Full text of DOI:10.1038/sj.jim.2900813

Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236
2000 Society for Industrial Microbiology 1367-5435/00 $15.00
www.nature.com/jim
Anomalies in the growth kinetics of Saccharomyces cerevisiae
strains in aerobic chemostat cultures
SH de Kock, JC du Preez and SG Kilian
Department of Microbiology and Biochemistry, University of the Orange Free State, PO Box 339, Bloemfontein 9300,
South Africa
Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132,
ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively
with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concen-
trations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism com-
menced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts.
However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066
produced 7–8 g ethanol L−1 from 20 g glucose L−1 with concomitant low levels of residual glucose, which increased
markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae
ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L−1) than S. cerevisiae CBS 8066, concomi-
tant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and
appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism.
Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose
assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236.
Keywords: Saccharomyces cerevisiae; aerobic; chemostat; growth kinetics
Introduction
atory region. A similar trend may be seen in the data for
S. cerevisiae CEN.PK113–7D grown in an aerobic glucose-
limited chemostat culture [5], suggesting that this phenom-
enon may also be found in various other strains of S. cerevi-
siae.
Saccharomyces cerevisiae is a glucose-sensitive yeast, also
termed Crabtree-positive, exhibiting aerobic ethanol pro-
duction in the presence of excess glucose [2,8,21]. In aero-
bic chemostat culture, glucose is metabolised oxidatively at
low dilution rates, resulting in a high biomass yield [11,21].
Biomass yield coefficients ranging from 0.47 to 0.50 g
g glucose−1 have been reported for different S. cerevisiae
strains [13,14]. At dilution rates above a critical value,
metabolism of the yeast shifts towards a respiro-fermenta-
tive (also termed oxido-reductive) type of metabolism, with
aerobic ethanol production [11].
Above this critical dilution rate, an ethanol yield of
0.32 g g glucose−1 was reported for S. uvarum [11] and
0.34 g g−1 for S. cerevisiae H1022 [14]. This respiro-fer-
mentative region of a chemostat culture is also character-
ised by a decrease in the biomass yield to values ranging
from 0.1 [11,13] to 0.21 g g glucose−1 [14]. Steady-state
residual glucose concentrations in the culture typically
remain low until the wash-out dilution rate is approached
[13,20].
Materials and methods
Organisms and maintenance
Saccharomyces cerevisiae strains NRRL Y132, ATCC
4126 (from the culture collection of the Dept of Micro-
biology and Biochemistry, UOFS, Bloemfontein, South
Africa) and S. cerevisiae CBS 8066 (Centraalbureau voor
Schimmelcultures, Delft, the Netherlands) were maintained
on Difco Yeast Nitrogen Base agar slants at 4°C.
Chemostat cultivation
Experiments involving continuous culture were conducted
in a 2-litre stirred tank reactor with a working volume of
700–800 ml (Multigen F-2000; New Brunswick Scientific,
Edison, NJ, USA), fitted with an exhaust gas condenser,
polarographic oxygen electrode (Ingold AG, Urdorf,
Switzerland) and a pH electrode (Mettler Toledo, Halstead,
UK) at 30°C and pH 5.5. Silicone rubber tubing with peri-
staltic pumps (Watson Marlow, Cornwall, UK) was used
for the medium feed and level control by withdrawal of
excess culture from the culture surface. The dissolved oxy-
gen tension (DOT) was regulated at or above 20% of air
saturation by maintaining an aeration rate of 1 L min−1 and
manually adjusting the stirrer speed of the bioreactor within
the range of 400–1000 rpm. The complex medium com-
prised (per litre distilled water): glucose, 20 g; (NH4)2SO4,
5.5 g; KH2PO4, 3.4 g; MgSO4·7H2O, 0.8 g; CaCl2·2H2O,
We found, however, that in aerobic cultures of S. cerevis-
iae strains ATCC 4126 and NRRL Y132 unusually high
steady-state residual sugar levels occurred in the respiro-
fermentative region. This was a significant deviation from
Monod kinetics, considering that the culture was clearly
glucose-limited when grown at dilution rates in the respir-
Correspondence: Professor JC du Preez, Department of Microbiology and
Biochemistry, University of the Orange Free State, PO Box 339, Bloem-
fontein 9300, South Africa. E-mail: dpreezjcȰmicro.nw.uovs.ac.za
Received 9 August 1999; accepted 18 December 1999

Growth kinetics of S. cerevisiae
SH de Kock et al
232
0.05 g; citric acid, 0.25 g; yeast extract (Biolab Diagnostics,
Midrand, South Africa), 3.0 g and trace elements as
reported elsewhere [7]. Silfoamex CF antifoam (SA Sili-
cones, Boksburg, South Africa) or Dow Corning 1520 Sili-
cone antifoam (Dow Corning, Seneffe, Belgium) was added
at 0.75 ml per litre to suppress foaming. Variations of the
above complex medium were used in some experiments,
namely: (a) using double distilled water instead of single
distilled water; (b) without the addition of trace elements,
also using double distilled water; and (c) without citric acid.
