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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1982, p.

757-763

Vol. 43, No. 4

0099-2240/82/040757-07$02.00/0

Effect of Sodium Chloride on Bakers' Yeast Growing in


Gelatin
CHIA-JENN WEI,1 ROBERT D. TANNER,1 AND GEORGE W. MALANEY2*
Departments of Chemical Engineering' and Civil and Environmental Engineering,2 School of Engineering,
Vanderbilt University, Nashville, Tennessee 37235
Received 6 August/Accepted 21 November 1981

The fermentation of sugars, especially glucose, by Saccharomyces cerevisiae in liquid


culture has been one of the most intensively
studied phenomena in the history of scientific
inquiry. Recently, interest in this area has shifted to solid and semisolid fermentations for two
primary reasons: certain products such as fermented foods like miso and soy sauce are becoming more popular worldwide, and stirring
and the separation of sugars from natural raw
materials are often not required with solid materials; therefore, much energy can be saved by
using the natural substrates directly.
We studied kinetic changes at a transition
point between the culture in liquid form and in
solid form by fabricating a gelatin medium as a
semisolid model. A 16% (wt/vol) gelatin concentration was selected as the maximum gelatin
level because preliminary studies had shown
that a higher concentration (i) made the separation of yeast cells from the gel medium (for
measurement of biomass) very difficult, (ii)
made pH measurements in the fermenting mash
unreliable, and (iii) caused concern about possible aeration of the anaerobic culture after the
removal of sampling plugs since the holes made
in the gelled medium by the sampling did not
close completely, allowing diffusion of ambient
air into the openings.
Historical review. The high concentrations of
electrolytes in fermented foods and in raw materials such as molasses-enriched sugar cane
stalks has motivated studies of the inhibitory

effects of high levels of inorganic salts on sugar


fermentations of industrial importance. This inhibitory action was first reported by Tajima et
al. (12) and Umemoto et al. (18). Tajima et al.
(12) have suggested that salt tolerance be added
to the list of desirable characteristics of yeast
strains used for alcoholic fermentation of molasses. For more convenient experimental methodology, subsequent researchers have replaced the
heterogeneous mixture of inorganic salts found
in molasses with pure sodium chloride.
To date, however, few kinetic studies have
been reported on the effects of sodium chloride
on entrapped yeasts growing in semisolid media
(16), mainly because it is difficult to track cells
attached to solids. Gelatin medium provides an
easy way to measure the mass of cells by simply
melting the gelatin at 40 to 45C. The still viable
cells are then easily separated from the gel, and
the cell-free liquid is handled as a conventional
liquid medium.
Summary of previously reported salt effects.
Elevated levels of inorganic electrolytes in an
otherwise satisfactory liquid growth medium
have been found to influence several parameters
of yeast activity. (i) Cell growth and multiplication: (a) the number of viable yeast cells per unit
volume of liquid growth medium decreases as
salt content increases, (b) the biomass of the
culture (i.e., the total weight of yeast cells per
unit volume of liquid growth medium) decreases
as salt content increases, and (c) the length of
the lag phase (i.e., the incubation period be757

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In recent years, industrial fermentation researchers have shifted their attention


from liquid to solid and semisolid culture conditions. We converted liquid cultures
to the semisolid mode by adding high levels of gelatin. Previous studies on liquid
cultures have revealed the inhibitory activity of mineral salts, such as NaCl, on
the fermentation of sugars by yeasts. We made a kinetic study of the effects of 1 to
5% (wt/vol) NaCl on the alcoholic fermentations of glucose by Saccharomyces
cerevisiae in a growth medium containing 16% gelatin. Our results showed that
the effect of high salt content on semisolid culture is essentially the same as the
effect on liquid culture; i.e., as the salt content increased, the following occurred:
(i) the growth of yeasts decreased, (ii) the lag period of the yeast biomass curve
lengthened, (iii) the sugar intake was lowered, (iv) the yield of ethanol was
reduced, and (v) the production of glycerol was increased. We observed a new
relationship correlating the area of kinetic hysteresis with ethanol production rate,
acetaldehyde concentration, and the initial NaCl concentration.

