APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb.

1976,
Copyright 1976 American Society for Microbiology

p.

158-162

Vol. 31, No. 2

Printed in U.SA.

Influence of the Rate of Ethanol Production and

Accumulation on the Viability of Saccharomyces
cerevisiae in "Rapid Fermentation" I
TILAK W. NAGODAWITHANA AND KEITH H. STEINKRAUS*
Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456

Received for publication 6 June 1975

By using 7 x 108 cells of brewers' yeast per

ml, 25 Brix honey solutions fortified with nitrogen, phosphate, and minerals were fermented
to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to
3 h at 30 C (6). Under these conditions of"rapid
fermentation," death rate of the cells was high,
with only 2.1% of the cells surviving at the end
of the fermentation. When the fermentation
temperature was decreased to 15 C and the
content of dissolved oxygen in the medium was
maintained at 13%, fermentation time to 9.5%
(wt/vol) ethanol increased to 6 h, but the yeast
cells retained their viability (6). Rapid fermentations using high cell populations require
Journal Paper no. 2128, New York State Agricultural
Experiment Station.

higher levels of vitamins and minerals than

slower fermentations at lower temperatures or
those using smaller populations (Nagodawithana and Steinkraus, unpublished data).
However, even in the presence of the higher
levels of vitamins and minerals, brewers' yeast,
the most rapid fermenting yeast we have encountered, loses its viability much more rapidly
at 30 C than it does at lower temperatures.
The effect of ethanol on yeast metabolism
has been studied (1, 3, 9; F. F. Pironti, Ph.D.
thesis, Cornell Univ., Ithaca, N.Y., 1971). Under
Pironti's conditions, ethanol appeared to inhibit
yeast cell growth at relatively low concentration, whereas the fermentative activity of the
cells seemed to tolerate ethanol until its concentration approached approximately 20% (vol/
158

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Whereas "rapid fermentation" of diluted clover honey (25 Brix) fortified with
yeast nutrients using 8 x 10' brewers' yeast cells per ml resulted in an ethanol
content of 9.5% (wt/vol; 12% vol/vol) in 3 h at 30 C, death rate of the yeast cells
during this period was essentially logarithmic. Whereas 6 h was required to
reach the same ethanol content at 15 C, the yeast cells retained their viability.
Using a lower cell population (6 x 107 cells/ml), a level at which the fermentation
was no longer "rapid," the yeast cells also retained their viability at 30 C.
Ethanol added to the medium was much less lethal than the same or less quantities of ethanol produced by the cell in "rapid fermentation." It was considered
possible that ethanol was produced so rapidly at 30 C that it could not diffuse
out of the cell as rapidly as it was formed. The hypothesis was postulated that
ethanol accumulating in the cell was contributing to the high death rate at 30 C.
It was found that the intracellular ethanol concentration reached a level of
approximately 2 x 1011 ethanol molecules/cell in the first 30 min of fermentation at 30 C. At 15 C, with the same cell count, intracellular ethanol concentration reached a level of approximately 4 x 101" ethanol molecules/cell and
viability remained high. Also, at 30 C with a lower cell population (6 x 107
cells/ml), under which conditions fermentation was no longer "rapid," intracellular ethanol concentration reached a similar level (4 x 10"' molecules
ethanol/cell) and the cells retained their viability. Alcohol dehydrogenase
(ADH) lost its activity in brewers' yeast under conditions of"rapid fermentation"
at 30 C but retained its activity in cells under similar conditions at 15 C. ADH
activity was also retained in fermentations at 30 C with cell populations of 6 x
107/ml. It would appear that an intracellular level of about 5 x 101" ethanol
molecules/cell is normal and that this level does not damage either cell viability
or ADH activity. Higher intracellular ethanol concentrations, such as 2 x 1011
molecules ethanol/cell (a fourfold increase in intracellular ethanol concentration), are accompanied by inactivation of ADH and loss of cell viability.

VOL. 31, 1976

VIABILITY LOSS IN "RAPID FERMENTATION"

(9).

