Springer 2005

World Journal of Microbiology & Biotechnology (2006) 22: 177182

DOI 10.1007/s11274-005-9016-1

Synergism among lactic acid, sulte, pH and ethanol in alcoholic fermentation

of Saccharomyces cerevisiae (PE-2 and M-26)
C. Dorta*, P. Oliva-Neto, M.S. de-Abreu-Neto, N. Nicolau-Junior and A.I. Nagashima
Departamento de Ciencias Biologicas, Universidade Estadual Paulista, Av.Dom Antonio, 2100, CEP 1980000, Assis,
SP, Brazil
*Author for correspondence: Tel.: +55-18-33025850, E-mail: claudorta2000@yahoo.com.br
Received 2 May 2005; accepted 29 June 2005

Keywords: Ethanol, fermentative stress, lactic acid, low pH, Saccharomyces cerevisiae, sulte

Summary
The industrial production of ethanol is aected mainly by contamination by lactic acid bacteria besides others
factors that act synergistically like increased sulte content, extremely low pH, high acidity, high alcoholic content,
high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26
were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected
to values up to 200 mg NaHSO3 l)1, 6 g lactic acid l)1, 9.5% (w/v) ethanol and pH 3.6 during fermentative
processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain
produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic
bacteria. Both strains of yeasts showed similar performance during the fermentation, with no signicant dierence
in cell viability.

Introduction
The Brazilian commercial production of fuel alcohol is
carried out by fed-batch or continuous fermentation
process, using sugar cane juice or molasses as raw
material with Saccharomyces cerevisiae as promoter.
Lactic bacteria, mainly lactobacilli, are most common
responsible for the decrease of alcoholic yield and productivity in the factories (Oliva-Neto & Yokoya 1994,
Hynes et al. 1997). According to Maiorella et al. (1983)
40 g of lactic acid l)1 in synthetic medium was capable
of reducing 80% of the S. cerevisiae population. Daeshel
et al. (1988), using species of S. cerevisiae and S. rosei
with Lactobacillus plantarum as a contaminant, during a
cabbage juice fermentation, established inhibitory concentrations of 2 g lactic acid l)1 for cellular growth.
Oliva-Neto & Yokoya (1994) noticed that after the 15th
cycle of a fermentative process, the alcoholic eciency
suered an important reduction when the lactic acid
exceeded 6 g l)1 and the number of bacterial contaminants exceeded 1.2109 cells ml)1. Ngang et al. (1989)
showed that the toxic eect of this acid depended on the
osmotic pressure of the medium, organic acid concentration, and the yeast cell number. According to Cartwright et al. (1989) the toxic eect of the acid on
S. cerevisiae depended on the pH value. At low pH organic acids will not be predominantly dissociated, and
thus allows the acid to enter passively across the yeast
membrane.

Since 1990 the concentration of cane molasses in the

composition of fermentative must in Brazilian sugar and
alcohol factories has increased, with consequent increase
in the sulte content. Sulte is normally used in the
sugar clarication process and it is present in high level
in sugar cane molasses, contributing to the decrease in
alcoholic yield and yeast viability (Gibbons & Westby
1987).
Ethanol can become toxic for the yeast cell due to a
non-competitive inhibition (Leao & van Uden 1982).
Factors like temperature (Ramos & Madeira-Lopes
1990), by-products: acetaldehyde, formic, acetic and
lactic acids (Maiorella et al. 1983), octanoic and decanoic acids (Viegas et al. 1989) enhance the toxic eect of
ethanol.
It is important to study the synergetic eect of these
toxins and conditions in the cell recycle process of the
alcoholic fermentation, due to the problems of reduced
ethanolic yield. In this work, this study was done aiming
at improvement of the fuel ethanol technology and yeast
physiology.

