Theses and Dissertations at Montana State University (MSU)
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Item Chromatographic, spectroscopic and microscopic analyses reveal the impact of iron oxides and electron shuttles on the degradation pathway of 2,4,6- trinitrotoluene (TNT) by a fermenting bacterium(Montana State University - Bozeman, College of Agriculture, 2003) Borch, Thomas; Chairperson, Graduate Committee: William P. Inskeep and Robin Gerlach (co-chair)Contamination of surface and subsurface environments with explosives such as 2,4,6-trinitrotoluene (TNT) is a worldwide problem. The fate and analysis of TNT were investigated in numerous artificially contaminated model systems. We developed a unique high performance liquid chromatography gradient elution method for the analysis of commonly observed TNT metabolites and EPA explosives. Column temperature was identified as the key parameter for optimal separation. Iron (hydr)oxides play an important role in the reduction, sorption and fate of TNT in soil and sediment. Consequently, characterization of the nature and properties of natural and synthetic Fe (hydr)oxides is important for determining reaction mechanisms and surface-associated chemical processes. This work thus summarizes the potential applicability of imaging and spectroscopic techniques for eliciting chemical and physical properties of iron (hydr)oxides. TNT is persistent in soils due to its low redox potential and sorption. Batch and column studies revealed some of the first results on TNT desorption behavior in two well-defined model soil systems. Biosurfactants were found to be the most promising technique for enhanced TNT desorption. Batch studies with a Cellulomonas sp. in the presence of ferrihydrite and the electron shuttle anthraquinone-2,6-disulfonate (AQDS) were conducted to reveal biotic and abiotic mechanisms contributing to the degradation of TNT. Strain ES6 was found to reduce TNT and ferrihydrite with enhanced reduction in the presence of AQDS. Ferrihydrite stimulated the formation of more reduced TNT metabolites such as 2,4-diamino-6-nitrotoluene. Interestingly, a completely different degradation pathway was observed in AQDS-amended iron-free cell suspensions, showing a rapid transformation of TNT to 2,4-dihydroxylamino-6-nitrotoluene, which transformed into unidentified polar products. The influence of iron phases (i.e. hematite, magnetite, and ferrihydrite) and secondary Fe mineral formation on the degradation of TNT was also evaluated. The initial reduction of TNT was fastest in the presence of hematite; however, the further reduction of hydroxylamino-dinitrotoluenes was fastest, in the presence of magnetite and ferrihydrite (no AQDS). The impact of AQDS was predominant in the presence of hematite resulting in the formation of 2,4,6-triaminotoluene. Ferrihydrite underwent reductive dissolution with the formation of secondary hematite. The enhanced TNT reduction in ferfihydrite-amended systems was therefore most likely due to redox-active Fe(II) rather than secondary Fe phases.Item Alkaline hydrolysis of explosives(Montana State University - Bozeman, College of Engineering, 2010) VanEngelen, Catherine Elizabeth; Chairperson, Graduate Committee: Brent M. PeytonIn the United States, ammunitions testing and manufacturing facilities must transform unused explosives into non-hazardous materials for disposal. 2,4,6- trinitrotoluene (TNT) is an explosive that has been found as a soil and groundwater contaminant at numerous ammunitions testing sites. Unused quantities of nitrocellulose (NC), another explosive, have also been accumulating at ammunitions manufacturing facilities. Transformation of both TNT and NC to non-explosive compounds has been studied using either chemical or biological approaches, each with limited success. With respect to TNT, the use of alkaline hydrolysis (degradation at high pH) as a chemical treatment had been tested at room temperature (20°C) under conditions where the hydroxide concentration exceeded that of TNT (pH > 10). These high hydroxide conditions were not directly amenable to biological treatment of the hydrolysis products. This study found that alkaline hydrolysis was effective for complete degradation of TNT at elevated temperatures (60°C and 80°C) when the concentration of TNT was less than the hydroxide concentration (pH 9 and 10). The resulting solution, or hydrolysate, contained no TNT. This hydrolysate was used as the carbon and nitrogen source for an aerobic bacterial enrichment from the Bozeman wastewater treatment plant. With respect to NC, the back-log of accumulated NC necessitates a degradation method that will process high NC concentrations (200g/L). Alkaline hydrolysis at 60°C was used with very high hydroxide concentrations to rapidly degrade high concentrations of NC, producing high nitrate and nitrite concentrations. The NC hydrolysate was neutralized and spiked