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Adaptive Extremum-Seeking Control Applied to Productivity Optimization

in Yeast Fed-Batch Cultures

Conference Paper · July 2008

DOI: 10.3182/20080706-5-KR-1001.01643

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 Adaptive extremum-seeking control applied to
productivity optimization in yeast fed-batch
cultures
L. Dewasme ∗ A. Vande Wouwer ∗
∗ Service d’Automatique, Faculté Polytechnique de Mons, Boulevard Dolez
31, B-7000 Mons, Belgium (e-mails : Laurent.Dewasme,
Alain.VandeWouwer@fpms.ac.be)

Abstract: In this study, we consider the problem of optimizing the productivity of fed-batch cultures
of S. cerevisiae, which are characterized by strongly nonlinear kinetic models based on the bottleneck
assumption of Sonnleitner and Käppeli [1986] and ethanol inhibition resulting from the fermentation
of a possible excess of substrate feeding. In contrast with most published studies where the critical
substrate level is assumed constant, we investigate the situation where this critical substrate level depends
on the yeast respiratory capacity, and in turn on the oxygen and etahnol concentration in the culture
medium. The challenge is thus to maintain the process at a high level of productivity by avoiding the
accumulation of ethanol. To this end, an adaptive extremum seeking control scheme, coupled to an
asymptotic observer, is developed based on Lyapunov stability arguments. Copyright 2008 c IFAC

Keywords: Extremum seeking, nonlinear adaptive control, asymptotic observer, fermentation process,
biotechnology.

1. INTRODUCTION of the inhibitory effect of ethanol on this yeast respiratory

capacity. Compared to previous studies, the underlying mod-
els therefore significantly depart from the classical Monod or
Yeasts are one of the most important host microorganisms in Haldane laws and a new analysis is required. In particular, an
manufacturing of biopharmaceuticals. Industrial vaccine pro- adaptive extremum-seeking strategy including two adaptation
duction is usually achieved using fed-batch cultures of geneti- laws is developed and a simplified more robust version of this
cally modified yeast strains, which can express different kinds strategy is also proposed. Moreover, the use of an asymptotic
of recombinant proteins. From an operational point of view, it observer is considered so as to limit the number of required
is necessary to determine an optimal feeding strategy (i.e. the on-line measurements (Chen et al. [1995], Bastin and Dochain
time evolution of the input flow rate to the fed-batch culture) [1990]).
in order to maximize productivity. The main problem that can This paper is organized as follows. The next section introduces
be encountered at this stage is due to fermentation of an excess the macroscopic model of yeast fed-batch cultures used in this
of substrate (glucose), which can lead to the accumulation of study and defines the optimal operating conditions. Section 3
ethanol in the culture medium, and in turn to the inhibition of presents the adaptive extremum seeking algorithm, the design
the cell respiratory capacity. of an asymptotic observer and a Lyapunov stability analysis. In
To avoid this undesirable effect, a closed-loop optimizing Section 4, the performance of the algorithm is tested in simula-
strategy is required, which could take various forms (Chen tion and discussed, whereas Section 5 draws some conclusions
et al. [1995], Akesson [1999], Renard [2006], Dewasme et al. and perspectives.
[2007]). In particular, the use of extremum seeking strategies
for bioprocess optimization has received an increasing attention
in recent years (Ariyur and Krstic [2003], Guay and Zhang 2. MODELING YEAST FED-BATCH CULTURES
[2003], Guay et al. [2004], Marcos et al. [2004], Titica et al.
[2004]). 2.1 Nonlinear dynamic model
In this study, we develop an adaptive extremum-seeking strat-
egy based on Lyapunov stability arguments (in a similar way
as in (Guay et al. [2004], Titica et al. [2004])). The main The yeast strain S. cerevisiae presents a metabolism that can
challenge is to consider the dependence of the critical substrate be macroscopically described by the following three main reac-
level on the respiratory capacity, itself influenced by the oxy- tions:
genation level and the ethanol inhibition. A second difficulty
rises from the strong nonlinearity of the kinetic laws, which is 1r
Substrate oxidation : S + k5 O → k1 X + k7 P (1a)
due to the switch between a respirative regime and a respiro-
r2
fermentative regime, depending again on the yeast respiratory Substrate fermentation : S → k2 X + k4 E + k8 P (1b)
capacity and, in turn, on the substrate concentration in the r3
culture medium (i.e. the bottleneck assumption of Sonnleitner Ethanol oxidation : E + k6 O → k3 X + k9 P (1c)
and Käppeli [1986]). In addition, the kinetic laws take account
where X, S, E, O and P are, respectively, the concentration in
the culture medium of biomass, substrate (typically glucose),
ethanol, dissolved oxygen and carbon dioxide. ki are the yield
coefficients and r1 , r2 and r3 are the nonlinear reaction rates
given by:
 
