Kinetics of toluene degradation by toluene-oxidizing bacteria as a function of oxygen concentration, and the effect of nitrate

Abstract

The kinetics of toluene degradation as a function of oxygen concentration were compared for six strains of toluene-oxidizing bacteria using initial rate assays. The effect of nitrate was also examined. Rates of degradation and the relative effect of oxygen on the degradation rate were correlated with the pathway for toluene oxidation. Strains which synthesize toluene dioxygenases, Pseudomonas putida F1, P. fluorescens CFS215, and Pseudomonas sp. strain W31, degraded toluene at significantly higher rates (151–166 nmol/mg per min) than strains synthesizing toluene monooxygenases, Burkholderia cepacia G4 (23 nmol/mg per min) and B. pickettii PKO1 (14 nmol/mg per min), or a methylmonooxygenase, P. putida PaW1 (12 nmol/mg per min). Rates declined 30–48% for the dioxygenase strains and 25% for PaW1 as the oxygen concentration was decreased from 240 to 50 μM, but declined less than 10% for G4 and PKO1. Nitrate enhanced toluene degradation by the denitrifying strains PKO1 and W31 at oxygen concentrations below 30 μM, but had no significant effect on any of the other strains. Biphasic kinetics were observed for all of the strains, with double-reciprocal plots of the data exhibiting an inflection point at a ‘critical oxygen concentration’ between 20 and 30 μM. Below this concentration, the rate of toluene degradation was inhibited to a greater extent than predicted by the kinetic data for higher oxygen concentrations. For the denitrifying strains PKO1 and W31, however, monophasic kinetics were observed when nitrate was provided as an alternative electron acceptor. These observations suggest that biphasic kinetics result when rates of toluene degradation are limited by the availability of electron acceptor at the critical oxygen concentration, and that this limitation is overcome by denitrifying strains able to respire nitrate. Taken together, our findings suggest that the synthesis of monooxygenases and the ability to denitrify represent independent adaptations for toluene utilization in low oxygen environments. Moreover, these data support the use of nitrate in mixed electron acceptor strategies for the bioremediation of contaminated aquifers, as well as the targeted use of monooxygenase and dioxygenase strains in settings in which their physiological traits can be best exploited.