Date of Degree
Access restricted until 07/03/2019
PhD (Doctor of Philosophy)
Chemical and Biochemical Engineering
Peeples, Tonya L.
First Committee Member
Just, Craig L.
Second Committee Member
Salem, Aliasger K.
Third Committee Member
Horswill, Alexander R.
Fourth Committee Member
Murhammer, David W.
Bacterial strain Pseudomonas sp. ADP is capable of degrading atrazine via an enzymatic pathway in six sequential steps to yield carbon dioxide and ammonia. Atrazine is a persistent herbicide that frequently contaminates soil, drinking water, and ground water throughout areas of heavy use in the United States. A biological remediation approach using Pseudomonas sp. APD is considered as an effective, cost-efficient, and environmentally conscious method of decreasing atrazine concentration in areas of high contamination. Each enzyme in the degradation pathway is encoded by a corresponding gene, AtzA-AtzF, and is located on a self-transmissible 108-kb plasmid.
Due to their prevalence in nature, and their unique genetic and physical characteristics, biofilms are of great interest in the field of bioremediation. Biofilms exhibit high tolerance for harsh environmental stressors/conditions, prodigious potential for recalcitrant compound entrapment via an extracellular polymeric matrix, quorum sensing, and increased horizontal gene transfer compared to their planktonic counterparts. Despite frequent genetic and chemical analyses performed on atrazine-degrading genes on planktonic cells of strain Pseudomonas sp. APD, the genetics and degradation potential of Pseudomonas sp. ADP biofilms is relatively unexplored.
Real-time quantitative PCR was used to differentiate the expression of six genes involved in the process of atrazine degradation. Relative expression experiments revealed no statistically significant difference in the expression of atrazine-degrading genes in Pseudomonas sp. ADP cells grown as biofilms relative to Pseudomonas sp. ADP cells grown as planktonic cells. In biofilms alone, the expression of genes AtzDEF was differentiated via temperature of biofilm growth in cells grown at 25, 30, and 37 degrees.
Analytical techniques, including GC-MS and HPLC, were used to elucidate atrazine remediation potential of Pseudomonas sp. ADP biofilms and our previously collected genetic data. Stable decreases in atrazine degradation following a first-order kinetic model have been demonstrated for planktonic cells compared to a complex degradation pattern, including transient increases, observed for corresponding biofilm-mediated cells. This is attributed to the unique structure of the biofilm and the potential of atrazine to be entrapped in the substances of the extracellular polymeric matrix and subsequently released into the effluent. Overall, the biodegradation efficiency was higher for Pseudomonas sp. ADP biofilm-mediated cells compared to their planktonic counterparts.
A novel methodology of using confocal microscopy and in situ reverse transcription was proposed for optimization to visualize the expression of atrazine-degrading genes in fixed Pseudomonas sp. ADP biofilms. The sugar composition of Pseudomonas sp. ADP was evaluated using fluorescent lectin binding analysis and was determined to exhibit a prominent level of diversity and dependent upon growth medium. The results from these experiments will play a role in application of biofilms grown in bioreactors for atrazine remediation throughout areas of persistent and high contamination throughout the US. The new step in methodology development of an in situ visual gene expression technique can be extended to bioremediation of alternate recalcitrant compounds. The results may also be aid progress in alternate biofilm-related studies in medicine & human health, metallurgy, and engineering.
Biodegradation, Biofilms, Bioreactors, Bioremediation, Genetics, Kinetics
xxi, 181 pages
Includes bibliographical references (pages 173-181).
Copyright © 2018 Michael Asher Delcau
Delcau, Michael Asher. "Differentiation of Pseudomonas sp. strain ADP biofilms and planktonic cells using methods in gene expression analysis." PhD (Doctor of Philosophy) thesis, University of Iowa, 2018.