The experiments reported were undertaken

to attempt to determine the part rate of ethanol
production or accumulation contributes to
loss of viability in "rapid fermentations" with
brewers' yeast at 30 C.
MATERIALS AND METHODS
Yeast strains. A brewers' strain of Saccharomyces
cerevisiae was kindly supplied by Mario Frati,
Genesee Brewing Co., Rochester, N.Y. The brewers'
yeast was obtained as a solid paste containing from
3.85 x 109 to 4.04 x 109 cells/g. The yeast was
washed with citrate-phosphate buffer, pH 6.8, and
centrifuged to recover the cells to be used in the
experiments.
Yeast counts. Based upon the number of yeast
cells desired in the inoculated medium, a given
weight, generally 1 kg of yeast paste, was slurried
with 1 liter of the medium. After thorough dispersion, a 1-ml sample was diluted serially and plated
in duplicate to obtain the initial viable count. The
culture medium used for pour plates contained 1.5%
maltose, 1.5% malt extract (Difco), and 1.5% agar
(Difco). The plates Wvere incubated at 30 C for 48 h,
and the final colony count was taken as the average
of the two plates for the dilution containing 30 to
300 colonies/plate. Calculations involving cell populations were based upon viable plate counts. The
percentage of viability was determined by dividing
the viable count at the desired fermentation time of
the initial viable count. If multiplication occurred,
the percentage of viability thus rose above 100%.
Fermentation medium. Clover honey stored at
1 C was diluted with water to provide the 250 Brix
sugar substrate. A basal level of 0.25 g of Actiferm
per liter (Budde & Westermann, New York, N.Y.),
a yeast vitamin mixture, was added to the fermentation medium and this level is referred to as X. The
0.25 g of Actiferm per liter contained 12.5 ug of
biotin, 250 Mg of pyridoxine, 1.87 mg of meso-inositol,
2.5 mg of calcium pantothenate, 5 mg of thiamine,
25 mg of peptone, and 215 mg of ammonium sulfate.
The medium was also supplemented with 1.0 g of
(NH4)2SO4, 0.5 g of K:1P04, 0.2 g of MgCl2, 0.05 g of
NaHSO4, and 5.0 g of citric acid per liter as per
formula I (10). The basic level of mineral saltscitric acid supplement was referred to as Y. Certain

fermentations in this study were conducted using a

2Y level of mineral salt-citric acid supplement and
4X concentration (1 g of vitamins [Actiferm] per
liter). The pH of the fermentation medium was
adjusted to 4.2. It then was pasteurized at 76.5 C
for 30 min and cooled to fermentation temperature
before inoculation.
Apparatus. A Microferm laboratory fermentor
model 214 (New Brunswick Scientific Co., New
Brunswick, N.J.) with 14-liter fermentors (working
capacity, 7 liters) was used. The fermentations
were carried out at 15 or 30 C and with 13% dissolved oxygen (unless otherwise specified) with
agitation at 300 rpm. A fermentor sampler (New
Brunswick model S21) was used to sample medium
during fermentation. A dissolved oxygen controller
model DO-60 by New Brunswick Co., was used in
conjunction with the above to measure and control
oxygen level.
Preparation of cell-free extract. Samples (25 ml)
were withdrawn from the fermenting medium at
desired time intervals and chilled immediately using
a dry ice-ethanol mixture; all subsequent operations
were carried out at 0 to 4 C. Cells were harvested
by centrifugation at 9,000 x g for 10 min in Sorvall
centrifuge, model SS-3, washed twice with 12.5-ml
portions of 0.1 M citrate-phosphate buffer (pH 6.8),
and centrifuged at 9,000 x g for 10 min. The volumes
of all three supernatants were determined, and 1-ml
samples were set aside for ethanol analysis.
The cell pellet was then suspended in 12.5 ml of
0.1 M citrate-phosphate buffer, pH 6.8, and 1 ml of
the thoroughly mixed suspension was suitably
diluted for determination of cell number by direct
microscopic count using an American Optical Bright
Line hemacytometer. The remaining yeast suspension was disintegrated by sonic oscillation (Sonifier
cell disruptor, model W 185D, Heat Systems-Ultrasonics Inc., Plainview, N.Y.) for a total of 6 min,
subjecting the cell to a period of 30 s of sonic oscillation followed by a period of 1 min of cooling. A 1-ml
amount of the sonicated suspension was suitably
diluted to determine the population of unruptured
cells by direct microscopic count. The treatment
ruptured approximately 60% of the cells in the
suspension. The cell debris was removed by centrifuging at 32,800 x g for 15 min. Having measured the volume of the cell extract, it was held
at 1 C until further use. The extract was subsequently analyzed for ethanol by gas-liquid chromatography. The ethanol content of the extract was
used to calculate the ethanol content of the ruptured cells. Moles of ethanol were converted to
molecules of ethanol per cell by multiplying by
Avogadros number and dividing by the total number
of ruptured cells.
A Carle gas chromatograph, model 9000, equipped
with a flame ionization detector was used for the
separation and quantitation of ethanol using procedures described earlier (6).
ADH assay. Alcohol dehydrogenase (ADH) activity was measured by a procedure based on previous
methods (2, 7), modified as follows. A 3-ml amount
of buffer, pH 8.8 (2), containing 10 Ml of 1.5 mg of
ethanol per ml and 0.05 ml of the cell-free extract,

