Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e7, 2012

www.elsevier.com/locate/jbiosc

Kinetics of D-glucose and D-fructose conversion during the alcoholic fermentation

promoted by Saccharomyces cerevisiae

Angela Zinnai, Francesca Venturi, Chiara Sanmartin, Mike F. Quartacci, and Gianpaolo Andrich*

Department of Crop Plant Biology, University of Pisa, Via del Borghetto 80, I-56124 Pisa, Italy

Received 11 June 2012; accepted 10 August 2012

Available online xxx

Although many studies on the different aspects of alcoholic fermentation are available in the literature, it is still
difficult to identify the possible causes of the slowing-down or stuck of fermentations, even if the change of some
compositional parameters (D-glucose/D-fructose and glycerine produced/hexoses converted ratios) could be assumed as
sound signals of a possible deviation from the usual Saccharomyces metabolic pathways. The reason why alcoholic yeasts
preferably metabolise D-glucose rather than D-fructose was investigated by a kinetic model based on six functional
parameters having a well-defined chemicalephysical meaning. The time evolution of different initial concentrations of
D-glucose and D-fructose, dissolved in a model solution simulating a must (citrate buffer at pH 3.4 inoculated by
a commercial strain of Saccharomyces cerevisiae), was investigated adding or not ethanol to the reaction medium. When
a reduced amount of ethanol was dissolved in the reaction medium, the time evolution of the fermentation rates of
these two sugars did not differ significantly, to diversify rather strongly when the alcoholic concentration increased. The
hypothesised mathematical model accounts for this particular kinetic behaviour. In fact, only the sensitivity to ethanol
showed by the enzymatic protein involved in the limiting steps of the fermentation process of these two sugars differed
significantly, the enzymatic transformation of D-fructose being more sensitive to ethanol than D-glucose. This difference
was able to justify the different kinetic behaviours shown by the two sugars when ethanol concentration in the reaction
medium increased.
Ó 2012, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Hexose metabolisation; Kinetic model; Stuck of fermentation; Saccharomyces cerevisiae; Wine; Alcoholic fermentation; Yeast]

As widely reported in the literature, the lack of micro- and
macronutrients necessary for yeasts, unsuitable reaction tempera-
tures, too low pH values, the presence of significant concentrations
of inhibitors (ethanol, phenols, etc.) in the reaction medium, the
development of dangerous microorganisms as well as the alteration
of ionic equilibrium can induce a deep modification of the alcoholic
fermentation kinetics (1,2).
Over the past two decades, wine producers aimed to produce
grapes with increased sugar to total acid ratios to obtain higher
concentrations of phenols and aromatic compounds in order to
increase wine quality. As a consequence, the musts obtained by
these grapes are more difficult to process because they present
unsuitable conditions for yeast reproduction, particularly in the
warmer climates (3). The factors above cited could justify the
increasing number of stuck of fermentations that occurs in several
wine-producing countries, similarly to what observed for beer
fermentation (4).
The cause of the decline of the fermentation rate is not fully
understood. The increase in the alcoholic fermentation rate ob-
tained by the addition of selected yeast strains to the must/wine
* Corresponding author. Tel.: þ39 050 2216624; fax: þ39 050 2216636.
E-mail addresses: azinnai@agr.unipi.it (A. Zinnai), fventuri@agr.unipi.it
(F. Venturi), csanmartin@agr.unipi.it (C. Sanmartin), mfquart@agr.unipi.it
(M.F. Quartacci), gandrich@agr.unipi.it (G. Andrich).
could often result ineffective if the residual sugars are used by
contaminating microorganisms able to carry on unwanted meta-
bolic pathways (5,6). In these conditions some heterofermentative
lactic acid bacteria strains could significantly increase volatile
acidity with a consequent loss of the sensory quality of the alco-
holic beverage. Moreover, favourable conditions for yeasts lysis and
release of intracellular compounds e as it often occurs at the end of
the alcoholic fermentation when reduced amounts of molecular
SO2 are dissolved in the liquid phase e may strongly stimulate the
growth of Brettanomyces spp. (5).
Although many studies on the different aspects of alcoholic
fermentation are available in the literature, it is still difficult to
identify the possible causes of the slowing-down or stuck of
fermentations (7), even if the change of some compositional
parameters (D-glucose/D-fructose and glycerine produced/
hexoses converted ratios) or an accumulation of intermediates of
sugar catabolism could be assumed as sound signals of
a possible deviation from the usual Saccharomyces metabolic
pathways (6).
In particular, it is interesting to find the reason why alcoholic
yeasts preferably metabolise D-glucose rather than D-fructose
(2,8e11), and to investigate the kinetic aspects related to their
conversion in order to optimise specific substrate consumption
rates. Moreover, this kinetic approach appears to be potentially able
to clarify many metabolic aspects related to hexoses conversion

