Bioprocess Biosyst Eng (2012) 35:605614

DOI 10.1007/s00449-011-0633-9

ORIGINAL PAPER

Use of sugarcane molasses B as an alternative for ethanol

production with wild-type yeast Saccharomyces cerevisiae
ITV-01 at high sugar concentrations
C. L. Fernandez-Lopez B. Torrestiana-Sanchez
M. A. Salgado-Cervantes P. G. Mendoza Garca
M. G. Aguilar-Uscanga

Received: 10 July 2011 / Accepted: 20 September 2011 / Published online: 5 October 2011
Springer-Verlag 2011

Abstract Molasses B is a rich co-product of the

sugarcane process. It is obtained from the second step of
crystallization and is richer in fermentable sugars
(5065%) than the final molasses, with a lower non-sugar
solid content (1833%); this co-product also contains good
vitamin and mineral levels. The use of molasses B for
ethanol production could be a good option for the sugarcane industry when cane sugar prices diminish in the
market. In a complex medium like molasses, osmotolerance is a desirable characteristic for ethanol producing
strains. The aim of this work was to evaluate the use of
molasses B for ethanol production using Saccharomyces
cerevisiae ITV-01 (a wild-type yeast isolated from sugarcane molasses) using different initial sugar concentrations
(70291 g L-1), two inoculum sizes and the addition of
nutrients such as yeast extract, urea, and ammonium sulphate to the culture medium. The results obtained showed
that the strain was able to grow at 291 g L-1 total sugars in
molasses B medium; the addition of nutrients to the
culture medium did not produce a statistically significant
difference. This yeast exhibits high osmotolerance in this
medium, producing high ethanol yields (0.41 g g-1). The
best conditions for ethanol production were 220 g L-1
initial total sugars in molasses B medium, pH 5.5, using
an inoculum size of 6 9 106 cell mL-1; ethanol production was 85 g L-1, productivity 3.8 g L-1 h-1 with 90%
preserved cell viability.
C. L. Fernandez-Lopez
B. Torrestiana-Sanchez

M. A. Salgado-Cervantes
P. G. M. Garca

M. G. Aguilar-Uscanga (&)
Instituto Tecnologico de Veracruz, UNIDA (Unidad de
Investigacion y Desarrollo en Alimentos), Bio-Engineering
Laboratory, Av. Miguel A. de Quevedo 2779, Col. Formando
Hogar, CP 91860 Veracruz, Ver., Mexico
e-mail: maguilaruscanga@yahoo.com.mx

Keywords Ethanol
Molasses B
Osmotolerance

Inoculum size
Abbreviations
lmax Maximum specific growth rate (h-1)
Yx/s
Biomass yield (g biomass g-1 substrate)
Yet/s Ethanol yield (g ethanol g-1 substrate)
Yet/x Ethanol specific yield (g ethanol g-1 biomass)
Pet
Ethanol productivity (g ethanol L-1 h-1)
%V
Cell viability (%)

Introduction
Bio-fuel production such as ethanol from renewable raw
materials has increased from 15,000 to 65,000 million liters
from 1990 to 2010 [1]. Ethanol presents advantages over
petrol derivatives like methyl terbutyl ether (MTBE); it has
lower toxicity, higher combustion when combined with
other gases, it can be a gasoline oxygenator, it diminishes
polluting emissions, and its ignition temperature is higher
than for gasoline (420 C compared to 220 C) amongst
others [2]. Globally, the production of ethanol comes
mainly from the United States of America (48%), Brazil
(42%), and China (4%) [1]; they use corn, sugarcane, beet
juice and sugarcane and beet molasses as raw materials
[2, 3]. With the decrease in fossil fuel sources, the search for
alternatives in bio-ethanol production has been encouraged.
One of the principal factors that has a very important impact
on this process is feedstock availability, because it affects
final ethanol price to a great degree [4].
For many years, sugarcane and beet molasses have been
used as excellent options for ethanol production [5, 6] and

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recently sweet sorghum juice, and other amylose rich

