Characterization of carbon and hydrogen isotopic fractionation during anaerobic benzene biodegradation using enrichment cultures under sulfate-reducing, nitrate-reducing and methanogenic conditions, suggested that the initial steps in the benzene biodegradation pathway may determine the extent of isotopic fractionation.  Enrichment factors (e) calculated for carbon (between –1.9 to –3.6‰) and hydrogen isotopic fractionation (between -29 to –79‰) showed significant differences between cultures using the three terminal electron accepting processes (TEAP). These differences were not related to initial biomass levels or rates of biodegradation. 

Re-evaluation of the hydrogen enrichment factors in terms of kinetic isotope effects (KIE) resulted in values of ^Hk/^Dk=1.88, 1.56 and 1.24 for the sulfate-reducing, methanogenic and nitrate-reducing cultures respectively.   The KIE value for the nitrate-reducing culture is consistent with a Friedal-Crafts alkylation reaction, where benzene is methylated to toluene in the initial degradation steps.  The KIE value for the methanogenic culture is inconsistent with a hydroxyl radical reaction or an oxidation reaction with molecular oxygen, but may be consistent with an addition/elimination reaction, for example where benzene is converted to phenol.  Linear regressions of the carbon versus the hydrogen isotope ratios for each of the cultures with different TEAP’s revealed that the nitrate-reducing cultures produced fractionation with a distinct slope for the regression compared to the methanogenic and sulfate-reducing cultures that had similar slopes.  These results suggest that the methanogenic and sulfate-reducing cultures used a similar initial mechanism for benzene biodegradation, while the initial reaction in the nitrate-reducing cultures was different. Results from this study show that carbon and hydrogen isotope analysis can be used to provide evidence of benzene biodegradation in the field and to help elucidate biodegradation pathways and mechanisms.