1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2012.08.008

Please cite this article in press as: Zinnai, A., et al., Kinetics of D-glucose and D-fructose conversion during the alcoholic fermentation promoted
by Saccharomyces cerevisiae, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.08.008
2 ZINNAI ET AL.
with the aim to better control alcoholic fermentation and to avoid
that unwanted dangerous transformations take place.
To reduce the large number of variables able to influence the
kinetics of alcoholic fermentation, the time evolution of different
initial concentrations of D-glucose and D-fructose, dissolved in
a model solution simulating a must (citrate buffer at pH 3.4
inoculated by a commercial strain of Saccharomyces cerevisiae),
have been investigated in the presence or not of ethanol.
MATERIALS AND METHODS
The kinetic runs were carried out at 27.0  1.5 C using a 500 mL batch reactor. To
ensure anaerobic and sterilised conditions the whole experimental apparatus was
autoclaved and subjected to three cycles of vacuum following by replacement with
nitrogen sterilised by filtration. Thus, the presence of undesired microorganisms and
the aerobic utilisation of sugars by yeasts were ruled out.
The characteristics of this bioreactor, realised at the Department of Crop Plant
Biology of the University of Pisa, were already reported in a previous paper (12). The
fermentation temperature was maintained constant by a heat exchanger, whereas
the homogeneity of the reaction medium was ensured by a magnetic stirrer. The
bioreactor was initially filled with 250 mL of a citrate buffer aqueous solution
(pH 3.4) containing D-glucose and/or D-fructose (at three different concentrations:
900, 1111, and 1667 mmol L1) added or not with 1120 or 1340 mmol L1 of ethanol,
respectively, that was sterilised by filtration. To the reaction medium containing only
buffer and sugars (and ethanol when added) about 3.1 g (6.2 g/L) of a lyophilised
yeast commercial strain (S. cerevisiae Actiflore BJL source p1, Laffort Oenologie) were
directly added to ensure a number of colony forming units (CFU) ranging from 1010
to 1011. This addition represented the initial time of all kinetic determinations.
The time evolution of both CFU and concentrations of both reagents (D-glucose
and/or D-fructose) and their products (glycerine and ethanol) were evaluated by
a total plate count or utilising specific commercial enzymatic kits (Megazyme),
respectively (13).
The identification of the best values to be assigned to the model parameters was
carried out by the specific statistical programme BURENL (14) able to identify in
a space of j-dimensions (where j is equal to the number of model parameters) the
minimum value of the F function, which is given by the sum of squares of differences
occurring among experimental (Yi, exper.) and calculated (Yi,calc.) data:
X
N 
2
F ¼ Yi;calc:  Yi;exper: (1)
i¼1
where N represents the total number of experimental determinations. The values
assumed by the model parameters at the minimum of the F function represent the
best values.
For each experimental run the calculation of the three parameters related to the
time evolution of the yeast cells (kY, kinetic constant of yeast population inactiva-
tion; [Y]t¼0, yeast density at the initial run time; KY$E, constant related to the
equilibrium occurring between alcoholic yeasts and ethanol) was carried out using
the experimental data deriving from the determination of the microbial density. To
evaluate the kinetic constants related to the time evolution of the hexose under
investigation ([H]t¼0, concentration of hexose initially added to the reaction
medium; kH, specific kinetic activity shown by a single cell; KH$E, constant related to
the equilibrium occurring between ethanol and the enzymatic protein (Penz.)
involved in the rate limiting step of sugar fermentation) the experimental data
concerning hexose (D-glucose) decrease and both ethanol and glycerine accumula-
tions were used.
RESULTS AND DISCUSSION
The kinetics of sugar utilisation by S. cerevisiae during fermen-
tation is largely driven by sugar transport, and glucose is typically
consumed at a faster rate than fructose (15). According to the