materials such as corn, wheat and cassava have been used,
and some studies are also being made with potatoes [7, 8].
In these processes the polysaccharides have to be hydrolyzed into simpler sugars. The lignocellulosic residues such
as cane bagasse [9]; grape waste [10], lignocellulosic
biomass [11], steam pretreated spruce [12] are promising
options, but in these cases it is necessary to use enzymes or
pretreatments such as alkaline or acid hydrolysis in a previous step to fermentation, making the process expensive at
an industrial scale [2]. In Mexico, the sugarcane industry
has suffered an economic crisis due to competition with
high fructose corn syrup as a substitute for cane sugar in
the soda industry. Therefore, one alternative to help
diminish this problem is the diversification of production
lines to produce ethanol and sugar simultaneously from
sugarcane [13]. Molasses B is a co-product of cane sugar
production, 15% richer in sugar content than the final
molasses and with good vitamin and mineral contents,
making it a better option than the final molasses [12, 13].
Mexico has 59 sugar mills, processing on average
29,300,000 tons sugarcane per year [14], with a cane sugar
production of 3,370,750 tons. For each ton of cane sugar
produced, 13,250,000 tons final molasses and 168,537.5
tons molasses B can be produced from which 235 and
275 L ethanol can be obtained per ton, respectively, that is
17% more from molasses B than final molasses [13, 14].
The most used microorganism for ethanol production is
Saccharomyces cerevisiae, but some studies have also been
made with bacteria like Zymomonas mobilis or some
Eschericia coli mutants [15]. They have some limitations
in their capacity to use several substrates [2] and also in
ethanol and sugar concentration resistance compared to
S. cerevisiae yeast. S. cerevisiae ITV-01 is a wild-type strain
isolated from sugarcane molasses exhibiting interesting
characteristics for ethanol production, such as osmotolerance and ethanol resistance in a synthetic medium. The
best conditions reported for ethanol production were pH
3.5, 150 g L-1 glucose, a temperature of 30 C, and
81 g L-1 ethanol tolerance [1618]. Several factors can
affect cell growth and ethanol production; carbon source,
inoculum size, initial sugar concentration, pH, temperature
and oxygen supply are the most important [19, 20]. When
molasses is used as the culture medium, one of the limiting
factors is the high solid content and high osmotic pressure
to which the cell is exposed. The stress caused by high
osmotic pressure activates self-adjusting mechanisms to
re-establish internal equilibrium so that cell activities are
not damaged [21, 22]. These mechanisms include a signal
system throughout the membranes and osmolite production
such as glycerol that plays an important role in osmoregulation [2224]. In order to evaluate the advantage of
using molasses B over other raw materials containing

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Bioprocess Biosyst Eng (2012) 35:605614

carbon sources such as glucose and sucrose, this work

studies of the effect of initial sugar concentrations in a
molasses B medium on ethanol production at two
inoculum sizes, taking into consideration the effect of
nutrient addition to the culture medium.

Materials and methods

Microorganism
Saccharomyces cerevisiae ITV-01, a regional wild-type
yeast isolated from sugarcane molasses obtained from
several sugar mills in Veracruz (Mexico), was used [16].
Culture medium
The strain was maintained in stock cultures at 4 C using a
culture medium containing (g L-1): glucose (Sigma) 70;
yeast extract (Dibico) 10; and agar (Difco) 25. An activation medium was prepared with the following components
(g L-1): commercial sucrose, 150; yeast extract (Dibico) 1;
ammonium sulphate [(NH4)2SO4] (J.T. Baker) 2; magnesium sulphate heptahydrate (MgSO47H2O) (J.T. Baker)
0.4; and potassium phosphate monobasic (KH2PO4) (J.T.
Baker) 5. The pH was adjusted to 5.5 using ortho-phosphoric acid solution (J.T. Baker) (after referred as synthetic
medium). Flasks (250 mL) containing 100 mL medium
were used for activation. The culture medium was sterilized for 15 min at 121 C.
After activation, to evaluated the effect of carbon
sources cells were transferred to pre-culture medium, made
in a fashion similar to the activation medium, supplemented with 150 g L-1 of glucose, sucrose or molasses
B (in this case dilutions with distilled water were made
to molasses B medium in order to achieve the desired
initial total sugar concentration). The same procedure was
used to evaluate the effect of initial molasses concentration
(70, 186, 220, 291 g L-1 initial total sugars).
Each medium (300 mL) was poured into a 500 mL flask
and pH was adjusted to 5.5 using ortho-phosphoric acid
solution. The culture medium was sterilized for 15 min at
121 C.
Nutrient supplementation of molasses B medium
The medium was supplemented with the following composition (g L-1): yeast extract 1; ammonium sulphate
[(NH4)2SO4] 2; urea [CO(NH2)2] 2; yeast extract 1 with
ammonium sulphate 2; yeast extract 1 with urea 2;
ammonium phosphate [(NH4)2SO4)] 2 with potassium
phosphate monobasic [(KH2PO4)] 5. They were compared
with a control medium (molasses B without nutrients).

Bioprocess Biosyst Eng (2012) 35:605614

Initial pH was adjusted to 5.5 with ortho-phosphoric acid.

All experiments were performed with the same initial total
sugars in duplicate.

