Design of a control system for biotransformation of toxic substrates: toluene hydroxylation by Pseudomonas putida UV4

Using the hydroxylation of toluene to toluene cis-glycol by Pseudomonas putida UV4 as an example, the design of a feed-back control system for the addition of a toxic, poorly water-soluble substrate to a fed-batch biotransformation is described. In kinetic studies the reaction followed Michaelis–Menten behavior until toxic toluene concentrations were reached (2.4 mM), above which irreversible denaturation of the biocatalyst was observed. An algorithm, based on a system mass balance, was used to maintain the aqueous phase toluene concentration in the desired range for zero order kinetics. The mass balance required accurate and rapid analysis of the product and reactant in both the liquid and the vapor phase. Various analytical methods were considered and the effect of the sampling and analysis time on the response of the control system was examined.