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Physiological consequences of trichloroethylene degradation by the toluene-oxidizing bacterium Burkholderia cepacia G4Yeager, Chris M. 24 July 2001 (has links)
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
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Measuring in situ reductive dechlorination rates in trichloroethene-contaminated groundwaterHageman, Kimberly J. 14 April 2003 (has links)
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
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Inhibition, kinetic and modeling studies of acetylene and 1-chloro-1-fluoroethene on reductive dechlorination of TCE and vinyl chloridePon, George 17 December 2003 (has links)
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
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