758

WEI, TANNER, AND MALANEY

tween inoculation of the culture and detectable

initiation of cell growth) lengthens as salt concentration increases. (ii) Utilization of the primary carbon and energy source is reduced. (iii)
Change in concentration of metabolic products:
(a) there is a decrease in the production of
ethanol as salt content increases and (b) there is
an increase in the concentration of other fermentation products (such as glycerol, acetaldehyde,
etc.) as salt content increases.

room

temperature.

Fermentation start-up. Maxon-Johnson medium (600


ml) with selected salt content (0.0 to 5.0% [wt/vol]
NaCI) was poured into a fermentor covered with
aluminum foil and steam sterilized at 15 pounds per
square inch gauge for 30 min.
The unit was cooled to 60 to 65C and gelatin was
added. With the temperature held in this range, the
contents of the fermentor were mixed with a magnetic
stirrer until the gelatin dissolved. At this point the
growth medium was adjusted to pH 5.0 with sterile 1.0
N HCI or 1.0 N NH40H solution.
The fermentor and contents were cooled to 40C and
the 20-ml suspension of yeast cells was added. The
loaded fermentor was then placed in a 25C constant
temperature bath, and the mixture was stirred at 500
rpm to keep the cells dispersed while gelation took
place. When gel formation was complete, stirring was
stopped and fermentation was continued at 25C.
pH monitoring. The pH of the fermenting mash was
measured by using a miniature combination electrode
(Sargent-Welch Scientific Co.) positioned about 5 cm
below the surface of the growth medium and 2 cm from
the fermentor wall. The probe was moved occasionally
so that the tip would not be immersed in a trapped gas
pocket, which would lead to faulty readings.
Sampling technique. After selected incubation periods, 20-ml samples of the semisolid fermenting mixture were removed by means of a sterile stainless steel
spatula. Each sample was transferred to a 50-mi flask
which was closed with a rubber septum to minimize
the loss of volatile components. The sample was
liquefied at 40 to 45C. After being mixed thoroughly,

a 1.0- or 2.0-ml portion of the melted sample was


removed and diluted with warm distilled water in
various proportions, depending upon the concentration of the component being measured and the sensitivity of the analytical method being employed.
Biomass of yeast cells. The concentration of yeast
cells in the fermenting mash was measured by the
turbidimetric (absorbancy) method. A 2.0-ml portion
(1.0-ml portions in later stages of fermentation) of the
liquefied sample described above was diluted 10-fold
with 40 to 45C distilled water in a test tube. Dilution
was crucial to the separation of yeast cells from the gel
matrix in the subsequent centrifugation at 1,800 rpm
for 10 min. The supernatant cell-free culture medium
was stored for determination of glucose, ethanol,
acetaldehyde, glycerol, and L-lysine.
The cell pellet was washed with 10 ml of distilled
water at 40 to 45C, succeeded by a second centrifugation. The supernatant was discarded. Finally, the
washed cells were suspended in 10 ml of distilled
water. The optical density of the washed cell suspension at 610 nm was measured in a Bausch & Lomb
Spectronic 20 spectrophotometer that had a red light
filter.
A standard curve of optical density versus yeast dry
weight (grams per liter) that covered the appropriate
range of concentrations was made with a series of six
suspensions prepared from the dry, packaged yeast
cells.
Glucose determination. After appropriate dilution of
the cell-free fermentation mash, glucose was measured
by the Somogyi-Nelson method (4a) with optical density measurements at 425 nm in the Spectronic 20. The
gelatin in the fermentation medium interfered with the
assay. Correction for this interference was made by
running the appropriate blank. Results were expressed
as grams of glucose per liter of fermenting medium.
Ethanol determination. Ethanol in the diluted cellfree fermentation mash was estimated by the alcohol
dehydrogenase method of Kaplan and Ciotti (5) with
readings made in the Spectronic 20 at 340 nm. Results
were expressed as grams of glucose per liter offermentation growth medium.
Acetaldehyde determination. Assay of acetaldehyde
was made on undiluted cell-free fermentation mash by
using aldehyde dehydrogenase (catalog no. 171-832)
purchased from Boehringer Mannheim Biochemicals.
Readings of NADH were made in the Spectronic 20 at
340 nm. Results were reported as grams of acetaldehyde per liter of fermentation mash.
Glycerol determination. Glycerol content of the diluted cell-free fermentation mash was measured by the
glycerol kinase method (1) with the Boehringer Mannheim Biochemicals glycerol UV test kit. Readings
were made in the Spectronic 20 at 340 nm. Results
were recorded as grams of glycerol per liter offermentation mash.
Lysine determination. The intracellular free L-lysine
content of the yeast cells was measured by microbiological assay after extraction by boiling.
Lysine was extracted from an appropriate weight of
yeast cells by suspending the cells in 5 ml of distilled
water in a test tube and holding the suspension in a
boiling water bath for 20 min. The cell debris was
removed by centrifugation.
The microbiological assay used was that described
in the Difco manual (4), with Pediococcus cerevisiae

