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Research Article

Received: 8 February 2010

Revised: 30 April 2010

Accepted: 1 May 2010

Published online in Wiley Interscience:

(www.interscience.wiley.com) DOI 10.1002/jctb.2441

Kinetic study on ethanol production using


Saccharomyces cerevisiae ITV-01 yeast
isolated from sugar cane molasses
a Octavio Carvajal-Zarrabal,b
Benigno Ortiz-Muniz,
Beatriz Torrestiana-Sanchezc and Maria Guadalupe Aguilar-Uscangac
Abstract
BACKGROUND: Bio-ethanol production from renewable sources, such as sugar cane, makes it a biofuel that is both renewable and
environmentally friendly. One of the strategies to reduce production costs and to make ethanol fuel economically competitive
with fossil fuels could be the use of wild yeast with osmotolerance, ethanol resistance and low nutritional requirements. The
aim of this work was to investigate the kinetics of ethanol fermentation using Saccharomyces cerevisiae ITV-01 yeast strain in a
batch system at different glucose and ethanol concentrations, pH values and temperature in order to determine the optimum
fermentation conditions.
RESULTS: This strain showed osmotolerance (its specic growth rate (max ) remained unchanged at glucose concentrations
between 100 and 200 g L1 ) as well as ethanol resistance (it was able to grow at 10% v/v ethanol). Activation energy (Ea) and
Q10 values calculated at temperatures between 27 and 39 C, pH 3.5, was 15.6 kcal mol1 (with a pre-exponential factor of
3.8 1012 h1 (R2 = 0.94)) and 3.93 respectively, indicating that this system is biologically limited.
CONCLUSIONS: The optimal conditions for ethanol production were pH 3.5, 30 C and initial glucose concentration 150 g L1 . In
this case, a maximum ethanol concentration of 58.4 g L1 , ethanol productivity of 1.8 g L1 h1 and ethanol yield of 0.41 g g1
were obtained.
c 2010 Society of Chemical Industry

Keywords: temperature; activation energy; pH; glucose; ethanol; S. cerevisiae ITV-01

NOTATION
max :
0 :
Ea:
R:
T:
Q10 :
T2 :
T1 :
2 :
1 :
Yx/s:
Yet/s:
Yet/x:
Pet:

Maximum specic growth rate (h1 )


Pre-exponential factor (h1 )
Activation energy (kcal mol1 )
Ideal gas constant (kcal mol1 K1 )
Temperature (K)
The activity of a microorganism.
The higher temperature.
The lower temperature.
The specic growth rate at the higher temperature.
The specic growth rate at the lower temperature.
Biomass yield (g biomass g1 sustrate).
Ethanol yield (g ethanol g1 sustrate).
Ethanol specic yield (g ethanol g1 biomass).
Ethanol productivity (g ethanol L1 h1 ).

recent years, several investigations have been carried out in order


to establish some yeast characteristics such as osmotolerance,
ethanol resistance, tolerance to low pH and high temperature in
order to improve alcohol fermentation yields.6
Like any other microorganism, yeasts have high water requirements in order to grow and maintain metabolic activity.
Osmotolerance is the ability of yeast to grow in media with high
solute concentrations or in a low water activity environment.
There are reports on yeast strains able to grow at high substrate
concentration (around 250 g L1 ), however, the specic ethanol
production rates were rather low.6 8 A maximum substrate concentration of 200 g L1 has been recommended9 because under

INTRODUCTION
Alcohol fermentation has been widely studied owing to its
economic relevance in beverage, chemical and biofuel industries.
However, technical issues during ethanol production such as
those related to contamination by lactic acid bacteria and
Brettanomyces yeasts,1,2 inhibition by ethanol and high substrate
concentration3 5 still remain unclear. In addition, temperature
is not controlled at an industrial scale because of high costs. In

J Chem Technol Biotechnol (2010)

Correspondence to: Maria Guadalupe Aguilar-Uscanga, Instituto Tecnologico


de Veracruz-UNIDA. Av. Miguel A. de Quevedo 2779, Col. Formando Hogar. CP.
91860. Veracruz, Ver. Mexico. E-mail: gaguilar@itver.edu.mx

a Instituto Tecnologico Superior de Tierra Blanca, Av. Veracruz s/n. Col. PEMEX,
95180 Tierra Blanca, Veracruz, Mexico
b Universidad Veracruzana, Area Bioqumica y Qumica de la Nutricion, Juan
Pablo II s/n, Boca del Ro, Ver. C.P. 94294, Mexico
c Instituto Tecnologico de Veracruz, Unidad de Investigacion y Desarrollo en
Alimentos (UNIDA), Veracruz, Mexico