607

cell dry weight, using a Thoma Chamber. Viability was

obtained using the methylene blue staining method [25].
Substrates and products

Preculture and culture conditions

Strain activation was carried out in a 250 mL Erlenmeyer
flask with 100 mL liquid mineral medium containing glucose. After inoculation, each Erlenmeyer flask was incubated at 35 C for 12 h, and stirred at 250 rpm (New
Brunswick Scientific classic series C24KC Refrigerated
Incubator Shaker, Edison NJ, USA). A preculture was then
prepared using the same medium to be used in fermentation
(with glucose, sucrose or molasses B). The pre-culture
was inoculated with 6 9 106 cell mL-1 from the activation
medium and incubated for 8 h or until the exponential
growth phase was reached (depending on initial total sugar
concentration in the medium).
For the comparison of carbon sources, cells were taken
from the pre-culture medium (mineral medium with glucose, sucrose, or molasses B) and experiments were
carried out in 500 mL flasks containing 300 mL medium
inoculated with 6 9 106 viable cells mL-1. Fermentation
was monitored every 2 or 3 h. Experiments were performed
in duplicate, with the same temperature and stirring speed
specified above.
Inoculum size evaluation
Two inoculum sizes were tested, 6 9 106 cells mL-1 and
1 9 107 cells mL-1. Cells grew under the same conditions as
described above and were then centrifuged. The cells obtained
were re-suspended in 10 mL medium, similar to that to be
used in the fermentation, in order not to affect bioreactor
medium volume. Experiments were done by duplicated.
Initial substrate concentration evaluation
To evaluate initial substrate concentrations, several dilutions of molasses B were prepared in order to obtain
initial total sugar concentrations between 70 and 291
g L-1. Batch experiments were performed in an Applikon
bioreactor equipped with an ADI 1010, and ADI 1025
controller (Applikon, Holland) 3 L capacity and 1.5 L
working volume.
Analytical methods
Biomass
Yeast growth was measured by two techniques: (a) in
synthetic media by optical density (620 nm) against cell
dry weight correlation; and (b) direct count correlation to

Glucose, fructose, acetic acid, glycerol and ethanol were

analyzed using an HPLC (Waters 600, TSP Spectra System, Waters, Milford, MA, USA) with a Bio-Rad Aminex
HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,
CA, USA). Samples were filtered through 0.45 lm nylon
filter. H2SO4 (J.T. Baker) 5 mM was used as the mobile
phase, 0.6 mL min-1, 50 C. A refraction index detector
(Waters 2414, TSP Refractometer Monitor V, Milford,
MA, USA), injection volume 20 lL, was used. For
molasses B medium samples, a defecation pre-treatment
was used: 800 lL sample were mixed in a microtube with
100 lL barium oxide (BaO) (J.T. Baker) 0.3 N solution,
and 100 lL zinc sulphate (J.T. Baker) solution (ZnSO4) 5%
w/w. The samples were kept at 4 C for 5 min, and then
centrifuged at 10,000 rpm for 10 min. The supernatant was
recovered and dilutions were made in order to obtain a
maximum sugar concentration of 100 g L-1. An acid
hydrolysis of the sugars was made, one volume of sample
to one volume of hydrogen chloride solution (HCl)
(J.T.Baker) 1.2 N. The mixture was maintained at 65 C
for 15 min in closed tubes, and subsequently placed in a
cold bath for 10 min. Acid hydrolysis was carried out for
sucrose and molasses B media. Samples were then ready
to be filtered and injected for HPLC analysis.

Results and discussion

Effect of carbon source
Glucose, sucrose and molasses B were used in order to
investigate the influence of carbon source on Saccharomyces cerevisiae ITV-01 growth and ethanol production.
The effect of different sugars as carbon sources presented
differences in growth and ethanol production; yields are
shown in Table 1.
A 20% loss in cell viability was observed after 36 h
when cells were grown on synthetic medium. This could be
due to a decrease in pH (to 2.14) and/or to some nutrient
deficiency in the synthetic medium. It has been reported
[21] that intracellular pH and the H? pump play an
important role in yeast growth and the glycolysis/gluconeogenesis relationship. Plasma membrane ATPase regulates intracellular pH and this is essential to cell growth.
The transmembrane H? gradient is the driving force in the
uptake of nutrients, so that when intracellular pH is
affected, all these functions are also affected, as well as the
physiology of the yeast [26]. This effect was not observed

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Bioprocess Biosyst Eng (2012) 35:605614

Table 1 Kinetic parameters for ethanol production by S. cerevisiae ITV-01 with different carbon sources
Medium used

Yet/s (g g

-1

Yx/s (g g

-1

Pet (g L-1 h-1)

Biomass (g L-1)

Viability (%)