literature the ability shown by the yeast population to metabolise
the two sugars largely depends on the temperature and the
compositions of culture media (sugar levels, D-glucose to D-fructose
ratio as well as yeast-assimilable nitrogen) (10,16). In particular, the
synthesis of proteins involved in the transport of sugars into the cell
deeply affects their following utilisation (15).
During winemaking, sugars are consumed mainly during the
stationary phase when the available nitrogen gradually becomes
less available. Since nitrogen is an essential nutrient involved in the
transport of sugars into the cell via protein synthesis, this partially
explains why both yeast replication and fermentation activity slow
Please cite this article in press as: Zinnai, A., et al., Kinetics of D-glucose and D-fructose conversion during the alcoholic fermentation promoted
by Saccharomyces cerevisiae, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.08.008
J. BIOSCI. BIOENG.,
down (16e18). In addition, the alcohol level gradually increases
becoming toxic to the yeast cells, and the use of fructose is even
more compromised (15).
To avoid that yeast replication in the different experimental
conditions could significantly affect the rate of sugar consumption
a high concentration of lyophilised yeasts (6.2 g compared to
0.2 g L1 suggested by the manufacturer) was initially added to the
reaction medium without any preventive rehydration. Thus, the
lack of oxygen and above all the absence of essential nutrients
significantly reduced the yeast replication rate, while the high CFU
number ensured a sensible conversion of sugars added. The
absence of a sigmoidal evolution of the experimental data related to
sugar consumption validates the previous statement. This proce-
dure was adopted to evidence whether ethanol accumulation could
be able to explain the different rates of utilisation of the two sugars.
According to the stoichiometry of alcoholic fermentation, the
sum of the analytical data related to the concentrations of uncon-
verted sugars, accumulated glycerine and half of ethanol formed
did not vary significantly with time, assuming values very close to
the initial concentration of sugar used. As a consequence, a possible
significant accumulation of a different intermediate reaction can be
ruled out.
The analytical points describing the decrease of concentrations
of the two monosaccharides (D-glucose and D-fructose) as a func-
tion of fermentation time when initial concentrations of 200 g L1
(1111 mmol L1) were used are reported in Fig. 1. While during the
first phases of the fermentative process no significant differences
between the metabolisation rates of the two sugars could be
observed, when reaction time increased and ethanol accumulation
became more relevant D-fructose consumption run more slowly
compared to D-glucose, although it reached very low concentration
values, consistent with an almost complete conversion of this
monosaccharide. This phenomenon was more evident when an
initial value of 300 g L1 (1667 mmol L1) of the two sugars was
tested (Fig. 2). In these conditions also D-glucose needed a reaction
time more than double to reach concentration values close to zero
(200 vs. 80 h necessary with an initial amount of 1111 mmol L1).
D-fructose conversion became even more difficult and its concen-
tration assumed with time an asymptotic minimum value so that
about 20% of the initial amount remained unconverted in the
reaction medium. According to the literature, these preliminary
results confirm that ethanol inhibited the activity of the yeast
microbial population which, in its turn, induced an analogous
reduction of the conversion of both sugars. When the concentration
of ethanol in the fermentation medium increased the conversion
rates of the two sugars diversified significantly, D-fructose being
converted more slowly than D-glucose (Figs. 1 and 2).
FIG. 1. Time evolution of the ratio between the concentration of D-glucose and
D-fructose at a random time t ¼ t ([H]t¼t) and the initial value ([H]t¼0) of 200 g L
1
(1111 mmol L1).
VOL. xx, 2012 KINETICS OF HEXOSE CONVERSION IN WINE BY S. CEREVISIAE 3