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c 2010 Society of Chemical Industry




et al.
B Ortiz-Muniz

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this stress condition stimulation of glycerol production, needed to
maintain water activity inside the cell, has been observed.8
On an industrial scale it is recommended to perform yeast cell recycling in order to improve process productivity. However, ethanol
is a toxic compound that increases permeability and uidity of
the plasma membrane, causing viability loss.10 Additionally, high
ethanol concentration can cause important metabolic changes in
yeasts such as ATPase inhibition, denaturation of several glycolitic
enzymes11 and changes in the cell wall.12
Tolerance to low pH values and high temperature are related,
since pH changes can increase temperature tolerance to the
maximum.13 Therefore, it is important to study the independent
effect of temperature, as well as the synergistic temperaturepH
effect. Temperature tolerance has also been related to media
composition and other physical factors such as water activity.
High temperatures cause a decrease in cell viability, as well as
changes in mitochondria and uidity of the plasma membrane.14
In this regard, the use of an osmotolerant, ethanol resistant, low
pH value tolerant and thermotolerant strain can help the control
of alcohol fermentation spoilage, due to the fact that on a large
scale this process is carried out under non-sterile conditions.
Therefore the aim of this research was to study the kinetics of
ethanol fermentation from glucose by Saccharomyces cerevisiae
ITV-01 isolated from sugar cane molasses. The effect of various
fermentation variables such as pH, temperature, initial sugar and
ethanol concentration on the kinetics of ethanol fermentation was
also investigated.

EXPERIMENTAL
Microorganism
The wild type yeast S. cerevisiae ITV-01 was previously isolated
from sugar cane molasses.7
Culture media
S. cerevisiae was stored at 4 C using a culture media with the
following composition (g L1 ): glucose, 150; yeast extract, 20;
and agar, 20. Preculture media was (g L1 ): glucose, 100250,
at 50 intervals; yeast extract, 12.5, at 0.5 intervals; KH2 PO4 , 8.0:
(NH4 )2 SO4 , 5.0; and MgSO4 7H2 0, 1.0. In order to evaluate ethanol
resistance, two initial ethanol concentrations were tested (3 and
6% w/v), and added after media sterilization. The initial pH was
adjusted to the desired value from 2.0 to 6.5, at intervals of 0.5 pH
units, using 80% (v/v) orthophosphoric acid solution. The culture
medium was sterilized for 15 min at 121 C.
Preculture conditions
The preculture was made in a 250 mL Erlenmeyer ask with
100 mL liquid medium and stirred at 150 rpm. After inoculation,
each Erlenmeyer ask was incubated at the test temperature (27,
30, 33, 36 and 39 C) for 12 h. Two precultures were prepared to
obtain inoculums of 6 106 viable cells mL1 .
Culture conditions
Fermentations were also carried out in 250 mL Erlenmeyer asks
with 100 mL medium, for 36 h. The agitation was xed at 150 rpm
(New Brunswick Scientic classic series C24KC Refrigerated
Incubator Shaker Edison NJ, USA). The asks were inoculated
with 6 106 viable cells mL1 . All experiments were carried out in
duplicate.

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Analytical techniques
Yeast growth was measured by two techniques: (a) correlation
optic density (620 nm) against cell dry weight; and (b) direct count
using a Thoma Chamber. Viability was obtained using the methylene blue staining method.15 In addition, the culture medium
was centrifuged for 10 min at 10 000 rpm using an Eppendorf Centrifuge 5424 (Germany). The supernatant was stored at 20 C until
analysis. Glucose, glycerol, acetic acid and ethanol were measured
by high performance liquid chromatography (Waters 600,TSP
Spectra System, Waters, Milford, MA, USA) using a Biorad Aminex
HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
The temperature was 40 C, mobile phase sulfuric acid 5 mmol L1 ,
ow rate 0.4 mL min1 and an index refraction detector (Waters
2414, TSP Refracto Monitor V, Waters) was used.

