APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1988, p. 2439-2446 Vol. 54, No.

10
0099-2240/88/102439-08$02.00/0
Copyright © 1988, American Society for Microbiology

Influence of Calcium Ion on Ethanol Tolerance of Saccharomyces

bayanus and Alcoholic Fermentation by Yeasts
REGINA C. NABAIS, ISABEL SA CORREIA,* CRISTINA A. VIEGAS, AND JULIO M. NOVAIS
Laboratorio de Engenharia Bioquimica, Instituto Superior Tecnico, 1000 Lisbon, Portugal
Received 29 March 1988/Accepted 8 July 1988

The addition of Ca2' (as CaCl2) in optimal concentrations (0.75 to 2.0 mM) to a fermentation medium with
a trace contaminating concentration of Ca2+ (0.025 mM) led to the rapid production of higher concentra-
tions of ethanol by Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus. The
positive effect of calcium supplementation (0.75 mM) on alcoholic fermentation by S. bayanus was explained by
the increase in its ethanol tolerance. The ethanol inhibition of growth and fermentation followed the equation
,UXi = FL0i [1 - (X/Xmj)J", where Foi and P-xi are, respectively, the specific growth (i = g) and fermentation (i
= f) rates in the absence or presence of a concentration (X) of added ethanol, and Xmi is the maximal
concentration of ethanol which allows growth or fermentation. The toxic power is given by n,. In Ca2+-
supplemented medium (0.75 mM), ng = 0.42 for growth and nf = 0.43 for fermentation compared with 0.52
and 0.55, respectively, in unsupplemented medium; for both media, Xmg = 10% (vol/vol) and Xmi = 13% (vol/
vol). For lethal concentrations of ethanol, the specific death rates were minimal for cells that were grown and
incubated with ethanol in medium with an optimal concentration of Ca2 , maximal for cells grown and
incubated with ethanol in unsupplemented medium, and intermediate for cells grown in unsupplemented
medium and incubated with ethanol in calcium-supplemented medium. The effect of Ca2+ on the acidification
curve of energized cells in the presence of ethanol was found to be closely associated with its protective effect
on growth, fermentation, and viability.

