Acta Biotechnol.

35 (1993) 4, 31 1- 322 Akademie Verlag

Kinetics of the Selective Fermentation of Glucose from Glucose/

Fructose Mixtures Using Saccharomyces cereuisiae ATCC 36859

KOREN,D. W., DUVNJAK,

Z.*

University of Ottawa
Faculty of Engineering
Department of Chemical Engineering
Ottawa, Ontario, Canada

Summary
The kinetics of batch fermentation during the growth of S. cereuisiae ATCC 36859 was studied in
various glucose/fructose mixtures. It was found that the growth is inhibited equally by glucose and
fructose even though fructose is not consumed to any large extent by the yeast under the conditions
tested here. The inhibition of growth by the substrate and ethanol is represented by linear equations.
These equations were combined with the MONODexpression in order to formulate equations for the
biomass growth, glucose and fructose consumption and ethanol production. Parameter estimates were
obtained by fitting these equations to batch fermentation data and so developing models which indicate
that the growth is completely inhibited when 62 g/l ethanol is produced by the yeast, while glucose
consumption and ethanol production continue up to an ethanol concentration of 152 g/l. Products
containing a high concentration of fructose. are best produced by using a high initial biomass
concentration.

Introduction

It has already been shown that S. cereuisiue ATCC 36859 is capable of selectively fermenting
glucose from glucose/fructose mixtures [I, 2, 31. Using this method glucose is converted to
ethanol which is more easily separated from fructose than glucose. It was shown that the
presence of fructose in the medium along with glucose inhibits growth and ethanol
fermentation. As expected, ethanol also inhibits the growth of the yeast [4]. In order to
quantitate these relationships and thus determine the capabilities of this process, it is
necessary to establish kinetic equations for the growth, the substrate consumption and
product formation rates.
In the literature many kinetic studies on ethanol fermentation have been reported [5, 6, 71.
These studies have provided expressions for the growth and ethanol production rates which
account for the inhibitory effects of added ethanol. These authors have found that inhibition

* Adress for correspondence: University of Ottawa

Faculty of Engineering
Department of Chemical Engineering
770 King Edward Ave
Ottawa, Ont., Canada, K I N 9B4
312 Acta Biotechnologica 13 (1993) 3

was of the non-competitive type. AIBAet al. [5] included an exponential ethanol inhibition
term with the MONODexpression in order to model the kinetics of ethanol production; the
following relationships were obtained

In addition to the product, the substrate can have an inhibitory effect on the ethanol
fermentation. STREHAIANO et al. [8] and MOULINet al. [9] have reported on the inhibition
by the substrate. The effect is related in part to osmotic phenomena in which plasmolysis
of the yeast cell begins to occur in low water content media (10). GHOSEand TYAGI[7]
observed a strong inhibition when hydrolyzed bagasse was fermented. The effect was
attributed to the presence of large amounts of glucose as well as unfermentable substances
such as xylose, cellobiose and cellotriose in the medium which lowered the water activity.
Therefore, a sugar inhibition term was included in the final expression for the ethanol
fermentation of bagasse hydrolyzate. A linear ethanol inhibition term was also necessary.
The rate equations were

v = v, (1 - +)(
pmax K:
S
+ s + s2/Kfw’ (4)

Bearing in mind that S. cerevisiae ATCC 36859 is used for the conversion of glucose from
glucose/fructose mixtures and that a strong inhibition by ethanol and sugars was noticed,
the proposed objective of this work is to fomulate kinetic equations for the biomass, glucose,
fructose, and ethanol concentration. There has been no work on the kinetics of fermentation
by S. cerevisiue ATCC 36859 and since it is a mutant it may be different from that of the
wild strain already reported. The relationships will take into account the inhibitory effects
of the substrate (glucose + fructose) and ethanol.