d½Ht¼t =dt ¼ kP $½Penz: t¼t $½Ht¼t (8)

where kP is the kinetic constant, [Penz.]t¼t is the concentration of

the free form of the enzyme involved in the rate limiting step at the
reaction time t ¼ t, and [H]t¼t represents the concentration of the
hexose at the reaction time t ¼ t. If this inhibitory effect can be
regarded as competitive, the following equilibrium reaction can be
introduced:

E þ Penz: 4E$Penz: (9)

and KH$E (KG$E for D-glucose and KF$E for D-fructose), the constant
related to this equilibrium, will be equal to:
 
KH$E ¼ ½E$Penz t¼t = ½Et¼t $½Penz: t¼t (10)

FIG. 2. Time evolution of the ratio between the concentration of D-glucose and from which:
1
D-fructose at a random time t ¼ t ([H]t¼t) and the initial value ([H]t¼0) of 300 g L
(1667 mmol L1). ½E$Penz: t¼t ¼ ½Penz: t¼t $½Et¼t $KH$E (11)

The total concentration of the enzyme dissolved in the reaction

In the experimental conditions used, the density of total cells medium will be given by the sum of the free enzyme fraction and
([Y]t¼t) able to promote the alcoholic fermentation inevitably the one bound to ethanol:
decreased with time as the reaction medium was devoid of many
components essential for promoting the growth of the yeast pop- ½Penz: tot;t¼t ¼ ½Penz: t¼t þ ½E$Penz: t¼t
ulation (e.g., nitrogen sources for protein synthesis). If a first order ¼ ½Penz: t¼t þ ½Penz: t¼t $½Et¼t $KH$E
kinetic equation is used to describe the time evolution of microbial  
¼ ½Penz: t¼t $ 1 þ ½Et¼t $KH$E (12)
inactivation, the following mathematical relation can be introduced:
As the total concentration of the enzyme involved in the limiting
d½Yt¼t =dt ¼ kY $½Yt¼t (2)
step is proportional to the density of the active fraction of yeasts
This differential equation can be easily integrated and the ([Y*]) present in the reaction medium at that time:
following relation obtained:  
½Penz: tot;t¼t ¼ kPenz $½Y*t¼t ¼ ½Penz: t¼t $ 1 þ ½Et¼t $KH$E (13)
½Yt¼t ¼ ½Yt¼0 $eky$t (3)
it follows that:
Ethanol (E) produced by the alcoholic fermentation might  
½Penz: t¼t ¼ kPenz $½Y*t¼t = 1 þ ½Et¼t $KH$E (14)
interact with yeasts and induce their progressive inactivation by
the following equilibrium reaction: and the rate related to hexose conversion (d[H]t¼t/dt) will be
* equal to:
Y þ E4Y$E (4)
d½Ht¼t =dt ¼ kP $½Penz: t¼t $½Ht¼t
If KY$E represents the constant related to the proposed equilib-  
rium [Y$E]/([Y*]$[E]), and [Y*] the density of yeast cells still active in ¼ kP $kPenz $½Y*t¼t = 1 þ ½Et¼t $KH$E $½Ht¼t (15)
the alcoholic medium, the number of yeast cells inactivated by
As the product of two constants is equal to a new constant
ethanol will be equal to:
(kP$kPenz ¼ kH), the following expression can be obtained:
½Y$Et¼t ¼ ½Y*t¼t $½Et¼t $KY$E (5)  
d½Ht¼t =dt ¼ kH $½Y*t¼t = 1 þ ½Et¼t $KH$E $½Ht¼t (16)
Thus, the total number of microbial cells able to form colonies,
present in the reaction medium at a random time t ¼ t ([Y]t¼t), will In order to describe the time evolution of the concentrations of
be equal to the sum of the active fraction ([Y*]t¼t) and the one reagents (D-glucose and D-fructose) and their products (ethanol and
which interacted with ethanol ([Y$E]t¼t): glycerine) as well as that of yeast density, the following system of
three equations (Eqs. 3, 17 and 18) should be solved:
½Yt¼t ¼ ½Y*t¼t þ ½Y$Et¼t ¼ ½Y*t¼t þ ½Y*t¼t $½Et¼t $KY$E
   
¼ ½Y*t¼t $ 1 þ ½Et¼t $KY$E (6) ½Yt¼t ¼ ½Yt¼0 $eky$t 3

   
and the following relation can be obtained: ½Y*t¼t ¼ ½Yt¼t = 1 þ 2a0E $ ½Ht¼0  ½Ht¼t $KY$E (17)
   
½Y*t¼t ¼ ½Yt¼t = 1 þ ½Et¼t $KY$E ¼ ½Yt¼0 $eky$t = 1 þ ½Et¼t $KY$E    
d½Ht¼t =dt ¼ kH = 1 þ 2a0E $ ½Ht¼0  ½Ht¼t $KH$E $½Y*t¼t $½Ht¼t
(7)
(18)
Ethanol, interacting with an enzymatic protein (Penz.) involved
in one of the several metabolic steps of sugar fermentation (active where a0E represents the fraction of hexose converted to ethanol
transport into the cell, glycolysis, pyruvate decarboxylation and (selectivity to ethanol), whereas the difference ([H]t¼0  [H]t¼t) is
acetaldehyde reduction), may give rise to an inactive form (E$Penz.) the amount of sugar converted at the reaction time t ¼ t. As,
and slow down the rate of a specific step so to make it the limiting according to the stoichiometry of this fermentation, from every unit
step of all the fermentative process. In this case, the metabolisation of hexose converted 2 mol of alcohol are produced, the amount of
rate (d[H]t¼t/dt) of the hexose under investigation would coin- ethanol produced can be easily calculated:
cide with that of the limiting step, and the following equation could  
be introduced: ½Et¼t ¼ 2a0E $ ½Ht¼0  ½Ht¼t (19)

Please cite this article in press as: Zinnai, A., et al., Kinetics of D-glucose and D-fructose conversion during the alcoholic fermentation promoted
by Saccharomyces cerevisiae, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.08.008
4 ZINNAI ET AL. J. BIOSCI. BIOENG.,