The ethanol tolerance of yeasts is strongly dependent on
the composition of the medium in which it is evaluated (3,
12, 20, 22, 23). It is now admitted that the maximal concen-
tration of ethanol that is characteristic of different alcoholic
fermentations (for the production of beer, wine, or sake or
for large-scale production of ethanol) is more dependent on
medium composition than on the intrinsic ethanol tolerance
of the industrial strains (12).
It has been shown that several compounds lead to im-
provements in alcoholic fermentation productivity: unsatu-
rated fatty acids and sterols, proteins, amino acids, vitamins,
and metal ions (for a review, see reference 3). Complex
media such as Jerusalem artichoke juice (22, 23) or complex
supplements such as soy flour and peptone have been found
to lead to improvements in alcoholic fermentations by in-
creasing yeast ethanol tolerance (29; unpublished data). It is
likely that additives such as oryzenin, albumin, and koji
mold mycelia (3, 5) may act in a similar way.
For optimal growth and fermentation, yeasts require mi-
cro- and millimolar concentrations of different inorganic
cations (for a review, see reference 9). These ionic species
could either act on the enzymatic activity by participating on
the catalytic center or as activators or stabilizers, or they
could have a structural role by shielding the negatively
charged membrane phospholipids and cell wall phosphoman-
nans (9). The optimal concentrations of various cations,
which have been determined for several species of the genus
Saccharomyces, have been given in a review by Jones and
Greenfield (9). The supplementation of yeast fermentations
with 0.5 mM Mg2+ proved to prolong exponential growth
and to reduce the decline in fermentative activity (4), and
Dombek and Ingram (4) have suggested that some of the
*
Corresponding author.
2439
reported beneficial effects of complex nutrients (soy flour,
yeast extract) on ethanol production could partially result
from the correction of a simple inorganic ion deficiency,
such as a deficiency in magnesium. The addition of magne-
sium salts has been proposed to reverse the ethanol-induced
leakage that has been reported to occur in Zymomonas
mobilis (21). This is particularly true for magnesium, the
cation that is required for nucleotide cofactors of glycolytic
enzymes (21).
The presence of calcium in optimal concentrations (range,
2.5 to 10 mM) was found to increase the thermostability of
Bacillus stearothermophilus (11, 19) by stimulating growth at
supraoptimal temperatures and increasing the maximum
growth temperature, but it had no effect on growth within the
optimal range (11). This protection appeared to result from
increased membrane stability as its addition to a cellular
suspension at 60°C, in buffer, was found to prevent the
release of cytoplasmic compounds (19).
Ethanol and high temperatures interfere with membrane
organization, increasing its fluidity and permeability to ions
and small metabolites (6, 26) and inhibiting the transport of
nutrients (28). Therefore, it is possible that the increase in
the thermostability by the presence of optimal concentra-
tions of Ca2+ could be extensible to an increase in ethanol
tolerance in fermenting yeasts. In this study we tested this
hypothesis after confirming the significant improvement in
the performance of alcoholic fermentations by Saccha-
romyces cerevisiae, Saccharomyces bayanus, and Kluyve-
romyces marxianus in unsupplemented medium (with 0.025
mM Ca2+) and in medium supplemented with CaCl2 in
concentrations ranging from 0.75 to 2.0 mM. Studies on the
ethanol tolerance of S. bayanus were concentrated on the
inhibition of growth and fermentation (Warburg respirom-
eter) by ethanol and the decrease in viability by the presence
2440 NABAIS ET AL.
of lethal concentrations of ethanol in media presenting
optimal and suboptimal concentrations of calcium. The
effect of Ca2" on the acidification curve of energized cells in
the presence of ethanol was also evaluated as a measure of
the ethanol toxicity in S. bayanus in calcium-supplemented
and -unsupplemented media (7).
MATERIALS AND METHODS
Microorganisms. The main strain used in this study was
the industrial strain S. bayanus IST 154 (now S. cerevisiae
[30]), which is used in the production of sparkling wines (29).
For comparison, S. cerevisiae IGC 3507 III (26, 28) and K.
marxianus (22, 23) were also tested.
Alcoholic fermentations. Batch fermentations were carried
out in conical flasks that were closed with needle-perforated
rubber bungs in an orbital shaker at 30°C. They were
undertaken with strains of S. bayanus, S. cerevisiae, and K.
marxianus in base medium containing yeast extract (5 g/liter;
Difco Laboratories, Detroit, Mich.), MgSO4- 7H20 (1
g/liter; BDH, Poole, England), (NH4)2SO4 (5 g/liter; Merck
AG, Darmstadt, Federal Republic of Germany), KH2PO4 (5
g/liter; BDH), and glucose (320 g/liter; Merck). This base
fermentation medium was supplemented with calcium (0 to 2
mM as CaCl2; Merck) before the beginning of the process or
after 48 h of fermentation. Fermentation medium was inoc-
ulated with cells of S. bayanus that were obtained by
rehydration of the dry active industrial granules (105 viable
cells per ml) or obtained from the exponential phase of a
growth in Ca2+-unsupplemented medium (5 x 106 viable
cells per ml). In fermentations with S. cerevisiae and K.
marxianus, the inoculation was done with cells in the expo-
