Carbon and energy flux constraints in continuous cultures of Alcaligenes eutrophus grown on phenol

Centre de Bioingenierie Gilbert Durand, UMR CNRYINSA and Lab. Ass. INRA, lnstitut National des Sciences Appl iq uCes, Complexe Scientifique de Rangueil, 31077 Toulouse cedex 4, France The growth behaviour of AIcaligenes eutrophus on phenol was investigated in continuous cultures to identify the phenomena limiting both growth efficiency and substrate degradation rates. It was shown that the fixed stoichiometry of the meta pathway of phenol degradation, leading to equimolar quantities of pyruvate and acetate, and the structure of the central pathways, which do not allow gluconeogenesis of acetate during growth on phenol, provoke the accumulation of polyhydroxybutyrate (PHB) under certain growth conditions. Acetate is predominantly used as an energy source and PHB accumulates when the cells are carbon-limited rather than energy-limited. The maximum rates of phenol degradation can be attributed to the expression of the enzymes of the catabolic pathway. This is particularly true of phenol hydroxylase and 2hydroxymuconate semialdehyde (2-hms) dehydrogenase, whose substrates accumulated to physiologically significant concentrations at high growth rates. Indeed the concentration of 2-hms that accumulated in the medium indicated that this enzyme was substrate-saturated at maximum growth rates. However, the specific activity profiles of other catabolic enzymes associated with phenol degradation were close to the estimated flux through the pathway. This suggests a complex control structure in which several enzymes contribute to the control of pathway flux, as would be expected in a catabolic pathway.