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MATERIALS AND METHODS


Fermentors. The fermentations were carried out in
1-liter Pyrex glass jars with approximately 600 ml of
working volume. Closure was with aluminum foil. No
stirring, aeration, or pH-control devices were used
during the runs.
Growth medium. Maxon and Johnson Synthetic
Medium C (6) was the basal fermentation medium.
Sodium chloride was added as American Chemical
Society-grade NaCl (catalog no. S-271, Fisher Scientific Co.). This liquid culture medium was converted to
the semisolid mode by the addition of 16% (wt/vol)
gelatin (BBL Microbiology Systems). The final fermentation medium contained 10% glucose.
Organism. The fermenting organism was S. cerevisiae (bakers' yeast) purchased as Fleischmann's dry
yeast in foil packets. Each yeast package was used
only on the day it was opened.
The inoculum for 600 ml of growth medium was
prepared by the suspension of 1.2 g of dry yeast in 20
ml of sterile Maxon-Johnson Synthetic Medium C at

APPL. ENVIRON. MICROBIOL.

EFFECT OF NaCl ON BAKERS' YEAST IN GELATIN

VOL. 43, 1982

I'o-

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0If
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wz 6.0

>_x

-5.0
a3

70
60
50
40
30
20
FERMENTATION PERIOD, hr
Effect of NaCl levels on yeast biomass in

10

FIG. 1.
semisolid growth cultures of S. cerevisiae in ca. 10%
glucose. All points represent data points.

(NRRL B-1116) as the test organism. The assay was


made on the extract at 37C with incubation for 18 h.
The final cell concentrations of the test organism were
measured in optical density units in the Spectronic 20
at 660 nm. The lysine results were reported as specific
free lysine, i.e., % (wt/wt) L-lysine per unit cell mass
of yeast.

RESULTS AND DISCUSSION


Number of yeast cells per unit volume of liquid
growth medium. Combs et al. (3) have studied
the effects of changing NaCl levels on the multiplication of Candida albicans, as measured by
conventional plate counts with Sabouraud agar.
NaCl in 1.0% concentration has essentially no
effect on the 96-h viable cell count. This is not
surprising since 1.0% NaCl solution is essentially isotonic physiological saline solution. On the
other hand, 3.5% NaCl reduces the 96-h count
by 70 to 80%, and 5.0% NaCl by about 90%.
Biomass of yeast cells per unit volume of liquid
growth medium. In the study of several yeasts
isolated from marine environments, Ross and
Morris (8) have found that as the concentration
of NaCl in the culture medium increases, the
production of yeast biomass decreases. Norkrans (7) has reported that 4.0% NaCl reduces the
growth of S. cerevisiae by 10 to 15% compared
with that of the control (0.0% NaCl), whereas
8.0% NaCl cuts the biomass to about 90% of the
control level. These researchers measured biomass by the absorbancy (turbidimetric) method.
Combs et al. (3) have investigated changes in
biomass by monitoring dry cell weight per liter
after a 48-h incubation period. They have reported that biomass falls from 5.61 g/liter at 0.0%
NaCl, to 3.40 g/liter at 1.0% NaCl, and to 1.90 g/
liter at 5.0% NaCl.