nential phase of growth that were obtained from Ca2+-
unsupplemented medium (3 x 106 viable cells per ml).
Growth conditions. The growth of cells to be used in
alcoholic fermentations or in the quantification of ethanol-
induced inhibition or stimulation of several biological pro-
cesses was done in a base medium, which was identical to
that referred to above for alcoholic fermentations, but with
30 g of glucose per liter supplemented or not supplemented
with 0.75 mM calcium (as CaCI2). Growing suspensions of
yeasts were incubated in shaken flasks at 30°C in order to
obtain exponentially growing cells.
Ethanol-induced inhibition of growth. Specific growth rates
were calculated by least-squares fitting to the linear part of
semilogarithmic growth curves that were drawn by measur-
ing the culture optical density at 640 nm. The growth of S.
bayanus IST 154 in the presence of increasing concentra-
tions of ethanol (0 to 10% [vol/vol]) was followed for 24 to 40
h, depending on the ethanol concentration, in growth me-
dium supplemented or not supplemented with calcium (0.75
mM). Inoculation was done with exponentially growing cells
in unsupplemented medium after centrifugation and two
washes with deionized water (initial concentration, 40 mg
[dry weight]/liter).
Ethanol-induced death rates. The specific death rates (Kd)
were calculated by least-squares fitting to the linear parts of
the semilogarithmic survival plots. Cells of S. bayanus IST
154 to be incubated with lethal concentrations of ethanol
were harvested exactly in the same phase of the exponential
growth phase either in calcium-supplemented (0.75 mM) or
-unsupplemented growth medium. After centrifugation and
two washes with deionized water, cells were incubated with
lethal concentrations of ethanol added to calcium-supple-
mented (0.75 mM) or -unsupplemented growth medium. The
initial concentration of viable cells was 104/ml, and at
APPL. ENVIRON. MICROBIOL.
suitable intervals, samples were taken in order to follow the
concentrations of viable cells.
Respirometry measurements. Warburg flasks were loaded
with a S. bayanus IST 154 cell suspension (120 mg [dry
weight]/liter) in phosphate buffer (0.1 M, pH 4.5) with 10 g of
glucose per liter and increasing concentrations of ethanol (0
to 14% [vol/vol]) supplemented or not supplemented with
CaCl2 (0.75 mM). Cells were previously grown in the unsup-
plemented medium, centrifuged, and washed twice with
deionized water. Cell suspensions were flushed with nitro-
gen gas during the 10 min that was taken to equilibrate them
at 30°C. Rates of CO2 production were measured with a
differential respirometer. Those values were used to calcu-
late the specific fermentation rates (as microliters of CO2
evolved per minute and per optical density unit at 640 nm as
a measure of dry biomass concentration) after they were
corrected from the value obtained from endogenous respira-
tion.
Acidification curves. Cells of S. bayanus IST 154 grown in
calcium-supplemented (0.75 mM) or -unsupplemented
growth medium were washed twice with deionized water and
suspended (200 mg [dry weight]/liter) in a glucose solution
(20 g/liter) with different concentrations of ethanol (0 to 16%
[vol/vol]) that was supplemented or not supplemented with
CaCl2 (0.75 mM). Those suspensions (100 ml) were placed in
250-ml Erlenmeyer flasks that were closed with needle-
perforated rubber bungs and incubated in a water shaker at
30°C for 5 h. After sampling (10 ml), each cellular suspension
was quickly centrifuged and the pH of its supernatant was
measured.
Analysis. The concentration of glucose was determined by
the dinitrosalicylic acid method (18), and the concentration
of ethanol was determined by gas chromatography (n-pro-
panol was used as the internal standard).
Viable cells. Viable cells were counted by plating, after
appropriate dilution, on the surface of petri dishes with a
solidified medium composed of 2% glucose, 1% peptone,
0.5% yeast extract, and 2% agar. Plates were incubated at
30°C for 2 to 3 days.
RESULTS
Fermentation profiles of calcium-supplemented and -unsup-
plemented media. Suboptimal concentrations of the calcium
ion (0.025 mM) that was present in a laboratory fermentation
medium with 320 g of glucose per liter did not allow complete
fermentation of the sugar by S. bayanus, despite the size (1
x 105 viable cells per ml or 5 x 106 viable cells per ml) and
the physiological state (nongrowing or exponentially grow-
ing cells, respectively) of the inoculum being used (Fig. 1 and
2, respectively). The addition of 0.75 mM (30 mg/liter)
calcium ion (as CaCl2) led to the improvement of alcoholic
fermentation (Fig. 1 and 2). When the initial concentration of
exponentially growing cells was higher (5 x 106 viable cells
per ml; Fig. 2), the improvements caused by calcium sup-
plementation did not appear to be so expressive, because
without supplementation almost all the sugar became ex-
hausted (Fig. 1 and 2). Therefore, the presence of an optimal
concentration of calcium must be significant when a higher
concentration of ethanol can potentially be reached or when
the concentration of the inoculum, or any other fermentation
condition, does not allow the complete fermentation and
leads to premature stoppages.
The addition of calcium after 48 h of fermentation, when it
suffered an important deceleration, led to its restart and,
with delay, to a higher final concentration of ethanol (Fig. 1).
VOL. 54, 1988 INFLUENCE OF Ca2+ ON ETHANOL TOLERANCE 2441

