DOI

10.17077/etd.he3zt8hy

Document Type

Dissertation

Date of Degree

Fall 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Chemical and Biochemical Engineering

First Advisor

Peeples, Tonya L

Second Advisor

Jessop, Julie L

First Committee Member

Peeples, Tonya L

Second Committee Member

Jessop, Julie L P

Third Committee Member

Murhammer, David W

Fourth Committee Member

Haes, Amanda J

Fifth Committee Member

Horswill, Alexander R

Abstract

Microbial biofilms are significant in a variety of settings including the human microbiome, infectious disease, industrial processes, and environmental remediation. Due to the ubiquitous nature of biofilms, there is a great interest in understanding cellular activities within the biofilm matrix. Biofilm cells are able to better withstand environmental stress, experience increased horizontal gene transfer, and live longer. The purpose of this research is to grow Pseudomonas sp. strain ADP as a biofilm and examine the chemical and physical characteristics the microbe undertakes in a sticky extracellular matrix.

ADP is the organism of choice because of its ability to metabolize atrazine. Cells are grown in a drip biofilm reactor and flow cells under varying time lapse to gain insight to biofilm formation. Some cells are grown with atrazine as the sole nitrogen source, while others are grown in a nutrient-rich medium to compare cells response under nutrient-limited conditions with atrazine particles in the matrix. As a positive control, Escherichia coli are grown in a similar manner.

Raman spectroscopy was the main analytical technique used to evaluate the chemical and molecular characteristics of this system. Scanning electron microscopy is used to examine cellular distribution, and several assays are performed for molecular composition analysis. Raman analysis in the fingerprint region revealed distinct differences between free cells and cells in biofilm. Soluble extracellular polymeric substances (EPS) were found to be more prevalent than tightly bound EPS and lightly bound EPS in the biofilm matrix. Comparison of relative peak intensity ratios suggests that it is possible to track atrazine degradation by means of intermediates using

Raman spectroscopy. SEM micrographs revealed EPS role as an immobilizing agent when in contact with compounds, such as atrazine.

Further research is needed to determine if atrazine can bind to EPS fractions outside the presence of cells and whether its affinity to EPS is mostly attributed to physical conditions, due to the architecture of biofilm, or chemical, based on functional groups presents.

The results obtained from this research will contribute to the development of a less invasive microscale approach to address the acquisition and induction of biotransformation activity occurring in xenobiotic degrading systems. The extracellular interactions observed can be used to further characterize biofilm-mediated bioremediation. Results have contributed to the Raman spectra library for microorganisms and organic compounds.

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Keywords

Atrazine, Biofilm, Bioremediation, Extracellular polymeric substances, Raman spectroscopy, SEM

Pages

xvii, 137 pages

Bibliography

Includes bibliographical references (pages 130-137).

Copyright

Copyright © 2015 Victoria Azula Henry

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