| In this thesis, mechanisms of carbon and hydrogen isotopic fractionation were investigated during biodegradation of petroleum hydrocarbons, benzene and toluene, in order to evaluate the potential to use Compound Specific Isotope Analysis (CSIA) to assess in situ biodegradation under various field conditions. Variation in carbon and hydrogen enrichment factors, determined using a Rayleigh model, was observed for benzene degradation under different terminal electron accepting processes in enrichment cultures derived from three field sites. This variation was controlled by different biodegradation pathways for the nitrate-reducing cultures, and the methanogenic and sulfate-reducing cultures, as determined by a two-element isotopic approach comparing delta 13C and delta2H values between degradation experiments. Significant differences in carbon and hydrogen enrichment factors were also observed in Pseudomonas putida mt-2 cultures grown under low and high iron concentrations during aerobic toluene biodegradation. These differences were related to changes in the rate-limiting steps of the enzyme-controlled reaction whereby a slower enzyme-catalyzed substrate conversion step (k 2) relative to the enzyme-substrate binding step (k-1) was suggested to occur. No significant differences in carbon and hydrogen isotopic fractionation were observed between a methanogenic enrichment culture and cell free extract experiments during toluene degradation. These results indicated that substrate transport across the cell membrane of the microorganisms involved in biodegradation did not influence isotopic fractionation in this culture.;Overall, the results of this thesis demonstrated that resolvable differences in carbon and hydrogen isotopic fractionation were not random but occurred for different microbial communities, biodegradation pathways and growth conditions. Significant 13C and 2H enrichment occurred in all experiments carried out in this thesis, indicating that carbon and hydrogen CSIA has the potential to identify in situ biodegradation of benzene and toluene. In addition, the variability in carbon and hydrogen enrichment factors did not significantly impact the ability to use stable isotope analysis to quantify in situ biodegradation. The measurement of both carbon and hydrogen isotope analysis in laboratory experiments and a field study demonstrated that a two-element isotopic approach is the best approach to differentiate between in situ biodegradation and sources of groundwater contamination, as well as potentially delineate between biodegradation pathways. |