RESULTS AND DISCUSSION


Glucose and ethanol inuence on growth and fermentative
activity
In order to evaluate glucose and ethanol effects, four different
initial glucose concentrations without ethanol (100, 150, 200 and
250 gL1 ) and three initial ethanol concentrations (0, 3 and 6%
v/v) were tested. In these experiments, the initial pH was set at 4.5
and fermentation kinetics were performed at 30 C. Results show
that the duration of S. cerevisiae ITV-01 lag growth phase increased
from 4 to 18 h when initial ethanol concentration was increased
in culture media (Fig. 1). On the other hand, during fermentation
with no initial ethanol added, there was no difference in the length
of latency phases at the different initial glucose concentrations
evaluated.
When the initial glucose and ethanol concentration was
increased, lower nal biomass concentrations were obtained
(from 4.5 to 1.2 g L1 weight dry) compared with initial glucose
concentration of 100 g L1 and ethanol concentration of 0% (v/v).
This is also shown in Fig. 2 where biomass productivities obtained
for each treatment are presented. Based on these results, it is
possible to establish that the inhibition effect of ethanol on yeast
growth is higher than that induced by glucose.
Nevertheless, the specic growth rate (max = 0.2525 to
0.2671 h1 ) (Table 1) was not affected at 100 and 250 g L1 initial
glucose concentrations when no ethanol was added. These results
suggest that initial glucose concentrations do not affect specic
growth rate and this might be related to the osmotolerant ability
of S. cerevisiae ITV-01.8
On the other hand, at higher initial glucose concentrations
glycerol production increased, while glycerol production was
lower (1.4 gL1 ) when the initial ethanol concentration was
increased in culture media (Fig. 3). This was probably due to a
decrease: (a) in biomass production; or (b) in the water activity of
the culture medium. Glycerol production was not stimulated by
the presence of ethanol in the culture medium, probably because
glycerol does not carry out a physiological function when the
yeast strain is submitted to the stress caused by the presence of
ethanol in the fermentation medium (Fig. 3). These results agree
with those previously reported16 for S. cerevisiae grown on ethanol,
where glycerol production was lower than when using glucose.
The inability of ethanol to stimulate glycerol production is not due
to an inhibitory effect, but is related to the osmotically mediated
regulation of glycerol 3-phosphate dehydrogenase (NAD+ ).17
When glucose and ethanol initial concentrations were higher,
ethanol production decreased (Fig. 4), probably because of a
synergistic inhibition effect of glucose and ethanol, even though

c 2010 Society of Chemical Industry




J Chem Technol Biotechnol (2010)

Kinetic study on ethanol production using S. cerevisiae ITV-01 yeast

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Figure 1. Effect of initial glucose and ethanol concentrations on S. cerevisiae ITV-01 growth. Initial glucose: 100 (), 150 (), 200 () and 250 () g L1 .

Figure 2. Biomass (Px ), glycerol (Pg ) and ethanol (Pe ) productivities at different initial glucose and ethanol concentrations at 36 h. Initial glucose: 100 (a),
150 (b), 200 (c) and 250 (d) g L1 .

the inhibitor effect of ethanol on its own production is higher


than that of glucose. Previously, it was established18 that glucose
inhibition was observed in S. cerevisiae UG5 at a concentration
above 162 g glucose L1 , in S bayanus above 70 g glucose L1
and in C. pseudotropicalis at 100 g glucose L1 .19 This shows that
glucose resistance in yeasts is related to the species and its ability to
adapt under stress conditions, such as low water activity caused by
high levels of substrate concentration. Ethanol decreases biomass
and alcohol production by denaturation of glycolitic enzymes and
also by increasing plasma membrane uidity.8
As shown in Fig. 5, glucose was totally consumed at an
initial glucose concentration of 100 g L1 without ethanol added.
However, at an initial glucose concentration of 150 g L1 , a residual
glucose concentration of 50 g L1 was obtained. The presence of
residual glucose indicates an inhibition effect of glucose and/or
ethanol. Considering that S. cerevisiae ITV-01 is an osmotolerant
strain,7 the incomplete substrate consumption might rather be
due to the presence of either a combined inhibition effect or