Samples were taken at steady state, usually after three to
five residence times, except when shifts from the respir-
atory region to the respiro-fermentative region were carried
out, in which case at least eight residence times were
allowed before sampling. Steady state was defined as a vari-
ation of less than 7% in the culture turbidity for at least
three consecutive residence times with no upward or down-
ward trend. In some experiments exhaust gas analysis was
also used to verify steady state conditions.
The critical wash-out dilution rate, numerically equival-
ent to the maximum specific growth rate (␮max), was deter-
mined by partially washing out a culture adapted to a high
growth rate and measuring the growth rate after interruption
of the medium feed. The specific rates of ethanol pro-
duction (qp) were calculated according to qp = D.p/x, where
p and x are the ethanol and biomass concentrations, respect-
ively, at steady state. The specific rate of glucose assimi-
lation (qs) was calculated as qs = D (sr − s)/x, where s is the
steady-state residual glucose concentration and sr the glu-
cose concentration in the sterile medium reservoir.
ple bottles, each containing 500 ␮l 5 N HCl, and immedi-
ately put on ice. Ethanol concentrations were determined
with a gas chromatograph (model 5710A; Hewlett-Packard,
Atlanta, GA, USA) equipped with a glass column
(1.5 m × 1.5 mm ID), packed with 80–100 mesh Porapak
N (Waters Associates) and with 50 ml nitrogen carrier gas
min−1 at an oven temperature of 165°C. The composition
of the exhaust gas was determined using an infrared Uras
10E CO2 analyser and a paramagnetic Magnos 6G O2 ana-
lyser (Hartmann & Braun AG, Frankfurt, Germany). The
rates of gas exchange were calculated by means of a nitro-
gen balance.
Results and discussion
In agreement with previous reports [3,9], at low dilution
rates respiratory growth occurred with a high biomass yield,
no or very little ethanol production (Ͻ80 mg L−1) and very
little residual glucose in aerobic glucose-limited steady-
state cultures of S. cerevisiae NRRL Y132 (data not
shown), ATCC 4126 and CBS 8066 (Figures 1 and 2,
Shift and pulse experiments
The effect of a nutrient pulse was investigated by addition
of the following nutrients dissolved in 20 ml distilled water
directly into the culture at steady-state in the respiro-fer-
mentative region: (a) (NH4)2SO4, 4.4 g and yeast extract,
2.4 g; (b) KH2PO4, 2.72 g and MgSO4·7H2O, 0.64 g; (c)
trace elements (FeSO4·7H2O, 0.028 g; MnSO4·H2O,
0.0056 g; ZnSO4·7H2O, 0.0088 g; CuSO4·5H2O, 0.0008 g;
CoCl2·6H2O, 0.0016 g; NaMoO4·2H2O, 0.00104 g; KI,
0.0028 g; H3BO3, 0.0016 g; Al2(SO4)3·18H2O, 0.00128 g)
and CaCl2·2H2O, 0.04 g.