To describe the decrease of hexose concentration and the hexose fermentation (CFU L1); [Y]t¼0 is density of yeast viable
accumulation of both ethanol and glycerine with fermentation cells initially (t ¼ 0) present in a liter of fermentation medium
time, the numerical integration of the differential equation intro- (CFU L1); kY is kinetic constant related to yeast inactivation (h1);
duced was carried out. According to this approach the following t is reaction time; a0E is hexose fraction converted to ethanol;
relation can be written: [H]t¼0 is sugar concentration initially (t ¼ 0) present in the
 . reaction medium (mmol L1); [H]t¼t is sugar concentration
d½Ht¼t =dt ¼ ½Ht¼t2  ½Ht¼t1 ðt2  t1 Þ present in the reaction medium at a random time t ¼ t (mmol L1);
n  KY$E is constant related to the equilibrium occurring between
0
¼ kH = 1 þ 2aE $ ½Ht¼0
ethanol and alcoholic yeasts (L mmol1); kH is kinetic constant
 o
 ½Ht¼t1 $KH$E $½Y*t¼t2 $½Ht¼t1 (20) related to hexose conversion (h1 L CFU1); KH$E is constant
related to the equilibrium occurring between ethanol and the
hence: enzymatic protein (Penz.) involved in the limiting step of sugar
n  fermentation (L mmol1); and a00Gly is hexose fraction converted to
½Ht¼t2 ¼ ½Ht¼t1  kH = 1 þ 2a0Ε $ ½Ht¼0 glycerine.
 o When a further decrease of the Dt value does not induce any
 ½Ht¼t1 $KH$E $½Y*t¼t2 $½Ht¼t1 $ðt2  t1 Þ (21) significant difference on the time evolution of the active fraction of
yeast as well as on the concentrations of reagents and products, the
If t2  t1 ¼ Dt, then t2 ¼ t1 þ Dt. Thus, the following sequence of numerical integration can be assumed to supply a realistic picture
equations is involved in the iterative calculation connected to the of the process. Thus, knowing the initial conditions (t1 ¼ 0;
numerical integration: ½Yt¼t1 ¼ ½Yt¼0 ; ½Ht¼t1 ¼ ½Ht¼0 ) it is possible to determine the
evolution of all the involved components ([Y]t¼t, [H]t¼t, [E]t¼t and
t2 ¼ t1 þ Dt (22) [G]t¼t).
The identification of the best values to assign to the eight
    functional parameters involved in the proposed kinetic model was
½Yt¼t2 ¼ ½Yt¼0 $eky$t2 = 1 þ 2a0E $ ½Ht¼0  ½Ht¼t1 $KY$E (23) carried out using the experimental data describing the time course
of yeast population ([Y]t¼t), hexose consumption ([H]t¼t), as well
n   o
½Ht¼t2 ¼ ½Ht¼t1  kH = 1 þ 2a0E $ ½Ht¼0  ½Ht¼t1 $KH$E $½Yt¼t2 as ethanol ([E]t¼t) and glycerine ([Gly]t¼t) accumulations (Figs. 1
and 2). The values of the eight functional parameters calculated
 ½Ht¼t1 $Dt (24) by BURENL are reported in Table 1 as a function of the hexose
utilised and of its initial concentration.
  Although the kinetic constant of the time evolution of yeast
½Et¼t2 ¼ 2a0E $ ½Ht¼0  ½Ht¼t2 (25)
population (kY) was not affected by a high variability, the squares of
the correlation coefficients of the microbial evolution showed
 
½GY t¼t2 ¼ a00Gly $ ½Ht¼0  ½Ht¼t2 (26) a remarkable variation ranging from 0.30 to 0.96 (Table 1). The
number of yeast cells did not change significantly during some
experimental runs and this could be the main reason of the rela-
½Ht¼t1 ¼ ½Ht¼t2 (27) tively low values of the squares of correlation coefficients. On the
other hand, the high values assumed by the squares of correlation
coefficients (rG 2 , r 2 , r 2 and r 2 ) related to the time evolution of
t1 ¼ t2 F E Gly
D-glucose, D-fructose, ethanol and glycerine concentrations
where [Y]t¼t is density of yeast viable cells present in a liter of (Table 1) confirm the suitability of the kinetic equations as well as
fermentation medium at a random time t ¼ t able to promote the validity of the hypothesis introduced.

TABLE 1. Values assigned to the functional parameters involved in the kinetic model used to describe alcoholic fermentation following the addition to the reaction medium of
glucose and fructose at two different initial concentrations in the absence of ethanol.

