THE JOURNAL OF BIOLOGICAL

CHEMISTRY
Vol. 235, No. 5, May 1960
Printed
in U.S.A.

The Aerobic

Assimilation

of Glucose

by Yeast

Cells*

W. FALES

FRANK

From the Department of Biochemistry, Emory University, Atlanta, Georgia

(Received

for publication,

* This work was supported

ence Foundation.

by a grant from the National

Sci-

2, 1959)

the period of rapid oxygen consumption.

The oxygen concentration in the shaker experiments was well above its limiting
concentration
at all times and the same maximal oxygen consumption rate was observed in both the shaker and bubbler experiments.
The metabolic pattern was strikingly different under the two experimental
conditions.
Under the highly aerobic
conditions of the bubbler, aerobic fermentation was strongly inhibited, and the maximal increase in cellular carbohydrate
was
equivalent to 40% of the added sugar. This represents an increase in cellular carbohydrate
synthesis of about 100% compared with the synthesis previously observed with this yeast
under anaerobic conditions (7), and an increase of about 40%
compared with the synthesis observed in the shaker experiments.
In the shaker, there was a vigorous aerobic fermentation and the increase in the fatty acid content of the cells was
more than 100% in excess of the fatty acid synthesis observed in
the bubbler experiments.
The synthesis was equivalent to the
conversion of 11 y0 of the added sugar to fatty acids plus carbon
dioxide, if it is assumed that two-thirds
of the glucose carbons
are converted to fatty acid with an obligatory formation of carbon dioxide from one-third of the glucose carbons.
The estimated total oxygen consumption in the bubbler was equivalent
to the oxidation of about 30% of the added sugar whereas about
43% of the sugar was oxidized in the shaker.
It is evident that
the changes induced by varying the mode of aeration represent
major shifts in the metabolic pattern of the cells, with both the
catabolic and anabolic reactions being strongly modified.
EXPERIMENTAL

Materials and Methods

Fresh Fleischmann bakers yeast was used in all the experiments to be reported.
The suspension was prepared as previously described (5). The washed yeast was suspended in 0.1 M
NaHzP04 solution and the suspension was aerated for one hour
before each experiment.
The reactions were carried out in a
constant temperature
water bath at 30. In the Warburg experiments, 2 ml of 1.25% yeast suspension were placed in each
vessel and 0.5 ml of 1% glucose solution (0.1 M NaHtPOJ was
placed in the side arms. The shaking speed was 120 complete
strokes per minute and the gas phase was air. The vessels were
of about 15-ml capacity.
Oxygen consumption and carbon dioxide production were determined by the direct Warburg method
with the use of duplicate vessels containing base and duplicate
vessels without added carbon dioxide absorbent.
For the chemical determinations,
either separate Warburg vessels were provided for each analysis or the conditions in the Warburg vessels
were simulated by adding 60 ml of yeast suspension and 15 ml of

1255

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The study of the effect of oxygen on carbohydrate assimilation

has been largely neglected although innumerable
studies have
Obbeen made concerning its effect on the catabolic reactions.
viously, there is an interdependency
among the assimilatory,
in the
glycolytic, and oxidative enzyme systems. Modifications
oxidative metabolic pattern induced by changes in the oxygen
tension may be reflected by changes in the assimilatory pattern
as well as by changes in the glycolytic pattern as exemplified by
the Pasteur effect. Several papers have appeared which have
dealt directly or indirectly with the effect of oxygen on the assimilation of glucose by nonproliferating
yeast cells. Meyerhof
(l), in his extensive study of the effect of oxygen on alcoholic
fermentation,
observed that with pressed yeast, about 500/, of
the sugar that disappeared under aerobic conditions was not accounted for by the sum of the aerobic fermentation and oxidation, whereas under anaerobic conditions the discrepancy was
about 25%. He assumed that the additional amount of sugar
unaccounted
for under aerobic conditions
was indicative of
an increased assimilation.
Also, Winzler and Baumberger
(2)
found that under aerobic conditions there was a much greater
discrepancy between the predicted and measured heat production
than there was under anaerobic conditions, and they attributed
this to an increased synthesis of cellular carbohydrate
in the
However, conflicting reports have appeared
presence of oxygen.
concerning the effect of aeration on the synthesis of cellular carbohydrate, when direct measurements have been made. Neither
Gottschalk (3) nor Stier (4) found a greater synthesis of cellular
carbohydrate under aerobic conditions, but Fales (5) observed a
significant increase.
The lower recovery of catabolic products
under aerobic conditions also may be partially explained by an
increased fat synthesis.
Stier (4) observed a considerable increase in the fat content of the cells under aerobic conditions
whereas a negligible increase was observed under anaerobic conditions.
Klein (6) has confirmed the synthesis of fat by nonproliferating
yeast cells during aerobic metabolism, and a tracer
study indicated that the fat was derived from the glucose.
Early in the course of the present study, it was realized that
the metabolic pattern of the yeast was considerably modified
It is the puropse of this
under different conditions of aeration.
In one syspaper to report the results of a comparative study.
tem, air was rapidly bubbled through the suspension from a
fritted glass base. The oxygen tension in this system was mainIn the other system, the
tained at about 150 mm of mercury.
aeration was accomplished by shaking with the conventional
Warburg apparatus with air as the gas phase. In the latter system, the oxygen tension fell to about 50 mm of mercury during

