Document Type

Dissertation

Date of Degree

Fall 2009

Degree Name

PhD (Doctor of Philosophy)

Degree In

Civil and Environmental Engineering

First Advisor

Timothy E. Mattes

Abstract

Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene, chlorinated solvents often used in plastics and dry cleaning solvent manufacturing. The treatment of VC-contaminated sites by bacteria that can biodegrade VC has been demonstrated to be a practical and potentially cost-effective alternative to traditional "pump and treat" site cleanup options. However, little is known about the biochemical pathways involved in VC-assimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment.

The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified.

Techniques to extract proteins from various environmental samples such as activated sludge, sediments, soils, and water samples were developed based on preliminary experiments with protein extraction from strain JS614. The results of these studies demonstrated the successful extraction and identification of proteins involved in VC-assimilation from ethene-enriched groundwater samples. The presence and diversity of VC-assimilating bacteria in several ethene-enriched groundwater samples were examined using tandem mass spectrometry analysis to identify the protein biomarkers EtnC and EtnE.

VC-assimilating organisms can evolve in vitro from bacteria that grow on ethene but very little is known about the molecular changes involved. Proteomic investigations comparing three strains of Mycobacterium strain JS623, a wild type and two VC-adapted strains, validated previous studies indicating that protein expression changes are involved in VC-adaptation. Tandem mass spectrometry and spectral counting were used to identify proteins and semi-quantitatively estimate protein expression levels in the three ethene-grown JS623 variants. The results of this study suggest that multiple VC-adaptation mechanisms are involved in the two VC-adapted strains

Keywords

bioremediation, mass spectrometry, proteomics, vinyl chloride

Pages

xii, 128 pages

Bibliography

Includes bibliographical references (pages 111-121).

Copyright

Copyright 2009 Adina Shiang Chuang

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