Process Biochemistry 38 (2003) 1451
/1456

www.elsevier.com/locate/procbio

Ethanol and ethyl acetate production during the cider fermentation

from laboratory to industrial scale
Carlos de la Roza, Adriana Laca, Luis A. Garca, Mario Daz *
Department of Chemical Engineering and Environmental Technology (I.U.B.A.), University of Oviedo, c/Julian Clavera 8, 33006 Oviedo, Spain
Received 23 July 2002; received in revised form 6 December 2002; accepted 24 December 2002

Abstract
Biomass, sugars and ethanol are the main compounds involved in beverage fermentation processes, although many other minor
components, such as esters, also play important roles in the flavour and taste of the final product. These compounds were monitored
during cider fermentations carried out on laboratory, semipilot and pilot plant scales (100 ml, 13 and 125 l, respectively) and even
during a cider industrial fermentation (45 000 l). The behaviour was similar except for fermentations carried out in Erlenmeyer flasks
whose ethanol/sugar yield factor was slightly lower and the ethyl acetate concentration achieved was too high due to the different
geometry that gave rise to different metabolic pathways. Simple models are developed to predict the evolution of these compounds
during the process and they are successfully applied to simulate experimental results.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Ethanol; Ethyl acetate; Cider fermentation; Modelling; Saccharomyces cerevisiae ; Scale-up

1. Introduction
During the cider fermentation process, the apple must
suffers a large amount of biochemical transformations
whose control is fundamental in obtaining cider of high
quality. The two main bioprocesses are the alcoholic
fermentation and the malolactic fermentation [1,2].
During the alcoholic fermentation, yeast transforms
the majority of sugars (fructose, glucose and sucrose)
into ethanol and CO2 by the Embden
/Meyerhof
/
Parnas pathway [3]. This is the fundamental bioreaction,
but it is not the only one and at the same time numerous
secondary products are formed. From an organoleptic
point of view, the most important of these compounds
are the organic acids, the higher alcohols and the esters
[4]. The last ones have a significant influence on cider
aroma, although some of these are present only in small
concentrations. Most of these esters are formed at the
beginning of the fermentation and decrease or remain
stable towards the end of the fermentation [4].

* Corresponding author. Tel.: /34-98-5103439; fax: /34-985103434.

E-mail address: mdf@sauron.quimica.uniovi.es (M. Daz).

Quantitatively, the main ester is ethyl acetate derived

from the ethanolysis of acetyl Co-A [3,5] (Fig. 1a). In
contrast to other ethyl esters of higher molecular weight,
which are desirable elements of the aroma of wines,
ethyl acetate confers a disagreeable odour [4]. Amerine
et al. [6] have reported that an ethyl acetate concentration over 200 mg/l negatively influences wine quality.
The same occurs in cider, with ciders that contain large
amounts of ethyl acetate which seems to enhance the
intensity of the acetic aroma and flavour [3,5,7] being
considered of low quality. The biosynthesis of esters is
affected by several factors [4], such as the aeration of the
must, the fermentation temperature, the technique of
fermentation and even the maturity of the fruits. Taking
all the above comments into account, it seems clear that
the production of these volatile compounds should be
controlled, and that good models to predict the ester
production would be useful in selecting the best operational conditions, for fermentation.
Several models have been developed over the last 20
years to predict ethanol production by yeasts from
different natural or synthetic media [8
/12]. However,
regarding real industrial fermentation processes the
number of existing works decreases. In this sense, beer
is probably the beverage whose production process has