ro
r1 = min rS , (2)
k5
 
ro
r2 = max 0, rS − (3)
k5
  
ro − k5 rS
r3 = max 0, min rE , (4)
k6
where the kinetic terms associated with the substrate consump-
tion rS , the oxidative or respiratory capacity ro and the ethanol
oxidative rate rE are given by:

S
rS = µS (5a)
S + KS
O
ro = µO (5b)
O + KO
E Fig. 1. Illustration of Sonnleitner’s bottleneck assumption for
rE = µE (5c)
E + KE yeast limited respiratory capacity.
These expressions take the classical form of Monod laws where
µS , µO and µE are the maximal values of specific growth OT R = kL a(Osat − O) (7a)
rates and KS , KO and KE are the saturation constants of the CT R = kL a(P − Psat ) (7b)
corresponding element.
This kinetic model, which is often encountered in the literature, where kL a is the volumetric transfer coefficient and, Osat and
is based on Sonnleitner’s bottleneck assumption (Sonnleitner Psat are respectively the dissolved oxygen and carbon dioxide
and Käppeli [1986]) (Figure 1). During a culture, the yeast cells concentrations at saturation.
are likely to change their metabolism because of their limited Ethanol has a detrimental effect on the cells growth because it
respiratory capacity. When the substrate is in excess (concen- directly inhibits the cells respiratory capacity (Pham [1999]).
tration S > Scrit ), the yeast cells produce ethanol through fer- Taking this remark into account, a more detailed expression of
mentation, and the culture is said in respiro-fermentative (RF) ro is given by:
regime. On the other hand, when the substrate becomes limit-
ing (concentration S < Scrit ), the available substrate (typically
glucose), and possibly ethanol (as a substitute carbon source), O KiE
ro = µO (8)
if present in the culture medium, are oxidized. The culture is O + KO KiE + E
then said in respirative (R) regime.
where KiE is the inhibition constant of ethanol.
Component-wise mass balances give the following differential
equations : It is common in the literature to consider the critical substrate
dX level Scrit constant. However, this level is actually not constant
= (k1 r1 + k2 r2 + k3 r3 )X − DX (6a) and depends on the respiratory capacity which is limited by
dt a lack of oxygen and inhibited by an excess of ethanol. For
dS
= −(r1 + r2 )X + DSin − DS (6b) illustration purposes, Figure 2 shows a simulation of the process
dt where the substrate concentration in the culture medium is reg-
dE ulated around a constant theoretical value of Sset = 0.0226g/l.
= (k4 r2 − r3 )X − DE (6c)
dt It is apparent that ethanol is produced during the batch and
dO that the biomass growth rate is lower than expected. A simple
= −(k5 r1 + k6 r3 )X − DO + OT R (6d)
dt substrate regulation does not allow to avoid the production of
dP ethanol, which in turn, reduces the respiratory capacity and the
= (k7 r1 + k8 r2 + k9 r3 )X − DP − CT R (6e) critical substrate level.
dt
dV Considering we are in the optimal operating conditions (S =
= Fin (6f)
dt Scrit ), the fermentation and ethanol oxidation rates are equal
where Sin is the substrate concentration in the feed, Fin is the to zero and the substrate consumption rate rS is equal to kro5 .
inlet feed rate, V is the culture medium volume and D is the
Consequently, after a trivial mathematical manipulation of (5a),
dilution rate (D = Fin /V ). OT R and CT R represent respectively
a relation between the critical substrate concentration level and
the oxygen transfer rate from the gas phase to the liquid phase the cell respiratory capacity is obtained as:
and the carbon transfer rate from the liquid phase to the gas
phase. Classical models of OT R and CT R are given by:
 40
0.025
Biomass [g/l]

