A number of bacterial species are capable of degrading the widespread environmental pollutant trichloroethylene (TCE) via aerobic cometabolism, but cytotoxic effects that can debilitate the microorganism often accompany this transformation. In this dissertation the effects of TCE degradation on the well-studied, toluene-oxidizing bacterium Burkholderia cepacia G4 were investigated at the physiological and genetic level and compared and contrasted to the effects elicited by several nonhalogenated, short chain alkenes and alkynes. Linear alkynes (C���-C������) were classified as strong mechanism-based inactivators of toluene 2-monooxygenase activity in B. cepacia G4, with 2- and 3-alkynes providing a more potent effect than their 1-alkyne counterparts. The C��� alkyne, acetylene, was weak inactivator of toluene 2-monooxygenase activity presumably because it does not bind efficiently to this oxygenase. Toluene-grown cells of B. cepacia G4 cells oxidized ethylene and propylene to their respective epoxides with no observable effect on cell culturability or general respiratory activity. In contrast, TCE oxidation was accompanied by a myriad of cytotoxic effects. Accumulation of general cellular damage, manifested as a loss of cell culturability and general respiratory activity, outpaced loss of toluene 2-monooxygenase activity during TCE oxidation. Measures of the culturability of TCE-injured cells varied up to 3 orders of magnitude (depending on the method of assessment), and it was found that TCE-injured cells were ultra sensitive to H���O��� on the surface of agar plates. It was proposed that a toxicity threshold exists for B. cepacia G4 during TCE oxidation, and once cells have degraded ���0.5 ��mol of TCE (mg of cells�����) the likelihood of recovery decreases significantly. Tn5 mutants of B. cepacia G4 with disruptions in genes putatively encoding enzymes involved in DNA repair (including UvrB, RuvB, RecA, and RecG) were ultra susceptible to killing by TCE, as well as the known DNA damaging agents, UV light, mitomycin C, and H���O���. Physiological and genetic analysis of the mutants provided suggestive evidence that nucleotide excision repair and recombinational repair activities are linked to the survivability of TCE-injured B. cepacia G4. / Graduation date: 2002

Trichloroethene (TCE) is the most frequently detected organic contaminant in groundwater, is classified as a probable human carcinogen, and exhibits toxicological effects on the human endocrine, immune, developmental, and reproductive systems. While significant research efforts have been devoted to the development of strategies for remediating TCE-contaminated groundwater, their advancement is currently hindered by limitations in current methodologies for measuring in situ reductive dechlorination rates, especially for sorbing solutes. This dissertation describes the development, evaluation, and demonstration of a method for measuring in situ reductive dechlorination rates that utilizes single-well, "push-pull" test technology. Initial field tests indicated that trichlorofluoroethene (TCFE) could be used as a surrogate for TCE in push-pull tests since (a) TCE and TCFE were transported similarly and (b) TCFE underwent reductive dechlorination by a pathway analogous to that of TCE while retaining the fluorine label. Because TCFE and TCE experienced sorption at the selected field site, a novel data analysis technique called "forced mass balance" (FMB) was developed to obtain in situ transformation rates of sorbing solutes from push-pull test data. The FMB technique was evaluated by quantifying errors in rates derived by applying FMB to push-pull test data generated by a numerical model. Results from simulated tests indicated that an example in situ rate for the reductive dechlorination of TCFE, which was obtained by applying FMB to field data, was underestimated relative to the true in situ rate by 10%. The utility of the rate-determination method presented in this dissertation was demonstrated by using it to evaluate the effectiveness of a chemical amendment, namely fumarate, at enhancing in situ reductive dechlorination rates in TCE-contaminated groundwater. Reductive dechlorination rates increased following three consecutive additions of fumarate in all five of the tested wells. The development of the rate-determination method described in this dissertation advances the state of bioremediation technology because methods for measuring in situ transformation rates are needed to both assess the potential for natural attenuation and to quantify the effects of bioremediation techniques in the field. / Graduation date: 2003

Laboratory and modeling studies were performed with a mixed-anaerobic-culture obtained from the Evanite site in Corvallis, Oregon. The culture completely transforms trichloroethene (TCE) to cis-dichloroethene (c-DCE), vinyl chloride (VC), and finally to ethene. Acetylene inhibition studies were used to examine the culture's microbial activities. Kinetic studies determined the half-saturated constant (K[subscript s]), the maximum utilization rate (k[subscript max]X), and inhibition constants (K[subscript I]). The kinetic constants were used to model the results of inhibition studies using competitive and uncompetitive inhibition models. Acetylene was found to function as a reversible inhibitor and was used to probe the activities of reductive dechlorination. Various acetylene concentrations were used to differentiate microbial processes, including methanogenesis, acetogenesis, and halorespiration. Acetylene concentrations of 48, 192, and 12 ��M, respectively, were required to achieve 90% inhibition in the rates of methanogenesis, TCE and VC transformation. H���-dependent acetate production was not inhibited by acetylene. K[subscript s] values for TCE and VC were 12 ��M and 63 ��M, respectively. Model fitting of acetylene inhibition constants (K[subscript IC]) for TCE and VC transformations yielded the same value (0.4 ��M) for a competitive inhibition model. However, for uncompetitive inhibition the estimated K[subscript IU] for TCE to c-DCE, TCE to 1,1-DCE and VC to ethene were 13.3, 14.1 and 2.2 ��M, respectively. Competitive and uncompetitive inhibition models simulated experimental data equally well for results obtained at high TCE and VC concentrations. The models were further verified to fit transient data of acetylene inhibition at lower TCE and VC concentrations, and competitive inhibition resulted in a better fit to the experimental data. 1-chloro-1-fluoroethene (1,1-CFE) was found to track the rate of VC transformation well, since VC and 1,1-CFE had similar maximum transformation rates and K[subscript s] values. A competitive inhibition model with the measured K[subscript s] values, 63 and 87 ��M. was used to predict the rates of VC and 1,1-CFE transformation, respectively. The similar rates and results of acetylene and compound inhibition studies indicated VC and 1,1-CFE were transformed by the same enzyme. 1,1-CFE transformation by three different cultures, clearly demonstrate that 1,1-CFE was an excellent surrogate to track rates of VC transformation. / Graduation date: 2004