Materials and methods

Strains and culture conditions
Saccharomyces cerevisiae Pedra-2 (PE-2) was obtained
from Department of Science and Agroindustrial

178

C. Dorta et al.

Technology, University of Sao Paulo, Piracicaba/Brazil;

and S. cerevisiae M-26 was isolated by Laboratory of
Biochemistry and Microbiology, Sao Paulo State University, Assis/Brazil. The strains were stored at )75 C
in Eppendorfs with medium YM (with 10% v v)1
glycerol) at pH 5.0. These two yeasts are used in the
ethanolic fermentation industry in Brazil.
The inoculum for the fermentative process of
S. cerevisiae PE-2 and M-26 strains was prepared
aseptically through successive cultivation in the formulated medium: 2% sucrose (Difco-P.A), 1% yeast extract (Difco), 0.1% ammonium sulfate, 0.114%
K2HPO43H2O, 0.024% MgSO47H2O, 0.00104%
MnSO4H2O, 0.0028% ZnSO47H2O, pH 5.0, incubated
at 30 C for 24 h by continuous shaking at 135 rev min)1 on an orbital shaker (Tecnal TE421-Sao PauloBrazil). The yeasts were centrifuged (Fanen-SP-Brazil)
after nal growth at 4 C, 3500g for 25 min.
To start the fermentation 27 g (wet biomass) of yeasts
was suspended in 75 ml distilled water in 500 ml
Erlenmeyer asks. The compositions of semi-synthetic
media (must) with stressing conditions is shown in Table 1. The fed batch process was carried out by adding
15 ml of the must at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 9 h of
fermentation. The nal volume of each fermentative
cycle was 225 ml. The asks were shaken at 80 rev min)1 on an orbital shaker (Tecnal TE421-Sao PauloBrazil) at 32 C for 12 h. The yeasts were centrifuged
after the end of the fermentative cycle at 4 C, 3500g
for 25 min and the pitching cell suspension (inoculum)
was prepared for the next cycle. This system was performed for seven cycles. The samples were collected for
analysis at the end of fermentation.

column (SCR 101H) and oven (CTO-10AVP). Perchloric acid was used as solvent (10 mM) with a ow
rate of 1.2 ml min)1 at 50 C and acid standards (HPLC
grade): aconitic, citric, tartaric, malic, succinic, lactic
and acetic (Merck-USA). The samples were thawed,
diluted to the required strength with deionised water
and 20 ll aliquots were injected into HPLC.
Trehalose and protein residue
Yeast endogenous trehalose was determined by the
anthrone method (Brin 1966), after selective extraction
with trichloroacetic acid at 0 C performed according to
Trevelyan & Harrison (1956). Protein residue was
determined by the Lowry method using bovine serum
albumin as standard.
Determination of sugar and ethanol
One ml diluted sample of wine (fermented must) was
hydrolysed with 1 ml (2 M) HCl in boiling water for
15 min. After cooling, 1 ml (2 M) NaOH was added and
the total sugars were determined by Somogyi and Nelsons method (Nelson 1944). The concentration of ethanol was determined by gas chromatography (GC,
Mod. CG 37, Instrumentos CG Ltd.) equipped with a
column (PAD 2499 CG). Temperatures of column,
vaporiser and ionization ame were 96 C, 150 C and
250 C, respectively. Ethanol concentration was converted to ethanol productivity (g ethanol produced/litre
of total fermented must/h) and ethanol yield (g ethanol
produced  100/g sugar consumed  0.511), where 0.511
is the conversion factor of sugar to ethanol based on the
theoretical maximum yield).

Viability and budding of yeasts

Statistical methods
The percentage of budding and live cells (viability) to
the total cells was determined by light microscope with a
Neubauer chamber. The yeast cells were dyed with
erythrosin solution (dilution 5000 times), in samples
after 12 h of each fermentative cycle.
Determination of organic acids
Organic acids were determined by HPLC (Shimadzu)
equipped with pump (LC-10AUV), detector (SPD-10A),

The experiments were performed three times. The samples of the third to the seventh fermentative cycle were
analysed by variancy (ANOVA) and the averages
compared by Tukey and Kramer tests through the
Graphpad Instat program (Rutgers University, Camden, New Jersey). The t-test was applied in the averages
between two sample groups by the same program. The
treatments were considered signicantly dierent with
P<0.05.