Umemoto et al. (18) have reported that the


high concentrations of molasses electrolytes reduce yeast cell growth. Furthermore, they have
showed that individual pure inorganic salts have
a similar effect.
Tanner et al. (16) have found that 40 g of NaCl
per liter reduces cell production in a semisolid
(40 g of gelatin per liter) growth medium. Figure
1 shows results of our studies on the effects of
NaCl on S. cerevisiae growth in gelatin. These
kinetic trajectories show that inhibition of yeast
cell growth occurs in semisolid ferme'ntation as
it does in liquid fermentation. At virtually every
time point, the trend is clear; i.e., NaCl quantitatively reduces the cell content up to about 5%
NaCl, where growth becomes negligible.
Length of lag phase. Ross and Morris (8) and
Norkrans (7) have plotted absorbancy (as a
measure of cell growth) versus incubation time.
The early portions of their curves suggest that
increasing NaCl content proportionately lengthens the lag period in the yeast growth curve in
liquid culture. The curves in Fig. 1 show that
similar inhibition occurs in semisolid cultures.
Utilization of primary carbon and energy
source. During an investigation of the liquid
alcoholic fermentation of Okinawa molasses,
Umemoto et al. (18) have observed a so-called
"sugar defect"; i.e., sugar in the molasses mash
is not converted to alcohol in the expected yields
based on glucose equivalents. The higher the
concentration of electrolytes in the mash, the
larger the "sugar defect," i.e., the poorer the
"fermentation efficiency." A similar inhibitory
action has been noted in liquid fermentations
utilizing six different yeasts.
Spencer (9) has reported that glucose consumption by the yeast Saccharomyces rouxii is
reduced by the presence of 3.1 M (18% wt/vol)
NaCl in the liquid growth medium. Brown (2),
studying S. cerevisiae, has found that 1.73 M
(10% wt/vol) NaCl in liquid culture increases
sugar utilization. Tanner et al. (14) have found
that a concentration of NaCl as low as 0.3 M (ca.
1.5% wt/vol) initiates a reduction in the rate of
glucose uptake for liquid cultures of S. cerevisiae.
It is interesting that when Umemoto et al. (18)
added 10-3 M sodium azide to the growth medium, the growth of a yeast identified as alcoholic
yeast Hakken no. 1 was strongly inhibited,
whereas sugar uptake was only slightly reduced
from that of the control, suggesting that the
sugar is used less in cell synthesis than for the
synthesis of noncellular products, such as glycerol.
In semisolid culture, an increase in the NaCl
level in the growth environment above 2.0%
markedly reduced the rate of glucose uptake by
S. cerevisiae, although eventually (by 69 h) the

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759

APPL. ENVIRON. MICROBIOL.

WEI, TANNER, AND MALANEY

760

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-J

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--

10

20

40

30

50

60

70

FERMENTATION PERIOD, hr

10

50
60
40
30
20
FERMENTATION PERIOD, hr

70

FIG. 2. Effect of NaCi levels on glucose uptake in


semisolid growth cultures of S. cerevisiae in ca. 10%
glucose.

FIG. 4. Effect of NaCl levels on glycerol production in semisolid growth cultures of S. cerevisiae in ca.
10% glucose.

glucose was entirely used (Fig. 2).


Decrease in the concentration of ethanol produced. Tajima and Yoshizumi (10) have reported
that as the concentration of NaCi in the growth
medium increases from 0.0 M to 1.0 M, the
amount of ethanol produced falls from 5.95 to
5.05 ml per 100 ml of liquid growth medium.
0' Results obtained in the present semisolid
study (Fig. 3) support the results of Tajima and
Yoshuzumi (10) for salt levels above 2% up to 30
h. After 30 h, the differences tend to be negligirates
production
ble.30.
7
5
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40
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slowed proportionately by increasing salt levels,
it appears that ultimate ethanol content is essentially the same, regardless of salt concentration.
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reported
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o10M
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the "sugar
in liquid culture cause
electrolytes
defect," but also high concentrations of electro.5t
amuto taolpoue alsfo

lytes in several yeasts promote high accumulations of polyhydric organic compounds, such as
glycerol, 2,3-butanediol, arabitol, and erythritol.
Tajima and Yoshizumi (10) have found that
Saccharomyces formosensis Nakazawa in 0.25
to 1.00 M NaCl converted much of the sugar
substrate into glycerol, 2,3-butanediol, mannitol, erythritol, organic acids, vicinal diketone,
acetaldehyde, and CO2.
Our gelatin study results corroborate previous
reports on the effect of salt in stimulating the
production of glycerol during the fermentation
of glucose by S. cerevisiae in liquid culture (Fig.
4). However, it should be noted that the control
(0.0% NaCl) curves, after reaching a plateau,
subsequently show a gradual drop in glycerol
beginning at about 24 h, at which time the
glucose substrate has been exhausted (Fig. 2),
suggesting the shift to glycerol as substrate for
the yeast cells. This suggestion is reinforced by
the diauxie curves (Fig. 1) for low salt levels.
The same sequence of events is seen in the 1.0,
2.0, and 3.0% NaCl curves, with the reduction in
glycerol beginning after 30 to 35 h. The concentration of glycerol continued to build up in the
4.0 and 5.0% NaCl cultures in which (Fig. 2)
glucose was not exhausted during the length of