Final pH

Glucose

0.39 0.001

0.037 0.011

1.62 0.131

5.29 0.073

82 2.21

2.0

Sucrose

0.28 0.011

0.048 0.001

0.81 0.040

4.98 0.160

81 1.83

2.1

Molasses B

0.40 0.013

0.054 0.010

1.31 0.220

11.2 1.012

90 1.61

4.7

when intermediate molasses B was used. The pH stayed

around 4.7, favoring cell viability (90%), due to the buffer
effect of molasses B medium and also to other nutrients
(such as vitamins) present in the medium (Table 2).
Biomass in molasses B medium reached 11.2 g final
concentration, compared to 5.29 g L-1 obtained in synthetic medium experiments (Table 1), which is an advantage as long as ethanol production is a growth associated
product. Ethanol yields are similar to those using molasses
B and synthetic medium with glucose (0.40 g ethanol
g-1 substrate) and to synthetic medium with sucrose (0.28
g g-1); however, there are differences in residual sugars,
these being higher in synthetic media than in molasses B
medium. Therefore, molasses B medium was therefore
the best carbon source. Disaccharide consumption depends
on the strain and environmental conditions. In the case of
sucrose, a unique plasma membrane sucrose-binding protein is present in S. cerevisiae which mediates sucrose
uptake in a non-saturable, linear manner [27, 28]. In the
case of the medium using sucrose as the carbon source,
invertase activity determines hexose availability and
therefore their consumption. Once hexoses are available,
the strain shows a glucose preference over fructose (data
not shown) also previously reported for Saccharomyces
cerevisiae [29, 30]. Hexose presence in the medium controls plasmatic membrane transport rate and can also affect
catabolic repression [18, 20]. However, these differences
between the preferences of the type of sugar consumed can
vary depending on several factors: strain type, initial substrate concentration, nitrogen availability, all of which are
Table 2 Composition of molasses B [22, 36]
Compound

Dried matter (non sugars)

18

Dried matter (sugars)

56

Water

17

Nx6.25

Ashes
Biotine (mg/kg)
Choline (mg/kg)
Pantothenic acid (mg/kg)

7
[0.36
[745.0
[21.0

Rivoflavine (mg/kg)

[1.8

Tiamine (mg/kg)

[0.9

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suspected to affect fructose more than glucose consumption. The presence of ethanol in the fermentation medium
[20] might affect the consumption rate of the different
types of sugars. Figure 1 shows the behavior of Saccharomyces cerevisiae ITV-01 growing in different media and
a similar lag phase in the three carbon sources can be
observed. Sugar consumption was slightly higher when the
strain was grown in sucrose rather than glucose medium.
When using molasses B medium, a higher biomass
production was observed. The preference of the strain to
consume glucose over fructose was also confirmed.
Ethanol production was 8.9% lower in the synthetic
medium (52.7 g L-1) than when using molasses B
medium (58 g L-1). These results were similar to those
reported previously for sugarcane juice and molasses
media [15] where final ethanol concentrations were
3942 g L-1 when modified strains of Escherichia coli
KO11 and Klebsiella oxytoca P2 were used. Low ethanol
production obtained with sucrose and glucose could be due
to a lack of nutrients in the medium, while molasses B,
with its higher vitamin and mineral contents, gave better
results. Nevertheless, a stress response, observed as an
increase in glycerol production, was found in molasses
B medium but not in synthetic medium. Experiments
demonstrated cell viability maintenance in molasses B
medium, a big advantage for its use as an industrial
medium.
Effect of nutrient supplementation to intermediate
molasses B medium
In order to evaluate the need of nutrients in molasses B
medium, several nitrogen sources were added to dilute
molasses B as described above and Fig. 2 shows the
effects observed. Control treatment (?) and all the experiments supplemented with nutrients do not exhibit differences in ethanol production (from 78 to 82 g L-1).
Maximum specific growth rates were similar in supplemented media (around 0.27 h-1), compared to 0.29 h-1 in
the control medium; but in the (j) ammonium sulphate
and the (h) yeast extract and urea treatments, maximum
specific growth rates were higher (0.38 and 0.40 h-1,
respectively); however, biomass production at steady state
was similar in all cases (8.611.2 g L-1). Molasses B
medium was enough in itself for good ethanol and biomass

Bioprocess Biosyst Eng (2012) 35:605614

609

Fig. 1 Sugar consumption,

biomass and ethanol production
in mineral media with glucose
(asterisks), sucrose (open
squares) and intermediate
molasses B media (open
circles)

Fig. 2 Effect of nutrient

addition to molasses B
media: sugar consumption, and
ethanol and biomass production.
Treatment codes (g L-1): (open
circles) yeast extract 1; (filled
squares) ammonium sulphate
[(NH4)2SO4)] 2; (straight lines)
urea [CO(NH2)2] 2; (multi
symbols) yeast extract 1 with
ammonium sulphate 2; (open
squares) yeast extract 1 with
urea 2; (filled circles)
ammonium phosphate
[(NH4)2SO4)] 2 with monobasic
potassium phosphate (KH2PO4)
5; (plus sign) control medium
molasses B without nutrients

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Bioprocess Biosyst Eng (2012) 35:605614

