Date of Award

5-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School

College of Science and Mathematics

Department/Program

Earth and Environmental Studies

Thesis Sponsor/Dissertation Chair/Project Chair

Dibyendu Sarkar

Committee Member

Rupali Datta

Committee Member

Huan Feng

Committee Member

Edward Landa

Committee Member

Mark Chappell

Abstract

Urban sprawl in big cities often encroaches on military land, where residual toxic explosive compounds like 2, 4, 6-trinitrotoluene (TNT) in soil could pose a serious health risk. Additionally, in demilitarized areas, lack of sustainable remediation techniques hinder the much needed residential development.. Phytoremediation is an environmentally safe and cost effective solution; however, the characterized low aqueous solubility of nitroaromatic compounds resulting in limited availability of TNT for plant uptake is a major constraint. To overcome this limitation, we propose a new innovative phytoremediation technique using urea, a common nitrogen fertilizer, as a chaotropic agent, to enhance the solubility of TNT in the soil solutions and thus enhancing the TNT uptake by plant. A multi-process approach was used which included (1) sorption studies to understand the retention/release of TNT in soil solutions in the presence and absence of urea, followed by (2) a greenhouse study to fully characterize the urea catalyzed uptake of TNT using vetiver grass from TNT contaminated soils. This study also aimed to investigate the enzyme-mediated plant detoxification activities and changes in the plant-proteomic profile, to provide important clues to the mechanism of stress response and the TNT-tolerance in vetiver grass. Results showed that the extent of TNT sorption and chaotropic effectiveness of urea varies with the soil properties, predominately with the soil organic matter. Urea significantly (p<0.0001) catalyzed TNT extraction from all soils, suggesting that it mobilizes soil-TNT by increasing its solubility at the solid/liquid interface. Vetiver grass showed high uptake (73%) and significant root-to-shoot translocation (38%) of TNT. Urea significantly enhanced (p<0.0001) the vetiver-TNT uptake and translocation. Within the limits of agronomic fertilizer N application rates, 125 mg kg-1 of urea was considered optimum for TNT uptake by vetiver grass (82%). However, increasing the urea rate to 1000 mg kg-1 further increased the TNT removal (91%). Three metabolites of TNT, such as 2-ADNT, 4-ADNT and 1,3,5-TNB were detected in the plant tissues. The enhanced nitroreductase (NR) enzyme activity in TNT treated vetiver grass suggests the role of NR mediated biochemical mechanism in transforming TNT. The optimum kinetic parameters of the NR enzymes were determined. To the best of our knowledge, this study is the first attempt to investigate the proteomic profiling of a plant under TNT stress. Root proteins showed a significant (p<0.0001) negative correlation (r=-0.97) with TNT. Proteomics technique with integrated bioinformatics approach revealed downregulation of growth-related proteins and key functional proteins involved in important cellular mechanisms like transcription, translation, ribosome biogenesis, nucleocytoplasmic transport, and protein glycosylation. Plant defense related proteins were upregulated at lower TNT treatments suggesting vetiver’s innate defense mechanism against TNT stress. The highly encouraging results of the current study showed the potential of using chaotropically enhanced phytoremediation of TNT contaminated soils using vetiver grass.

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