0 _\ -400 -

C:
a,
LU
-0 200 -

300

a,~~~~~~~

o 00 X
0

100 .

cn 200 -

0 24 48 72 96 120 144
20 20 40 60 8
Time (h)
FIG. 1. Fermentation profiles by S. bayanus IST 154 at 30°C of
fermentation media with 320 g of glucose per liter supplemented
with Ca2+ (0.75 mM) from the beginning of the fermentation (0),
supplemented after 48 h of fermentation (A), and unsupplemented 0 40
20 60 ~8
(0) (inoculum, by rehydration of dry granulated yeasts; 105 viable Time (h)
cells per ml).
FIG. 2. Fermentation profiles by S. bayanus IST 154 at 300C of
fermentation media with 320 g of glucose per liter supplemented
with Ca2+ (0.75 mM) (0) and unsupplemented (0) (inoculum,
The concentration of the viable cells during the fermenta- exponentially growing cells; 5 x 106 viable cells per ml).
tion justified the differences observed between the profiles of
supplemented and unsupplemented media (Fig. 2). In fact,
during the entire process the concentration of viable cells in
calcium-supplemented medium remained the highest, de- medium decreased the inhibition by ethanol of both the
spite the production of a higher concentration of ethanol specific growth and fermentation rates of S. bayanus (Fig.
(Fig. 2). This suggests that the ethanol tolerance of S. 4). In the absence of ethanol, the rates were identical in
bayanus in the medium supplemented with an optimal con- calcium-supplemented or -unsupplemented medium (Fig. 4).
centration of calcium must be higher than that which is This indicates that calcium, in optimal concentrations, some-
possible in unsupplemented medium, in terms of the inhibi-
tion of growth and fermentation and the decrease of cellular
viability by ethanol. This hypothesis was tested and proved
in this study. 14
The role of calcium in improving alcoholic fermentation
performance appeared to be a general effect in fermenting
yeasts. It was also detected in strains of K. marxianus
(unpublished data) and S. cerevisiae (Fig. 3). The optimal
concentration of calcium for batch alcoholic fermentation 13C
was found to be 0.75 mM for S. bayanus IST 154 (inoculum
by rehydration of dry granulated yeasts; 105 viable cells per
ml). Above 0.75 mM and up to 1.5 mM, the maximal 12
concentration of ethanol decreased but its value was above
that reached with the unsupplemented medium (Fig. 3). For
S. cerevisiae IGC 3507 III (inoculum with exponentially 0 0.4 0.8 1.2 1.6 2.0
growing cells; 3 x 106 viable cells per ml), the optimal range
of calcium concentrations was higher (1.6 to 2.0 mM) (Fig. Ca2+ concentration (mM)
3). FIG. 3. Effect of the Ca2" concentration added to a fermentation
Effect of calcium on the inhibition of growth and fermenta- medium (320 g of glucose per liter) on the maximal concentration of
tion by ethanol. The presence of an optimal concentration of ethanol reached during the fermentation at 30°C by S. bayanus IST
calcium (0.75 mM) in the growth or in the fermentation 154 (A) and S. cerevisiae IGC 3507 III (0).
2442 NABAIS ET AL.
0 2 4 6 8 10 12 14
Etha noL % ( v/v)
FIG. 4. Inhibition by ethanol of the specific growth (-, Ol) and
fermentation rates (0, 0) of S. bayanus IST 154 at 30°C in
Ca2+-supplemented (0.75 mM) (-, *) and -unsupplemented media
(OI, 0)-
how protects S. bayanus from the toxic effects of ethanol,
despite the fact that suboptimal concentrations (0.025 mM)
are adequate to sustain maximum growth and fermentation
in the absence of ethanol (Fig. 4).
Ethanol was found to inhibit growth more drastically than
fermentation either in supplemented or in unsupplemented
medium (Fig. 4), in accordance with results of several other
studies (3, 28).
The kinetics of growth and fermentation inhibition by