Analytical procedures
Periodic samples (20 ml) were taken aseptically and kept
on ice until analyzed. Growth was monitored with a Klett-
Summerson colorimeter (Klett Mfg Co, New York, USA)
fitted with a red filter (No. 66). The cell concentration was
gravimetically determined in duplicate by drying centri-
fuged and washed samples at 105°C. The glucose content
of the culture supernatants was determined with a Sugar
Analyser
equipped with a refractive index detector and a Sugarpack
column (Waters Associates, Milford, MA, USA)
operating at 85°C with an eluent (degassed water) flow rate
of 0.5 ml min−1. Glucose concentrations below 1 g L−1 were
analysed with a d-glucose enzymatic bioanalysis kit (Cat.
No. 716251, Boehringer Mannheim, Mannheim, Germany).
For these enzymatic assays, rapid harvesting of the cells
was essential; therefore, biological activity in the samples
was rapidly inactivated by aspirating them into 30-ml sam-
I
high-performance liquid chromatograph
Figure 1 Steady-state concentrations of biomass (᭿), glucose (ᮀ) and
ethanol (᭜) in aerobic glucose-limited chemostat cultures of S. cerevisiae
ATCC 4126 in a complex medium containing 20 g glucose L−1. The respir-
atory quotient (RQ, ̄), the yield coefficients for biomass (̆) and ethanol
(᭝), as well as the specific rates of glucose (qs, ̃) and oxygen uptake
(qO2, ᭹) and CO2 evolution (qCO2, ᭺) are indicated. The broken line
indicates the residual glucose values predicted by the Monod equation,
using an arbitrary ks value of 100 mg glucose L−1. The wash-out dilution
rate (Dw) is indicated by an arrow. The data points are from eight inde-
pendent experiments.
I
Journal of Industrial Microbiology & Biotechnology

Growth kinetics of S. cerevisiae
SH de Kock et al
233
Table 1 Kinetic and stoichiometric parameters of S. cerevisiae strains
grown in aerobic glucose-limited chemostat cultures at different dilution
rates in a complex medium
Parameter
S. cerevisiae S. cerevisiae S. cerevisiae
ATCC 4126 NRRL Y132 CBS 8066
Respiratory region
D (h−1
)
0.15
0.54
0
0.28
0
0.14
0.52
0
0.27
0
0.19
0.5
0
0.38
0.044
100
Yx/s (g g glucose−1
)
)
Yp/s (g g glucose−1
qs (g g−1 h−1
)
)
qp (g g−1 h−1
E (%)
100
100
Respiro-fermentative region
D (h−1
)
0.38
0.18
0.37
2.15
0.79
47.81
0.44
0.1
0.33
4.59
1.54
36.62
0.41
0.13
0.39
3.09
1.21
98.77
Yx/s (g g glucose−1
)
)
Yp/s (g g glucose−1
qs (g g−1 h−1
)
)
qp (g g−1 h−1
E (%)
ks (mg L−1
Dc (h−1
(h−1
)
60
0.29
0.54
ND
0.29
0.45
40
0.29
0.5
)
␮
)
max
D, Dilution rate.
Dc, Critical dilution rate; the highest D value where the metabolism was
completely oxidative.
E (%), Efficiency of glucose utilisation, (g glucose assimilated g−1 glucose
supplied) ×100.
ks, Saturation constant in Monod equation.
qs, Specific rate of substrate assimilation.
qp, Specific rate of ethanol production.
Yx/s, Biomass yield coefficient.
Y
␮
p/s, Ethanol yield coefficient.
max, Maximum specific growth rate.