0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0032-9592(03)00026-8

1452

C. de la Roza et al. / Process Biochemistry 38 (2003) 1451
/1456

Nomenclature
Ce
ethanol concentration (mol/l)
Cea
ethyl acetate concentration (mol/l)
Cs
sugar concentration (mol/l)
Cx
biomass concentration (mol/l)
ke
kinetic parameter (Eqs. (2) and (3)) (l/mol day)
kea
kinetic parameters (Eqs. (2)
/(4)) (l2/mol2 day)
kx
kinetic parameter (Eq. (1)) (day 1)
re
ethanol generation rate (mol/l day)
rea
ethyl acetate generation rate (mol/l day)
rs
sugar consumption rate (mol/l day)
rx
biomass generation rate (mol/l day)
t
kinetic parameter (Eq. (1)) (l/mol)
Ye/s
proportion of sugar transformed into ethanol and ethyl acetate (mol/mol)
been most studied and several models have even been
proposed to simulate the production of other minority
products such as esters, diacetyl or fusel alcohols [8,13
/
15]. Nevertheless, mathematical models of this kind
have not been specifically prepared for describing a cider
elaboration process. In fact, although a large number of
investigations regarding the microbiological process
have been carried out [1
/3,5,7], the number of works
regarding modelling of cider fermentation is almost nonexistent. Taking into account that a model is a fundamental tool to control the process and thus the final
quality of cider, in the present paper the modelling of
biomass growth, sugar consumption, ethanol and ethyl
acetate production has been raised in a simple way. The

model has also been tested on different scales, from the

laboratory to the industrial level.

2. Materials and methods

Four different types of bioreactors were employed
[16]: 250 ml capacity Erlenmeyer flasks containing 100
ml of apple must were placed in an orbital incubator at
100 rpm; semipilot and pilot plant bioreactors, with a
capacity of 13 and 125 l and a height of 1.4 and 4 m
respectively, made of glass with a cylindrical geometry,
with no mechanical agitation; and finally an industrial
cylinder
/conical bioreactor placed horizontally, with a

Fig. 1. (a) Scheme of some processes that take place during cider fermentation; (b) Simplified scheme of sugar consumption, ethanol and ethyl
acetate production during cider fermentation.

C. de la Roza et al. / Process Biochemistry 38 (2003) 1451
/1456

capacity of 45 000 l and a height of 4 m. This last

fermentation was carried out in the cider factory
Escanciador, S.A. (Villaviciosa, Principado de Asturias,
Spain). In the last three cases, samples were taken from
the point of the fermenter that was considered the most
representative. The temperature was maintained at
15 8C, except for the industrial fermentation, where
the temperature evolved freely in the range 15
/27 8C.
The culture medium was prepared from concentrated
apple juice supplied by Escanciador, S.A. This was
reconstituted with distilled water (1:6) to a final density
of approximately 1060 g/l, and sterilised in a tangential
flow filtration device (0.33 mm pore diameter) except for
the industrial fermentation.
The microorganisms used were a commercial active
dry yeast strain of Saccharomyces cerevisiae subsp
bayanus (strain Pasteur Institute, Paris, 1969, Champagne, supplied by Novo Ferment, Switzerland) and a
malolactic bacterium (strain Lc2), previously isolated in
the cellars of the collaborating factory, identified as an
Oenococcus oeni strain, selected on the basis of its ability
to degrade malic acid [16]. An active dried preparation
of yeast was rehydrated in sterile apple juice and grown
under aerobic conditions at 250 rpm, 25 8C, for 18 h.
The apple must was then inoculated with yeast to a final
concentration of 107 CFU/ml (colony forming units per
ml), this inoculation level being similar to that employed
in the industrial fermentation. Malolactic bacteria were
also grown in apple juice for 7 days, but at 37 8C and
without shaking due to their microaerophilic nature. To
start malolactic transformation the medium was inoculated with malolactic bacteria, adjusted to 107 CFU/ml.
In the fermentations carried out in Erlenmeyer flasks
and semipilot plant bioreactor the inoculation of the
lactic acid bacteria was sequential, taking place at the
end of the alcoholic fermentation (after about 3 weeks).
The pilot plant bioreactor was inoculated simultaneously with the yeast and the bacteria at the beginning
of fermentation, while in the case of the industrial
fermentation, only the yeast was inoculated since the
malolactic bacteria were already present inside the
fermenter.
Yeast growth was monitored by measuring the optical
density at 660 nm. The fermentable sugars of the liquid
medium were analysed by HPLC (Waters, Milford, MA,
USA, Alliance 2690), with a differential refractometer
(Waters 410). A Spherisorb-NH2 analytical column
(20 /0.4 cm2, 5 mm, Technokroma, Barcelona, Spain)
was used under the following conditions: column
temperature 30 8C, mobile phase 80/20 acetonitrile/
water, 0.9 ml/min, detector temperature 45 8C and
injection volume 10 ml.
Ethanol and ethyl acetate were analysed using a gas
chromatograph (GC-14B, Shimadzu) equipped with a
FID detector and an auto injector (AOC-20i, Shimadzu), fitted with a Supelcowax 10 column (Supelco)