Materials and Methods

The media used for the growth of the inoculum of S. cereuisiae ATCC 36859 consisted of glucose
(10.0 g), yeast extract (30.0 g), peptone (3.5 g), KH,PO, (2.0g), MgSO, (1.0 g). (NH,),SO, (1.0 g)
and distilled water (up to 1 litre). The fermentations were carried out in a medium similiar to that
used for inoculum preparation except that various glucose and fructose concentrations were used.
Tests were carried out in ERLENMEYER flasks in a shaker which was kept at 33 “C.
Fructose and glucose concentrations were determined with a Waters High Performance Liquid
Chromatograph. A Sugar Pak I column was used; the mobile phase was deionized water while the
column was maintained at 80 “C. The ethanol concentration was determined enzymatically using
alcohol dehydrogenase [I I]. Biomass concentration was measured after overnight drying in an oven.
Rate equations for the biomass, glucose, fructose, and ethanol concentrations were formulated after
considering the inhibitory effects of the substrate and the product. Since some of the parameters are
common to two or more of the responses they are estimated by combining information from all the
responses and applying “Multiresponse Parameter Estimation” procedures. Software developed at
the University of Ottawa’s Chemical Engineering Department [ 121 has been used to perform this
analysis.
KOREN,D. W., DWVNJAK,
Z., Kinetics of the Selective Fermentation of Glucose 313

Results and Discussion

Firstly, the effect of various sugar and product concentrations on the specific growth rate
is studied. This is done by carrying out a series of batch experiments in media containing
different concentrations of glucose, fructose and ethanol. The specific growth rates are
calculated from the logarithmic phase of growth. The relationships found, are then used
to formulate rate equations for the biomass, glucose, fructose, and ethanol concentrations.
Finally, estimates of the parameters are obtained by analyzing batch fermentation data
which were measured in media containing different glucose/fructose mixtures.

Specific Growth Rate

Effect of Carbohydrate Concentration

Increased glucose concentrations result in a decrease in the growth rate of the yeast (Tab. 1).
A similiar result occurs when the fructose concentration in media containing z 65 g/1
glucose is increased from 0.0 to 129 g/1 (Tab. 1). Even though fructose is not consumed to
any large extent by the yeast in glucose/fructose mixtures [l], it exerts an inhibitory effect
on the growth similiar to that caused by an increase in the glucose concentration.
In order to establish a more quantitative relationship between the specific growth rate and
the glucose and fructose concentrations, further experiments were performed in various
glucose and glucose/fructose mixtures. The experimentally determined specific growth rates
are plotted as a function of the total initial carbohydrate (glucose + fructose) concentration
in Fig. 1. The relationship between the two variables appears linear; therefore, the following
relationship is proposed
jl = pm(1 - As, - Bs,) (5)
where p and j l m are the specific and maximum specific growth rates, sg and sf are the initial
glucose and fructose concentration and A and B are the empirical constants. This model
will quantitatively determine the inhibition due to glucose and that due to fructose. When
the equation was fitted to the data, the parameter estimates and their 95% confidence
intervals are: pm = 0.249 (+ 0.012) h-'; A = 1.57 x (k6.47 x I/g and B = 1.68
x l o v 3(k 2.31 x lo-"). From a consideration of these parameter estimates, it can be
suggested that glucose and fructose inhibit the growth equally. This was also shown to be
the case for a wild strain of S. cerevisiae which consumes both glucose and fructose at
approximately the same rate [13]. In the latter case this was explained in terms of the

Tab. 1. Specific growth rates calculated in various glucose and glucose/fructose media

Initial Total Specific growth rate

Glucose Fructose
[SPI [g/ll [gPl [h - 'I
70.1 - 70.1 0.255
96.4 - 96.4 0.241
125.4 - 125.4 0.214
187.9 - 187.9 0.198
65.8 33.9 99.7 0.23 1
67.3 66.5 133.8 0.216
62.6 129.3 191.9 0.190
314 Acta Biotechnologica 13 (1993) 3

.u
=” 0.10
0)
n
v)
0.05

0 100 200 300 400 500 600

Total carbohydrate I g / l l

Fig. 1. Specific growth rate as a function of total initial carbohydrate concentration

decrease in the water activity caused by the increased amount of solids in the medium.
Glucose and fructose cause similiar decreases in the water activity.
With the assumption that glucose and fructose inhibit the growth equally, the following
simplified expression can be formulated which models the relationship between the specific
growth rate and the total carbohydrate concentration

where s, is the total carbohydrate concentration and s, is the total carbohydrate

concentration above which growth no longer occurs. The parameter estimates obtained
when the data are fitted to Eq. 6 are: p m = 0.251 (+ 0.013)h-’ and smax= 602.8
(+ 66.0) g/l. This model is shown in Fig. 1 as the solid line. If extrapolated, the model
predicts that no growth will occur when the total carbohydrate concentration is equal to
or higher than 603 g/l.

