Rational medium design for Bordetella pertussis: basic metabolism

In current Bordetella pertussis media ammonium accumulates because of an im balance in the nitrogen:carbon ratio of the substrates used, which is one o f the factors limiting cell density in fed-batch cultures. The aim of this study was to map B. pertussis' catabolic and anabolic capabilities, in orde r to design a medium that avoids ammonium accumulation, while substrates ar e metabolised completely. Besides the known dysfunctional glycolysis, B. pe rtussis also possessed a partially dysfunctional citric-acid cycle. Althoug h ammonium accumulation was avoided by adding various carbon sources to med ium with glutamate, nuclear magnetic resonance (NMR) showed excretion of ac etate, acetoacetate and beta-hydroxy-butyrate, thereby reducing the biomass yield. Acetoacetate and beta-hydroxy-butyrate were also formed in Verwey, B2 and modified Stainer-Scholte medium. Electron microscopy in combination with NMR showed that cells early on in these cultures contained poly-hydrox y-butyrate (PHB) globules, which disappeared later during the culture, coin ciding with the appearance of beta-hydroxy-butyrate and/or acetoacetate. No globules nor metabolite excretion was detected when lactate in combination with glutamate were used as substrates. Thus, metabolite excretion and amm onium accumulation were avoided, while the yield of 8.8 g C-mol(-1) compare d favourably with literature values, averaging 6.5 g C-mol(-1). Optimisatio n of this medium for pertussis toxin production will be reported in a separ ate article. (C) 1999 Elsevier Science B.V. All rights reserved.