The role of microorganisms in the degradation of xenobiotics in the environment is well established. Bacteria from the genus Pseudomonas are particularly well adapted to the degradation of hydrocarbons, aromatics, and numerous other natural and introduced substrates. We have isolated a strain of Pseudomonas putida, designated PC2P15, that uses toluene, phenol, benzene, and a number of other substrates as its sole sources of carbon and energy.

Utilization of toluene by Pseudomonas putida as its sole carbon and energy source affects morphology, outer membrane protein composition, and glycolipid production. Two strains of P. putida were found to utilize toluene and to coexist in continuous and batch culture. The two strains were designated translucent and opaque, based upon their readily identifiable coloration when grown on Luria agar. The translucent strain was the dominant strain in continuous culture conditions. The outer membrane proteins of P. putida were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. When toluene is the carbon and energy source, the trend in protein composition was towards a general increase in concentration of lower molecular weight proteins (wt). A similar decrease occurred in the concentration of higher molecular weight proteins in the range of 70X104-9X104 mol wt. P. putida produces glycolipids when grown on toluene as a sole carbon and energy source. Three glycolipids have been isolated from chemostat and batch culture spent media, using thin layer chromatography on silica gel GF254· The glycolipids are believed to be previously reported mono- and di-rhamnolipids that function as biosurfactants. The release of glycolipid into the media is believed to function to emulsify toluene, aiding in toluene uptake.

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

Groundwater contamination by man-made chemicals is increasingly being reported in the United States. The potential for detrimental health effects is substantial and has been addressed by the environmental engineering profession. Typically, contaminated groundwater is pumped to the surface and treated in a variety of methods including air stripping, carbon adsorption, and biodegradation. In situ biodegradation is increasingly being considered as an alternative to pump-and-treat technology. The primary goal of this research was to determine the fate of an organic chemical adsorbed to a subsurface soil when exposed to acclimated bacteria. Toluene was chosen as a representative compound because it is a major constituent of groundwater contaminated by gasoline. In addition, toluene is known to be both biodegradable and adsorbable. Sybron Biochemical, Inc. supplied the aerobic bacteria Psgudomonas gutjga known to readily transform toluene. Soil microcosms were established in test-tubes and conditions simulated those of a saturated, aerobic aquifer. Gas chromatography was used to quantify changes in toluene concentration due to adsorption and biodegradation. The addition of an aqueous toluene solution to sterile microcosms resulted in the rapid and extensive adsorption of toluene to the soil. Subsequent analysis revealed the slow adsorption of an additional small fraction of toluene. Biodegradation studies entailed the addition of acclimated bacteria to sterile soil microcosms in which substantial toluene adsorption had occurred. Addition of small doses of hydrogen peroxide effectively maintained aerobic conditions for biodegradation. As a result, E, putjda was able to transform all measurable toluene in the microcosms. Additional desorption studies revealed that a "resistant" component of toluene remained adsorbed to the soil during biodegradation. This component was neither acted upon by bacteria nor readily extractable by methylene chloride. However, slow desorption of toluene was shown to occur at a rate comparable to slow adsorption. To achieve complete removal, groundwater treatment methods must address the rate-controlled desorption of the resistant toluene component. / Master of Science