Effect of Ethanol Concentration

Fig. 2 shows that an increase in the initial ethanol concentration results in a decrease in
the growth rate. The experimentally determined specific growth rates are plotted as a
function of the initial ethanol concentration in Fig. 3. The results suggest a linear relationship
between the growth rate and the initial ethanol concentration. The following GHOSEand
TYAGI[7] model was fitted to the experimental data

where p is the ethanol concentration and pmaxis the maximum ethanol concentration above
which growth no longer occurs. The parameter values obtained when Eq. 7 was fitted to
the data were: pm = 0.188 (+ 0.019) h-’ and pmax = 87.5 (k 11.8) g/l. This equation is
shown in Fig. 3 as the solid line. The dashed line in Fig. 3 was obtained by fitting an
exponential equation of the type proposed by AIBAet al. [5]. GHOSEand TYAGI’S equation
KOREN, Z., Kinetics of the Selective Fermentation of Glucose
D. W., DUVNJAK, 315

10’

0 7 14 21 20 35
Time Chl

Fig. 2. Effect of ethanol concentration on the growth of S. cereuisiue ATCC 36859 in

media containing 90 g/l glucose
Ethanol
0 = 0.7 g/l
0 = 31.9g/l
A = 47.9 g/1
+ = 64.1 g/l

I I I I
0 15 30 45 60 75 90
Initial ethanol I g / l I

Fig. 3 . Specific growth rate as a function of initial ethanol concentration

(-) GHOSE and TYAGI[7]
(--) AIBAet al. [5]

predicts that little or no growth will occur when the initial ethanol concentration is above
88 g/l, this is in good agreement with the values of 87 and 93 g/1 which were reported by
GHOSEand TYAGI[7] and BAZUAand WILKE[6], respectively, using a wild strain of S.
cerevisiae. AIBA’Smodel predicts an assymptotic decrease in the growth rate as the initial
ethanol concentration is increased. In this respect the linear equation is more realistic.
316 Acta Biotechnologica 13 (1993) 3

Differential Analysis of Data

From the results presented thus far it is evident that increases in the glucose, fructose and
ethanol concentrations result in a linear decrease in the specific growth rate. Using this
information, equations for the growth, glucose consumption and product formation rates
can be formulated.
After a brief lag phase
dx
- -- P X
dt
where x = biomass concentration. Combining the MONODequation

with Eq. 8., the following expression is obtained for the growth rate

In the present case, inhibition by the substrate and product can be accounted for by linear
terms

as was suggested by the previous results (Eqs. 6 and 7).

In ethanol fermentations the formation of product results directly from the energy
metabolism; therefore, a reasonable kinetic approximation to the fermentation is

where %/: is the product yield coefficient and yXIs the cell yield coefficient. Using these
relationship, the rate equations for the glucose consumption and ethanol production rates are

where shax and pkaxare the maximum total carbohydrate and ethanol concentrations above
which glucose consumption and ethanol production ceases. The extra parameters are
introduced since it has been reported by others [5,7] that glucose consumption and ethanol
production rates are inhibited differentlythan the growth rate by the substrate and product.
Experimental continuous process results [3] have shown that the ratio of the glucose to the
fructose consumption rate is relatively constant when the glucose concentration is higher
than 20g/l. A proportionality constant (R) is thus introduced to relate the fructose
consumption rate to the glucose consumption rate
KOREN,D. W., DUVNJAK,
Z., Kinetics of the Selective Fermentation of Glucose 317