into a denitrifying culture which was able to reduce both nitrate and nitrite. The goal of this work was to develop a dual component chemical-biological system for complete degradation of the explosives TNT and NC, which was achieved using alkaline hydrolysis as the chemical component and bacterial wastewater treatment enrichments as the biological component.Item Reductive transformation of 2,4,6-Trinitrotoluene by Yarrowia lipolytica AN-L15 under conditions of different initial pH of the culture medium or in the presence of ferrihydrite(Montana State University - Bozeman, College of Letters & Science, 2009) Pannier, Andy Joseph; Chairperson, Graduate Committee: Robin GerlachBatch and column studies were conducted to examine the difference in the transformation pathways of 2,4,6-trinitrotoluene (TNT) reduction by a hemiascomycetous yeast (Yarrowia lipolytica AN-L15) under conditions of different initial pH of the culture medium or in the presence or absence of ferrihydrite. Using high performance liquid chromatography (HPLC), it was observed that Y. lipolytica AN-L15 was able to transform TNT at three different initial proton concentrations of the culture medium: pH 7.0, pH 6.5, and pH 4.5. In the presence of TNT, Y. lipolytica AN-L15 showed preferential growth (OD₆₀₀) at the lower initial pH of 4.5. The increased growth (OD₆₀₀) resulted in increased reduction of TNT-metabolites in the culture medium with an initial pH of 4.5, as compared to, the culture medium with an initial pH of 6.5 or the culture medium with an initial pH of 7.0. TNT transformation via aromatic ring reduction was the major transformation pathway observed, with the major metabolite being 3-H -̄TNT. 4-hydroxylaminodinitrotoluene (4-HADNT) was the major metabolite of the nitro-group reduction pathway. In the presence of ferrihydrite at a pH of 7.0, the transformation of TNT by Y. lipolytica AN-L15 showed a change in the transformation pathway. Nitro-group reduction was the major pathway of TNT transformation in the presence of ferrihydrite with 4-HADNT and 2-aminodinitrotoluene (4-ADNT) being the major metabolites formed. The time it took to reduce TNT was longer and the concentrations of TNT-metabolites were lower in the presence of ferrihydrite than in its absence. This may have been due to competition for available electrons between TNT and TNT-metabolites and Fe(III). It is also possible that some of the intermediate products of TNT transformation were oxidized back to TNT-metabolites by Fe(III) resulting in lower concentrations of TNT-metabolites and increased concentrations of Fe(II). This study demonstrates the complexity of the interactions of various environmental parameters, under controlled laboratory conditions, in the transformation of TNT by Y. lipolytica AN-L15.Item Impact of a model soil on the biotransformation of 2,4,6-trinitrotoluene and its amine metabolites(Montana State University - Bozeman, College of Engineering, 2004) Walker, Diane Kathryn; Chairperson, Graduate Committee: Alfred B. CunninghamThe end of the Cold War resulted in the closure of many sites where explosives were manufactured, processed, and stored, and packaging practices left behind highly contaminated surface waters, groundwater and soils. Chief among the explosives contaminating these sites is the xenobiotic, 2,4,6- trinitrotoluene (TNT) whose electron-withdrawing nitro-groups make this aromatic compound highly resistant to biodegradation. An alternative option to mineralization as a bioremediation strategy, however, is immobilization. TNT can be biotransformed under reducing conditions to 2,4,6-triaminotoluene (TAT), a compound that researchers are currently investigating due to its potential to become irreversibly bound to soil components. The objectives of this research were to conduct TNT biotransformation studies under planktonic conditions and compare the results to those under slurry conditions. These objectives would also contribute to the overall goals of a DEPSCoR-sponsored project entitled "Biofilm-Induced Changes in Soil Organic Matter Structure and the Resulting Impact on the Bioavailability of Sorbed 2,4,6-Trinitrotoluene and its Amine Metabolites". Three experiments were performed for this research. The first was to create a model soil that was both well-characterized yet chemically representative of real soil constituents. The second was to add TNT to actively growing Desulfovibrio sp. strain SHV and monitor TAT formation. The third was a combination of the first two experiments, adding strain SHV to the model soil to both observe TAT formation and its disappearance from solution over time. The results of the TNT biotransformation studies indicated that TNT was transformed to TAT, which became the dominant metabolite in four weeks under planktonic conditions. Under slurry conditions, TAT became the dominant metabolite in two days and disappeared from solution by day three.