ethanol either in unsupplemented or in calcium-supple-
mented medium are expressed by equations 1 and 2, respec-
tively (14) (Fig. 5),
PLxg = PXog[1 - (XIXmax)]n
PLXf = XJVLXf1 - (XIX max)]n
where Puxog and lxg are the specific growth rates in the
absence or presence of a concentration (X) of ethanol,
respectively; [LXof and Pxf are the specific fermentation rates
in the absence or presence of a concentration (X) of ethanol,
respectively; Xmax and X'max are the maximal concentra-
tions of ethanol that allowed growth and fermentation,
respectively (10% [vol/vol] and 13% [vol/vol], respectively);
and n and n' are the ethanol toxic power calculated for
growth and fermentation inhibition, respectively.
In calcium-supplemented medium, the toxic powers (n and
n') were lower than those calculated in unsupplemented
medium (Table 1) and partially characterize the ethanol
tolerance of S. bayanus under both conditions.
Effect of calcium on ethanol-induced death. When sub-
jected to lethal concentrations of ethanol at 30°C, the viabil-
ity of S. bayanus cells grown in a base medium not supple-
mented with calcium decreased exponentially during the
time of contact, despite the addition of calcium to the
medium of incubation with ethanol (Fig. 6). The specific
death rates indicated that calcium has a protective effect
toward ethanol-induced lethality (Fig. 6 and 7). Moreover,
cells grown in a medium supplemented with optimal concen-
trations of calcium had the highest ethanol tolerance (Fig. 6
and 7).
The stimulation of ethanol-induced death by increasing the
concentration of this toxic substance was also studied. It
was found that, despite calcium supplementation of either
the growth medium or the medium in which the cells were
incubated with ethanol, the specific death rate was an
APPL. ENVIRON. MICROBIOL.
' 4
2 (b) _
0.7 xX
max
FIG. 5. Inhibition by ethanol of growth (a) and fermentation (b)
of S. bayanus IST 154 in Ca2"-supplemented (0.75 mM) (0) and
-unsupplemented (0) media, based on Equations 1 and 2; Xmax =
10% (vol/vol) and X'max = 13% (vol/vol). ODO40, Optical density at
(1) 640 nm.
(2)
exponential function of ethanol concentration (Fig. 7, equa-
tion 3),
ln udX = ln RdXm + KdX
(3)
where pudX and pLdxm were the specific death rates in the
presence of a concentration (X) of ethanol or the minimal
lethal concentration of ethanol used (13% [vol/vol]), respec-
tively. Kd was the constant of stimulation by ethanol of
ethanol-induced death.
The presence of an optimal concentration of calcium
either in the growth or in the incubation medium decreased
the specific death rate in the presence of the minimal lethal
concentration of ethanol used (13% [vol/vol]) for the three
situations described so far but did not affect the stimulation
constant Kd in the range of 13 to 17% (vol/vol) added ethanol
(Table 1).
Effect of calcium on the extracellular acidification in the
presence of ethanol. When cells of S. bayanus, which were
grown in the unsupplemented medium, were suspended in an
aqueous solution of glucose, protons were actively extruded
(Fig. 8). Therefore, the extracellular pH dropped to a final
value, pHf, after about 2 h of incubation (Fig. 8). By that
time, at the final pH, the active extrusion of H+ was
balanced by passive proton influx. In the absence of ethanol,
the pHf attained an identical value whether or not Ca2+ (0.75
mM) was added to the incubation medium (Fig. 9). In the
presence of ethanol, the pHf increased, and that has been
explained by Leao and van Uden (13) for S. cerevisiae by the
ethanol-induced exponential enhancement of the diffusion
VOL. 54, 1988 INFLUENCE OF Ca2+ ON ETHANOL TOLERANCE 2443