1453

(60 m/0.25 mm i.d., phase thickness 0.25 mm). The

chromatographic conditions were as follows: initial
temperature 40 8C for 10 min; program rate 4 8C/min
to 80 8C, 80 8C for 10 min; program rate 35 8C/min;
final temperature 200 8C for 15 min. Finally, the
column temperature was equilibrated at the initial
temperature for 20 min until the next injection. Injector
and detector temperatures were 200 and 230 8C, respectively; the carrier gas being He at 150 kPa; the volume
injected, 5 ml.

3. Results and discussion

3.1. Results at the laboratory level and modelling
Cider fermentations were carried out in Erlenmeyer
flasks containing 100 ml of must, these are the fermentation conditions usually employed when the process is
studied on a laboratory scale [2] (temperature, 15 8C;
agitation, 100 rpm; inoculation level, 107 CFU/ml). Fig.
2 show the evolution of biomass, ethanol, sugars and
ethyl acetate concentrations expressed in mol/l. For
biomass, it was considered that the molecular weight of
the yeast was 23.9 g/mol [17] and for sugars it was
considered 180 g/mol, since at the pH of the medium,
sucrose breaks down readily into glucose and fructose.
The stationary phase of cell growth was reached at the
fifth hour, increasing approximately one logarithmic
unit from the inoculation level [16]. However, it is not
before the 20th hour that all the sugar is consumed and

Fig. 2. Evolution of some important compounds during a cider

fermentation carried out in Erlenmeyer flasks. (a) Cell growth (");
(b) Sugar consumption (j), ethanol production (') and ethyl acetate
production (m). Experimental data (symbols) and model results (line).

C. de la Roza et al. / Process Biochemistry 38 (2003) 1451
/1456

1454

the maximum ethanol concentration achieved, thus

completing the alcoholic fermentation.
Regarding ethyl acetate production, it seems that it is
produced at the beginning of the fermentation remaining stable afterwards [4]. The amount of ethyl acetate
produced during this fermentation was high when it was
compared to values reported elsewhere. Beech et al. [5]
and Williams [3] have reported, ethyl acetate concentrations of 35 and 15 ppm, respectively in cider and LafonLafourcade [4] reported 190 ppm as the maximum
concentration of ethyl acetate found in wines. These
values are much lower than the ethyl acetate concentration of 450 ppm achieved in the present experiment. This
difference will be discussed later.
3.1.1. Kinetic model
A simplified model for the kinetic description of sugar
consumption and ethanol and ethyl acetate production
was established. The hypotheses of the model are: (a) the
fermentation medium is homogeneous; (b) no substrate
or products are accumulated significantly inside the cell;
(c) the concentration of intermediate compounds remain
approximately constant (steady-state approximation).
Thus, it can be assumed that ethanol and ethyl acetate
are synthesised directly from the monosaccharides.
Besides, it is assumed that ethanol concentration
influences the ethyl acetate formation rate, since it
seems logical if Fig. 1a is observed. From these
hypotheses, a simplified scheme that describes the
process is shown in Fig. 1b.
Regarding cell growth, the experimental results fit the
denominated Ricattis equation [13]. Therefore, the
kinetic equations describing the system will be:
Biomass

rx kx Cx (1tCx )

Sugar rs (ke Cs Cx kea Cs Ce Cx )

