Title

Enhancing aerobic biological degradation of trichloroethylene and metal lubricants

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Microbiology|Engineering, Chemical|Engineering, Environmental

Degree

Ph.D.

Abstract

Bioremediation is a widely-used method to utilize bacteria to degrade environmental pollutants. This study had two main objectives to improve bioremediation. The first objective was to increase the enzyme activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for trichloroethylene (TCE) degradation and 1-naphthol synthesis. The second objective was to characterize and design a biological metal-cleaning process to eliminate the use of hazardous metal cleaners, such as high alkaline solutions or toxic solvents. For the first objective, directed evolution (DNA shuffling) was used to produce a large number of Escherichia coli colonies containing slight genetic alterations of the genes that encode TOM. 96-well plate screening was used to choose mutants with enhanced enzyme activity from more than ten thousand colonies. The best colonies were characterized further using gas chromatography (GC), high-pressure liquid chromatography (HPLC), and DNA sequencing. This quantitative method identified the first enhanced non-heme monooxygenase, improved the degradation of chlorinated compounds (e.g., 2.8-fold for TCE), and enhanced 1-naphthol synthesis (7.6-fold) from naphthalene for green chemistry. For the second objective, an aqueous biological metal cleaning process (42°C) was characterized in terms of initial degradation rates of the test lubricant TUFDraw 2812RP (TUFDraw) and phylogenetic identification of the active bacteria at the species level using 16S ribosomal RNA (rRNA) sequences. This biological process is environmentally safe, economical, and holds promise for replacing current hazardous metal cleaners used in several industries. The original 42°C process was improved by utilizing a consortium of soil bacteria that enabled operation at a lower temperature (30°C) saving heating costs. Additional improvements in the process were obtained by identifying thermophilic bacteria (60°C) that are active on TUFDraw. Consequently, high (60°C), medium (42°C), and low (30°C) temperature biological metal cleaning processes are now achievable, which may allow a wider variety of metal lubricants to be degraded. ^

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