Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors () for carbon (range of ؊1.9 to ؊3.6‰) and hydrogen (range of ؊29 to ؊79‰) fractionation were observed during biodegradation of benzene under nitratereducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.

Compound-specific carbon and hydrogen isotope analysis was used to investigate biodegradation of benzene and ethylbenzene in contaminated groundwater at Dow Benelux BV industrial site. delta13C values for dissolved benzene and ethylbenzene in downgradient samples were enriched by up to 2+/-0.5 per thousand, in 13C, compared to the delta13C value of the source area samples. delta2H values for dissolved benzene and ethylbenzene in downgradient samples exhibited larger isotopic enrichments of up to 27+/-5 per thousand for benzene and up to 50+/-5 per thousand for ethylbenzene relative to the source area. The observed carbon and hydrogen isotopic fractionation in downgradient samples provides evidence of biodegradation of both benzene and ethylbenzene within the study area at Dow Benelux BV. The estimated extents of biodegradation of benzene derived from carbon and hydrogen isotopic compositions for each sample are in agreement, supporting the conclusion that biodegradation is the primary control on the observed differences in carbon and hydrogen isotope values. Combined carbon and hydrogen isotope analyses provides the ability to compare biodegradation in the field based on two different parameters, and hence provides a stronger basis for assessment of biodegradation of petroleum hydrocarbon contaminants.

1,2-Dichloroethane (1,2-OCA) is a widespread groundwater contaminant known to be biodegradable under aerobic conditions via enzymatic oxidation or hydrolytic dehalogenation reactions. Current literature reports that stable carbon isotope fractionation of 1,2-DCA during aerobic biodegradation is large and reproducible (-27 to -33/1000). In this study, a significant variation in the magnitude of stable carbon isotope fractionation during aerobic biodegradation was observed. Biodegradation in experiments involving microcosms, enrichment cultures, and pure microbial cultures produced a consistent bimodal distribution of enrichment factors (epsilon) with one mean epsilon centered on -3.9 +/- 0.6/1000 and the other on -29.2 +/- 1.9/1000. Reevaluation of epsilon in terms of kinetic isotope effects 12k/13k gave values of 12k/13k = 1.01 and 1.06, which are typical of oxidation and hydrolytic dehalogenation (S(N)2) reactions, respectively. The bimodal distribution is therefore consistent with the microbial degradation of 1,2-DCA by two separate enzymatic pathways. This interpretation is further supported in this study by experiments with pure strains of Xanthobacter autotrophicus GJ10, Ancylobacter aquaticus AD20, and Pseudomonas sp. Strain DCA1 for which the enzymatic degradation pathways are well-known. A small fractionation of -3.0/1000 was measured for 1,2-DCA degradation by Pseudomonas sp. Strain DCA1 (monooxygenase enzyme), while degradation by the hydrolytic dehalogenase enzyme by the other two pure strains was characterized by fractionation of -32.3/1000.

The initial metabolic reactions for anaerobic benzene biodegradation remain uncharacterized. Isotopic data for carbon and hydrogen fractionation from nitrate-reducing, sulfate-reducing, and methanogenic benzene-degrading enrichment cultures and phylogenic information were used to investigate the initial reaction step in anaerobic benzene biodegradation. Dual parameter plots of carbon and hydrogen isotopic data (deltadelta2H/ deltadelta13C) from each culture were linear, suggesting a consistent reaction mechanism as degradation proceeded. Methanogenic and sulfate-reducing cultures showed consistently higher slopes (m = 29 +/- 2) compared to nitrate-reducing cultures (m = 13 +/- 2) providing evidence for different initial reaction mechanisms. Phylogenetic analyses confirmed that culture conditions were strictly anaerobic, precluding any involvement of molecular oxygen in the observed differences. Using published kinetic data, we explored the possibility of attributing such slopes to reaction mechanisms. The higher slopes found under methanogenic and sulfate-reducing conditions suggest against an alkylation mechanism for these cultures. Observed differences between the methanogenic and nitrate-reducing cultures may not represent distinct reactions of different bonds, but rather subtle differences in relative reaction kinetics. Additional mechanistic conclusions could not be made because kinetic isotope effect data for carboxylation and other putative mechanisms are not available.

The effects of iron concentration on carbon and hydrogen isotopic fractionation during aerobic biodegradation of toluene by Pseudomonas putida mt-2 were investigated using a low iron medium and two different high iron media. Mean carbon enrichment factors (epsilonc) determined using a Rayleigh isotopic model were smaller in culture grown under high iron conditions (epsilonc = -1.7+/-0.1%) compared to low iron conditions (epsilonc = -2.5+/-0.3%). Mean hydrogen enrichment factors (epsilonH) were also significantly smaller for culture grown under high iron conditions (epsilonH = -77 +/-4%) versus low iron conditions (EpsilonH = -159+/-11%). A mechanistic model for enzyme kinetics was used to relate differences in the magnitude of isotopic fractionation for low iron versus high iron cultures to the efficiency of the enzymatic transformation. The increase of carbon and hydrogen enrichment factors at low iron concentrations suggests a slower enzyme-catalyzed substrate conversion step (k2) relative to the enzyme-substrate binding step (k-l) at low iron concentration. While the observed differences were subtle and, hence, do not significantly impact the ability to use stable isotope analysis in the field, these results demonstrated that resolvable differences in carbon and hydrogen isotopic fractionation were related to low and high iron conditions. This novel result highlights the need to further investigate the effects of other trace elements known to be key components of biodegradative enzymes.