(1)
1

Ye=s
Ethanol re 2ke Cs Cx 2kea Cs Ce Cx
Ethyl acetate rea 2kea Cs Ce Cx

(2)
(3)
(4)

where rx, re and rea are biomass, ethanol and ethyl

acetate generation rates, respectively; rs is sugar consumption rate; Cx, Cs, Ce and Cea are biomass, sugar,
ethanol and ethyl acetate concentrations, respectively;
kx, t , ke and kea are kinetic parameters. The t constant is
the reciprocal of the stationary phase biomass concentration. The parameter Ye/s has been introduced in Eq.
(2) to take into account the sugar that is consumed to
produce biomass or other products and it corresponds
to the proportion of sugar (moles per 1 mol) that is
transformed into ethanol and ethyl acetate. It is
supposed that Ye/s remains constant during fermentation. Concentrations are expressed in mol/l and time in
days.
The Ethanol generation rate from sugars is much
higher than the ethyl acetate generation rate, since

ethanol concentrations in cider are much higher than

ethyl acetate concentrations. Therefore, it can be
assumed that ke will be much higher than kea, and
Eqs. (2) and (3) can be simplified resulting Eqs. (5) and
(6):
Sugar rs ke Cs Cx

(5)

Ye=s

Ethanol re  2ke Cs Cx

(6)

Experimental data have been simulated by solving the

system of Eqs. (1), (4)
/(6). As can be observed in Fig. 2,
the model fits the experimental results well, with the
exception of the first ethyl acetate data, since the model
predicts concentrations lower than those found experimentally. The fitting parameter values are reported in
Table 1. The ke value is approximately 100-fold the kea
value, thus the simplification carried out in Eqs. (2) and
(3) can be assumed as being correct.
A simpler model has also been tested, considering that
ethanol concentration does not affect ethyl acetate
generation rate and results were also rather good.

3.2. Scaling-up and model application

The same parameters have been monitored during
cider fermentations carried out on semipilot, pilot and
industrial scales (13, 125 and 45 000 l, respectively). Fig.
3a shows biomass evolution. In pilot and semipilot
fermentations, stationary cell growth was achieved
around the fifth hour, as occurred in flask fermentations. Industrial fermentation was faster, with the
stationary phase of cell growth being reached at the
third hour and a higher cell concentration being
achieved. The explanation seems to be the slightly
higher inoculation level that tooks place in the latter
together with the fact that the temperature was also
higher. Cell concentration increased approximately one
logarithmic unit from the inoculation level as in the
other cases. Small differences observed between cell
growth when fermentations were carried out in flasks,
semipilot and pilot reactors, may be due to differences in
the agitation power and in the particular geometry of
each system [16].
Table 1
Fitting parameters for the Erlenmeyer flask, semipilot, pilot and
industrial reactor fermentations

kx (day 1)
t (l/mol)
ke (l/mol day)
kea (l2/mol2 day)
Ye/s

Erlenmeyer

Semipilot

Pilot

Industrial

1.77
2.21
0.268
0.00203
0.88

0.58
2.78
0.755

/
0.99

0.86
7.12
0.930
0.000162
0.93

1.70
1.34
0.810
0.000272
0.95

C. de la Roza et al. / Process Biochemistry 38 (2003) 1451
/1456

1455

Fig. 3. Evolution of some important compounds during cider fermentations carried out in semipilot ("), pilot (') and industrial (j) reactors. (a)
Cell growth; (b) Sugar consumption; (c) Ethanol production; (d) Ethyl acetate production. Experimental data (symbols) and model results (line).

The evolution of sugar and ethanol concentrations

can be observed in Fig. 3b and c for the three scales. In
fermentations carried out in semipilot and pilot reactors,
sugar was depleted at around the 15th hour and
maximum alcohol concentration was also achieved at
the same time, the process being slightly faster than in
the flask fermentations. In the industrial case, alcoholic
fermentation finished around the seventh hour. As
expected, the process was faster, since temperature and
cell concentration were higher as previously observed.
Regarding ethanol/sugar yield factors (2Ye/s), it was 1.76
(mol/mol) for the flask fermentations, being higher than
1.85 for the rest of the cases [16].
When fermentation was carried out in pilot and
industrial reactors, ethyl acetate was produced until
sugars were totally depleted (Fig. 3d), remaining stable
afterwards. The ethyl acetate concentrations achieved
were 12 and 37 ppm, respectively, for the pilot and the
industrial fermentation, values similar to those reported
by other authors [3
/5]. The abnormally high ethyl
acetate values observed in the flask fermentations, and
the fact that the yield factor was the lowest in this case,
might be due to the different geometry of the fermenter.
As in Erlenmeyer flasks the air/must ratio was higher
than in the other fermenters, it would be possible for a
part of the yeast to develop an oxidative metabolic
pathway, giving rise to lower ethanol and higher ethyl
acetate concentrations [4,16].
The model previously developed has been applied to
semipilot, pilot and industrial scales. The model fits
experimental results well as can be observed in Fig. 3,
except for the sugar concentration in the pilot plant
reactor where the model predicts a slightly slower
consumption. The fitting parameter values are reported