Table 1. Formulation of the fermentative media with stress factors: ethanol, lactic acid, sulte and pH.
Medium

T (C)

Sulte (mg l)1) (NaHSO3)

Lactic acid (g l)1)

Ethanol (%)

pH*

Toxicity level

1
2
3
4
5
6 (control)

32
32
32
32
32
32

200
50
200
200
200
0

6.0
6.0
2.0
6.0
6.0
0.0

9.5
9.5
9.5
7.5
9.5
7.5

3.6
3.6
3.6
3.6
4.5
4.5

maximum
low sulte
low lactic acid
low ethanol
normal pH

*Sucrose at concentration of 16.37% or 20.65% (w/v) was used as carbon source; theoretical conversion to 7.5% or 9.5% (w/v) ethanol,
respectively.

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Synergism in ethanol fermentation

Results and discussion
Viability and budding
The low pH and the high ethanol concentration were the
main stress factors to cell viability of the S. cerevisiae
PE-2 and M-26 (Figure 1). The M5 medium (pH 4.5)
increased signicantly (Tukey, P<0.05) cell viability,
43.5% and 31% by PE-2 and M-26 strains, respectively,
when compared with cell viability of the M1, M2 and
M3 media (pH 3.6). Ethanol at 7.5% (w/v) (M4 medium) caused a signicant increase in the cell viability of
the PE-2 and M-26 strains, 38% and 29%, respectively,
when compared with 9.5% (w/v) ethanol used in the
M1, M2 and M3 media. It has been reported that the
very low pH increased the toxic eect of ethanol,
organic acids and SO2 (Cartwright et al. 1989). High
concentrations of ethanol (10%, w/v) decreased the
alcoholic yield during the industrial fermentation
(Walker-Capriolo et al. 1985). The toxic eects of ethanol are known and involve modication of membrane
lipid composition, the synthesis of stress proteins,
modulation of ion exchange processes, as well as
reduction of metabolic activity which cause the inhibition of glucose uptake, decreasing of growth rate and
product formation (Martini et al. 2004), and water
stress (Hallsworth 1998).
Low pH was the main factor in the decrease of budding rate in the two strains (Figure 2). The budding rate
of the M5 and control media were about 80% greater
than the M1 medium (higher stress).

Figure 2. Budding cells during dierent fermentation conditions by

Saccharomyces cerevisiae PE-2 (h) and M-26 (n) strains.

media as compared to PE-2 strain (Table 2). According

to Oliva-Neto et al. (2004) the supernatants obtained
after the cultivation of M-26 showed a bacteriostatic
eect on Lactobacillus fermentum. It could be due to
higher organic acids production especially succinic acid
(Basso et al. 1997). The production of the organic acids
(mainly succinic, tartaric, and acetic acids) were greater
in M-26 (Table 3) and it could explain the inhibitory
eect of M-26 on growth of lactic acid bacteria.
Trehalose and residual protein

Total organic acids

The highest production of total organic acids by strains
PE-2 and M-26 occurred in M2 medium which was
about 50% higher than control, a signicant dierence
(Tukey, P<0.05). The M-26 strain showed a signicant
increase in the total organic acids in all fermentative

The fermentative processes stimulated trehalose production for both strains (Figure 3). The M4 medium
and the control (both with the least sugar concentration)
were responsible for the highest trehalose contents in the
microorganisms. This is in agreement with Panek (1975),
Lillie & Pringle (1980) and Francois et al. (1991) that
related high concentration of trehalose to the low sugar
concentration in the medium. The PE-2 strain showed
higher accumulation of trehalose and higher cell
Table 2. Total organic acids produced during alcoholic fermentation
by Saccharomyces cerevisiae M-26 and PE-2 in dierent stress conditions.
Medium

Total organic acids (g l)1)

M-26

M1
M2
M3
M4
M5
M6
Figure 1. Cell viability during dierent fermentation conditions by
Saccharomyces cerevisiae PE-2 (h) and M-26 (n) strains. See Table 1
for media (M) compositions.