November

Aerobic Assimilation

1256

CARBON

of Glucose by Yeast Cells

DIOXIDE

EXTRACELLULAR
CARBOHYDRATE

= 80
E
\ 40

RESULTS

CARBOHYDRATE
6 70
L,
z 20
s

I6
I?
.-

2
REACTJON

TIME.

3
HOURS

FIG.
1. The aerobic metabolism of glucose (2.0 mg per ml) by a
1% suspension of yeast cells : O---O,
aerated by rapidly passing
air through the suspension; O-0,
aerated by Warburg shaker;
broken
line, anaerobic.
The limiting concentration
for glucose
oxidation of this yeast was 1.6 X 10-a M or 29 mg per 100 ml (see
text).
The values recorded for fatty acid and cellular carbohydrate are the averages obtained with six separate experiments.
In these experiments, the differences obtained between the shaker
and bubbler were significant at all time intervals.
The statistical
probability
of no difference for the 30-minute cellular carbohydrate determinations
was p< 0.02; for the 75-minute fatty acid
determinations,
p = 0.02; and for the other determinations,
p<
0.01. The values recorded for the carbon dioxide, oxygen, and
extracellular
carbohydrate were those obtained in single experiments with each point being the average of duplicate determinations.

glucose solution to a 500-ml Erlenmeyer flask. When the Erlenmeyer flask was used, the shaking speed was diminished to 90
strokes per minute since the oxygen tension was maintained
at
the level observed in the Warburg vessels at this shaking rate.
A 200-ml chromatography
tube with a fritted glass base was
used as the reaction vessel in the experiments in which air was
The air was brought to the
bubbled through the suspension.
temperature of the water bath and saturated with water before
it entered the reaction vessel. Air was passed through the suspension at a rate of about 350 ml per minute.
Yeast suspension
and glucose solution were added in the volumes of 80 and 20 ml,

The results of the experiments are shown in Fig. 1. There

was a small increase in the glucose utilization
rate in the bubbler
over that observed in the shaker which is the reverse of what
might be expected with an enhancement of the Pasteur effect.
The glucose consumption rate in both instances was considerably
slower than that observed under anaerobic conditions.
The Warburg measurements are of some interest.
After a
lo-minute lag period, a respiratory quotient (R.Q.) of 2 was observed for the following 35 minutes, indicating a vigorous aerobic
fermentation.
During the following 25-minute transition period,
the R.Q. rapidly shifted to about 0.67 which is the theoretical
R.Q. for ethanol catabolism.
The phase of rapid ethanol catabolism continued for 55 minutes.
A complete cessation of glucose
catabolism is not necessarily indicated by the R.Q. since there
was a concomitant
fat synthesis.
The transition from glucose
to alcohol catabolism was initiated considerably before the glucose concentration
became limiting.
A polarographic
study of
the oxygen consumption rate of the yeast as affected by the concentration of ethanol, glucose, and pyruvate showed that the
same maximal oxygen consumption rate was obtained with the
three substrates, and indicated that at appropriate
concentrations they were all capable of saturating the same limiting reaction of the yeast cells. However, the limiting concentrations
(minimal
concentrations
giving maximal oxygen consumption
rates) of the three substrates were markedly different, being
3.5 X 1O-4 M for ethanol, 1.6 X 10-s M for glucose, and 1.7 X
Thus, it is not surprising that the alcohol
10 M for pyruvate.
successfully competed as a substrate before the glucose concentration became limiting.
At the conclusion of the period of
rapid alcohol oxidation, a second transitional period was observed
during which the R.Q. increased.
At 3 hours the R.Q. was close
to unity, indicating that the principal substrate was reserve carbohydrate.
However, the total carbon dioxide production
for
the three hours was 13.7 y0 in excess of the total oxygen consumption. This suggests that a portion of the alcohol initially formed
was not oxidized to carbon dioxide and water, but was converted
to other products.
The fatty acid data are consistent with the
hypothesis of a considerable conversion of alcohol to fat. There
was a continued rapid fatty acid synthesis during the phase of al-