nutrient limitation. In order to discover the answer to this question,


experiments were carried out using enriched media with 150 g L1
of initial glucose concentration.
Yeast extract effect on growth and fermentative capability
In order to discard the glucose inhibition effect, fermentations using enriched media were performed. The experimental conditions
are summarized in Table 2. In these experiments, yeast extract
concentration was increased until no differences were found in
growth and glucose consumption, because the inhibition effect of
yeast extract has not been reported.20
For Media 3 and 4, a lower residual glucose concentration
(around 15 g L1 ) was observed (Table 3). This result suggests
that yeast extract (YE) stimulates glucose consumption because
it provides important cofactors like biotin and riboavin. It has
been reported previously that yeast extract is the most important
agent20,21 and that high concentrations of this component do not
affect either growth or fermentative ability in yeasts. When media

Table 1. Specic growth rates, (h1 ) of S. cerevisiae ITV-01 under different glucose and ethanol initial concentrations
Initial ethanol (%v/v)

Initial glucose (g L1 )

a,b,c

Specic growth rate, max (h1 )


0.2671 0.0108a
0.2581 0.0020a
0.2525 0.0016a
0.2586 0.0015a

100
150
200
250

0.2545 0.0278a
0.2561 0.0406a
0.2253 0.0152a
0.2348 0.0027a

0.2350 0.0211a
0.2333 0.0165a
0.2297 0.0066b
0.1927 0.0071c

: statistically different. ANOVA P = 0.95.

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et al.
B Ortiz-Muniz

Figure 3. Glycerol production by S. cerevisiae ITV-01 at different initial glucose and ethanol concentrations. Initial glucose: 100 (), 150 (), 200 () and
250 () g L1 .

Figure 4. Effect of initial glucose and ethanol concentrations on ethanol production in S. cerevisiae ITV-01. Initial glucose: 100 (), 150 (), 200 () and
250 () g L1 .

with yeast extract concentrations of 2.0 and 2.5 g L1 were used,


no important differences in ethanol volumetric productivity (DMS
0.05) were found, although there was a lower residual glucose
concentration with respect to fermentations carried out with 1.0
and 1.5 g L1 yeast extract. This result suggests that when YE
concentration is 2 g L1 no inhibition effect is observed due to
nutritional requirements. However, there is an inhibition effect
or limitation by other factors like pH or temperature. Based on
these results, further fermentation experiments were conducted in
order to evaluate the pH and temperature effect, at constant yeast
extract concentration of 2 g L1 . This yeast extract concentration
is lower than those used for other S. cerevisiae strains: 3.5 g L1
and 6 g L1 .21,22 Yeast extract as a nitrogen source increases
fermentation capability in S. cerevisiae,23 resulting in diminished
residual glucose in culture media,20 as observed in the results
shown in Table 3.

pH effect
The effect of pH on the metabolism of S. cerevisiae ITV-01 was
analyzed at pH levels between 2.0 and 6.5 at intervals of 0.5. Results
show that when the initial pH was 2.0, a high concentration of
residual glucose was left, suggesting a powerful inhibitor effect.
When the initial pH was between 3.0 and 3.5, this provoked
a decrease in residual substrate to a minimum value (Table 4)
which rose again at pH 6.5. These results indicated that the
best substrate consumption occurred in the pH range 3.03.5.
Biomass yield did not vary signicantly in the pH range between
3.0 and 6.5, indicating that yeast growth is not affected by initial
pH. Ethanol yield was higher at pH values between 3.0 and
3.5. The highest ethanol yield was obtained at pH values lower
than 4.5, an advantage for S. cerevisiae ITV-01, because under
these conditions the risk of bacterial contamination is minimum.1
Bacterial contamination is an ongoing problem in commercial fuel

Figure 5. Combined effect of glucose and ethanol on substrate (glucose) consumption by S. cerevisiae ITV-01. Initial glucose: 100 (), 150 (), 200 ()
and 250 () g L1 .