TABLE 1. Ethanol toxicity in S. bayanus IGC 154 for several ethanol-sensitive rate processes'
KPHf (M-1) with ethanol ate
Condition b
Conditionnn (13%F [volvl)d (13% [Vol/Vol])d Kdd (M-1)e PHf (10%f
[vol1vol]f 0 to 10% 13 to 16%
(vol/vol) (vol/vol)
A 0.52 0.55 1.5 4.9 4.4 0.37 0.60
B 0.42 0.43 1.3 4.7 4.2 0.30 0.60
C 1.00 4.9 4.1 0.25 0.58
a Ethanol toxicity was quantified for cells that were grown in a medium with a suboptimal concentration of calcium (0.025 mM) and incubated with ethanol in
the same medium (condition A) or in a medium supplemented with 0.75 mM Ca2+ (condition B) and for cells grown and incubated with ethanol in a calcium (0.75
mM)-supplemented medium (condition C).
b From equation 1; Xmax = 10% (vol/vol) (correlation coefficients greater than 0.99).
c From equation 2; X'.ax 13% (vol/vol) (correlation coefficients greater than 0.99).
d Specific death rate in the presence of 13% (vol/vol) ethanol (exponential death).
From equation 3.
f Final extracellular pH in the presence of 10% (vol/vol) ethanol.
g From equation 4 (correlation coefficients greater than 0.99).

constant of the proton influx. They proposed and tested an
equation in which pHf increased as a linear function of
the ethanol concentration. The constant of ethanol-enhanced
proton influx was later correlated, for several yeast strains,
with the ethanol concentration that reduced their specific
growth rate to 50%. Therefore, the evaluation of the extra-
cellular acidification was proposed as a rapid test for ethanol
tolerance in yeasts (7). More recently, Cartwright et al. (2)
have reported that ethanol noncompetitively inhibits the
E
'I-,
a,
activity of the plasma membrane ATPase of S. cerevisiae.
Therefore, the ethanol-induced enhancement of pHf might
be explainable by the inhibition of the ATPase activity and
by the increase of the proton influx, both of which in
the same direction and increase the pHf as a linear function
of ethanol concentration, as reported for S. cerevisiae (13).
For S. bayanus IST 154, we found two linear relationships
between pHf and the concentration of added ethanol (Fig. 9;
Equation 4 [see below]) for two ranges of ethanol concen-
trations: the range of 0 to 10% (vol/vol) (concentrations that
allowed growth) and above 13% (vol/vol) ethanol (lethal
concentrations) (Fig. 9 and Table 1).
Equation 4, which follows, expresses the linear relation-
ship among pHf, the final extracellular pH, and the con-
centration X (M) of ethanol present, where C is a constant
and KpHf is the constant of pHf stimulation by ethanol:
pHf = C + KpHf X.
For lethal concentrations of ethanol (equal to or above
13% [vol/vol]), the pHf increased above the initial pH value
(Fig. 9), which suggests that passive influx surpassed the
activity of the proton pump. For concentrations of ethanol

-T
-o

m .--

C-
(U)
'U

(it

a,
0._
a)

Time (h) Ethanol concentration ( M )

FIG. 6. Viability plots at 30°C of cells of S. bayanus IST 154
grown and incubated with lethal concentrations of ethanol in Ca2"
(0.75 mM)-supplemented medium (0, Y, *, *), grown in unsupple-
mented medium but incubated with ethanol in supplemented me-
dium (*, V, U, *), and grown and incubated with ethanol in
unsupplemented medium (0, V, O, K).
FIG. 7. Specific death rates induced by lethal concentrations of
ethanol in S. bayanus IST 154 cells grown and incubated with
ethanol in Ca2"-supplemented media (-), grown in unsupplemented
medium and incubated with ethanol in supplemented medium (0),
and grown and incubated with ethanol in unsupplemented medium
(0).
2444 NABAIS ET AL. APPL. ENVIRON. MICROBIOL.

