Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
NOTATION
max :
0 :
Ea:
R:
T:
Q10 :
T2 :
T1 :
2 :
1 :
Yx/s:
Yet/s:
Yet/x:
Pet:
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
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
www.soci.org
et al.
B Ortiz-Muniz
www.soci.org
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.
www.interscience.wiley.com/jctb
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.
www.soci.org
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 .
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
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
www.interscience.wiley.com/jctb
www.soci.org
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 .
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 .
www.interscience.wiley.com/jctb
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
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
www.soci.org
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 =
2
1
10
T2 T1
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
www.interscience.wiley.com/jctb
www.soci.org
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
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-
www.interscience.wiley.com/jctb
REFERENCES
1 Saithong P, Nakamura T and Shima J, Prevention of bacterial
contamination using acetate-tolerant Schizosaccharomyces pombe
during bioethanol production from molasses. J Biosci Bioeng
108:216219 (2009).
H, Studies on the wine spoilage capacity
2 Silva P, Cardoso H and Geros
of Brettanomyces/Dekkera spp. Am J Enol Vitic 55:6572 (2004).
3 Atteld P V and Ketsas S, Hyper-osmotic stress response by strains
of bakers yeast in high sugar concentration medium. Lett Appl
Microbiol 31:323327 (2000).
4 Malacrino P, Tosi E, Caramia G, Prisco R and Zapparoli G, The
vinication of partially dried grapes: a comparative fermentation
study of Saccharomyces cerevisiae strains under high sugar stress.
Lett Appl Microbiol 40:466472 (2005).
5 Sanchez-Gonzalez Y, Cameleyre X, Molina-Jouve C, Goma G and
Alfenore S, Dynamic microbial response under ethanol stress
to monitor Saccharomyces cerevisiae activity according to initial
biomass physiological states. Bioprocess Biosyst Eng 32:459466
(2009).
6 Panchal C, Yeast Strain Selection. Biotechnology and Bioprocessing
Series, Marcel Dekker Inc (1990).
7 Ortiz-Zamora O,
Cortes-Garca R,
Ramrez-Lepe M,
GomezRodrguez J and Aguilar-Uscanga MG, Isolation and selection
of ethanol-resistant and osmotolerant yeasts from regional
agricultural sources in Mexico. J Food Process Eng 32:775786
(2009). DOI: 10.1111/j.17454530.2008.00244.x.
8 Walker MG, Yeast Physiology and Biotechnology. John Wiley & Sons,
Chichester (1998).
9 Alfenore S, Molina-Jouve C, Guillouet SE, Uribelarrea JL, Goma G
and Benbadis L, Improving ethanol production and viability of
Saccharomyces cerevisiae by a vitamin feeding strategy during
feed-batch process. Appl Microbiol Biotechnol 60:6772 (2002).
de lethanol, resistance
10 Charpentier C, Les arrets de fermentation: role
de la levure. RF de nologie 140:4952 (1993).
11 Petrov VV and Okorokov LA, Increase of the anion and proton
permeability of Saccharomyces carlsbergensis plasmalemma by nalcohols as a possible cause for de-energization. Yeast 64:311318
(1990).
12 Francois JM and Aguilar-Uscanga B, A study of the yeast cell wall
composition and structure in response to growth conditions and
mode of cultivation. Lett Appl Microbiol 37:268274 (2003).
13 Coote PJ, Cole MB and Jones MV, Induction of increased
thermotolerance in Saccharomyces cerevisiae may be triggered
by a mechanism involving intracellular pH. J Gen Microbiol
137:17011708 (1991).
14 Swan TM and Watson K, Membrane fatty acid composition and
membrane uidity as parameters of stress tolerance in yeast. Can J
Microbiol 43:7077 (1997).
15 Lange H, Bavouzet JM, Taillander P and Delorme C, Systematic error
and comparison of four methods for assessing the viability
of Saccharomyces cerevisiae suspensions. Biotechnol Techniques
7:223228 (1993).
www.soci.org
16 Andre L, Hemming A and Adler L, Osmoregulation in Saccharomyces
cerevisiae: studies on the osmotic induction of glycerol production
and glycerol 3-phosphate dehydrogenase (NAD+ ). FEBS Lett
286:1317 (1991).
17 Thamas JM, Rep M, Thevelein JM and Hohmann S, Stimulation of the
yeast osmolarity glycerol (HOG) pathway: evidence for a signal
generated by a change in turgor rather than by water stress. FEBS
Lett 472:159165 (2000).
18 Strehaiano P, Phenomenes dinhibition et fermentation alcoolique.
Sciences I N P Toulouse, France (1984).
These Dr es
19 Moulin G, Boze H and Galzy P, Inhibition of alcoholic fermentation.
Biotechnol Gen Eng Rev 2:365382 (1984).
20 Aguilar-Uscanga MG, Delia ML and Strehaiano P, Nutritional
requirements of Brettanomyces bruxellensis: growth and physiology
in batch and chemostat cultures. Can J Microbiol 46:10461050
(2000).
21 Jrgensen H, Effect of nutrients on fermentation of pretreated wheat
straw at very high dry matter content by Saccharomyces cerevisiae.
Appl Biochem Biotechnol 153:4457 (2009).
22 Gaber ZB, Production of 16% ethanol from 35% sucrose. Biomass and
Bioenergy (2010). DOI:10.1016/j.biombioe.2010.03.017.
23 Madhavan A, Tamalampudi S, Srivastava A, Fujuda H, Bisaria VS and
Kondo A, Alcoholic fermentation of xylose and mixed sugars
using recombinant Saccharomyces cerevisiae engineered for xylose
utilization. Appl Microbiol Biotechnol 82:10371047 (2009).
24 Skinner KA and Leathers TD, Bacterial contaminants of fuel ethanol
production. J Ind Microbiol Biotechnol 31:401408 (2004).
www.interscience.wiley.com/jctb