Figure 2 Steady-state concentrations of biomass (᭿), glucose (ᮀ) and
ethanol (᭜) in aerobic glucose-limited chemostat cultures of S. cerevisiae
CBS 8066 in a complex medium containing 20 g glucose L−1. The respir-
atory quotient (RQ, ̄), the yield coefficients for biomass (̆) and ethanol
(᭝), as well as the specific rates of glucose (qs, ̃) and oxygen uptake
(qO2, ᭹) and CO2 evolution (qCO2, ᭺) are indicated. The broken line
indicates residual glucose values predicted by the Monod equation, using
an arbitrary ks value of 100 mg glucose L−1. The wash-out dilution rate
(Dw) is indicated by an arrow. Data points are from seven independent
experiments.
ND, Not determined.
0.1 and 0.18 with ethanol yield coefficients of 0.33–0.39
(Table 1). However, in the respiro-fermentative region of
cultures of strains NRRL Y132 and ATCC 4126, up to 10 g
glucose L−1 (ca 50% of the glucose concentration in the
feed) remained unutilised at steady state, resulting in pro-
duction of only 3–4 g ethanol L−1. In anoxic batch cultures
both of these strains produced up to 83 g ethanol L−1 from
200 g glucose L−1 (data not shown); an abnormally low
ethanol tolerance could, therefore, not be the explanation
for this observation.
Table 1). The biomass yield coefficients of the strains were
slightly higher than the theoretical maximum of 0.511 g
g glucose−1 [19], probably because yeast extract in the
medium also served as carbon source for biomass forma-
tion. The cell concentration in the outflow from the vessel
was identical to that of the culture in the vessel, indicating
homogeneous culture removal. The scatter in the biomass
data points resulted mainly from variations in the glucose
content of the many batches of medium used.
At a critical dilution rate, the limited respiratory capacity
of the cells apparently became saturated [17] and with a
further increase in dilution rate the metabolism of the yeasts
shifted towards a respiro-fermentative (oxido-reductive)
metabolism with aerobic ethanol production taking place.
This critical dilution rate, up to where the metabolism was
completely oxidative, namely 0.29 h−1, was similar for
these yeast strains (Table 1). These values were, therefore,
The steady-state residual glucose values predicted for
cultures of S. cerevisiae ATCC 4126 and CBS 8066 by a
transformation of the Monod model, s = ks[D/(␮max − D)],
using an arbitrary ks value of 100 mg L−1 [10] and the ␮
max
values as determined by wash-out experiments (Table 1),
are indicated by broken lines in Figures 1 and 2. Although
with strain CBS 8066 the steady-state residual glucose con-
centrations at high dilution rates were higher than predicted,
this was in sharp contrast to the very high residual glucose
concentrations noted in cultures of the other two strains.
Due to the sharp increase in residual glucose, non-linear
regression using the Monod model could not be used to
determine the ks values. The residual glucose concentration
at a dilution rate equivalent to 0.5 ␮max was, therefore, used
as an estimate of the ks value (Table 1).
between 50% and 65% of the ␮
values of these strains,
max
as determined by wash-out experiments. A previous report
indicated that the critical dilution rate of S. cerevisiae CBS
8066 was 0.38 h−1 [13]. Due to ethanol production, the
respective biomass yield coefficients decreased to between
Trends in the specific rates of oxygen consumption (qO2)
and CO2 production (qCO2) by S. cerevisiae ATCC 4126
and CBS 8066 as a function of dilution rate were similar,
Journal of Industrial Microbiology & Biotechnology

Growth kinetics of S. cerevisiae
SH de Kock et al
234
but the respiratory quotient (RQ) value of strain CBS 8066
in the respiro-fermentative region was about double that of
strain ATCC 4126 (Figures 1 and 2). The qO2 and qCO2
values of both strains gradually increased with an increase
in dilution rate up to the critical dilution rate, with a respir-
atory quotient of just above one (1.19 ± 0.18), indicating
respiratory metabolism. Above the critical dilution rate the
qCO2 value increased sharply, resulting in a high RQ value
which coincided with the formation of ethanol. The slight
decrease in the qO2 values above the critical dilution rate
might have been due to insufficient adaptation or due to the
presence of ethanol. It has been shown that the ‘respiratory
repression’ at high dilution rates could be eliminated after
sufficient adaptation, which required about 50 generations
[1]. It was also shown that ethanol exerted a negative effect
on respiration and could possibly suppress the upper limit
of respiration below the maximal value [16].