(about 300 g/liter) led to the production by S. bayanus of

~4.5
2 -j-A..
40 l some ethanol-sensitive biological processes (Table 1). Cells
Time (h)
FIG. 8. Evolution in time of the pH of a medium with
(20 g/liter), ethanol at 0% (vol/vol) (0, *), 6% (vol/vol) (A,
A), 8%
(vol/vol) (O, *), 9% (vol/vol) (0, 0), 10% (vol/vol) ( 0, 0 ), 13%
(vol/vol) (*, *), 14% (vol/vol) (*, 0), 15% (vol/vol) (V, V), 16%
(vol/vol) (0, 0) supplemented (closed symbols) or not supple-
mented (open symbols) with calcium (0.75 mM). pH variation was
caused by the presence of S. bayanus IST 154 cells grown in
unsupplemented medium.
up to 10% (vol/vol), the constant for the ethanol-induced
linear increase of pHf, KpHf was lower than the equivalent
constant calculated for higher ethanol concentrations in the
range of lethal concentrations (Fig. 9 and Table 1). The
addition of calcium to the growth medium or to the medium
of incubation with ethanol led, in both cases, to a lower
increase in pHf (Fig. 8 and 9).
DISCUSSION
The presence of optimal concentrations of calcium in
fermentation media with high concentrations of sugars
5.0
4.5-
L- 4.0
0 2 4 6 8 10 12 14
Ethanol concentration % (v/v)
FIG. 9. Final extracellular pH (pHf) as a function of the concen-
tration of added ethanol for cells of S. bayanus IST 154 grown in
Ca2+- supplemented medium (0.75 mM) and incubated with ethanol
in media with 20 g of glucose per liter and 0.75 mM calcium (a), and
grown in unsupplemented medium and incubated with ethanol in
Ca2+-supplemented (0) and -unsupplemented (0) media.
higher final concentrations of ethanol and to a lower percent-
age of unfermented sugars during batch alcoholic fermenta-
tions. The reported calcium effect was generalized to strains
of the species S. cerevisiae and K. marxianus. The trace
contaminating concentration of 0.025 mM total Ca2+ was
found to be suboptimal for the batch alcoholic fermentation
by the three strains tested. The optimal calcium concentra-
tion was variable with the strain tested, but it was in the
range of 0.75 to 2 mM. Moreover, calcium had a dose-
dependent effect in S. bayanus, as has been reported by
other investigators (1, 8, 9).
The increase in the ethanol tolerance of S. bayanus cells
grown or incubated with ethanol in calcium-supplemented
medium (0.75 mM) proved to underlie the positive effect of
calcium in alcoholic fermentation. This tolerance was char-
acterized by quantifying the inhibition or the stimulation of
grown and incubated in medium with an optimal concentra-
tion of Ca2+ (0.75 mM) presented a higher ethanol tolerance.
However, note that the ethanol tolerance of cells grown in
unsupplemented medium increased with the addition of 0.75
mM calcium to the medium of incubation with ethanol.
glucose Among the important roles played by the calcium ion in
yeasts, we point out its action in the maintenance of the
membrane permeability barrier under adverse conditions by
shielding the charged structural membrane phospholipids
and regulating the lipid-protein interactions (17). Calcium is
also one of the typical cations that make complexes with
yeast wall phosphomannans. At growth temperatures, the
target sites for ethanol-induced death are probably located in
the plasma membrane (25, 27).
Ethanol may interact with membranes by insertion into
the hydrophobic interior, increasing the polarity of this
region, weakening the hydrophobic barrier to the free ex-
change of polar molecules, and weakening the hydrophobic
interactions and affecting the positioning of proteins within
the membranes (6). The loss of membrane integrity de-
creases the ability of the cell to maintain a concentration
gradient across the plasma membrane, and several systems
involved in solute transport should be affected (6, 28).
The association of Ca2+ with the surface of yeasts is well
documented, and this association has been used to protect
the plasma membrane from the action of polyene antibiotics
and butanol (15, 16, 26a). It was shown that the presence of
Ca2+ prevents the release of nucleotidic material by cells of
Saccharomyces carlsbergensis suspended in glucose solu-