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respectively.
Measurements
of the fluid volume indicated that
there was no net evaporation or condensation during the course
Also, there was no significant trapping of
of the experiments.
the cells in the fritted glass base since a constant cell count was
observed.
For each analysis, the bubbler and shaker experiments were carried out simultaneously
with aliquots of the same
yeast suspension and glucose solution.
The oxygen consumption
rate in the bubbler experiments was determined polarographically
by the method of Baumberger
(8). Aliquots of the suspension
were transferred to the polarographic
vessel for the measurements.
The total cellular and extracellular
carbohydrate
were determined by the anthrone method as previously described (5). The
supernatant fluid was immediately
separated from the cells by
centrifugation.
This immediate separation was found necessary
because there was a gradual diffusion of trehalose from the cells
after the reaction was stopped with HgC12. The total fatty acid
content of the cells was determined by the method of Klein (6).
Palmitic and oleic acids were utilized as the standards for the
calorimetric assay method of Kibrick and Skupp (9).

160

Vol. 235, No. 5

May

F. W. Fazes

1960

cohol oxidation, a period during which the rate of glycolysis was

minimal.
In contrast to the shaker experiments, no appreciable aerobic
fermentation was evident in the bubbler experiments.
No alcohol or acetaldehyde could be found in the suspension fluid during
the course of the reaction and only traces of these materials could
be recovered from the effluent air. There was a rapid decrease
in the oxygen consumption rate from the time the sugar became
limiting in concentration,
indicating that there was no appreciable accumulation of intermediates that could be rapidly oxidized.
Also, there was a marked reduction in the rate of fatty acid synthesis after the sugar became limiting in concentration.
DISCUSSION

the inhibition of the synthesis by the uncoupling of phosphorylation is not readily understandable
since hexose phosphates are
intermediates
for both the synthesis and the fermentation
and
since the fermentation
was not inhibited.
It is of interest that
in the present study, there was a greater synthesis of both trehalose and glycogen in the bubbler than in the shaker experiments, and that the increased synthesis of the former was much
more marked than that of the latter.
Several hypotheses may be advanced for explaining the greater
fat synthesis that was observed in the shaker. More of the required reduced coenzyme may have been available for the fatty
acid synthesis at the lower oxygen tension (15). Also, the data
suggest that a portion of the alcohol, which accumulated in the
shaker, acted as a precursor for fat synthesis.
It is also possible
that the higher carbon dioxide tension in the shaker stimulated
fat synthesis (16, 17).
SUMMARY

A comparative study of the aerobic metabolism of glucose by

nonproliferating
yeast cells with the use of two different modes
of aeration, has been carried out. In one instance, air was rapidly bubbled through the suspension and in the other instance
the aeration was accomplished with the Warburg shaker. Under
the higher oxygen tension extant in the bubbler there was an inhibition of aerobic fermentation,
an increase in the synthesis of
cellular carbohydrate,
and a decrease in the synthesis of fat.
Thus both the catabolic and anabolic reactions were strongly
modified.
REFERENCES
1. MEYERHOF,
O., Biochem.
Z., 162, 43 (1925).
2. WINZLER.
R. J.. AND BAUMBERGER.
J. P.. J. Cellular
and ComD.
Physioi.,
21,77
(1943).