in Table 1. The kinetic parameters for ethanol formation, ke, are similar for the semipilot, pilot and industrial
fermentations, however the values differ from the flask
fermentations one. The same occurs with kea that
presents a value much higher for the flask fermentations. The values of these parameters seem to confirm
the different behaviour previously observed that took
place in flask fermentations.
The proposed model allows, for a determined geometry and temperature, predicting in an approximate
way the evolution of the main fermentation compounds,
cells, sugar and ethanol, and also the evolution of an
important minority compound, such as ethyl acetate,
only by knowing the initial sugar concentration and the
cell inoculation level.

References
[1] Beech FW. English cider making: technology, microbiology and
biochemistry. Prog Ind Microbiol 1972;11:133
/213.
[2] Herrero M, Roza C, Garca LA, Daz M. Simultaneous and
sequential fermentations with yeast and lactic acid bacteria in
apple juice. J Ind Microbiol Biot 1999;22:48
/51.
[3] Williams AA. Flavour research and the cider industry. J Inst Brew
1974;80:455
/70.
[4] Lafon-Lafourcade S. Wine and brandy. In: Rehm HJ, Reed G,
editors. Biotechnology, vol. 5. Germany: Weinheim, 1983.
[5] Beech FW, Carr JG. Cider and perry. In: Rose AH, editor.
Economic microbiology. Alcoholic beverages, vol. I. London:
Academic Press, 1971:139
/314.
[6] Amerine MA, Berg HW, Kunkee RE, Ough CS, Singleton UL,
Webb AD. Technology of wine making. Westport, Connecticut:
The AVI Publishing Company, 1982.
[7] Beech FW, Davenport RR. The role of yeast in cider making. In:
Rose AH, Harrison JS, editors. The yeast, vol. 3. London:
Academic Press, 1970:11.

1456

C. de la Roza et al. / Process Biochemistry 38 (2003) 1451
/1456

[8] Engasser JM, Marc I, Moll M, Duteurtre B. Kinetic modelling of

beer fermentation. Proc EBC Congress 1981;59:579
/83.
[9] Jones ST, Korus RA, Admassu W, Heimsch RC. Ethanol
fermentation in a continuous tower fermentor. Biotechnol Bioeng
1984;26:742
/7.
[10] Oliveira SC, De Castro HF, Visconti AES, Giudici R. Continuous
ethanol fermentation in a tower reactor with flocculating yeast
recycle: scale-up effects on process performance, kinetic parameters and model predictions. Bioprocess Eng 1999;20:525
/30.
[11] Pons MN, Pfeil J, Muller M, Corriou JP. Optimal control of a
fedbatch alcoholic fermentation on molasses under uncertain
initial conditions. Adchem 1991;91:252
/6.
[12] Starzak M, Krzystek L, Nowicki L, Michalski H. Macroapproach
kinetics of ethanol fermentation by Saccharomyces cerevisiae :

[13]
[14]
[15]

[16]

[17]

experimental studies and mathematical modelling. Chem Eng J

1994;54:221
/40.
Garca AI, Garca LA, Daz M. Fusel alcohols production in beer
fermentation processes. Process Biochem 1994;29:303
/9.
Garca AI, Garca LA, Daz M. Modelling of diacetyl production
during beer fermentation. J Inst Brew 1994;100:179
/83.
Garca AI, Garca LA, Daz M. Prediction of ester production in
industrial beer fermentation. Enzyme Microb Tech 1994;16:66
/
71.
de la Roza C, Laca A, Garca LA, Daz M. Stirring and mixing
effects at different cider fermentation scales. Trans IChemE
2002;80(Part C):129
/34.
Atkinson B, Mavituna F. Biochemical engineering and biotechnology handbook. Surrey, England: McMillan Publisher Ltd,
1983.