Cometabolic oxidation of TCE (trichloroethylene) by Pseudomonas putida F1 in biofiltration

Author

Hae-Jin Woo

Date of Award

5-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Civil Engineering

Major Professor

Chris D. Cox

Committee Members

Mriganka Ghosh, Gary Sayler, Kevin Robinson

Abstract

Biofiltration is a technology that utilizes microorganisms supported on porous media such as compost, peat, or soil to remove gas-phase organic and inorganic compounds. The biodegradation of many chlorinated compounds requires a primary substrate to provide an energy and carbon source and to induce the necessary enzymes responsible for metabolism of the target compounds. In this research, a toluene degrading bacteria, Pseudomonas putida F1, was used. Toluene induces the formation of the toluene dioxygenase enzyme (Tod) system, a non-specific enzyme capable of oxidizes TCE to formic and glyoxylic acid.Batch and column experiments were performed to understand the biokinetics of TCE cometabolic oxidation with respect to competitive inhibition, NADH limitation,enzyme activity, and toxicity. A fluorescence -based assay was developed and used to monitor Tod activity during TCE degradation. Suspended-growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with pure indigo production (R²=0.89) as measured spectrophotometrically. In batch experiments, the normalized enzyme activity decreased without toluene, but remained relatively constant when toluene was present.For the suspended cells, the maximum specific toluene utilization rate and half-saturation constant were 2.674 µg toluene (min mg biomass)^-1 and 3 mg L^{-1, respectively.The degradation rate of toluene by suspended cells was not significantly affected by TCE.At toluene concentrations greater than 2.15 mg L-1, significant inhibition of TCEdegradation was observed during the first 20 to 35 minutes of the experiment; however,once the toluene decreased below 2.15 mg L-1, TCE degradation was rapid.A differential volume reactor was constructed to measure the growth rate ofimmobilized cells and the rate of toluene utilization. The maximum specific growth rate(µ_max) of immobilized cells was estimated to be 0.029 hr-1 Growth was stronglydependent upon toluene concentration up to 4000 lig L-1 . Growth did not increase significantly above 5000 µ/L because the maximum specific growth rate was achieved.The maximum specific toluene utilization rate (k_T) and half saturation constant (K_sT)were found to be 1.250 µg toluene (min mg biomass)-1 and 1988 µg toluene L-1,respectively.Normalized NADH was relatively unchanged when toluene and TCE were fed to the biofilter simultaneously. A steady-state NADH concentration of about 0.4 µmol (mg protein)-1 was maintained whenever toluene was fed to the column. NADHconcentrations in the biofilm were observed during TCE degradation with no toluene present. While the rate of TCE removal and the normalized enzyme activity decreased during each experiment, the average concentration of NADH remained above 0.477 µmol(mg protein)-1.The rate of enzyme inactivation in both suspended and immobilized cells appears to be correlated with the rate of TCE degradation, suggesting that enzyme inactivation is related to intermediates produced during TCE degradation. A dynamic biofiltration model was developed to predict the concentration profile and effluent history for the case when TCE alone is feed to the biofilter. The model demonstrated that the principal factor resulting in the decrease in TCE removal with time is the inactivation of Tod enzyme byTCE metabolic intermediates.The values of TCE reaction rate constant, k_s' = 1428.6 µg TCE (mg indigo L)-1,TCE half-velocity constant, K_s = 2620 µg TCE L-1 and inactivation constant of enzyme activity, k₂ = 0.0004 mg indigo (µ TCE L)-1 were determined and used for prediction the changes in effluent TCE concentration and enzyme activity during TCE degradation via biofiltration.In most cases, the model gives a reasonable prediction of TCE degradation in the biofilter. The model effectively predicted the diminishing rate of TCE degradation because of inactivation caused by TCE intermediates. Sensitivity testing of the model showed that TCE degradation increased with decreasing K_s and that the rate of increase in the TCE effluent concentration with time increased with decreasing K_s. The model became less sensitive to K_s as the value of this parameter increased. TCE degradation was not sensitive to k2, but enzyme activity was relatively sensitive to this parameter. The Slope of the plot of effluent concentration as a function of time increased with TCE feed concentration. The normalized enzyme activity decreased rapidly for higher TCE feed concentrations.}

Recommended Citation

Woo, Hae-Jin, "Cometabolic oxidation of TCE (trichloroethylene) by Pseudomonas putida F1 in biofiltration. " PhD diss., University of Tennessee, 1999.
https://trace.tennessee.edu/utk_graddiss/8947