Rational medium design for Bordetella pertussis: basic metabolism.
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.
Thalen
M
M
National Institute of Public Health and the Environment (RIVM), Laboratory for Product and Process Development, Bilthoven, The Netherlands. marcel.thalen@rivm.nl
van den IJssel
J
J
Jiskoot
W
W
Zomer
B
B
Roholl
P
P
de Gooijer
C
C
Beuvery
C
C
Tramper
J
J
D001886:Bordetella pertussis / Q000254:growth & development* / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009584:Nitrogen / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism
D003470:Culture Media; D000644:Quaternary Ammonium Compounds; D019344:Lactic Acid; D018698:Glutamic Acid; D002244:Carbon; D009584:Nitrogen