Scrit = f(ro)
20
α ro

0 0.02
0 5 10 15 20 25 30

S
Substrate [g/l]

0.04
S
crit
0.015
0.02

[g/l]
crit
0
0 5 10 15 20 25 30

S
100 0.01
Ethanol [g/l]

50

0.005
0
0 5 10 15 20 25 30
Time [h]

0
0 1 2 3 4 5 6 7 8
Fig. 2. Simulation of a fed-batch process controlled at a con- r [g/g/s] −5
o x 10
stant Scrit value.
Scrit = f(ro) Fig. 4. Scrit as a function of ro and linear approximation.
0.025
in the classical work of (Ariyur and Krstic [2003]). It consists
in a permanent estimation of the system dynamics through the
0.02 analysis of a ”control error signal” (which in the present case
is a function of the difference between S and the set point
(r
o
,S
crit
) Scrit ) following the injection of a periodical excitation signal d
max max
0.015 into the adaptive system. This allows the convergence of the
[g/l]

parameter estimates to their true values and the stabilization

of the error signal around zero (but not exactly zero as the
crit
S

0.01 excitation is permanent).

3.2 Controller design

0.005

We first define the main parameters to estimate. Then, we derive

adaptation laws and a control law from the consideration of a
0 candidate Lyapunov function ensuring system stability.
0 1 2 3 4 5 6 7 8
r [g/g/s] x 10
−5
o
First, equation (6b) can be rewritten as follows:
Fig. 3. Scrit as a function of ro .
dS
Ks ro = −θX − D(S − Sin ) (10)
Scrit = (9) dt
k5 µs − ro
where θ = r1 + r2 is considered as an unknown kinetic parame-
Figure 3 shows a plot of this relation where the point [0, 0] ter.
corresponds to a totally inhibited respiratory capacity, prevent- As we wish to control the substrate concentration around its
ing any growth, and the point [romax , Scritmax ] corresponds to critical point, we need to assess its value at every moment.
maximum productivity (i.e. absence of ethanol in the culture Unfortunately, equation (9) contains several unknown (or at
medium and a sufficient level of oxygenation). Obviously, the least uncertain) parameters (µs , Ks , k5 , Ko , µo and KiE ), which
presence of ethanol in the culture medium can decrease the can be detrimental to the control quality. To avoid this possible
respiratory capacity and in turn the value of the critical substrate lack of robustness, as the order of ro is clearly smaller than k5 µS ,
concentration S = Scrit . In order to maintain the system at the we propose to approximate expression (9) by the following one:
edge between the respirative and respiro-fermentative regimes,
it is necessary to determine on-line the critical substrate con-
KS
centration (Scrit ) and to control the substrate concentration in Scrit ≈ ro = αro (11)
the culture medium around this value. k5 µS
where α is a positive parameter which has to be adapted during
3. AN ADAPTIVE EXTREMUM-SEEKING STRATEGY the batch (as a modeling exercise, a residual mean error of 0.2%
is obtained after a linear regression applied to (9) demonstrating
3.1 Introduction and main principles the quality of a first-order approximation - see Figure 4 ).
The adaptive extremum-seeking strategy that is chosen in this Defining:
study is related to the techniques developed in Titica et al.
[2004], Betancur et al. [2004], Guay and Zhang [2003], Guay Z
et al. [2004] and Marcos et al. [2004], which take their roots Zs = k p (S − Scrit ) + ki (S − Scrit )dt − d (12)
the control error variable, where d is the periodical ”dither
signal” and, k p and ki are positive tuning parameters, ξ = Kφ − Dξ + F − Q (22)
θ̃ = θ − θ̂ (13) where ξ is the state vector, K the yield coefficient matrix, φ the
the estimation error on θ, and reaction rates vector, F the inlet flux vector when the external
components are diluted in the culture medium and Q the outlet
α̃ = α − α̂ (14)
flux vector for gas components.
the estimation error on α, we consider the following Lyapunov In the present case, the rank of K is equal to 3 so that 3
candidate function: measurements are necessary in order to estimate all the states.
1 1 1 2
V = Zs2 + θ̃2 + α̃ (15) We propose here to estimate the biomass and ethanol concen-
2 2γ 2γs trations from measurements of glucose, dissolved oxygen and
where γ and γs are strictly positive tuning parameters. carbon dioxide:

