Rational medium design for Bordetella pertussis: basic metabolism

Publication Type:

Journal Article

Source:

Journal of Biotechnology, Volume 75, Number 2-3, pp. 147-159 (1999)

ISBN:

0168-1656

Keywords:

bordetella pertussis; medium design; ammonium accumulation; waste metabolism; nmr; poly-hydroxy-butyrate; phb; toxin; protein

Abstract:

In current Bordetella pertussis media ammonium accumulates because of an imbalance in the nitrogen:carbon ratio of the substrates used, which is one of the factors limiting cell density in fed-batch cultures. The aim of this study was to map B. pertussis' catabolic and anabolic capabilities, in order to design a medium that avoids ammonium accumulation, while substrates are metabolised completely. Besides the known dysfunctional glycolysis, B. pertussis also possessed a partially dysfunctional citric-acid cycle. Although ammonium accumulation was avoided by adding various carbon sources to medium with glutamate, nuclear magnetic resonance (NMR) showed excretion of acetate, 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-hydroxy-butyrate (PHB) globules, which disappeared later during the culture, coinciding 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 ammonium accumulation were avoided, while the yield of 8.8 g C-mol(-1) compared favourably with literature values, averaging 6.5 g C-mol(-1). Optimisation of this medium for pertussis toxin production will be reported in a separate article. (C) 1999 Elsevier Science B.V. All rights reserved.

28/10/2009