PE-2

M*

SD

M*

SD

14.97
15.88
12.02
13.30
14.65
10.62

1.76
0.67
1.37
1.00
1.00
1.15

10.86
12.25
8.50
8.06
11.00
8.00

3.68
2.60
1.96
2.90
2.28
2.00

*M=mean, SD=standard deviation. The means were obtained of

samples of the third, fth and seventh fermentative cycles.

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C. Dorta et al.

Table 3. Organic acids produced during alcoholic fermentation by Saccharomyces cerevisiae M-26 and PE-2 in dierent stress conditions.
Organic acid (mg l)1)

Aconitic
M-26
PE-2
Citric
M-26
PE-2
Tartaric
M-26
PE-2
Malic
M-26
PE-2
Succinic
M-26
PE-2
Lactic
M-26
PE-2
Acetic
M-26
PE-2

M1

M2

M3

M4

M5

M6

M*

SD

M*

SD

M*

SD

M*

SD

M*

SD

M*

SD

0.81
0.62

0.19
0.17

1.00
0.75

0.20
0.23

0.80
0.60

0.29
0.18

0.73
0.50

0.20
0.13

1.50
1.18

0.70
0.39

1.30
0.92

0.08
0.20

0.19
0.09

0.02
0.06

0.26
0.12

0.23
0.12

0.10
0.07

0.11
0.10

0.19
0.03

0.23
0.04

0.00
0.03

0.00
0.06

0.08
0.03

0.05
0.14

0.67
0.43

0.05
0.12

0.72
0.60

0.12
0.23

0.42
0.36

0.50
0.05

0.42
0.39

0.05
0.07

0.44
0.32

0.05
0.15

0.31
0.15

0.03
0.03

5.08
3.70

0.57
1.00

4.90
4.25

0.42
1.26

3.00
2.63

0.42
0.60

0.34
3.07

0.13
0.98

5.14
3.85

0.68
0.91

2.42
2.21

0.25
0.39

3.89
2.86

0.25
1.03

4.20
3.12

0.85
1.04

3.87
2.84

0.54
0.92

4.38
3.04

0.33
1.03

4.30
3.17

0.28
1.00

4.20
3.06

0.43
1.00

1.39
1.10

0.44
0.43

1.55
1.27

0.21
0.39

1.02
0.85

0.36
0.31

1.62
1.11

0.29
0.19

1.21
1.50

0.07
0.25

1.00
1.11

0.07
0.12

3.18
2.13

0.91
0.83

3.18
2.39

0.53
0.90

2.83
1.78

0.47
0.43

2.40
1.58

0.29
0.37

1.90
1.14

0.29
0.58

1.36
0.74

0.17
0.18

*M=mean, SD=standard deviation. The means were obtained of samples of the third, fth and seventh fermentation cycles.

viability, suggesting that these two parameters were

associated. According to Thevelein (1984), low trehalose
concentration decreased cell membrane integrity and cell
viability.
Must fermented by PE-2 strain showed a marked
increase (up to 83%) in residual protein in M1 and M2
media as compared with M5 and control must (Figure 4). This showed that pH 4.5 provided lower cellular
lysis, higher membrane integrity and higher protein
consumption than at pH 3.5 by the PE-2 strain. However, the dierent treatments had no signicant eect on
protein release by M-26 strain.

Figure 3. Trehalose concentration in Saccharomyces cerevisiae PE-2

and M-26 strains during dierent fermentation conditions.(h) PE-2,
rst cycle-zero time; ( ) PE-2, seventh cycle-12 h; (n) M-26, rst cyclezero time; (N) M-26, seventh cycle-12 h.