production. This could be due to the vitamin and salt

contents in molasses B medium. As regards to industrial
needs and the impact that medium composition can have on
production costs, this is an interesting result.
In the case of yeast, it is well known that medium
composition, primarily nitrogen source, affects growth rate
and product formation. Ammonium, glutamic acid and
mixed amino acid supplemented media have been tested
using Saccharomyces cerevisiae to prevent the loss of raw
material in ethanol production through a diversion into
glycerol formation [31]. In other studies it was reported
that specific growth rate and metabolite formation was
affected when several nutrients were added. The best
specific ethanol production was obtained when a mixture of
amino acids (as in yeast extract) was used, followed by
ammonium supplemented medium and finally medium
supplemented with glutamic acid. The addition of diammonium phosphate and ammonium phosphate has also
been tested in grape juice experiments [32] in order to
evaluate the impact of nitrogen source on fermentation
with Saccharomyces cerevisiae PYCC 4072. The results
showed that initial nitrogen concentrations in the media
had no effect on specific growth rates, although the time to
complete fermentation and fermentation rate were dependent on nitrogen source availability.
Effect of inoculum size and initial total sugar
concentration on biomass and ethanol production
Inoculum size effect on ethanol and biomass production
The effect of inoculum size on ethanol and biomass production was evaluated using two concentrations (6 9 106
viable cells mL-1 and 1 9 107 cells mL-1) in batch fermentations using molasses B medium with 230 g L-1
initial total sugars. The inoculum sizes tested were between
the optimum values previously reported [32]. The results
obtained showed that there are differences between sugar
consumption rates when using different inoculum sizes
(Table 3), this being faster when the greater inoculum size
of 1 9 107 cells mL-1 was used (5.56 compared to
1.47 h-1). Biomass production was slightly affected and
specific growth rate was similar (0.14 and 0.17 h-1,
respectively). It was also observed that when the greater

inoculum size was used, residual sugars increased from 3.6

to 19.7 g L-1 after 34 h fermentation.
Specific ethanol rate and ethanol productivity were
higher with the lower inoculum size (6 9 106 cells mL-1).
Final ethanol concentration was higher with the smaller
inoculum size, reaching a final concentration of 95 g L-1
after 27 h fermentation, compared with 87 g L-1 after 34 h
fermentation with the greater inoculum size tested; fermentation time was also reduced (7 h).
The initial inoculum or cell concentration in fermentation, also called the pitching rate in breweries, should be
around 1 9 107 viable yeast cells mL-1. In brewing, the
initial cell concentration added has important effects not
only on the speed of sugar conversion to ethanol but also
on the final cell concentration [33, 34]. Previous experiments using 3, 6, 9 and 12 9 106 cells mL-1 as inoculum
sizes [17] showed that increasing the inoculum size
reduced the lag phase, the best inoculum size being
6 9 106 cells mL-1. Experiments carried out using synthetic medium with glucose at an initial concentration of 70
g L-1, greatly differed when complex medium was used at
higher initial sugar concentrations (270 g L-1). It has been
reported [31, 32] that fermentation rate was improved with
higher inoculum sizes (1 9 106 to 1 9 107 cells mL-1)
but not final ethanol production. These authors also
reported that there was an increase in higher alcohols at
inoculum sizes of 1 9 106 cells mL-1 compared to
1 9 107 cells mL-1.
From these experiments, it can be concluded that the
best inoculum size for ethanol production in this case was
the smaller one resulting in a final concentration of 95
g L-1, and the highest productivity of 3.5 g L-1 h-1. This
is in good agreement with other studies [17]. This inoculum
size (6 9 106 cells mL-1) was therefore used in later
experiments to evaluate initial total substrate effect.
Effect of initial sugar concentration in molasses B media
on ethanol and biomass production
Experiments were carried out in a batch reactor in order to
evaluate initial total sugar concentration effect. The concentrations tested were 70, 186, 220, 291 g L-1 and an
inoculum size of 6 9 106 cells mL-1 was used. It was
observed that as the initial sugar concentration increased,

Table 3 Kinetic parameters for molasses B medium using two inoculum sizes
Inoculum
(cells mL-1)

Residual
substrate
(g L-1)

[Biomass]
(g L-1)

lmax (h-1)

Vs
(g L-1 h-1)

Vp
(g L-1 h-1)

Pet
(g L-1 h-1)

Px
(g L-1 h-1)

Ethanol
(g L-1)

6 9 106

3.60 0.581

11.71 0.131

0.14 0.001

1.47 0.014

0.68 0.014

3.51 0.007

0.43 0.007

95 0.233

1 9 107

19.73 0.560

13.27 0.102

0.17 0.007

5.56 0.241

0.59 0.007

2.55 0.212

0.31 0.014

87 1.555

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611

biomass concentration varied between 7.8 and 11 g L-1,

with a biomass production inhibition of 62% when initial
total sugars were higher than 187 g L-1. Specific ethanol
production rate increased as initial sugar concentration
increased from 70 to 220 g L-1; resulting in a final concentration of 85.57 g L-1, however above this value they
both started to decrease (Fig. 3). The inhibition of both
biomass and ethanol productions were calculated taking as
reference the yield values obtained at 70 g L-1 (Fig. 4); the
results revealed strain osmotolerance. The best maximum
specific growth rate (0.46 h-1) was when initial sugars were
220 g L-1. Biomass yield diminished from 0.14 to 0.03
g g-1, with 70 and 291 g L-1 initial sugars, respectively.
Inhibition due to initial sugars could be observed to be
stronger in growth rate than in ethanol production. Previous
studies with S. cerevisiae ITV-01 [19] showed that lmax was
not affected by initial substrate concentration in synthetic
media with glucose as the carbon source. However, in
complex media with high solid contents that could affect
specific growth rate, osmotic pressure in the medium
affected biomass growth, so that lag phase was delayed. The
best lmax obtained was 0.46 h-1 with initial sugars at 220
g L-1 in molasses B medium. This was higher than that
reported for synthetic medium [16] with the same strain
(0.25 h-1 for 200250 g L-1 initial glucose concentration).
Fig. 4 Effect of initial total sugar concentration on kinetic parameters lmax (h-1) (filled squares) and Vp (g g-1 h-1) (open squares).
Below: % inhibition of biomass (open squares) and ethanol (filled
squares) production based on yields