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J Chem Technol Biotechnol (2010)

Kinetic study on ethanol production using S. cerevisiae ITV-01 yeast

Table 2. Composition of evaluated enriched media


Component (g L1 )
Glucose
Ammonium sulfate
Magnesium sulfate
Potassium phosphate
Yeast extract

Media 1

Media 2

Media 3

Media 4

150
2
0.4
5.0
1.0

150
5.0
1.0
8.0
1.5

150
5.0
1.0
8.0
2.0

150
5.0
1.0
8.0
2.5

Table 3. Residual glucose, ethanol yield (Yet/s ) and volumetric


productivity (Pet ) of S. cerevisiae ITV-01 on different culture media
Culture
Media
1
2
3
4
a,b,c

Glucose residual
(g L1 )

Yet/s
(g g1 )

Pet
(g L1 h1 )

62.2 1.10a
20.3 0.54b
15.2 0.02c
15.1 0.01c

0.3976 0.0092a
0.3986 0.0103a
0.4001 0.0109a
0.3998 0.0085a

1.08 0.05a
1.16 0.05a
1.43 0.04b
1.42 0.06b

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The activation energy (Ea) for S. cerevisiae ITV-01 calculated in
the temperature range 2739 C at pH 3.5 was 15.6 kcal mol1
with a pre-exponential factor of 3.8 1012 h1 (R2 = 0.94). This
value suggests that the ITV-01 strain is less sensitive to temperature
than Schizosaccharomyces pombe, which has a higher activation
energy (26.2 kcal mol1 ) (Table 5) but it is more sensitive than P.
tannophilus (Ea = 13.59 kcalmol1 ). The activation energy value
indicates if the process is within a biological or diffusional regimen.
A biological regimen implies that temperature directly affects
kinetic growth parameters and a diffusional regimen indicates that
physical phenomena, like oxygen transfer, restrict the reaction. It
has been reported that when the activation energy is higher than
12 kcal mol1 , the process is in a biological regimen.25 Results
from this work indicate that ethanol fermentation with S. cerevisiae
ITV-01 is carried out under a biological regimen.
Another way to evaluate the effect of temperature on microbial
growth (or on the activity of a microorganism) is with the Q10
value, which represents the increase in the specic growth rate
when there is a 10 C increment.

Q10 =

: Statistically different. DMS P = 0.95.

ethanol production facilities. Lactic acid levels often rise during


contamination, suggesting that the most common contaminants
are lactic acid bacteria. Contaminants create a constant drain on
the carbon available for conversion to ethanol and compete for
growth factors needed by yeast. They also produce byproducts
that are inhibitory to yeast, particularly lactic acid.24 Lactic acid
bacteria are the primary bacterial contaminants of fuel ethanol
fermentations, therefore, using S. cerevisiae ITV-01 at initial pH 3.5
for ethanol production is a useful tool to avoid process spoilage.
Temperature effect
The effect of temperature on the growth of S. cerevisiae ITV-01
was evaluated in terms of activation energy using the Arrehenius
equation, which has been commonly used for thermodynamic
studies in bioprocesses. This equation describes the dependence
of growth rate on temperature:
Ea
max = 0 e RT

2
1

10
T2 T1

Like the activation energy, Q10 is used to indicate if the process


is physical (Q10 < 1) or biochemical (Q10 > 2). Q10 is higher
at low temperatures due to fact that the biochemical reactions
involved are limited by low enzymatic activity.25 In this study
the Q10 value was 3.93, as shown in Table 5. This value conrms
that this process is limited by the biological regime and not by
a diffusional regimen. This result agrees with the previous results
obtained for activation energy (Ea). When temperature increases,
Q10 decreases due to physical limitations such as reduction in
oxygen diffusion. Therefore, activation energy and Q10 are values
that make it possible to evaluate the effect of the temperature
on microbial growth. Nevertheless, Ea is more signicant, being
constant over a wide temperature range, while Q10 varies with
temperature.
Analysis of the synergistic effect of pH and temperature using
a statistic model
Statistical analysis using SPSS software was also conducted to
determine the existence of two possible optima (Fig. 6), using
a complete factorial 52 design, using pH values from 3.0 to

Table 4. Residual glucose, biomass yield (Yx/s ), ethanol yield (Yet/s ) and volumetric productivity (Pet ) in S. cerevisiae ITV-01 at different initial pH
values
Initial pH
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
a,b,c,d,e,f,g

Glucose residual (g L1 )

Yx/s (g g1 )

Yet/s (g g1 )

Pet (g L1 h1 )

133.1 3.2a
72.2 2.1b
6.25 1.2c
6.75 0.9c
19.9 2.3d
22.0 1.8d
23.2 2.2d
29.3 2.7e
45.0 3.1f
48.1 2.9f