Oscillations in the gas exchange values were observed
at low dilution rates. Synchronised metabolic oscillations
in aerobic chemostat cultures is a well-known phenomenon
[12,15,22]. As observed previously, the oscillatory growth
originated spontaneously after the chemostat was started
and it was maintained for as long as the chemostat was
operated [12,18]. This oscillatory behaviour was observed
between dilution rates of 0.126 h−1 and 0.2 h−1 with strain
ATCC 4126 and between 0.135 h−1 and 0.285 h−1 with
strain CBS 8066 (data not shown). An increase in the
dilution rate of the ATCC 4126 culture resulted in a longer
oscillatory cycle time. A similar phenomenon was also
observed with strain CBS 8066, but only up to a dilution
rate of 0.26 h−1, at a dilution rate of 0.285 h−1 the oscillatory
period decreased again.
Figure 3 The concentrations of biomass (᭹), glucose (̄) and ethanol
(̃), dissolved oxygen tension (DOT, ᭺) and RQ values (· · ·) and the
content of O2 (– – –) and CO2 (——) in the exhaust gas over time in an
aerobic, glucose-limited chemostat culture of S. cerevisiae ATCC 4126 at
a dilution rate of 0.167 h−1
.
Oscillations in the dissolved oxygen tension (DOT) and
ethanol concentration correlated with oscillations in the O2
and CO2 content of the exhaust gas (Figure 3). These oscil-
lations, therefore, exerted a slight but significant effect on
the steady-state values reported in this paper. Many cyclic
parameter changes have been reported during oscillatory
growth [6,15]. A 10% change in biomass concentration at
a dilution rate of 0.07 h−1 [22] and a variation of about 25%
in the biomass yield at a dilution rate of 0.1 h−1 [6] during
oscillatory growth have been reported. By contrast, others
found no variation in the biomass concentration under simi-
lar conditions [15].
The physiological difference between strains CBS 8066
and ATCC 4126 also was evident from the transients during
dilution rate shift experiments. A steady-state culture of S.
cerevisiae CBS 8066 at a dilution rate of 0.19 h−1 (in the
respiratory region) was subjected to a one-step shift-up to a
dilution rate of 0.38 h−1 (in the respiro-fermentative region)
(Figure 4). The immediate decrease in biomass concen-
tration, concomitant with an increase in ethanol concen-
tration, was in agreement with previous shift experiments
done with Saccharomyces uvarum H2055 [11]. However,
whereas in our experiments the glucose concentration
increased after about 7 h and again decreased to unde-
tectably low concentrations 8 h later, an almost immediate
increase in glucose concentration was observed with S. uva-
rum H2055 after a shift from a dilution rate from 0.14 h−1
to 0.21 h−1, followed by a decrease after about 6 min [11].
The response of S. cerevisiae ATCC 4126 in a similar
Figure 4 The effect of a dilution rate shift (at time zero) on an aerobic
chemostat culture of S. cerevisiae CBS 8066 from an initial dilution rate
of 0.19 h−1 to 0.38 h−1. The values at time zero are the steady-state values
at a dilution rate of 0.19 h−1. Symbols: biomass (᭹), glucose (᭺) and
ethanol (̄).
Journal of Industrial Microbiology & Biotechnology

Growth kinetics of S. cerevisiae
SH de Kock et al
235
experiment was quite different (Figure 5). The dilution rate
of a steady state culture was increased from 0.14 h−1 to
0.361 h−1. However, a slight lag in ethanol production was
evident. The glucose concentration increased after about
6 h, but in this instance it stabilised at about 8 g L−1,
resulting in the production of only 3.5 g ethanol L−1.
