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Biodegradation kinetics of toluene, m-xylene, p-xylene and their intermediates through the upper TOL pathway in Pseudomonas putida (pWWO)

¹ Institut für Biotechnologie, ETH Hönggerberg, HPT, CH 8093 Zürich, Switzerland
² Laboratory of Ecotoxicology, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands
³ School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, UK

ABSTRACT

Summary: Pseudomonas putida mt-2, harbouring TOL plasmid pWWO, is capable of degrading toluene and a range of di- and tri-alkylbenzenes. In this study, chemostat-grown cells (D = 0.05 h^-1, toluene or m-xylene limitation) of this strain were used to assess the kinetics of the degradation of toluene, m-xylene, p-xylene, and a number of their pathway intermediates. The conversion kinetics for the three hydrocarbons showed significant differences: the maximal conversion rates were rather similar [11-14 mmol h^-1 (g dry wt)^-1] but the specific affinity (the slope of the v vs s curve near the origin) of the cells for toluene [1300 I (g dry wt)^-1 h^-1] was only 5% and 14% of those found for m-xylene and p-xylene, respectively. Consumption kinetics of mixtures of the hydrocarbons confirmed that xylenes are strongly preferred over toluene at low substrate concentrations. The maximum flux rates of pathway intermediates through the various steps of the TOL pathway as far as ring cleavage were also determined. Supply of 0-5 mM 3-methylbenzyl alcohol or 3-methylbenzaidehyde to fully induced cells led to the transient accumulation of 3-methylbenzoate. Accumulation of the corresponding carboxylic acid (benzoate) was also observed after pulses of benzyl alcohol and benzaldehyde, which are intermediates in toluene catabolism. Analysis of consumption and accumulation rates for the various intermediates showed that the maximal rates at which the initial monooxygenation step and the conversion of the carboxylic acids by toluate 1,2-dioxygenase may occur are two- to threefold lower than those measured for the two intermediate dehydrogenation steps.

^* Author for correspondence: Wouter A. Duetz. Tel: +41 1 6333811. Fax: +41 1 6331051. e-mail: duetz@biotech.biol.ethz.ch

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