136
Chapter 5
parameters to be estimated, van Gulik and Heijnen (1995) and van Rolleghem and Heijnen
(1998) applied this approach for
S. cerevisiae
and Christiansen and Nielsen (2002) applied it for
analysis of
Bacillus clausii
(see Example 5.3).
Example 5.3
E nergetics o f
Bacillus clausii
D ue to the ability to secrete large am ounts o f protein, m em bers o f the genus
Bacillus
are w idely used as
host in the ferm entation industry for production o f industrial enzym es, fine chem icals, antibiotics, and
insecticides.
B. clausii
is a facultative alkalophilic
Bacillus,
w hich is com m ercially used in the
production o f the alkaline serine protease Savinase®, an enzym e used in detergents. In order to design
optim al industrial processes a detailed u n derstanding o f the bioenergetics and in particular the
m aintenance dem ands are im portant. P roduction o f proteases in
Bacilli
is g enerally a response o f poor
nutritional conditions and proteases are therefore typically p roduced at low specific grow th rates. M ost
industrial ferm entations pro d u cin g proteases are therefore operated in the fed-batch m ode w ith very low
specific grow th rates in the later stages o f the process. D u rin g these stages w ith low specific grow th rates
a large p art o f the carbon source is consum ed fo r m aintenance dem ands. A t these conditions the grow th
energetics are o f particular im portance since the energy m etabolism in the cells influences both the yield
o f biom ass and product on substrate, b u t it also determ ines the oxygen requirem ents and the rem oval o f
excess heat produced. T he oxygen consum ption is very closely related to the energy m etabolism in the
cell since oxygen is used as the final electron accep to r in the respiratory chain, w hich p lay s a central role
in the oxidative phosphorylation. T h e p aram eters w ith the m ost significant influence on grow th
energetics and oxygen consum ption are: T he am ount o f A T P needed for biom ass form ation (Y ,ATP), the
m oles o f A TP form ed per m ole o f oxygen consum ed (P /O -ratio), and the A T P needed for m aintenance
( m ATp)-
A s discussed in the text it is not possible to estim ate all three energetics param eters by using only data
from grow th on glucose, but by com bining data for grow th on m ore than one carbon source w ith a
detailed know ledge on the biom ass com position and the central carbon m etab o lism all the energetic
param eters can be estim ated. A n im portant assum ption in this approach is th at the param eters are
conserved properties and do not change w ith the carbon source used. C hristiansen and N ielsen (2002)
perform ed a study o f the grow th kinetics and energetics in glucose and citrate lim ited chem ostat cultures
o f
B. clausii
w ith special focus on the oxygen consum ption. T he true y ield coefficients o f glucose,
oxygen, and carbon dioxide w ere obtained from glucose lim ited chem ostats. From experim ental results
on the am ino acid com position o f the biom ass, the R N A content and literature data a detailed biom ass
com position w as determ ined and the b uilding blo ck requirem ents for biom ass form ation w ere calculated.
From this analysis the elem ental com position o f the biom ass w as found to be CH , 8:Oo
5
5No
i>
,
and the ash
content w as found to be
6
% . F urtherm ore, the A T P cost for biom ass synthesis w as calculated to be 16.2
m m oles A T P (g D W
) 1
(or 0.43 m oles A T P (C -m ole b io m a ss )') and 19.0 m m oles A T P (g D W
) 1
(or 0.73
m oles A T P (C -m ole b io m ass)'1) for grow th on glucose and citrate, respectively. T he cost o f biom ass
synthesis from p recursor m etabolites is obviously the sam e for grow th on glucose and citrate, but the
A TP cost for synthesis o f p recursor m etabolites is different for the tw o substrates. T hus, it is
energetically m ore
expensive to
synthesize pyruvate
and p hosphoenolpyruvate
(both precursor
m etabolites) from citrate than from glucose. In order to account for additional d rain o f A T P for biom ass
synthesis a constant value o f
Kx
m oles o f A T P per C -m ole biom ass is added to the above-calculated
values.
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