Doctoral Dissertations

Date of Award

5-2006

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemical Engineering

Major Professor

Paul R. Bienkowski

Committee Members

Robert M. Counce, Paul D. Frymier, Gary S. Sayler

Abstract

The process of sensing an attractant chemical by a motile bacterium and subsequent motion towards that attractant, known as chemotaxis, occurs in Escherichia coli, Pseudomonas putida G7, Rhizobium meliloti, and several other subsurface strains. To date, there have been no widely accepted experimental studies that demonstrate whether bacterial chemotaxis can enhance biodegradation of contaminants in the subsurface. This research investigates the effect of bacterial chemotaxis on degradation rate in an experimental model for in situ bioremediation. The novel experimental protocols of this work, developed to investigate bacterial chemotaxis and migration, have provided for the systematic evaluation of the effect of the chemotaxis phenomena in a porous medium. The hypothesis formulated is that a bacterium undergoing chemotaxis in a porous medium will be able to sense an attractant chemical, bias its motion towards it, and subsequently degrade the attractant at a higher rate than a strain exhibiting non-chemotactic behavior.

The experimental model has been developed to measure the degradation rate of serine, a simulated contaminant and chemoattractant. E. coli RP437 was used as a representative chemotactic in situ bacteria while E. coli RP5700, a tsr-mutant strain of RP437 that lacks the serine chemoreceptor, was used as the control strain. RP5700 exhibits random motility similar to RP437, regardless of serine gradients. These two strains were highly characterized for this work, a process which was rigorous and performed in more detail than in prior works. Chemotactic ability of RP437 toward serine was validated via capillary and swarm plate assays. Swimming speeds, run lengths, and turn angles were compared using a tracking microscope and were statistically similar. Serine uptake rates in liquid media were also statistically similar. These results show that these strains are suitable for investigating any enhancing effect of chemotaxis on biodegradation rate.

For in situ bioremediation experiments, a model aquifer has been designed to introduce RP437 and RP5700 bacteria to serine in saturated sand via a sharp gradient. The aquifer was used to compare serine degradation rates and migration rates through sand. Results showed that the degradation rate of serine was statistically similar for both strains over a 21 hour period, indicating that enhancement was not detected. The experimental parameters chosen for this study did not elucidate degradation or migration enhancements due to chemotaxis. However, the experimental methodologies developed to acquire these results represent novel contributions to the field of chemotaxis analysis in porous media. These methodologies can easily be extended for the variation of other sets of parameters, such as particle size, cell densities, growth conditions, and selection of chemoattractants.

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