To establish whether the above aberrant chemostat
behaviour might have been due to an unknown nutrient
limitation, several pulse additions of various nutrients (see
Materials and Methods) were made directly into a steady-
state culture of S. cerevisiae ATCC 4126 in the respiro-
fermentative region (data not shown). None of the pulses
elicited either an increase in the ethanol concentration or a
decrease in the high residual sugar concentration. Increas-
ing the glucose concentration in the feed resulted in an
increased biomass concentration with both the ATCC 4126
(Figure 6) and the CBS 8066 (data not shown) strains, con-
firming that the chemostat was operating under a glucose
limitation. Further experiments with variations of the
medium normally used (see Materials and Methods) did not
result in any significant improvement in ethanol production
or sugar assimilation (data not shown). In fact, the residual
glucose concentration decreased only slightly. These results
confirmed that the culture was not limited in respect to
nitrogen, minerals or growth factors (as supplied by the
pulse of yeast extract), nor were high residual glucose lev-
els due to inhibitory effects of citric acid (used as chelating
agent) or the trace elements added.
Figure 6 The steady-state biomass concentration in an aerobic chemostat
culture of S. cerevisiae ATCC 4126 as a function of the glucose concen-
tration in the feed at a dilution rate of 0.25 (±0.02) h−1
.
ted either with high growth rates or with a fermentative (or
respiro-fermentative) metabolism. These results show that
distinct differences in the kinetic behaviour exist between
strains of the same yeast species, which may impact on the
industrial applications of S. cerevisiae. This deviation in
Monod kinetics is obviously of importance in studies of the
kinetics or physiology of S. cerevisiae in carbon-limited
continuous culture. Further experiments are being conduc-
ted to determine whether this phenomenon was due to nutri-
ent limitations other than those tested here by pulse experi-
ments.
A similar increase in the steady-state residual glucose
concentration at dilution rates above 0.18 h−1 was found
with both the ATCC 4126 and CBS 8066 strains in anoxic
chemostat cultures using the same complex medium [4].
Thus, the above aberrant behaviour occurred under both
anoxic and aerobic conditions, and appeared to be associa-
Acknowledgements
This work was supported by a grant from the Foundation
for Research Development. The able technical assistance
of Piet Botes with the chromatographic analyses is grate-
fully acknowledged.
References
1 Barford JP and RJ Hall. 1979. An examination of the Crabtree effect
in Saccharomyces cerevisiae: the role of respiratory adaptation. J Gen
Microbiol 114: 267–275.
2 Barford JP and RJ Hall. 1981. A mathematical model for the aerobic
growth of Saccharomyces cerevisiae with a saturated respiratory
capacity. Biotechnol Bioeng 23: 1735–1762.
3 Beck C and HK von Meyenburg. 1968. Enzyme pattern and aerobic
growth of Saccharomyces cerevisiae under various degrees of glucose
limitation. J Bacteriol 96: 479–486.
4 De Kock SH. 1999. The relationship between nutritional requirements,
membrane transport and continuous culture kinetics of Saccharomyces
cerevisiae strains. PhD thesis. Department of Microbiology and Bio-
chemistry, University of the Orange Free State, Bloemfontein, South
Africa.
Figure 5 The effect of a dilution rate shift (at time zero) on an aerobic
chemostat culture of S. cerevisiae ATCC 4126 from an initial dilution rate
of 0.14 h−1 to 0.361 h−1. The values at time zero are steady-state values
at a dilution rate of 0.14 h−1. Symbols: biomass (᭹), glucose (᭺) and
ethanol (̄).
5 Diderich JA, M Schepper, P van Hoek, MAH Luttik, JP van Dijken,
JT Pronk, P Klaassen, HFM Boelens, MJT de Mattos, K van Dam and
AL Kruckeberg. 1999. Glucose uptake kinetics and transcription of
HXT genes in chemostat cultures of Saccharomyces cerevisiae. J Biol
Chem 274: 15350–15359.
Journal of Industrial Microbiology & Biotechnology

Growth kinetics of S. cerevisiae
SH de Kock et al
236
6 Duboc PH, I Marison and U von Stockar. 1996. Physiology of Sac-
charomyces cerevisiae curing cell cycle oscillations. J Biotechnol 51:
57–72.