Fig. 3 Total initial sugar concentrations effect on sugar consumption

and biomass production, in batch fermentations with molasses B
medium. (Multi symbols) 70 g L-1, (open squares) 186 g L-1,
(straight lines) 220 g L-1 and (open circles) 291 g L-1 total initial
sugars

With beet molasses medium at initial sugars of 187 g L-1, a

lmax of 0.214 was reported [7, 35], lower than that obtained
in the present work, which shows the advantage of using
molasses B as the culture medium. When initial sugars of
220 g L-1 were used for fermentation, the effect of ethanol
concentration on yeast growth can be observed as a
decrease in biomass concentration and also as an increase in
residual sugars (Fig. 3). Above 220 g L-1 initial sugars,
yeast growth decreased due to substrate inhibition, evident
as residual sugar at the end of fermentation (from 7 at 220
g L-1 initial sugars, to 82 at 291 g L-1 initial sugars in the
medium). The use of molasses B medium has advantages
as the vitamin and mineral concentrations are high [27, 35]
so that cells can get all the nutrients they need, but there is
also an important increase in osmotic pressure because of
the high solid non-sugar content.
Other authors [19] have reported that fermentation
inhibition by ethanol can be observed by following the
specific rate of ethanol production which is related to
ethanol concentration. Inhibitory concentration is strain
specific, and also dependent on nitrogen availability in the
medium, so that when ethanol concentration is above
50 g L-1 the Vp decreases greatly until total ethanol

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Bioprocess Biosyst Eng (2012) 35:605614

Fig. 5 Ethanol production in batch fermentation using several initial

total sugar concentration, in molasses B medium. (Multi symbols)
70 g L-1, (open squares) 186 g L-1, (straight lines) 220 g L-1,
(open circles) 291 g L-1

production is inhibited at 85 g L-1, which is in agreement

with that reported by other authors [19] where ethanol
specific rate decreased by half (between 46 and 57 g L-1).
Ethanol production increased as initial sugars increased.
Ethanol yields ranged from 0.35 g g-1 at 70 g L-1 initial
sugars to 0.43 g g-1 at 187 g L-1 initial sugars, but after this
concentration they fell to 0.39 and 0.39 g g-1 at initial sugars
of 220 and 291 g L-1, respectively, with ethanol specific
rates of 1.3 and 0.91. The maximum final ethanol concentration was at the highest initial total sugars (291 g L-1);
however, high glycerol production was also present (22
g L-1) and residual sugars were high (43.4 g L-1). Glycerol
production is related to stress response [20, 37] and it was
observed to increase when synthetic media were changed for
complex media. As initial total sugar concentration in
molasses B medium increased, so did glycerol production.

In this case, a synergistic effect of high initial total sugar

concentration and high ethanol concentration in the medium
exists; improvements, however, could still be made by other
strategies. Therefore, the best ethanol production without
residual sugars was when initial sugars were between 187
and 220 g L-1. The effect of initial sugar concentration on
ethanol production can be observed in Fig. 5.
The comparison between the development of S. cerevisiae ITV-01 in molasses B medium with other studies
where several carbon sources were used can be seen in
Table 3, where kinetic parameters (ethanol yield, efficiency of ethanol production or final ethanol concentration)
obtained with the strains used are presented. It can be
observed that when complex media are used, as compared
to synthetic media, ethanol yield decreased, as does final
ethanol concentration obtained. The highest substrate
concentration used in the reported studies was 257 g L-1
with beet molasses medium; a final ethanol concentration
of 59 g L-1 was obtained. Maximum final ethanol concentration was 70 g L-1 for cane molasses medium with
200 g L-1 initial sugars. As can be seen, the final ethanol
concentration obtained in this study was 8588 g L-1,
higher than those reported in previous studies (Table 4).
An inhibition was observed at certain substrate concentrations and this effect increases in complex media due
to the high concentration of solids. Ethanol production also
decreases, as can be appreciated in Table 4. This does not
occur with Saccharomyces cerevisiae ITV-01, however,
where greater substrate tolerance and higher ethanol production are observed.
We could also observe that ethanol resistance was
approximately 88 g L-1, because after this concentration, a
rapid loss in % viability of the yeast was observed. This is
in agreement with that reported previously [19]. It is

Table 4 Comparison of ethanol production by yeast and bacteria strains using different substrates in batch systems
Author

Strain

Substrate (S)

Ethanol (g L-1)

Yield (g g-1)

[36]

S. cerevisiae

Beet Molasses sugars

70

0.37

[14]

Modified E. coli KO11, Klebsiella oxytoca P2

Synthetic medium with Sucrose

27

0.4

30

0.22

Modified E. coli KO11, Klebsiella oxytoca P2

Cane juice

39.4
42.1

0.44

Modified E. coli KO11, Klebsiella oxytoca P2

Molasses medium

11.0

25.0

[15]

Saccharomyces ssp.