Parameter G G F F

[Y]t¼0 (3.41  0.01)$1011 (1.90  0.01)$1011 (4.72  0.01)$1011 (1.63  0.01)$1011

[G]t¼0 1023.9  0.1 1659.8  0.2 e e
[F]t¼0 e e 1159.2  0.1 1782.0  0.1
a0E 0.90  0.01 0.85  0.01 0.90  0.01 0.85  0.01
a00Gly 0.14  0.01 0.14  0.01 0.14  0.01 0.16  0.01
kY (5.61  2.23)$107 (5.61  4.01)$107 (5.62  2.32)$107 (5.61  2.23)$107
KY$E (4.48  3.08)$106 (4.48  3.36)$106 (4.54  1.75)$106 (4.54  2.15)$106
kG (8.70  0.04)$1014 (7.23  0.03)$1014 e e
KG$E (0.97  0.08)$104 (1.32  0.05)$104 e e
kF e e (8.75  0.01)$1014 (6.73  0.02)$1014
KF$E e e (9.57  0.03)$104 (9.34  0.05)$104
rY2 0.37 0.96 0.30 0.89
2
rG 0.95 0.98 e e
rF2 e e 0.97 0.98
rE2 0.99 0.98 0.99 0.99
2
rGly 0.96 0.98 0.93 0.94

Data are expressed as means  confidence intervals (p ¼ 0.05). Y, yeast active fraction; G, glucose; F, fructose; E, ethanol; Gly, glycerine; a0E , hexose fraction converted to
ethanol; a00Gly , hexose fraction converted to glycerine; kY, kinetic constant related to yeast inactivation; KY$E, constant related to the equilibrium occurring between ethanol and
yeasts; kG, kinetic constant related to glucose conversion; KG$E, constant related to the equilibrium occurring between ethanol and the enzymatic protein involved in the
limiting step of glucose fermentation; kF, kinetic constant related to fructose conversion; KF$E, constant related to the equilibrium occurring between ethanol and the
enzymatic protein involved in the limiting step of fructose fermentation; r, correlation coefficients related to the time evolution of D-glucose, D-fructose, ethanol and glycerine
concentrations.

Please cite this article in press as: Zinnai, A., et al., Kinetics of D-glucose and D-fructose conversion during the alcoholic fermentation promoted
by Saccharomyces cerevisiae, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.08.008
VOL. xx, 2012 KINETICS OF HEXOSE CONVERSION IN WINE BY S. CEREVISIAE 5

The kinetic constants representing the time evolution of the

yeast population (kY) did not change significantly as a function of
the monosaccharide used (Table 1) and showed a similar sensitivity
to ethanol (compare the values of the KY$E constant). In all runs
a slight decrease of yeast populations was observed (from an initial
1010e1011 to a final 108e109 CFU mL1). Also the kinetic constants
related to the fermentation rates of the two sugars (kG and kF) did
not vary significantly and assumed very similar values. As easily
predictable, the production of ethanol (a0E ) and glycerine (a00Gly ) did
not change with the fermentation substrate. Only the sensitivity to
ethanol showed by the enzymatic protein involved in the limiting
steps of the two sugars differed significantly, the enzymatic
transformation of D-fructose being more sensitive to ethanol than
D-glucose. This difference, by itself, would be able to justify the
different time evolutions shown by the two sugars as demonstrated
by the high degree of overlap between experimental and calculated
values (Figs. 3A, B and 4A, B) and by the high values (Table 1) of the
squares of correlation coefficients (rG2 , r 2 , r 2 and r 2 ) connected to
F E Gly
the reagents and products.
To verify how the presence of ethanol in the reaction medium
can justify the different experimental behaviours shown by the two
monosaccharides, ethanol was initially added to the aqueous
solution of the two sugars (1120 and 1340 mmol L1 for glucose and
fructose, respectively). Table 2 reports the values calculated for the
model parameters when yeasts were added to a hydroalcoholic
solution. The evolution with fermentation time of reagents and
products are shown in Fig. 5A, B and the good degree of overlap
between calculated and experimental values give a measure of the
capacity shown by the model to describe the time evolution of
reagents and products. Moreover, the high values of the squares of
correlation coefficients confirm the suitability of the mathematical
FIG. 4. Experimental points (squares, sugar; triangles, ethanol; circles, glycerine) and
model to describe the time evolution of the species involved in the theoretic values calculated by the kinetic model as a function of fermentation time. (A)
fermentation process also when ethanol was initially added to the [D-glucose]t¼0 ¼ 1667 mmol L1; (B) [D-fructose]t¼0 ¼ 1667 mmol L1.
reaction medium.

TABLE 2. Values assigned to the functional parameters involved in the kinetic model
used to describe alcoholic fermentation following the addition to the reaction
medium of glucose and fructose in the presence of ethanol.