7 Du Preez JC and JP van der Walt. 1983. Fermentation of d-xylose to
ethanol by a strain of Candida shehatae. Biotechnol Lett 5: 357–362.
8 Fiechter A, GF Fuhrmann and O Ka¨ppeli. 1981. Regulation of glucose
metabolism in growing yeast cells. Adv Microbial Physiol 22: 123–
183.
9 Ka¨ppeli O, M Arreguin and M Rieger. 1985. The respirative break-
down of glucose by Saccharomyces cerevisiae: an assessment of a
physiological state. J Gen Microbiol 131: 1411–1416.
10 Ka¨ppeli O and B Sonnleitner. 1986. Regulation of sugar metabolism
in Saccharomyces-type yeast: experimental and conceptual consider-
ations. CRC Crit Rev Biotechnol 4: 299–325.
11 Petrik M, O Ka¨ppeli and A Fiechter. 1983. An expanded concept for
the glucose effect in the yeast Saccharomyces uvarum: involvement
of short- and long-term regulation. J Gen Microbiol 129: 43–49.
12 Porro D, E Martegani, BM Ranzi and L Alberghina. 1988. Oscillations
in continuous cultures of budding yeast: a segregated parameter analy-
sis. Biotechnol Bioeng 32: 411–417.
13 Postma E, C Verduyn, WA Scheffers and JP van Dijken. 1989. Enzy-
mic analysis of the Crabtree effect in glucose-limited chemostat cul-
tures of Saccharomyces cerevisiae. Appl Environ Microbiol 55:
468–477.
metabolism of Saccharomyces cerevisiae in continuous culture. FEMS
Microbiol Lett 98: 261–268.
16 Sonnleitner B and U Hahnemann. 1994. Dynamics of the respiratory
bottleneck of Saccharomyces cerevisiae. J Biotechnol 38: 63–79.
17 Sonnleitner B and O Ka¨ppeli. 1986. Growth of Saccharomyces cerevi-
siae is controlled by its limited respiratory capacity: formulation and
verification of a hypothesis. Biotechnol Bioeng 28: 927–937.
18 Sonnleitner B, C Straessle and A Fiechter. 1987. A structured and
segregated model: explanation and description of the stable spon-
taneous synchronization of Saccharomyces cerevisiae at low dilution
rates. In: Proc 4th European Congress on Biotechnology 1987, Vol 3
(Neijssel OM, Van der Meer RR and Luyben KChAM, eds), pp 76–
79, Elsevier Science Publishers BV, Amsterdam.
19 Thomas KC, SH Hynes and WM Ingledew. 1996. Practical and theor-
etical considerations in the production of high concentrations of alco-
hol by fermentation. Process Biochem 31: 321–331.
20 Van Urk H, PR Mak, JP van Dijken and WA Scheffers. 1987. The
question of limited respiratory capacity in Saccharomyces cerevisiae.
In: Proc 4th European Congress on Biotechnology 1987, Vol 3
(Neijssel OM, Van der Meer RR and Luyben KChAM, eds), p 367,
Elsevier Science Publishers BV, Amsterdam.
21 Verduyn C, TPL Zomerdijk, JP van Dijken and WA Scheffers. 1984.
Continuous measurement of ethanol production by aerobic yeast sus-
pensions with an enzyme electrode. Appl Microbiol Biotechnol 19:
181–185.
22 Von Meyenburg HK. 1969. Energetics of the budding cycle of Sac-
charomyces cerevisiae during glucose limited aerobic growth. Arch
Microbiol 66: 289–303.
14 Rieger M, O Ka¨ppeli and A Fiechter. 1983. The role of limited respir-
ation in the incomplete oxidation of glucose by Saccharomyces cerevi-
siae. J Gen Microbiol 129: 653–661.
15 Satroutdinov AD, H Kuriyama and H Kobayashi. 1992. Oscillatory
Journal of Industrial Microbiology & Biotechnology