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vol; 16.26% wt/vol). The effect of ethanol on

growth rates and ethanol production was shown
to be noncompetitive inhibition, confirmed by
Lineweaver-Burk plots (1).
Inhibition by ethanol was investigated after
the concentration of yeast cells reached maximum value in the shochu-molded rice fermentation (5). The decrease in the rate of ethanol
production was shown to be directly related to
the decrease in the number of viable yeast
cells.
Added thiamine enabled yeast enzymes to
tolerate the amount of ethanol the cell would
normally produce but not higher concentrations

159

160

APPL. ENVIRON. MICROBIOL.

NAGODAWITHANA AND STEINKRAUS

100

0I5'C

90
8%

80

ETHANOL WN

70
Ua

60

\0

~~~~~~~~(EXTERNAL)

30C

10

7~@

V 3 0C

O~

30C

94% ETHANOI W/V

(FERMENTATLON)

10
0

3
2
HOURS
FIG. 2. Loss of viability of brewers' yeast cells at
30 C in 25 Brix honey solutions containing increasing concentrations of added (external) ethanol compared with loss of viability during rapid fermentation when ethanol is being produced by the cells.

HOURS

FIG. 1. Effect of initial cell population on viable

cell count during fermentation using brewers' yeast
at 15 and 30 C. 13% dissolved oxygen (D.O.).

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during this period was essentially logarithmic

(Fig. 1). Although it required 6 h to reach the
same ethanol content at 15 C, the yeast cells
retained their viability. Using a lower cell
population (6 x 107 cells/ml), a level at which
the fermentation was no longer "rapid" at 30 C,
the yeast cells also retained their viability.
It was found that 9.4% (wt/vol) ethanol produced by the cells during fermentation at 30 C
was much more lethal than even 13.8% (wt/vol)
ethanol added to a similar cell population in the
diluted honey base (Fig. 2). Only 15.5% of the
cells inoculated survived 3 h of fermentation to
9.4% ethanol (wt/vol) at 30 C, whereas 57.5%
of the cells inoculated survived 3 h in the same
medium in which the ethanol content was increased to 12% (wt/vol) by addition of ethanol.
The internal ethanol content of cells was
determined during fermentation using 8 x 108
cells/ml at 30 C. It was found that the higher
concentration of brewers' yeast cells (8 x 108
RESULTS AND DISCUSSION
cells/ml) contained a higher ethanol content
Whereas "rapid fermentation" (defined as (1.6 x 101" ethanol molecules/cell) after 30-min
fermentation in which the ethanol content fermentation at 30 C than the same concentrarises from 0 to 9.5% [wt/vol; 12% vol/vol] in 6 h tion of cells fermenting at 15 C, which conor less) using 8 x 108 cells/ml at 30 C resulted tained 3.9 x 10"' ethanol molecules/cell (Table
in an ethanol content of 9.5% (wt/vol; 12% 1). The lower population of cells (6.3 x 107
vol/vol) in 3 h, death rate of the yeast cells

was transferred directly to a cuvette (1-cm light

path), and the optical density at 340 nm was determined. Twenty microliters of 0.15 M nicotinamide
adenine dinucleotide (NAD) was transferred to the
cuvette with a syringe and mixed immediately with
a plastic rod, and the change in optical density at
340 nm was monitored every 30 s during the first 3
min. From the standard curve for reduced NAD
(NADH), the optical density reading at 340 nm was
directly converted to the amount of NADH plus H+
formed in the cuvette with time for the particular
run. The initial rate of NADH plus H+ formed per
minute was calculated from the graph, showing the
increase of NADH plus H+ with time for the particular run. Since NAD and ethanol were added in
excess, the reaction rate was dependent on the
activity of ADH in the 0.05-ml cell-free extract added
to the cuvette. Protein in the cell-free extract was
determined by the method of Lowry et al. (4) using
bovine albumin serum as the standard. The specific
activity of ADH = micromoles of NADH plus H+
formed per minute/milligram of protein.

TABLE 1. Comparison of the molecules of ethanol

found within yeast cells at different stages of
fermentation
Molecules of ethanol per average cell
x 101 cells/ 8 x 10' cells/ 6.3 x 107 cells/
ml at 30 C
ml at 30 C
ml at 15 C
(cells remain (cells die rap- (cells remain
