Simpson, Helen D. (1987) The metabolism of toluene by thermotolerant bacteria. PhD thesis, University of Warwick.

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Abstract

Several thermotolerant organisms capable of growth on toluene as the sole carbon and energy source, were isolated from various soil samples and liquid samples from a waste treatment works. Two isolates ware selected for further studies and tentatively characterized as Bacillus species. The isolates were designated strain HTB16 and strain AT50 and had optimum growth temperatures of 45°C and 50°C respectively.

An initial aim of the project was to investigate whether thermotolerant organisms could be exploited to produce aromatic cis-dihydrodiols. Both strains HTB16 and AT50 oxidized toluene using a dioxygenase-catalyzed reaction, thereby forming cis-toluene dihydrodiol as an intermediate. Therefore, a mutant of either strain, which lacked functional cis-toluene dihydrodiol dehydrogenase would accumulate cis-toluene dihydrodiol when grown in the presence of toluene and a co-substrate.

A mutant of strain AT50 would probably be the most suitable organism for an industrial process since it exhibited a higher optimum growth temperature, which was reflected by a greater thermostability of its enzymes. The enzymes detected in crude extracts of strain AT50 appeared to be more thermostable than those in extracts of HTB16. Cis-Toluene dihydrodiol dehydrogenase from AT5O was particularly stable. This enzyme was purified and partially characterized and amongst its properties, had a half-life of 100 min at 80°C.

Strains HTB16 and AT5O were subjected to various mutagenic procedures. NTG mutagenesis appeared to be the best method for obtaining a relatively high percentage of mutants amongst the survivors. Unfortunately, all the mutants obtained appeared to lack toluene dioxygenase.

In conclusion, it appears that enzymes from thermotolerant organisms are more thermostable than those from mesophiles. Consequently, it may be advantageous to use strain AT50 to produce cis-toluene dihydrodiol, once a suitable mutant is obtained. However, experiments with continuous cultures of strain AT5O suggested that one possible disadvantage of growing toluene-utilizing bacteria at 50°C is the low solubility of toluene in aqueous medium at this temperature.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Microbial metabolism, Toluene -- Biodegradation, Aromatic compounds -- Biodegradation, Bacillus (Bacteria), Mutagenesis
Official Date:	November 1987
Dates:	Date Event November 1987 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Dalton, Howard
Sponsors:	Science and Engineering Research Council (Great Britain). Biotechnology Directorate
Format of File:	pdf
Extent:	xviii, 256 leaves : illustrations, charts, photographs
Language:	eng

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