Since some of the parameters are common to two or more of the responses (e.g. A/,, K,,
sk,,, pkax, Y& better estimates of the parameters can be obtained if all the responses are
fitted to the data simultaneously [14] using “Multiresponse Parameter Estimation”.
Information required for this estimation procedure were: the rate equations (i.e. equations
11, 13, 14, 15) and the derivatives of these equations with respect to each of the responses
(i.e. dx/dt, ds$dt, ds,/dt, dpjdt) and with respect to each of the parameters (i.e. x, sg, sf,p ) .
Experimental data required were: initial biomass, glucose and fructose concentrations as
well as biomass, glucose, fructose, and product concentrations at various times during the
fermentation. This analysis is unique in that it uses biomass, substrate and product data
simultaneously to fit the rate equations. Traditional analysis involves using only biomass
data to fit a biomass rate equation, substrate data to fit a substrate rate equation and
product data to fit a product rate equation. In this respect, the approach taken here is
more realistic since it attempts to take into account interactions between these parameters.
The purpose of this analysis is to obtain a model for the biomass, substrate and product
concentrations as a function of time.
Data collected from a series of experiments, carried out in media containing glucose
(66- 161 g/l) and fructose (88-344 gp), were analyzed. The parameter estimates and the
95% confidence intervals, obtained when the responses were fitted to the data, are provided
in Tab. 2.
The value obtained for pm in this analysis (i.e. 0.241 h-I) is lower than the values of 0.4
and 0.448 reported by CHOSE and TYAGI[7] and BAZUAand WILKE[6], respectively, which
were obtained using the wild strain of S. cerevisiae. The saturation constant for the mutant
strain is 4.56 g/l; this is much higher than the values reported by GHOSEand TYAGI[7] and
BAZUAand WILKE161 of 0.48 and 0.24 g/l, respectively.
It was previously shown (Eq. 7) that the growth rate would be reduced to zero when the
initial ethanol concentration was increased to 88 g/l. In this analysis the ethanol
concentration above which growth is predicted to be zero (p,,,,,) is 62 g/l. This difference
may be due to the source of ethanol in the two cases. Previously, the inhibition due to
externally added ethanol was measured, while in the analysis carried out here the inhibition
caused by the ethanol produced by the yeast is measured. Externally added ethanol, therefore,
does not inhibit the growth as strongly as does ethanol produced by the yeast. This was
also reported by HOPPEand HANSFORD [15] using a wild strain of S. cereuisiae. It should
also be noted that the growth is more strongly inhibited by ethanol than the glucose
consumption and ethanol production rates (i.e. pmax< pkax).This was found to be the case
for most microbes [16]. GHOSEand TYAGI[7] reported values of 87 and 114 g/1 for pmax
and p’,,,,,, respectively.

Tab. 2. Converged parameter estimates and confidence intervals using data collected
from glucose/fructose experiments

Parameter estimates Confidence intervals

upper lower

Pln 0.241 h- * 0.260 0.222

K, 4.56 g/1 7.43 1.69
Pmax 62.0 pi1 65.8 58.2
Smax 488.4 g/l 510.6 466.3
K / P 0.140 g/g 0.157 0.124
YP/* 0.466 g/g 0.475 0.457
P k X 152.0 g/l 191.8 112.2
S A X 788.9 g/l 899.9 677.9
R 22.7 41.1 4.29
318 Acta Biotechnologica 13 (1993) 3
- 200

- 160:
\
m
Y

-
P 0)

-
>-120 ;
YI

-
0
2
L
Y
g- 80 o.
a f
-
-"a
40*
X

1 0
0 3 6 9 12 15 18 21 24
Time t h l

Fig. 4. Experimental (symbols) and predicted (lines) results from a fermentation

containing 161 g/l glucose and 91 g/l fructose

The total carbohydrate concentration above which growth ceases (smaJ obtained in this
analysis is 488 g/l, which is in good agreement with the value of 500 g/l reported by JONES
and GREENFIELD [ 131. As was the case for ethanol inhibition, total carbohydrates inhibits
the growth more strongly than the glucose consumption and ethanol production rates (i.e.
Smax < &ax).
The biomass yield (r,,)
reported here is higher than the values of 0.09 and 0.10 reported
by GHOSEand TYAGI[7] and BAZUAand WILKE[6],respectively. This may be due to the
higher yeast extract concentration that was used in the media of the present study. The

10
1250

8
-
c
\
m
- 6
bl
y1

0
E
.-
m 4
0

Jo
Fig. 5. Experimental (symbols) and predicted (lines) results from a fermentation
containing 68 g/1 glucose and 222 g/l fructose
KOREN,D. W., DUVNJAK,
Z., Kinetics of the Selective Fermentation of Glucose 319
10 50 - 300

8 40
- 250 --
-.
- =. - 200 $
\
cn
d

2 6 30 2
-
-”
0

a
2 - r=
Y)