Cane molasses

5070

0.46

[17]

S. cerevisiae ITV-01

Synthetic medium with Sucrose

29

0.39

Cane juice

33

0.40

Molasses B mediuma

53

0.42

Molasses B mediumb

85

0.40

This work

S. cerevisiae ITV-01

Initial sugars concentration of 70 g L-1

Initial sugars concentration of 220 g L-1

123

Bioprocess Biosyst Eng (2012) 35:605614

important to remark that there was a buffer effect presented

by molasses B medium, advantageous for maintaining
strain viability when compared to synthetic media, where
acetic acid production lowers pH values causing a sudden
loss in cell viability. Ethanol production might be
improved by testing aeration supplementation as reported
[38] and glycerol production could be diminished from 12
to 4 g L-1 by using an aeration strategy, an alternative for
improving the development of this wild-type strain.

613

9.

10.

11.

12.

Conclusion
S. cerevisiae ITV-01 preferentially consumes intermediate
molasses B medium. This medium was enough in itself
for growth and ethanol production; no medium supplementation was needed. Strain osmotolerance could be
verified as it was able to grow at 220 g L-1 initial total
sugars in this medium. Biomass production was higher than
in the synthetic medium (10 to 6 g L-1), with an ethanol
production of 85 g L-1. Stress, observed as glycerol production, increased proportionally with initial total sugar in
the media. Therefore, intermediate molasses B medium
is a good option to diversify the sugarcane process and
ethanol production. S. cerevisiae ITV-01 wild-type yeast is
a promising option for ethanol production, as it is well
adapted to environmental conditions.

13.
14.
15.

16.

17.

18.
Acknowledgments Authors acknowledge the National Council of
Science and Technology (CONACYT) for financial support (scholarship number 8765 and project No128052 FOMIX-CONACYT) and
also the critical reading of the text by Patricia M. Hayward Jones
MSc. and Dulce Mara Barradas Dermitz MSc.

19.
20.

References
1. Berg C (2004) World fuel ethanol analysis and outlook. Short
communication, F.O. Licht
2. Sanchez OJ, Cardona CA (2008) Trends in Biotechnological
production of fuel ethanol from different feedstocks. Bioresour
Technol 99:52705295
3. Balat M, Balat H (2009) Recent trends in global production and
utilization of bio-ethanol fuel. Appl Energy 86:22732282
4. Cardona CA, Sanchez OJ (2007) Fuel ethanol production: process
design trends and integration opportunities. Bioresour Technol
98:24152457
5. Ergun M, Ferda MS (2000) Application of a statistical technique
to the production of ethanol from sugar beet molasses by Saccharomyces cerevisiae. Bioresour Technol 73:251255
6. Pramanik K, Rao DE (2005) Kinetic Study on Ethanol Fermentation of Grape Waste using Saccharomyces cerevisiae yeast
isolated from toddy. Inst Eng Indian J 85:5358
7. Rhonda H, Winston AM (1989) Malt hydrolysis of sweet-potatoes and eddoes for ethanol production. Biol Wastes 29:263270
8. Kilpimaa S, Kuokkanen T, Lassi U (2009) Bio-ethanol production from waste potatoes. In: Paukkeri A, Yla-Mella J, Pongracz
E (eds) Energy research at the University of Oulu. Proceedings of

21.
22.

23.

24.

25.

26.

27.