%NaCI

.- 0.0
0-2.0

- 3.0

- 40 _

A- 5.0

o
4

10

20
30
60
50
40
FERMENTATION PERIOD, hr

70

FIG. 3. Effect of NaCl levels on ethanol production


in semisolid growth cultures of S. cerevisiae in ca. 10%
glucose.

the run.
Brown (2) has discussed evidence for considering the polyols synthesized by S. cerevisiae as
fulfilling several physiological functions in the
yeast, including the role of food reserves.
The acetaldehyde curves (Fig. 5) corroborate
the previously observed (10, 20) increasing
buildup of acetaldehyde in liquid cultures at high
NaCl levels, but only if measurements are made
between 10 and 17 h or after 40 h. All of the
curves, including the control curve, reach a
maximum and then fall off at variable rates.
Tempest et al. (17) have reported that the
presence of 4% (ca. 0.67 M) NaCl S. cerevisiae-

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CD

EFFECT OF NaCl ON BAKERS' YEAST IN GELATIN

VOL. 43, 1982

%NoCl
-0.0
3.0
0 -4.0
A - 5.0

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10

20

30

40

50

60

70

FERMENTATION PERIOD, hr
FIG. 5. Effect of NaCi levels on acetaldehyde pro-

duction in semisolid growth cultures of S. cerevisiae in


ca. 10% glucose.

glucose continuous liquid cultures produce no


observable change in intracellular amino acid
pool size or composition. Tanner et al. (15) have
reported that 0.15 to 0.6 M NaCl in batch liquid
conditions promotes an increase in free intracellular lysine. The results (Fig. 6) for semisolid
fermentation do not confirm any increase in the
production of intracellular lysine per cell over
that in the control, but rather, recalling the
biomass data in Fig. 1, total intracellular lysine
concentration decreases with increasing NaCl
levels. However, in their studies, Tanner et al.
(15) used aerated cultures, whereas this semisolid study used unaerated cultures. As in the low
NaCl salt cases of glycerol production, any
lysine produced, even in the control, appears to
be used later in the fermentation, probably for
cell synthesis.
The rise in pH of the cultures containing high
levels of NaCl is shown in Fig. 7. The increased
acidity at lower salt concentrations corresponds

0-

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liquid culture.
Mechanisms proposed to explain the inhibitory
effects of NaCI on yeast fermentations. Umemoto
et al. (19) have suggested a partial explanation of
the inhibitory effects of electrotytes in terms of
the inhibition of yeast (pyruvate) carboxylase,
the enzyme that catalyzes the decarboxylation
of pyruvate to acetaldehyde. Obviously, if acetaldehyde production is slowed down, the production of ethanol is reduced proportionately.
They concluded that in the absence of acetaldehyde, if the fermentation was to continue, a
hydrogen acceptor other than acetaldehyde
must become available to oxidize the NADH

-0.0
1.0

20
30
40
50
FERMENTATION PERIOD, hr

to higher cell levels (Fig. 1). What is particularly


interesting, however, is the fact that a micro pH
probe can be used for on-line pH monitoring of
cells growing in a gel system, as well as in a

x -0.0

3i

10

FIG. 7. Effect of NaCl levels on the pH of semisolid growth cultures of S. cerevisiae in ca. 10% glucose.

ON

%NOCI

0.8

0.6

0.4

0.2

14

0.2

0
0 .3

0.1
o.
U)

'0

10

20

30

40

50

60

70

FERMENTATION PERIOD, hr
6. Effect of NaCl levels on intracellular lysine

FIG.
production in semisolid growth cultures of S. cerevisiae in ca. 10%o glucose.

ACETALDEHYDE CONCENTRATION, g/L

FIG. 8. Typical kinetic hysteresis curve relating


the synthesized acetaldehyde concentration to the
ethanol production rate at 3% NaCl. Arrows on the
curve indicate time progression.