tions containing butanol and other membrane-damaging
agents that increased cell leakage (15, 16). It has been
reported that ethanol-induced leakage in S. cerevisiae may
underlie ethanol toxicity and that the ethanol tolerance of
yeasts could be related to the resistance of the plasma
membrane to ethanol-induced leakage (26). For S. cerevi-
siae, Salgueiro et al. (26) have reported that it is for
concentrations of ethanol above the maximum for growth,
Xmax, that the stimulation of the leakage of amino acids and
260-nm-absorbing compounds is significant, suggesting a
critical increase in membrane permeability near the Xmax.
16 These results could underlie the difference in the values of
KpHf for concentrations of ethanol above and below the
maximum concentration of ethanol which allowed growth.
Therefore, the protection exerted by calcium could be
related to its action in decreasing the extent of the release of
intracellular compounds stimulated by ethanol. The acidifi-
cation curves observed with S. bayanus cells in calcium-
supplemented and -unsupplemented media (Fig. 8 and 9)
VOL. 54, 1988
partially support our hypothesis. The calcium ion could
increase plasma membrane stability (9) either by decreasing
the ethanol-induced passive proton influx or stabilizing the
ATPase activity inhibited by ethanol. For concentrations of
ethanol below the maximum for fermentation (up to 13%
[vol/vol]), the effect of calcium on the pHf can also be
attributed to differences in the rate of production of C02, as
was mentioned above in relation to the calcium protection of
the fermentative activity inhibited by ethanol.
Ionic deficiencies do occur in some natural sources of
carbohydrates that are used as alcoholic fermentation feed-
stock. On the other hand, high salt concentrations in sub-
strates such as molasses are detrimental to growth and
ethanol production, and the addition of EDTA has been
found to improve their fermentation (24).
The optimization of Ca2+ (or any other ion) concentration
in complex industrial fermentation media can be difficult. In
fact, industrial media contain a range of chelating, seques-
tering, and adsorbing materials (amino acids, proteins, or-
ganic acids, polyphenols, polyphosphates, and insoluble and
colloidal materials) which act to reduce the effective avail-
able ionic concentration (9).
Three types of cane sugar molasses of different origins and
diluted up to 21 to 22°Brix were tested for calcium supple-
mentation (0.75, 1.5, and 5 mM in CaCl2) for batch fermen-
tation by S. bayanus IST 154 and S. cerevisiae IGC 3507 III
under industrial conditions (unpublished data). No improve-
ments were observed, and for S. bayanus the supplementa-
tion with 1.5 and 5 mM calcium had a negative effect.
These results were not surprising, because molasses pro-
vides a high concentration of total calcium (0.4 to 0.6% [wt/
wt]), and the maximal concentration of ethanol produced did
not surpass 9 to 10% (vol/vol). It remains to be seen whether
the concentration of free Ca2+ in molasses would be sub-
optimal if higher concentrations of ethanol could be pro-
duced. However, because of the presence of inhibitory
compounds in molasses, concentrations higher than 22°Brix
can hardly be fermented.
Based on our results (Fig. 1, 2, 4 and 6), the calcium-
positive effect is expected to become more significant in
continuous fermentations (cells fermenting continuously in
the presence of high concentrations of ethanol) or in batch
fermentations involving the production of high concentra-
tions of ethanol (for example, during the secondary fermen-
tation in the sparkling wine industry). It is possible that the
positive effect described in fermentations with immobilized
yeasts entrapped in calcium alginate and carragenate gels
could partially be attributable to the protection exerted by
calcium toward the toxic effects of ethanol.
ACKNOWLEDGMENTS
We acknowledge the Instituto Nacional de Investigaqao Cientifica
for financial support. This study was supported, in part, by contract
EN 3B-0081-P from the Commission of the European Communities.
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