Parameter GþE FþE

10
[Y]t¼0 (8.75  0.02)$10 (2.01  0.01)$1011
[G]t¼0 1099.5  0.1 e
[F]t¼0 e 1094.2  0.2
[E]t¼0 1120.9  0.1 1344.5  0.2
a0E 0.86  0.01 0.90  0.01
a00Gly 0.14  0.02 0.16  0.01
kY (6.40  3.27)$107 (6.40  4.23)$107
KY$E (5.49  2.32)$106 (4.51  3.32)$106
kG (8.55  0.03)$1014 e
KG$E (1.73  0.03)$104 e
kF e (8.34  0.03)$1014
KF$E e (9.47  0.03)$104
rY2 0.45 0.76
2
rG 0.99 e
rF2 e 0.96
rE2 0.98 0.95
2
rGly 0.91 0.93

Data are expressed as means  confidence intervals (p ¼ 0.05). Y, yeast active

FIG. 3. Experimental points (squares, sugar; triangles, ethanol; circles, glycerine) and
theoretic values calculated by the kinetic model as a function of fermentation time. (A)
[D-glucose]t¼0 ¼ 1111 mmol L1; (B) [D-fructose]t¼0 ¼ 1111 mmol L1.
fraction; G, glucose; F, fructose; E, ethanol; Gly, glycerine; a0E , hexose fraction con-
verted to ethanol; a00Gly , hexose fraction converted to glycerine; kY, kinetic constant
related to yeast inactivation; KY$E, constant related to the equilibrium occurring
between ethanol and yeasts; kG, kinetic constant related to glucose conversion; KG$E,
constant related to the equilibrium occurring between ethanol and the enzymatic
protein involved in the limiting step of glucose fermentation; kF, kinetic constant
related to fructose conversion; KF$E, constant related to the equilibrium occurring
between ethanol and the enzymatic protein involved in the limiting step of fructose
fermentation; r, correlation coefficients related to the time evolution of D-glucose,
D-fructose, ethanol and glycerine concentrations.

Please cite this article in press as: Zinnai, A., et al., Kinetics of D-glucose and D-fructose conversion during the alcoholic fermentation promoted
by Saccharomyces cerevisiae, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.08.008
6 ZINNAI ET AL. J. BIOSCI. BIOENG.,

initially added to the reaction medium, are the average of only three
values.
The two kinetic constant kG and kF, which were expected to
assume different values as a function of the sugar utilised, did not
differ significantly (Tables 1 and 2). This particular situation might
find a possible explanation assuming that in the presence of very
low concentrations of ethanol, the limiting step of both mono-
saccharides remained the same, being connected to one of the
sugar fermentation reactions. Only when the concentration of
ethanol in the reaction increased significantly, its effect on the
transformation rates of these two sugars became more relevant.
The hypothesised mathematical model accounts for this
particular kinetic behaviour. In fact, the kinetic constants related to
0 0
D-glucose (kG ) and D-fructose (kF ) conversions are equal to the ratio
between a constant kH connected to the limiting step common to
the two sugars, and the sum of 1 and the concentration of ethanol
multiplied by a constant the value of which varies significantly as
a function of the monosaccharide:
 
k0G ¼ kH = 1 þ ½EtOHt¼t $KG$E (28)

and
 
k0F ¼ kH = 1 þ ½EtOHt¼t $KF$E (29)

At low ethanol concentrations (1 þ [EtOH]t¼t$KG$E w 1) the rate

limiting steps of the two sugars coincide, and the following situa-
tion occurs:
 
k0G ¼ kH = 1 þ ½EtOHt¼t $KG$E ¼ kH =f1 þ 0$KG$E g ¼ kH (30)

 
FIG. 5. Experimental points (squares, sugar; triangles, ethanol; circles, glycerine) and k0F ¼ kH = 1 þ ½EtOHt¼t $KF$E ¼ kH =f1 þ 0$KG$E g ¼ kH (31)
theoretic values calculated by the kinetic model when ethanol was initially added to
the reaction medium. (A) [D-glucose]t¼0 ¼ 1111 mmol L1 plus 1120 mmol L1 ethanol;
(B) [D-fructose]t¼0 ¼ 1111 mmol L1 plus 1340 mmol L1 ethanol.
thus, according to what experimentally found, k0G ¼ k0F ¼ kH .
On the contrary, when ethanol concentration increases the rate
The parameters related to yeast evolution (kY and KY$E) and those limiting steps related to the transformations of the two sugars are
connected to the conversion of the two sugars (kG, KG$E, kF, and KF$E) connected to different partial transformations. In particular, the
did not differ significantly from the ones reported in Table 1. rate limiting step concerning D-fructose conversion might be linked
Ethanol addition was able to modify the kinetics of fructose either to the active transport of this sugar from the reaction
fermentation immediately after its addition when the replication medium into the yeast cell or to its following isomerisation to
rate of yeast cells can be surely disregarded. Thus, the effect of produce D-glucose. In this case:
ethanol is able to justify the alteration of fructose metabolisation  
before a significant modification of the protein synthesis respon- k0G ¼ kH = 1 þ ½EtOHt¼t $KG$E skH (32)
sible of an altered transport of this sugar could occur.
 
