Graduate Thesis Or Dissertation
 

Aromatic hydrocarbon contaminants : investigations of detection methods, bioremediation strategies, and toxicity impacts

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  • Aromatic hydrocarbons represent a large class of environmental contaminants that have a broad range of structures, physicochemical properties, and toxicities. Arising from the burning of organic matter, particularly fossil fuels, they are both widespread and abundant in all environmental compartments. Both monoaromatic hydrocarbons (BTEX) and polycyclic aromatic hydrocarbons (PAHs) are toxic to ecosystems and humans, with some exhibiting carcinogenic and mutagenic properties. As such, they are regulated by the EPA with maximum contaminant levels (MCLs) that reflect their toxicity and are often the target for remediation efforts at contaminated sites. This dissertation focuses on bioremediation, using bacteria to break down contaminants, as the primary remediation strategy. Although a valuable remediation method in terms of cost and required resources, bioremediation of aromatic hydrocarbons is not without its own unique set of challenges. Aromatic hydrocarbons exist as complex mixtures in the environment that can be difficult to biologically treat in their entirety. Finding microorganisms that can transform a wide range of aromatic hydrocarbon structures can be a bottleneck for bioremediation technology development. Furthermore, these mixtures are difficult to monitor analytically, particularly in a rapid manner. The ability to monitor treatment progress in real time is of great value for novel bioremediation strategy development, however this is not possible with traditional analytical techniques. Finally, aromatic hydrocarbon contaminants, particularly PAHs, present an interesting challenge where their transformation products can be more toxic that the parent compounds. This is a troublesome factor to consider in remediation studies, as it is typically only the parent compounds that are considered and regulated. The work presented in this dissertation was motivated by these challenges to support the progress of aromatic hydrocarbon bioremediation strategies and tools needed for their continued development. Chapter II presents an initial investigation of the ability of a bacterial pure culture, Rhodococcus rhodochrous ATCC 21198, to transform aromatic hydrocarbon contaminants using BTEX as model contaminants. Methyl-tertiary-butyl ether (MTBE) was studied as well, as it is a common co-contaminant of BTEX that can hinder biodegradation of the mixture. It was found that 21198 was able to degrade BTEX, and MTBE both individually and as a mixture, and that isobutane, 1-butanol, and 2-butanol all supported this activity. Furthermore, MTBE’s primary transformation product, tertiary-butyl alcohol (TBA), was also transformed by 21198, but at a slower rate compared to MTBE. This degradation was a combination of metabolic and cometabolic activity, as 21198 was observed to grow on benzene and toluene. This work supported further investigations of aromatic hydrocarbon treatment with 21198 and was the first demonstration of 21198’s ability to degrade and grow on aromatic hydrocarbon contaminants. Chapter III details the development of a rapid bioremediation monitoring tool using fluorescent spectroscopy and parallel factor analysis (PARAFAC). A microorganism previously studied for phenanthrene transformation, Mycobacterium Sp. Strain ELW1, was used to develop an experimental data set where phenanthrene was transformed to the primary transformation product trans-9,10-dihydroxy-9,10-dihydrophenanthrene (P1). The transformation of phenanthrene and the formation of P1 were monitored and quantified with the novel fluorescent spectroscopy/PARAFAC method. Results were validated with an established GC-MS method, demonstrating comparable results in a fraction of the time. This was the first use of a fluorescent spectroscopy/PARAFAC method to monitor, identify, and quantify PAH biotransformation and product formation. Chapter IV adapts the method presented in Chapter III to a new biological system, embryonic zebrafish, with the same motivation: reduce time and resources required for monitoring aqueous PAH concentrations. Embryonic zebrafish have been used extensively for screening chemicals for toxicity and can be used in a high-throughput system established at the Sinnhuber Aquatic Research Laboratory at Oregon State University. A fluorescent spectroscopy method was developed to accommodate a 96-well plate and measure PAH concentrations in embryo media over a 5-day incubation period. It was observed that measurements with a fluorescent plate reader did not impact embryo development, nor did normal embryo development significantly impact background fluorescence in the microwells. Chemical dose, uptake rate, and abiotic loss rates were all derived from fluorescent measurements for acridine and 2-hydroxynaphthalene, demonstrating the ability to use the fluorescent plate reader for rapid, non-destructive analysis of PAHs in high-throughput toxicity assays with embryonic zebrafish. Chapter V combined aspects from the previous three chapters to investigate 21198’s ability to transform a mixture of phenanthrene, anthracene, fluorene, and pyrene on its own and in combination with the surfactant Tween ® 80 and cell immobilization techniques. Transformation of PAHs was observed in all batch tests, with cell immobilization in PVA/alginate beads generally improving the rate and extent of PAH transformation, especially when combined with the surfactant. However, toxicity to zebrafish embryos increased after bioremediation, indicating that transformation products contributed to toxicity more so than the parent PAHs. A complex mixture of hydroxylated products, ring fission products, and quinones, were putatively identified using UPLC-MS, many of which have been demonstrated to elicit toxic responses in embryonic zebrafish. Despite this, 21198’s ability to rapidly transform such a broad range of environmentally relevant aromatic hydrocarbon contaminants suggests that it is a valuable bacterium in treating these contaminants and is amenable to use in combined treatment strategies that can improve treatment outcomes.
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  • Research reported in this dissertation was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number P42ES016465. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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