viable)
idly)
viable)

Time (h) 8

3.7 x 101"'
3.6 x 10"'
4.9 x 10"'
5.3 x 10"'

6.4
1.6
2.0
1.6

x
x
x
x

10"'
10'
10"
1011

4.4
3.9
4.5
5.0

.20
.18i
.16

.14

.12

.10

.08

1-

aU

u;

U)
0

10

20
HOURS

30

FIG. 4. ADH activity of brewers' yeast as influenced by internal ethanol molecules per cell at
30 C using an initial cell count of 6.3 x 107 cellslml.

fermentation, however, at this cell concentration was no longer rapid.

As the ethanol concentration increased inside
the cells in the 30 C fermentation carried out
with initial cell count of 8 x 10' brewers'
yeast cells/ml, the ADH activity rapidly decreased (Fig. 3). The concentration of ethanol
was much lower in the cells at 15 or 30 C using
a cell population of 6.3 x 107/ml, and there was
no inactivation of ADH (Fig. 3 and 4). Thus, it
can be concluded that ethanol accumulates
within the brewers' yeast cells under conditions
of "rapid fermentation" using a high cell population at 30 C.
It would appear that an intracellular level of
approximately 5 x 10"' ethanol molecules/cell
is in the normal range in brewers' yeast cells.
This level does not inactivate ADH or damage
cell viability. Higher intracellular ethanol concentrations such as 2 x 1011 molecules of ethanol per cell (a fourfold increase in intracellular
ethanol concentration) are accompanied by inactivation of ADH and loss of cell viability.

x 10"'
x 10"'
x 10"'
x 10"'

"Initial viable cell count of brewers' yeast cells.

4.x1011

06o

.04

LITERATURE CITED

.02

HOURS

FIG. 3. ADH activity of brewers' yeast

as

in-

molecules per cell at 15

and 30 C using an initial cell count of 8 101 cellsl
ml in a 250 Brix medium, 13% dissolved oxygen, with
adjunct nutrients added at 2Y + 4X levels.

fluenced by internal ethanol

1. Aiba, S., M. Shoda, and M. Nagatani. 1968. Kinetics

of product inhibition in alcohol fermentation. Biotechnol. Bioeng. 10:845-864.
2. Bonnichsen, R. 1965. Ethanol determination with
alcohol dehydrogenase and DPN, p. 285-287. In H. U.
Bergmeyer (ed.), Methods of enzymatic analysis.
Academic Press Inc., New York.
3. Holzberg, I., R. K. Finn, and K. H. Steinkraus. 1967.
A kinetic study of alcoholic fermentation of grape
juice. Biotechnol. Bioeng. 9:413-427.

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cells/ml) at 30 C had an internal ethanol content similar to the cells fermenting more slowly
at 15 C (3.9 x 101' molecules ethanol/cell).
This may be related to the relatively greater
volume of free liquid with the lower cell population, which would facilitate diffusion of ethanol
from the cells during fermentation.
At 15 C the intracellular ethanol concentration did not increase above 5.3 x 10"' molecules/
cell during the first 2 h of rapid fermentation,
whereas ethanol in the high cell population
fermenting at 30 C remained at the 2 x 1011
molecules/cell level. Nor was there any significant increase in the number of ethanol
molecules within the cells at the lower cell
population level during the fermentation. The

0
0.5
1
2

161

VIABILITY LOSS IN "RAPID FERMENTATION"

VOL. 31, 1976

162

NAGODAWITHANA AND STEINKRAUS

0. H., N. J. Rosebrough, A. L. Farr, and

R. J. Randall. 1951. Protein measurement with the
Folin phenol reagent. J. Biol. Chem. 193:265-275.
5. Nagatani, M., Y. Kuba, and S. Sugania. 1969. Kinetics
of product inhibition in shochu making. J. Ferment.
Technol. 47:723-728.
6. Nagodawithana, T. W., C. Castellano, and K. H.
Steinkraus. 1974. The effect of dissolved oxygen,
temperature, initial cell count, and sugar concentration on the viability of Saccharomyces cerevisiae in
rapid fermentations. Appl. Microbiol. 28:383-391.

4. Lowry,

APPL. ENVIRON. MICROBIOL.

7. Racker, E. 1955. Alcohol dehydrogenase from Baker's
yeast, p. 500-503. In S. P. Colowick and N. 0.
Kaplan (ed.), Methods in enzymology, vol. 1. Academic Press Inc., New York.
8. Rahn, 0. 1929. The decreasing rate of fermentation.
J. Bacteriol. 18:207-226.
9. Rahn, 0. 1952. Acid and alcohol tolerance imparted
by thiamine. Growth 16:59-63.
10. Steinkraus, K. H., and R. A. Morse. 1966. Factors
influencing the fermentation of honey and mead
production. J. Apic. Res. 5:17-26.

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