Table 3 reports the mean values of the parameters involved in the k0F ¼ kH = 1 þ ½EtOHt¼t $KF$E skH (33)
kinetic model calculated by the elaboration of the experimental runs
previously reported. While for the first four parameters (a0E , a00Gly , kY, Moreover, as the KF$E constant is greater than the one related to
KY$E) the values represent the average of six data, the last two D-glucose (KF$E/KG$E ¼ 9.46$104/1.34$104 w 7), the ratio between
(KG$E, KF$E), which are strongly related to the monosaccharide the two kinetic constants:
  
TABLE 3. Values of the functional parameters involved in the kinetic model used to k0F =k0G ¼ 1 þ KG$E $½EtOHt¼t 1 þ KF$E $½EtOHt¼t
describe alcoholic fermentation calculated by elaboration of experimental runs.   
¼ 1 þ KG$E $½EtOHt¼t 1 þ 7$KG$E $½EtOHt¼t (34)
Parameter Units
a0E 0.88  0.05 e will decrease when the concentration of ethanol increases and thus
a00Gly 0.15  0.02 e with fermentation time.
kY (5.87  0.82)$107 h1 L CFU1 The mean values of the model parameters were used to calcu-
KY$E (4.67  0.81)$106 L mmol1 lated the theoretical evolution of the components involved in an
kH (8.05  1.75)$1014 h1 L CFU1
experimental run where active cells of the commercial strain of
KG$E (1.34  1.11)$104 L mmol1
KF$E (9.46  0.34)$104 L mmol1 S. cerevisiae (w7$1011 CFU L1) were added to an aqueous solution
containing an equal amount of the two sugars (900 mmol L1). The
Data are expressed as means  confidence intervals (p ¼ 0.05). Y, yeast active
fraction; G, glucose; F, fructose; E, ethanol; Gly, glycerine; a0E , hexose fraction con- evolution with time of experimental and calculated points are re-
verted to ethanol; a00Gly , hexose fraction converted to glycerine; kY, kinetic constant ported in Fig. 6. The high degree of overlap and the high values of
related to yeast inactivation; KY$E, constant related to the equilibrium occurring the squares of correlation coefficients of the three species involved
between ethanol and yeasts; kH, kinetic constant related to hexose conversion; KG$E, 2 ¼ 0:99; r 2 ¼ 0:98; r 2 ¼ 0:98) give a measure of the suitability
(rG F E
constant related to the equilibrium occurring between ethanol and the enzymatic
protein involved in the limiting step of glucose fermentation; KF$E, constant related
of the kinetic model, which could be effectively used to describe the
to the equilibrium occurring between ethanol and the enzymatic protein involved in time evolution of reagents and products involved in the alcoholic
the limiting step of fructose fermentation. fermentation.

Please cite this article in press as: Zinnai, A., et al., Kinetics of D-glucose and D-fructose conversion during the alcoholic fermentation promoted
by Saccharomyces cerevisiae, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.08.008
VOL. xx, 2012
FIG. 6. Time evolution of both theoretical and experimental points (squares, D-fructose;
rhombus, D-glucose; triangles, ethanol) of equivalent amounts (900 mmol L1) of
D-glucose and D-fructose, and the corresponding accumulation of ethanol. The theo-
retical values were calculated by the kinetic model and using the mean values of the
functional parameters reported in Table 3.
In conclusion, ethanol accumulation in the reaction medium
was able to justify the different metabolisation rates observed for
the two sugars (D-glucose and D-fructose) involved in the alcoholic
fermentation, while at low ethanol concentration the rates related
to the two sugar conversion did not differ significantly. When
alcohol concentration increased the D-glucose metabolisation rate
became faster than fructose one. The hypothesised mathematical
model accounts for this particular kinetic behaviour. In particular,
at low ethanol concentration the two sugars have a same limiting
step located in the common part of their pathways, while when
alcohol concentration increased the rate limiting step of fructose
metabolisation appears to be located in a step different of that of
D-glucose. This step is likely located along the active transport of the
sugar through the cellular membranes of yeasts or in its isomer-
isation to produce D-glucose.
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by
ViewSaccharomyces
publication stats cerevisiae, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.08.008
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