The rate of removal of benzene, toluene, ethylbenzene and the xylene isomers (BTEX) from contaminated groundwater is needed to design remediation processes. Benzylsuccinic acid (BSA) and methyl-benzylsuccinic acid (methyl BSA) are unambiguous metabolites of anaerobic BTEX biodegradation. An analytical method for quantitative determination of BSA in groundwater samples was developed. Samples containing BSA and methyl BSA were extracted onto 0.5 g of styrene-divinylbenzene, eluted with ethyl acetate, and methylated with diazomethane. Gas chromatography coupled to mass spectrometry with electron impact ionization was used for separation and detection. The recovery from spiked 1 L groundwater samples was 88 to 100 %. The precision of the method, indicated by the relative standard error was ± 4% with a method detection limit of 0.2 μg/L. The method was then used to analyze samples from single-well push-pull tests conducted by injecting deuterated toluene and xylene into BTEX-contaminated wells in order to demonstrate in-situ biodegradation. Unambiguous evidence for deuterated toluene and xylene biodegradation was obtained with the observation of deuterated BSA and methyl BSA coupled with the utilization of nitrate presumably due to denitrification as terminal-electron-accepting process. Minimum first-order degradation rates for deuterated toluene estimated from formation of BSA were 0.0004 to 0.001 day⁻¹. Rates of methyl BSA formation were not calculated because methyl BSA, although detected, was not above the quantitation limit. Removal rates of deuterated toluene and o-xylene were not directly measurable because the rates were too low to measure significant changes in parent compound concentrations. Wells for which the formation of deuterated BSA and methyl BSA were observed had lower relative concentrations of toluene and xylenes relative to total BTEX than wells for which no deuterated BSA and methyl BSA were observed. Retardation factors for injected deuterated toluene and background toluene of 2 and 14, respectively, were obtained from push-pull tests conducted to determine toluene transport properties. Differences in retardation factors for injected and background toluene indicate differences between injected and background solute transport and is a topic that requires further study. / Graduation date: 2002

The increased use of alcohols as gasoline additives, and possible substitutes, has prompted the investigation of the fate of gasoline/alcohol mixtures in the environment. In situ bioremediation is one technique that can successfully be applied to remove ground water contaminants particularly in situations where the adsorptive capacity of the soil plays a major role. Frequently, enhanced in situ bioremediation techniques rely on indigenous microorganisms to degrade ground water contaminants; this technique may sometimes include the addition of acclimated bacteria. In this study, soil microcosms were constructed in order to simulate the conditions found in a saturated aerobic aquifer. The biodegradation potential of methanol, benzene, and m-xylene was investigated. Uncontaminated soil from the surface, 12, 16.5, and 18 foot depths was utilized to observe the differences in microbial responses throughout the soil profile. The biodegradation potential of the indigenous microbiota was determined and compared to that of benzene acclimated bacteria, for all the compounds in the mixture. To observe the impact that chemical and physical soil characteristics may have on microbial responses, soils from each depth were classified on the basis of their particle size, moisture content and pH. Substantial methanol, benzene, and m-xylene biodegradation by the indigenous microorganisms occurred in all subsurface soils. While methanol was readily biodegradable over concentrations ranging from about 80 mg/L to about 200 mg/L, benzene inhibited methanol biodegradation at about 125 mg/L in all soil depths. The addition of benzene acclimated bacteria considerably increased the biodegradation rates of all compounds in the mixture. Such increases in biodegradation rates may be attributed to the activities of both groups, the indigenous microorganisms and the benzene acclimated bacteria. The results obtained by this study suggest that biodegradation of methanol, benzene, and m-xylene can readily occur in a saturated aerobic subsurface environment. The physical and chemical properties of a ground water aquifer seem to have a marked effect on microbial responses, and consequently on the biodegradation potential of water contaminants. / Master of Science