0 150
5
z 4 20 3
z 0

0 a -roo:
-”
0
=I

2 1c - 50 x
0

0 - 0
320 Acta Biotechnologica 13 (1993) 3

0.8

Fig. 8. Specific ethanol production rate as a function of glucose concentration at different

fructose concentrations predicted by Eq. 14
Fructose
1 = 0.0 (g/l)
2 = 100.0 (g/l)
3 = 200.0 (g/l)
4 = 300.0 (g/l)

Some plots comparing experimental and predicted concentrationsof biomass, glucose, fructose,
and ethanol are shown in Figs. 4,5 and 6. The predicted values diverge from the experimen-
tal values when a lag phase is present; this is especially evident when the carbohydrate con-
centration is above 310 g/1 (Fig. 6). This is expected since the model used here assumes
exponential growth. The final biomass concentrations predicted by the model are within 12%
of the experimental values when the total carbohydrate concentration is below 3 10 g/l; above
this concentration an error of up to 21 YOis measured. The final ethanol and fructose concen-
trations predicted are within 12% of the experimental values for all the tests examined.
Since the kinetic models derived here provide reasonable estimates of the experimental
data, they can be used to predict the specific growth and ethanol production rates at
different glucose and fructose concentrations (Figs. 7 and 8). From Fig. 7 it can be seen
that an increase in the glucose concentration initially results in an increase in the specific
growth rate, as expected from MONOD’Sexpression. The specific growth rate eventually
levels off and decreases as the glucose concentration is increased further. This figure also
shows the effect of increasing fructose concentration. If an initial medium contains 150 g/1
glucose and either 100, 200 or 300 g/1 fructose, the specific growth rate will be reduced by
30, 59 and 89%, respectively, compared to the value predicted in a medium containing no
fructose. Under the same conditions, the specific ethanol production rate will be reduced
only by 16, 31 and 47%, respectively (Fig. 8). Therefore, although high fructose
concentrations significantly inhibit growth; ethanol production is affected much less.
In order to produce a syrup with a high concentration of fructose, the inhibitory effects on
the growth rate must be considered. Taking into account the models’ predictions, the
recommended scheme would involve the growth of a large amount of biomass in a medium
containing a low glucose concentration. The biomass is then transferred to a medium
containing a high glucose/fructose concentration where little or no growth will take place,
but the high biomass concentration will ensure a high production rate of ethanol.
KOREN, Z., Kinetics of the Selective Fermentation of Glucose
D. W., DUVNJAK, 321

Conclusion

Carbohydrates and ethanol inhibit the growth in a linear fashion. By combining the MONOD
equation with the inhibitory terms for carbohydrate and ethanol, rate equations were
formulated for the growth, glucose and fructose consumption and ethanol production rates.
Parameter estimates were obtained by fitting these equations to batch fermentation data
using a “Multiresponse parameter estimation” technique. The models predict that the
biomass growth and ethanol production rates will be zero when 62 and 152 g/l ethanol are
produced by the yeast. An initial total carbohydrate concentration of 488 g/l totally inhibits
the growth, while the ethanol production will cease when the total carbohydrate
concentration is 789 g/l.

Acknowledgements
The authors would like to thank Dr. D. MCLEAN, S . BOUDRICA
M. GUAYfor the advice and assistance
provided with the computer software used in this study.

Nomenclature

A, B empirical constants
K, saturation constant for growth [g/l]
K: saturation constant for ethanol production [g/l]
K,, K, empirical constants
ethanol concentration [g/l]
ethanol concentration above which growth no longer occurs [g/l]
ethanol concentration above which glucose consumption and ethanol production cease [g/l]
ratio of glucose to fructose consumption rates
glucose concentration [g/l]
fructose concentration [g/l]
carbohydrate concentration above which growth no longer occurs [g/l]
carbohydrate concentration above which glucose consumption and ethanol production cease
[gill
total carbohydrate (glucose + fructose) concentration [g/l]
time [h]
specific growth rate [h-’]
maximum specific growth rate [h-’]
specific ethanol formation rate [h-’1
maximum specific ethanol formation rate [h-’]
degree of carbohydrate inhibition of growth rate
degree of carbohydrate inhibition of product rate
biomass concentration [g/l]
cell yield coefficient [g biomass produced/g carbohydrate consumed]
product yield coefficient [g product produced/g carbohydrate consumed]

Received 4 January 1993

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