0.040 0.001a
0.031 0.002b
0.032 0.002b
0.033 0.001b
0.035 0.002bc
0.035 0.002bc
0.037 0.001c
0.040 0.001a
0.045 0.002d
0.051 0.001e

0.2043 0.0024a
0.3873 0.0021b
0.4100 0.0042c
0.4080 0.0035c
0.3900 0.0037d
0.3869 0.0012d
0.3845 0.0065d
0.3973 0.0079d
0.3846 0.0057d
0.3662 0.0041e

0.07 0.01a
0.93 0.03b
1.83 0.04c
1.84 0.03c
1.63 0.03d
1.58 0.03d
1.45 0.02e
1.25 0.05f
1.08 0.06g
1.00 0.04b

: Statistically different. ANOVA P = 0.95.

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Table 5. Values of Ea and Q10 for different yeasts


Yeast
B. bruxellensis B5d
P. tannophilus
Sch. pombe
S. cerevisiae
S. cerevisiae ITV01

Ea
(kcal mol1 )

Q10

Reference

16.61
13.59
26.20
13.59
15.60

3.76
1.86
3.33
2.05 (<25 C)
3.93 (>25 C)

25
26

et al.
B Ortiz-Muniz

the optimized temperature was 30 C29 and this value is lower than
that reported for fed-batch culture (33 C).30 Almost all reports of
S. cerevisiae strains5,7,9,19,27,31,32 used pH values from 4.5 to 5.5,
so the S. cerevisiae ITV-01 strain could be interesting owing to its
benecial alcoholic fermentation performance at pH values as low
as 3.5.

25
25

This study

5.0, at 0.5 intervals, and temperature from 27 to 39 C, at 3 C


intervals. Results showed one optimum between pH 3.0 and 3.5
and 27 and 30 C, and another at pH 4.55.0 and 3639 C.
These results indicate that the optimal conditions for performing
ethanol fermentation with S. cerevisiae ITV-01 are pH 3.5 and
30 C, since working at low pH values (3.03.5) limits lactic
acid bacteria growth, resulting in a lower residual glucose. It
is interesting to note that the optimum conditions reported in
previous work27 with S. cerevisiae ITV-01 using different substrates,
such as sucrose, claried cane juice and intermediate syrup B,
were pH 5.5 and 35 C which are nearly the same conditions
as for the alternative optimum found by statistical analysis.
The best ethanol production conditions were pH 3.5 and 30 C
with 1.8 g L1 h1 ethanol productivity, closed to the 2 g L1 h1
previously reported28 for a batch process; this suggests that the
use of feed batch or continuous cultures of this strain could result in
a competitive process for ethanol production. These results agree
with those previously established at high substrate concentration:

CONCLUSIONS
S. cerevisiae ITV-01 is an osmotolerant yeast, its specic growth
rate remaining unchanged at glucose concentrations between
100 and 250 g L1 . Nevertheless, total uptake of substrate was
not obtained at high glucose concentrations, probably because
of a synergistic effect caused by substrate concentration and
ethanol produced. S. cerevisiae ITV-01 is also resistant to low pH
values, which is an advantage for ethanol production, because
it prevents contamination with lactic acid bacteria. The Ea and
Q10 values indicate that a biological regimen limits alcoholic
fermentation, so that the culture medium must be optimized in
order to maximize ethanol production. Results from this work
showed that the optimal conditions for ethanol production with
S. cerevisiae ITV-01 were initial glucose concentration 150 g L1 ,
pH 3.5 and temperature 30 C. In this case, a maximum ethanol
concentration of 58.4 g L1 , ethanol productivity of 1.8 g L1 h1
and ethanol yield of 0.41 g g1 were obtained.

ACKNOWLEDGEMENTS
The authors acknowledge the nancial support from the National
Council of Science and Technology, Mexico (CONACYT) (schol-

Figure 6. Optimization prole generated by SPSS software. Temperature (), pH ().

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J Chem Technol Biotechnol (2010)

Kinetic study on ethanol production using S. cerevisiae ITV-01 yeast


arship 45161) and critical reading of the manuscript by Patricia
Hayward Jones MSc and Dulce Mara Barradas Dermitz MSc.

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