the EnePro conference, June 3rd, 2009, University of Oulu,

Finland pp 2123
Doran JB, Aldrich HC, Ingram LO (1994) Saccharification and
fermentation of sugarcane bagasse by Klebsiella oxytoca P2
containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway. Biotechnol Bioeng 44:240247
Bertolini MC, Ernandes JR, Laluce C (1991) New yeast strains
for alcoholic fermentation at high sugar concentration. Biotechnol Lett 3:197202
Asghari A, Bothast RJ, Doram JB, Ingram LO (1996) Ethanol
production from hemicellulosic hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KO11.
J Ind Microbiol 16:4247
Alkasrawi M, Andreas R, Gunnar L, Guido Z (2006) Influence of
strain and cultivation procedure on the performance of simultaneous saccharification and fermentation of steam pretreated
spruce. Enzyme Microb Technol 38:279286
Enriquez-Poy M (2005) Produccion de etanol anhidro en los ingenios azucareros. Short communication, CONAE. Mexico
Union Nacional de Caneros AC (2011) CNPR. Boletn de Prensa
Mar 007
Da Silva GP, de Araujo E, Daison OS, Guimaraes WV (2005)
Ethanolic fermentation of sucrose, sugar cane juice and molasses
by Escherichia coli K011 y Klebsiella Oxytoca P2. Braz J
Microbiol 36:395404
Ortiz-Zamora O, Cortes-Garca R, Ramrez-Lepe M, GomezRodrguez J, Aguilar-Uscanga MG (2009) Isolation and selection
of ethanol-resistant and osmotolerant yeasts from regional agricultural sources in Mexico. J Food Process Eng 32:775786
Campos-Pasteln JM (2008) Establecimiento de un proceso de
produccion de Etanol a partir de Jugo de cana y miel intermedia
B con Saccharomyces cerevisiae ITV-01 y S. cerevisiae EDV493. Masters Thesis, Instituto Unidad de Investigacion y Desarrollo de Alimentos, Tecnologico de Veracruz-Mexico
Ortiz-Muniz B, Carvajal-Zarrabal O, Torrestiana-Sanchez B,
Aguilar-Uscanga MG (2010) Kinetic study on ethanol production
using Saccharomyces cerevisiae ITV-01 yeast isolated from
sugar cane molasses. J Chem Technol Biotechnol 85:13611367
Moulin G, Boze H, Galzy P (1984) Inhibition of alcoholic fermentation. Biotechnol Gen Eng Rev 2:365382
Jones SRP, Pammen TN, Greenfirled PF (1981) Alcohol fermentation by yeast: the effect of environmental and other variables. Process Biochem 16:4245
Walker GM (1998) Yeast physiology and biotechnology. Wiley,
Chichester
Shen B, Hohman S, Jensen RG, Bohnert H (1999) Roles of sugar
alcohols in osmotic stress adaptation. Replacement of glycerol by
mannitol and sorbitol in yeast. Plant Physiol 21:4252
Schhuller C, Brewster JL, Alexander MR, Gustin MC, Ruis H
(1994) The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of Saccharomyces cerevisiae CTTI gene. EMBO J 13:43824389
Myers DK, Lawlor DTM, Attfield PV (1997) Influence of
invertase activity and glycerol synthesis and retention on fermentation of media with a high sugar concentration by Saccharomyces cerevisiae. Appl Environ Microbiol 63:145150
Lange H, Bavouzet JM, Taillander P, Delorme C (1993) Systematic error and comparison of four methods for assessing the
viability of Saccharomyces cerevisiae suspensions. Biotechnol
Tech 7:223228
Imai T, Ohno T (1995) The relationship between viability and
intracellular pH in the yeast Saccharomyces cerevisiae. Appl
Environ Microbiol 61(10):36043608
Gilis JF (1999) Etude de Contaminations de Fermentations Alcooliques Industrielles par les Levures Brettanomyces. Doctoral
Thesis. Institut National Polytechnique de Toulouse, France

123

614
28. Salmon JM (1989) Effect of sugar transport inactivation in Saccharomyces cerevisiae on sluggish and stuck enological fermentations. Appl Environ Microbiol 55:953958
29. Leao C, Van Uden N (1982) Effects of ethanol and other alkanols
on the glucose transport system of Saccharomyces cerevisiae.
Biotechnol Bioeng 24:26012604
30. Berthels NJ, Cordero-Otero RR, Bauer FF, Thevelein JM, Pretorius IS (2004) Discrepancy in glucose and fructose utilization
during fermentation by Saccharomyces cerevisiae wine yeast
strains. FEMS Yeast Res 4:683689
31. Albers E, Larsson C, Liden G, Niklasson C, Gustafsson L (1996)
Influence of the nitrogen source on Saccharomyces cervisiae
anaerobic growth and product formation. Appl Environ Microbiol
62:31873195
32. Mendes-Ferreira A, Mendes-Faia A, Leao C (2004) Growth and
fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry. J Appl Microbiol 97:540545

123

Bioprocess Biosyst Eng (2012) 35:605614

33. Erten H, Tanguler H, Cabaroglu T, Canbas A (2006) The influence of inoculum level on fermentation and flavour compounds of
white wines made from cv. Emir. J Inst Brew 112(3):232236
34. Mateo JJ, Jimenez M, Pastor A, Huerta T (2001) Yeast starter
cultures affecting wine fermentation and volatiles. Food Res Int
34(4):307314
35. Letourneau F, Villa P (1987) Saccharomyces yeast growth on
beet molasses. Effects of substrate concentration on alcohol
toxicity. Biotechnol Lett 9(1):5358
36. De Miniac M (1991) Utilisation des levures en fermentation alcoolique industrielle in Biotechnologie des levures. Doctoral
Thesis, Paris, France pp 335370
37. Hohmann S (2002) Osmotic stress signaling and osmosadaptation
in yeasts. Microbiol Mol Biol Rev 66:300372
38. Alfenore S, Cameleyre X, Benbadis L, Bideaux C, Uribelarrea J-L,
Goma G, Molina-Jouve C, Guillouet SE (2004) Aeration strategy: a
need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process. Appl Microbiol Biotechnol 63:537542