. The environmental applicability was proved by the analysis of groundwater samples containing BTEX and chlorinated contaminants at microgram per liter concentrations

Dual Carbon–Bromine Stable Isotope Analysis Allows Distinguishing Transformation Pathways of Ethylene Dibromide

The present study investigated dual carbon–bromine isotope fractionation of the common groundwater contaminant ethylene dibromide (EDB) during chemical and biological transformations, including aerobic and anaerobic biodegradation, alkaline hydrolysis, Fenton-like degradation, debromination by Zn(0) and reduced corrinoids. Significantly different correlation of carbon and bromine isotope fractionation (Λ_C/Br) was observed not only for the processes following different transformation pathways, but also for abiotic and biotic processes with, the presumed, same formal chemical degradation mechanism. The studied processes resulted in a wide range of Λ_C/Br values: Λ_C/Br = 30.1 was observed for hydrolysis of EDB in alkaline solution; Λ_C/Br between 4.2 and 5.3 were determined for dibromoelimination pathway with reduced corrinoids and Zn(0) particles; EDB biodegradation by <i>Ancylobacter aquaticus</i> and <i>Sulfurospirillum multivorans</i> resulted in Λ_C/Br = 10.7 and 2.4, respectively; Fenton-like degradation resulted in carbon isotope fractionation only, leading to Λ_C/Br ∞. Calculated carbon apparent kinetic isotope effects (¹³C-AKIE) fell with 1.005 to 1.035 within expected ranges according to the theoretical KIE, however, biotic transformations resulted in weaker carbon isotope effects than respective abiotic transformations. Relatively large bromine isotope effects with ⁸¹Br-AKIE of 1.0012–1.002 and 1.0021–1.004 were observed for nucleophilic substitution and dibromoelimination, respectively, and reveal so far underestimated strong bromine isotope effects

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 and Enantioselective Stable Isotope Analysis as Tools To Monitor Transformation of Hexachlorocyclohexane (HCH) in a Complex Aquifer System

Technical hexachlorocyclohexane (HCH) mixtures and Lindane (γ-HCH) have been produced in Bitterfeld-Wolfen, Germany, for about 30 years until 1982. In the vicinity of the former dump sites and production facilities, large plumes of HCHs persist within two aquifer systems. We studied the natural attenuation of HCH in these groundwater systems through a combination of enantiomeric and carbon isotope fractionation to characterize the degradation of α-HCH in the areas downstream of a former disposal and production site in Bitterfeld-Wolfen. The concentration and isotope composition of α-HCH from the Quaternary and Tertiary aquifers were analyzed. The carbon isotope compositions were compared to the source signal of waste deposits for the dumpsite and highly contaminated areas. The average value of δ¹³C at dumpsite was −29.7 ± 0.3 ‰ and −29.0 ± 0.1 ‰ for (−) and (+)­α-HCH, respectively, while those for the β-, γ-, δ-HCH isomers were −29.0 ± 0.3 ‰, −29.5 ± 0.4 ‰, and −28.2 ± 0.2 ‰, respectively. In the plume, the enantiomer fraction shifted up to 0.35, from 0.50 at source area to 0.15 (well T1), and was found accompanied by a carbon isotope enrichment of 5 ‰ and 2.9 ‰ for (−) and (+)­α-HCH, respectively. The established model for interpreting isotope and enantiomer fractionation patterns showed potential for analyzing the degradation process at a field site with a complex history with respect to contamination and fluctuating geochemical conditions

Carbon Stable Isotope Fractionation of Sulfamethoxazole during Biodegradation by <i>Microbacterium</i> sp. Strain BR1 and upon Direct Photolysis

Carbon isotope fractionation of sulfamethoxazole (SMX) during biodegradation by <i>Microbacterium</i> sp. strain BR1 (<i>ipso</i>-hydroxylation) and upon direct photolysis was investigated. Carbon isotope signatures (δ¹³C) of SMX were measured by LC-IRMS (liquid chromatography coupled to isotope ratio mass spectrometry). A new LC-IRMS method for the SMX metabolite, 3-amino-5-methylisoxazole (3A5MI), was established. Carbon isotope enrichment factors for SMX (ε_C) were −0.6 ± 0.1‰ for biodegradation and −2.0 ± 0.1‰ and −3.0 ± 0.2‰ for direct photolysis, at pH 7.4 and pH 5, respectively. The corresponding apparent kinetic isotope effects (AKIE) for <i>ipso</i>-hydroxylation were 1.006 ± 0.001; these fall in the same range as AKIE in previously studied hydroxylation reactions. The differences in SMX and 3A5MI fractionation upon biotic and abiotic degradation suggest that compound specific stable isotope analysis (CSIA) is a suitable method to distinguish SMX reaction pathways. In addition, the study revealed that the extent of isotope fractionation during SMX photolytic cleavage is pH-dependent