Determination of the residual sugar, ethanolic yield

and productivity
There was signicant increase in sucrose consumption in
the control, M4 and M5 media for both strains (Table 4). Once again pH was the most important factor
related to inhibition. In the fermented must of M5 there
was a sharp increase in the sugar consumption of 160%
and 84% when compared to the M1 medium in fermentation by PE-2 and M-26 strains, respectively.
The ethanolic yield by M-26 strain was 10% greater
than strain PE-2, although the dierence was not

Figure 4. Residual protein in medium after dierent fermentation

conditions by Saccharomyces cerevisiae PE-2 (h) and M-26 (n).

181

Synergism in ethanol fermentation

Table 4. Residual sugar in medium after dierent fermentation conditions by Saccharomyces cerevisiae PE-2 and M-26.
Medium

Residual sugar% (w/v)

M-26

M1
M2
M3
M4
M5
M6

PE-2

M*

SD

M*

SD

1.45
1.30
0.97
0.58
0.55
0.29

1.00
0.99
0.80
0.20
0.20
0.08

1.00
0.80
0.90
0.53
0.54
0.29

0.60
0.30
0.60
0.10
0.10
0.15

*M=mean, SD=standard deviation. The means were obtained of

samples of the third to the seventh fermentative cycles.

signicant (Figure 5). The best yield was found at pH

4.5 by yeasts studied, probably due the highest cell
viability, budding and the least residual sugar content.
The ethanolic yield in distilleries varies from 70 to 90%,
depending on the type of installation and the raw
materials and fermentation conditions used (Oliva-Neto
& Yokoya 1994). The ethanolic yield in this present
work varied between 72 and 85%, which agrees with the
industrial data (Figure 5).
M5 medium was responsible for the highest values of
ethanolic productivity for both microorganisms (Figure 6).
The low pH of 3.6 was shown to be markedly the
highest stress factor for both strains. It overcame the
high concentrations of lactic acid (6 g l)1), sulte
(150 mg SO2 l)1) and ethanol (9.5%, w/v). The stress
factors damage yeast metabolism resulting in cell lysis
and amino acid and vitamin release into the medium,
stimulating the growth of lactic acid bacteria (OlivaNeto & Yokoya 1997) and consequently leading to a
drop of alcoholic yield by S. cerevisiae. The low pH and
organic acid were related to yeast inhibition because
undissociated organic acid enters the cells by simple

Figure 6. Ethanolic productivity during dierent fermentation

conditions by Saccharomyces cerevisiae PE-2 (h) and M-26 (n) strains.

diusion through the plasma membrane (Cassio et al.

1987). Low pH also is related to sulte toxicity, since the
more toxic forms of sulte for yeast cells are SO2 and
HSO)3 which are found at pH 1.04.5, and not the SO2)
3
form which is more abundant at pH 5.07.0 (Macris &
Markatis 1974).
The organic acids, ethanol, and low pH cause the
acidication of intracellular pH, and it stimulates the
H+-ATPase of the plasma membrane to eliminate
intracellular H+ (Calahorra et al. 1987). When the
medium pH is less than 4.0 H+-ATPase activity is increased three times with expenditure of ATP (Eraso &
Gancedo 1987) and in this condition the yeast becomes
more stressed.
As a general conclusion, the data indicate that an
excessive use of sulfurous acid on the pitching yeast
during industrial fuel ethanol production, for control of
bacterial contamination, causes a decrease in the pH of
the fermented must with a negative eect on yield and
productivity. Hence, it should be done with caution for
the improvement of yeast physiology during cell recycling in the fermentation.
Acknowledgments
We thank Usina Nova America (Taruma/SP/Brazil) and
Imesa (Instituto Municipal de Ensino Superior de Assis/
SP/Brazil) for lending the HPLC and CG for the sample
analysis. This work was supported by Coordenacao de
Aperfeicoamento de Pessoal de N vel Superior
(CAPES), Brazil.
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Figure 5. Ethanolic yield during dierent fermentation conditions by

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