Ortho pathway of benzoate degradation in Pseudomonas putida: induction of meta pathway at high substrate concentrations

Abstract

The growth of Pseudomonas putida ATCC 49451 on various concentrations of sodium benzoate was examined under batch cultivation. At low initial concentrations (≤400 mgl −1), cell growth rate was described by the Monod model with parameters of maximum specific growth rate, μ m = 0.66 h −1 and saturation constant, K s = 10 mgl −1. At higher initial concentrations (≥500 mgl −1), product inhibition on specific growth and degradation rates was observed; specific growth rate decreased as sodium benzoate was degraded. A change in degradation pathway was observed with different initial concentrations of sodium benzoate. With initial concentrations up to 200 mgl −1, catechol was oxidized via the ortho ring cleavage with the induction of catechol 1,2-dioxygenase. The ortho pathway metabolites namely, catechol, cis, cis-muconate and muconolactone, were identified using gas chromatography/mass spectrometry (GC/MS) after trimethylsilyl (TMS) derivatization. When higher initial concentrations of sodium benzoate were used (≥300 mgl −1), the culture media showed absorbance at 375 nm (A 375) during the later stage of degradation after approximately 200 mgl −1 of sodium benzoate has been transformed. This was attributed to the accumulation of 2-hydroxymuconic semialdehyde (HMSA), which is an intermediate metabolite of the meta ring fission of catechol. It is concluded that cells grown on high enough concentrations of sodium benzoate simultaneously induced catechol 1,2-dioxygenase and catechol 2,3-dioxygenase, thus activating both the ortho and the meta catechol cleavage pathways. It was further found that catechol, a metabolic intermediate of sodium benzoate, did not effectively support the growth of P. putida when used as a sole substrate. Instead, a dark brown polymerization product was formed. We infer that the induction of the meta pathway is a consequence of the accumulation of 1,2-dihydro-1,2-dihydroxybenzoate (DHB) in the media.