A stabilizing controller is obtained if one can prove the strict

negativity of the Lyapunov function derivative. Differentiating ξ1 = [S O P]T
V , we obtain: ξ2 = [X E]T (23)

V̇ = Zs [k p (−θX − D(S − Sin ) − αr˙o ) + ki (S This partition induces the corresponding partition of the yield
matrix K, i.e., K1 and K2 .
θ̂˙ α̂˙
−α̂ro ) − d˙ + θ̃(− ) + α̃(− )

(16)
γ γs The definition of the auxiliary variables vector z = A1 ξA + A2 ξB
with A0 = A1 = −K2 K1−1 and A2 = I, leads to the asymptotic
Replacing (12), (13) and (14) in (16) and forcing V̇ to be observer structure:
negative as in:
V̇ = −kz Zs2 (17)
Sin D
    " #
ż1 z1
where kz is a strictly positive tuning parameter, we obtain: = −D + A0 OT R (24a)
ż2 z2
−CT R
−kz Zs = k p (−θ̂X − D(S − Sin ) − α̂r˙o )     S
" #
X̂ z
+ki (S − α̂ro ) − d˙ (18) = 1 − A0 O (24b)
Ê z2
P
provided that:
θ̂˙ = −γk p Zs X (19a)
The convergence speed of this observer is linked to the dilution
α̂˙ = −γs Zs (k p r˙o + ki ro ) (19b) rate:
Ŝcrit = α̂ro (19c)
Finally, the control law is given by d ξ̃2 d
= (ξ̂2 − ξ2 ) = −D(ξ̂2 − ξ2 ) (25)
h i dt dt
kz Zs −a+kd d
kp − θ̂X The dilution rate, given by (20), is persistently exciting and
D= (20) ensures the observer convergence.
S − Sin
with a dither signal d chosen as: In industrial practice, laboratory measurements are achieved
at the beginning of each run so that the error on the initial
state variables, ξ˜2 , is usually small and the dilution rate (which
d˙ = a + ki (S − α̂ro ) − k p α̂r˙o − kd d (21) evolves exponentially so as to follow yeast growth) ensures a
fast convergence of the asymptotic observer so that no stability
where a is a closed-loop excitation signal and kd is a new
problem of the closed-loop system (combining the asymptotic
strictly positive design parameter.
observer and the extremum-seeking controller) occurs.
The proposed control law (20) requires several on-line mea-
Rigorously, however, the stability of the whole control system
surements (X, S, O, E), which can nowadays be achieved using
should be analytically demonstrated through the derivation of
specific probes. However, these sensors are still quite expensive
a new Lyapunov candidate function taking into account the
and their use is not widespread. In the following we consider the
introduction of the observer in the closed-loop.
use of an asymptotic observer to provide estimation of biomass
and ethanol from glucose, dissolved oxygen and carbon dioxide
measurements (Chen et al. [1995]). The main advantage of the 1 1 1 1 1
asymptotic observer is that it provides an estimation indepen- V = Zs2 + θ̃ + α̃ + z˜1 2 + z˜2 2 (26)
2 2γ 2γs 2 2
dent of the kinetic laws.
The demonstration of stability is immediate considering that
3.3 Asymptotic observer z̃ = z − ẑ and (24), i.e.

The mass-balance equations (6) can be written in a compact

form: z̃˙ = −Dz̃ (27)
 Parameter Value Unit
0.03

γs 10 /

Substrate [g/l]
0.02
γ 10−7 / S
0.01 S
crit
kp 15 / S
crit
est

real
0
ks 0.004 / 0 5 10 15 20 25 30 35 40

kz 0.0015 X / 200

kd 0.01 / 150

Biomass [g/l]
ωi
100
2π i
4000 rad/s 50

Table 1. Tuning parameter values. 0

0 5 10 15 20 25 30 35 40

We are thus ensured that the introduction of the observer does 3

Ethanol [g/l]
not affect the stability as it preserves the negativity of the 2

1
Lyapunov function derivative. 0
0 5 10 15 20 25 30 35 40
Time [h]

4. SIMULATION RESULTS
Fig. 5. Substrate (S, Ŝcrit and Scrit ), biomass (X) and ethanol (E)
In this section, we apply the controller designed in the previous
concentrations evolutions.
section to a simulated case-study corresponding to classical
small-scale (20 l bioreactor) culture conditions. The initial and
operational conditions are: 0.04