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APPL. ENVIRON. MICROBIOL.

WEI, TANNER, AND MALANEY

762

parameter indicates greater alcohol dehydrogenase decay with time as the NaCl level increases.
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ACKNOWLEDGMENTS

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LITERATURE CITED

1. Boehringer Mannheim Biochemicals. 1979. Methods of


enzymatic food analysis. Boehringer Mannheim Biochem-

SODIUM CHLORIDE CONCENTRATION,% (w/v)

icals, Indianapolis, Ind.


2. Brown, A. D. 1978. Compatible solutes and extreme water

r. 9. Correlation between the area of the kinetic


hysteriesis curve (relating acetaldehyde concentration
to eth;anol production rate) and the initial NaCl concentraLtion. Hysteresis curve for the 3% NaCl case is
FIG

stress

during the metabolism of glucose in the


Embdlen-Meyerhof pathway. It was proposed
that tlhe substitute hydrogen acceptor was phoshnce the
thesubsequnt
Yceraldhyde,
hence
subsequent prophoglLyceraldehyde,
ducti( on of excess glycerol.
The :work oftoTajima and Yoshizumi (11) shows
no ea:.rlyr(24inhibition of
of acetaldehyde
48-h) inhibition
(24- to 48-h)
acetaldehyde
produiction by 1.0 M (6%) NaCl. Figure 5 also
show,s rapid production of acetaldehyde in the
early stages of glucose fermentation in the presence of 3.0, 4.0, and 5.0% NaCl. There is
itionwith
ventul
t
inhibiition
the 1.0 ad
and
with eventual recovery at the1.0
pro-

recvery

2.0% NaCl levels.

Taj:ima and Yoshizumi (11) have suggested


and actaldeacetalde-

isreducedand

hyde accumulation is increased under highly


salted growth conditions because the salt inhibits alcohol dehydrogenase (the enzyme which
reduc es

acetaldehyde
ethanol).
to ethaol).
acetldehydeto

This would

Ths

would

explaiin the

lengthening of the lag phase in the


salt urves in Fig. 3. This is consistent with the
rapid, early accumulation of acetaldehyde in the
curve for
for the 5% NaCl cultures shown in Fig.
Fig 5.
5
NaC*
*%
It wcluld also account for the fact that the
ethaniol production rate (which is proportional to
the a]lcohol dehydrogenase activity) is significantly less in the kinetics for late times compared with early times in the hysteresis curve
(Fig. 8). The direction of the kinetic hysteresis
curve has been shown to be useful as an indicaci

181-242. In A. H.

effect of sodium chloride on the lipid content and fatty


acid content composition of Candida albicans. Mycologia
60:1232-1239.
4. Difco

Laboratories.

1953. Difco manual of dehydrated

culture media and reagents, p. 229. Difco Laboratories,


Detroit, Mich. B. T. Hofreiter. 1962.

4a.Hodge, J. E.,
reducing

and
Determination of
and carbohydrates. Methods Carbohydr.

Chem. 1:386-388.
5.

Kaplan, N. O., and M. M. Ciotti. 1957. Enzymatic deter-

mination of ethanol,

p.

253-255. In S. P. Colowick and

N. 0. Kaplan (ed.), Methods in enzymology, vol. 3.


Academic Press, Inc., New York.

6. Maxon, W. D., and M. J. Johnson. 1953. Aeration studies


on propagation of baker's yeast. Ind. Eng. Chem.

45:2554-2560.

7.Norkrans,

B. 1966.

growth related

to

Studies

on

marine-occurring yeasts:

pH, NaCl concentration

ture. Arch. Mikrobiol.

that e thanol
production is reduced
,thanolproducion

p.

ology, vol. 17. Academic Press, Inc., New York.


3. Combs, T. J., J. J. Guarneri, and M. A. Pisano. 1968. The

shown in Fig. 8.

forme d

in eukaryotic microorganisms,

Rose and J. G. Morris (ed.), Advances in microbial physi-

and

tempera-

54:374-392.

8. Ross, S. S., and E. 0. Morris. 1962. Effect of sodium


chloride on the growth of certain yeasts of marine origin.
J. Sci. Food Agric. 13:467-475.
9. Spencer, J. F. T. 1968.

p.

