Denitration of nitroarene and nitrate ester pollutants

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He, Fei
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Nitroaromatics and other nitrated compounds are a group of industrial chemicals extensively used in the synthesis of dyes, pesticides, pharmaceuticals and explosives, and have become contaminants widespread in soil and groundwater during manufacturing, handling and storage. During the past decades, numerous reports have addressed the biodegradation and biotransformation of these toxic xenobiotic compounds and developed bioremediation strategies to clean up the contaminants in soil and ground water. The degradation of nitroglycerin (NG) has been reported but the degradation mechanism is not well established. Moreover, the biodegradability of some recalcitrant explosives such as such 2,6-dinitroxyelene (2,6-DNX) and 3,5-DNX and their environmental fate are poorly understood, which present challenges to ongoing efforts to bioremediate soils at contaminated sites. Therefore, the overall objectives of thesis were to investigate the degradability and the catabolic pathways of DNX isomers and NG. Chapter 1 rigorously established NG degradation pathway in Arthrobacter JBH1, which has been reported as the first bacteria that could use NG as a sole source of carbon, nitrogen and energy. This study demonstrates that by two flavoproteins from the Old Yellow Enzyme (OYE) family, PfvA and PfvC, are involved in the sequential denitration of NG to 1-mononitroglycerin (1-MNG) and 2-MNG, producing 1,2-dinitroglycerin (DNG) and 1,3-DNG as intermediates. The phosphorylation of 1-MNG by a glycerol kinase homolog facilitates one of the flavoproteins, PfvC, to remove the last nitrate ester group and produce 3-phosphoglycerol, which is readily to enter the central metabolism. The advance in understanding of the mineralization mechanism of NG sets stage for developing strategies for bioremediation of NG. Chapter 2 the thesis explored the possibility of biotransformation of DNX. Due to the structural similarity of DNX isomers and dinitrotoluene (DNT) isomers, this part investigated the potential of oxidation of DNX isomers by nitrobenezene dioxygenase and 2-nitrotoluene dioxygenase as well as their active-site mutants, which actively oxidize 2,4-DNT and 2,6-DNT to dimethylnitrocatechols. This study show evidence that dioxygenases are able to attack the aromatic ring of 2,6-DNX and 3,5-DNX and produce dimethylnitrocatechols with nitrite release. The oxidation efficiency is limited, however, likely due to the steric effects of an extra methyl group of DNXs compared to DNTs and the distince electronic properties of DNX smolecules are different. Efforts to achieve ring cleavage of the dimethylnitrocatechols by catechol cleavage enzymes from DNT degradation pathways were not successful. The study found that the dominant reaction of DNX isomers by Escherichia coli under aerobic conditions is reduction, regardless of the presence of dioxygenases. The reduction products are identified as aminonitroxylenes. Enzyme assays carried under different redox conditions (aerobic, microaerophilic and anaerobic) indicate that the nitroreductase of E.coli is oxygen insensitive. Microcosm studies with soil from contaminated sites were conducted to investigate the ability of microbial communities from DNX contaminated soils to transform these two isomers under aerobic or anaerobic conditions. Results showed that 2,6-DNX and 3,5-DNX were converted to aminonitroxylenes by soil bacteria when external carbon/energy sources are provided. The reduction of 2,6-DNX was not affected by oxygen concentration, on the contrary, the reduction of 3,5-DNX showed elevated levels under anaerobic conditions. These findings suggest that biostimulation could be an effective way to remediate DNX contamination. Since the DNX isomers are susceptible for reduction in biological systems, fate of DNX is likely a combination of biotic process and abiotic reactions by bulk reductants in soil. To better understand the fate of DNX in the environment, Chapter 3 studies the reduction of DNX by juglone in the presence of H2S under abiotic conditions. One electron-transfer potentials E1h’ were determined for 2,6-DNX, 3,5-DNX, and structure-activity relationships were established. The study on abiotic reductions of DNX isomers advances the understanding of reductive transformation process of DNX and the transformation products in a given natural system, and prediction of the fate of the compounds in soil. Another goal of this study described here is to understand the impact of the methyl or isoprenyl substituents of polyphenol compounds on enzyme substrate specificity (Chapter 5). Plants produce resveratrol, referedreferred to as alleochemicals, as a defense mechanism against fungal infection, and as microbes evolve catabolic pathways to degrade resveratrol and use it as a carbon source, plants modify the structure of resveratrol and produce the less biodegradable derivatives, such as pterostilbene by methylation, and arachidin-3 by isoprenylation. This study isolated a bacteria isolate, Massilia sp. JS1662, which could grow on arachidin-3 as a carbon source. The catabolic pathway was established, and the initial enzyme that catalyzes the transformation of arachidin-3 was identified as a member of the carotenoid cleavage oxygenase (CCO) family. Enzyme assays with E.coli cell extracts overexpressing CCO homologs from different stilbene degraders reveal that CCO from arachidin-3 degrader has a broader range of substrate specificity than those from resveratrol and pterostilbene degraders. The relationship between substrate structure and enzyme specificity suggests the emergence of novel chemicals is a drive force for evolution of biochemical pathways in plant-microbe competition, which in turn, foster the secretion of more effective alleochemicals by plants. In summary, this research advances the understanding of the metabolic diversity in nitroaromatic and nitrate ester compounds biodegradation or biotransformation, and provides a basis for development of remediation strategies.
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