Fin [l/s]
X0 = 0.4g/l, S0 = 0.5g/l, E0 = 3g/l, O0 = Osat = 0.035g/l, 0.02

P0 = Psat = 1.286g/l, V0 = 6.8l, Sin = 350g/l 0

0 5 10 15 20 25 30 35 40

360
For the kinetic and yield parameter values, the reader is referred 340

to Sonnleitner and Käppeli [1986].

α

320

300
0 5 10 15 20 25 30 35 40
The selection of an appropriate dither signal is based on a per- 1
x 10
−3

θ
sistence of excitation (PE) condition (Guay and Zhang [2003], est
θ
θ [s−1]

0.5 real

Marcos et al. [2004], Adetola and Guay [2006]) which, once

0
fulfilled, ensures the asymptotic convergence of the parameter 0
−5
5 10 15 20 25 30 35 40
x 10
estimates. 8
ro [g/g/s]

The excitation signal is here chosen as a simple sum of sinu- 6

5
soidal signals of the form: 0 5 10 15 20
Time [h]
25 30 35 40

5
a = ∑ Ai sin(ωi t) (28)
i=1 Fig. 6. Feed rate (Fin , α and θ parameters, and respiratory
capacity (ro ) evolutions.
where Ai are normally distributed random numbers contained
in [−0.0005, 0.0005] and ωi are the pulsations.
0.025
The initial substrate and ethanol concentrations are chosen at
high values, so as to challenge (in a difficult situation) the
controller convergence speed. Figures 5, 6 and 7 present the 0.02

simulation results. The substrate concentration evolution (Fig-

ure 5) shows that the presence of ethanol at the beginning of the
batch causes a decrease of the critical substrate concentration 0.015
Scrit [g/l]

level. An adaptation of this critical substrate concentration is

then needed so as to avoid an increased production of ethanol 0.01

(due to the excess of substrate) and a serious inhibition of cell

growth. At the end of the batch, ethanol is almost completely
consumed so that the system is driven close to the optimum (see 0.005

Figure 7). Figure 6 also shows the evolution of the feed rate
Fin . θ converges to its true value, so as α through a judicious 0

choice of the value of γs (see Table 1) as the convergence is

0 1 2 3 4 5 6 7 8
r [g/g/s] x 10
−5
o

generally very slow. The productivity is quite satisfactory as

more than 150g/l of biomass are obtained within less than 40
hours, despite the high initial concentrations in substrate and Fig. 7. Representation of the algorithm convergence through the
ethanol. evolution of Scrit as a function of ro .

The main drawback of this control strategy is the delicate controller works efficiently and Zs vanishes, the convergence of
choice of the tuning parameters, depending on the initial and α is significantly affected. In turn, if the critical substrate level
operating conditions. This problem originates in the presence is overestimated, the control action can lead to the production of
of the error control variable Zs as a factor in (19b). If the ethanol, and as a consequence, the inhibition of the respiratory
substrate concentration quickly converges to its setpoint, i.e. the capacity and a further decrease of the critical substrate level.
 0.025
influenced by the oxygenation level and the ethanol inhibitory
effect (Pham [1999]). Based on Lyapunov stability arguments,
0.02 original adaptation laws are derived to estimate on-line un-
known kinetic parameters. In addition, an asymptotic observer
[g/l]

0.015
is designed so as to limit the need for expensive hardware
crit

0.01 sensors of the culture component concentrations. Finally, a

S

simplified strategy is proposed, which provides a more robust

0.005
estimation of the critical substrate level.
0
0 1 2 3 4 5 6 7 8 ACKNOWLEDGEMENTS
r [g/g/s] −5
x 10
o

This paper presents research results of the Belgian Network

Fig. 8. Scrit as a function of ro (in green) and reduced approxi- DYSCO (Dynamical Systems, Control, and Optimization),
mation (in blue). funded by the Interuniversity Attraction Poles Programme, ini-
tiated by the Belgian State, Science Policy Office. The scientific
150 responsibility rests with its authors.
Biomass [g/l]

100
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