1-42. In D. J. D. Hockenhull

(ed.), Progress in industrial microbiology, vol. 7. J. and A.

Churchill Ltd., London.


10. inorganic
and H. Yoshizumi. 1972.
Effects of the
Tajina, K.,
salt concentration
metabolism in alcoon

yeast

fermentation. III. Metabolic pathway of abnormal


fermentation by yeast in the salted medium. J. Ferment.
holic

Technol. 50:764-769.
11. Tajima, K., and H. Yoshizumi. 1975. Mechanisms of
abnormal fermentation of distiller's yeast in salted media
(such

molasses media) from the point of

NAD(P) redox

balances. J. Ferment. Technol. 53:841-853.


12. Tajima, K., H. Yoshizumi, and Y. Terashima. 1966. Salt
and sugar tolerances of yeast on alcoholic fermentation. I.

slThe inhibition

of fermentation by the
highly concentrated
salts in molasses. J. Ferment. Technol. 44:77-84.

13. Tanner, R. D. 1978. Kinetic hysteresis

in

enzyme

and

fermentation systems, p. 73-89. In D. Perlman (ed.),


AnnualPress,
reportsInc.,
on fermentation processes, vol. 2. Acator of a decay
in enzyme
activity (13).
decay
enzym
activity
(13).demic
Richmond,
WoodThe relationship between the early and late 14. Tanner,
L.
Wei,
a
in

New York.

R. D.,

time e mnzyme activities becomes even more pronounc:ed when the area of hysteresis curve (another measure
measure of the difference bbetween
een

upper

lo

trajectorifferenes)is graphed

and lower trajectories)

functiion

of the salt level in

is

Fig.

9.

This

D.

C.-J.

ward. 1981. The effect of sodium chloride

and

on

J.

the intracel-

lular free lysine levels of growing baker's yeast. J. Chem.

N. T. Souki, and R. M. Russell. 1977.


athe( 15- Tanner, R. D., 31:290-294.

as

new

Tech.

Biotech.

fermentation

process

for

A
producing both ethanol and

lysine-enriched yeast. Biotechnol. Bioeng. 19:27-42.

Downloaded from http://aem.asm.org/ on December 21, 2016 by guest

This study developed from the United States-Taiwan Cooperative Science Program, which included the United StatesRepublic of China Seminar on Fermentation Engineering held
at the University of Pennsylvania on 30 May to 1 June 1978
and the Fermentation Engineering Research visit to Taiwan
between 6 and 21 June 1979 under the sponsorship of the
Division of International Programs of the National Science
Foundation (Project no. FCV-147).
S. Y. Huang and C. H. Lin collaborated in this effort. H. H.
Wang contributed significantly to the definition of the study
described in this report.

E-4

VOL. 43, 1982

EFFECT OF NaCl ON BAKERS' YEAST IN GELATIN

16. Tanner, R. D., C.-J. Wei, and J. Woodward. 1981. The


development of a semi-solid fermentation system for the
production of lysine-enriched yeast and ethanol, p. 323328. In M. Moo-Young, C. W. Robinson, and C. Verzina
(ed.), Advances in biotechnology, vol. 1. Pergamon Press,
Inc., Oxford.
17. Tempest, D. W., J. L. Meers, and C. M. Brown. 1970.
Influence of environment on the content and composition
of microbial free amino acid pools. J. Gen. Microbiol.
64:171-185.
18. Umemoto, S., Y. Irie, and T. Imai. 1967. The effect of
electrolytes concentrations on alcoholic fermentation of

763

molasses. I. Glycerol accumulation in the medium caused


by high concentrations of electrolytes. J. Ferment. Technol. 45:117-124.
19. Umemoto, S., Y. Irie, and T. Imal. 1967. The effect of
electrolytes concentrations on alcoholic fermentation of
molasses. II. Inhibitory effect of high concentrations of
electrolytes on yeast carboxylase. J. Ferment. Technol.
45:241-245.
20. Wei, C.-J., R. D. Tanner, and J. Woodward. 1981. Elucidating the transition between submerged culture and solid
state baker's yeast fermentations. Third Symposium on
Biotechnology in Energy Production, Gatlinburg, Tenn.

Downloaded from http://aem.asm.org/ on December 21, 2016 by guest