3. GOTTSCHALK,
A., Australian
J. Exptl.
Biol. Med. Sci., 19, 211
(1941).
4. STIER,
T. J. B., Cold Spring
Harbor
symposia
on quantitative
biology,
Vol. 7, Long
Island
Biological
Association,
Cold
Spring
Harbor,
Long Island,
New York,
1939, p. 385.
5. FALES,
F. W., J. Biol. Chem., 193, 113 (1951).
6. KLEIN,
H. P., J. Bacterial.,
69, 620 (1955).
7. FALES,
F. W., AND BAUMBERGER,
J. P., J. Biol.
Chem., 173,
1 (1948).
8. BAUMBERGER,
J. P., Cold Spring
Harbor
symposia
on quantitative biology,
Vol. 7, Long
Island
Biological
Association,
Cold Spring
Harbor,
Long Island,
New York,
1939, p. 195.
9. KIBRICK,
A. C., AND SKUPP,
S. J., Arch. Biochem.
Biophys.,
44, 135 (1953).
10. LELOIR,
L. F., AND CARBIB,
E., J. Am. Chem. Sot., 76, 5445
(1953).
11. LELOIR,
L. F., AND CARDINI,
C. E., J. Am. Chem. SOL, 79,634O
(1957).
12. T~EVE~YAN,
W. E., MANN,
P. F. E., AND HARRISON,
J. S.,
Arch. Biochem.
BioDhus..
60. 81 (1954).
13. FALES,
F. W., J. BioZ. chek.,
aO2, i57 (i953).
14. SPIEGELMAN,
S., KAMEN,
M. D., AND SUSSMAN,
M., Arch.
Biochem.,
18, 409 (1948).
15. CHANCE,
B., J. Biol. Chem., 217, 383 (1955).
16. BRADY,
R. O., Proc. Natl.
Acad. Xci. U. S., 44, 993 (1958).
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D. M., TITCHENER,
E. B., AND WAKIL,
S. J., Biochim.
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The large differences in the metabolic pattern that were observed with the two modes of aeration were apparently induced
by differences in the gas tensions in the two systems since it would
appear that these were the only variables in the experimental
conditions.
The increased cellular carbohydrate
synthesis together with the decreased total oxygen consumption that was observed in the bubbler experiments, suggests a higher over-all
efficiency of oxidative phosphorylation
at the higher oxygen tension. This increased efficiency may not be due to a direct action
of oxygen on the coupling of phosphorylation
with oxidation.
In
the shaker experiments, the data suggest that the oxidation of
alcohol accounted for about half of the total oxygen consumption,
but in the bubbler experiments, oxidative fermentation
was inhibited.
The over-all efficiency of oxidative
phosphorylation
may be decreased when alcohol is an intermediate because of the
energy required for bringing the 2-carbon compound into the
oxidative pathway.
The energy requirements for cellular carbohydrate synthesis may be much higher than previously supposed.
Leloir and Carbib (10) have presented evidence that UDP-glucase and hexose phosphate are precursors for trehalose synthesis
in yeast. Also, UDP-glucose
and hexose phosphate may be
precursors for glycogen synthesis in yeast since there is evidence
that this may be the case in mammalian tissue (11) and since
Trevelyan et al. (12) have found that during fermentation
the
orthophosphate
and glucose l-phosphate
levels are maintained
in the cells at an unfavorable ratio for glycogen synthesis by the
Since there is a considerable glyyeast phosphorylase reaction.
cogen synthesis during fermentation,
the UDP-glucose pathway
for glycogen synthesis is suggested.
If UDP-glucose is indeed a
precursor for cellular carbohydrate
synthesis, energy is required
for the reformation of the UDP-glucose
as well as for the phosphorylation
of the sugar. This proposed requirement
of additional energy for cellular carbohydrate
synthesis is helpful in
explaining the effect of azide on alcoholic fermentation.
At an
appropriate azide concentration, the synthesis of cellular carbohydrate is completely inhibited without reducing the fermentation
rate (13). Spiegelman et al. (14) have presented evidence that
azide uncouples fermentative
phosphorylation.
A mechanism
for the azide inhibition is readily deduced with an energy-requiring step for the conversion of hexose phosphate to cellular carbohydrate.
However, if there were no energy-requiring
step, then

1257

The Aerobic Assimilation of Glucose by Yeast Cells

Frank W. Fales
J. Biol. Chem. 1960, 235:1255-1257.

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