Dynamics of benzene and toluene degradation in Pseudomonas putida F1 in the presence of the alternative substrate succinate

In batch and continuous culture, the regulation of benzene and toluene degradation by Pseudomonas putida F1 was investigated in the presence of the alternative carbon and energy source succinate. In batch culture, benzene and toluene were used simultaneously, whereas succinate suppressed benzene consumption under carbon excess conditions resulting in diauxic growth. In carbon-limited continuous culture mixed substrate growth was observed. Since in nature low substrate concentrations and ever changing conditions prevail, this paper focuses on the dynamics of benzene/toluene degradation, biomass synthesis, and the regulation of benzene/toluene-degrading enzymes in cultures growing continuously at a dilution rate of 0.1 h(-1), when shifting the supply of the carbon and energy source from succinate to various mixtures of succinate and benzene/toluene, or to benzene only. When the succinate concentration was kept constant (1.25 mM) and the medium was supplemented with benzene (2 mM), growth with benzene began already two hours after the shift. In contrast, replacing succinate with benzene only led to a wash out of biomass for more then ten hours, before biomass production from benzene started. A striking and reproducible transition pattern was observed for all shifts where the succinate concentration was reduced or succinate was omitted. After an initial period of biomass production from benzene, the culture collapsed and a wash-out of biomass was observed. However, this wash-out was not accompanied by an increase in benzene in the cultivation liquid, indicating a benzene uptake without conversion into biomass. Another possibility is that in phases of low biomass concentrations, cells were only able to use the low amounts of benzene/toluene dissolved in the cultivation liquid yielding low biomass concentrations whereas in phases of high biomass concentrations, they were able to rapidly utilize the aromatic solvents so that additional benzene from the gas phase diffused into the cultivation liquid resulting in more biomass production. In most cases, growth resumed again after 10 to 80 h. Currently, the reasons for the decrease in biomass after the first rise are unknown. However, several indications rule out intoxication of the cells by either the solvents benzene or toluene themselves, or by toxic degradation intermediates, or by-products.