Jones, Richard Rhys; (1993) The effect of growth conditions upon the solvent tolerance and aromatic oxidation activity of Pseudomonas putida ML2. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Pseudomonas putida ML2 converts fluorobenzene to fluorocatechol, a compound of commercial value. The addition of a second immiscible solvent to the biotransformation could maintain low aqueous concentration of fluorobenzene (which inhibited growth and oxygen uptake of the bacterium). The impact of fermentation conditions on the solvent tolerance and aromatic oxidation activity was examined. Analytical techniques to quantify biocatalytic activity and solvent tolerance were developed. Growth of the bacterium at dissolved oxygen tensions (DOT) of 5 % air saturation resulted in specific aromatic oxidation activity increasing to approximately double that of cultures grown at 20 % DOT. Two phases to the growth phase were detected by online gas analysis and correlated to the changes in biocatalytic activity. A slow down in growth as a result of oxygen or iron limitation resulted in at least a doubling of aromatic oxidation activity, which was attributed to the change in the rate of growth. Fermentation samples lost biocatalytic activity within a few hours of harvesting, but shake flask cultures remained active for at least 24hr. Solvent tolerance was examined using chloroxylenol as a test solvent. The bacterial oxygen uptake rate (OUR) decreased exponentially reaching a steady value at the end of the assay. An equation was fitted to the data obtained for chloroxylenol, octanol and other solvents allowing specific decay rates to be calculated. The solvent tolerance of the bacterium was highest at the end of the fermentations controlled at 5 and 20 % DOT with a specific decay rate constant below 8 hr-1. A decrease in growth temperature from 30°C to 26°C resulted in a decrease in solvent biocompatibility when assayed at 30°C. At a growth temperature of 34°C the OUR was close to linearity signifying little loss of activity but bacterial growth at 34°C was slow and not reproducible. Addition of magnesium sulphate to the solvent tolerance assay increased tolerance of the bacterium to chloroxylenol, fluorobenzene and dichloroethane. A reduction in temperature of the solvent tolerance assay from 30°C to 23°C increased bacterial tolerance to dichloroethane. Significant improvement in solvent tolerance of the bacteria was dependent upon biotransformation conditions rather than changes in the growth conditions.

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