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THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 235, No. 5, May 1960

Printed in U.S .A.

The Aerobic Assimilation of Glucose by Yeast Cells*

FRANK

W.

FALES

From the Department of Biochemistry Emory University Atlanta Georgia

(Received for publication, November 2, 1959)

The study of the eff ect of oxygen on carbohydrate assimilation

has been largely neglected although innumerable studies have

been made concerning its eff ect on the catabolic reactions.

Ob-

viously , there is an interdependency among the assimilatory,

glycolytic, and oxidative enzyme systems.

Modifications in the

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 glycolyt ic pattern as exemplified by

the Pasteur eff ect . Several papers have appeared which have

dealt directly or indirectly with the eff ect of oxygen on the as-

similation of glucose by nonproliferating yeast cells.

Meyerhof

(l), in his extensive study of the effec t of oxygen on alcoholic

fermentation, observed that with pressed yeast, about 500/, of

the sugar that disappeared under aerobic conditions was not ac-

counted for by the sum of the aerobic fermentation and oxida-

tion, 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 o f cellular carbohydrate in the

presence of oxygen.

However, conflicting reports have appeared

concerning the eff ect of aeration on the synthesis of cellular car-

bohydrate, 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 in-

crease in the fat content of the cells under aerobic conditions

whereas a negligible increase was observed under anaerobic con-

ditions. Klein (6) has confirmed the synthesis of fat by non-

proliferating 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

under different conditions of aeration.

It is the puropse of this

paper to report the results of a comparative study.

In one sys-

tem, air was rapidly bubbled through the suspension from a

fritted glass base.

The oxygen tension in this system was main-

tained at about 150 mm of mercury.

In the other system, the

aeration was accomplished by shaking with the conventional

Warburg apparatus with air as the gas phase.

In the latter sys -

tem, the oxygen tension fell to about 50 mm of mercury during

* This work was supported by a grant from the National Sci-

ence Foundation.

the period of rapid oxygen consumption.

The oxygen concen-

tration in the shaker experiments was well above its limiting

concentration at all times and the same maximal oxygen con-

sumption rate was observed in both the shaker and bubbler ex-

periments. The metabolic pattern was strikingly different un-

der the two experimental conditions. Under the highly aerobic

conditions of the bubbler, aerobic fermentation was strongly in-

hibited, and the maximal increase in cellular carbohydrate was

equivalent to 40 of the added sugar. This represents an in-

crease in cellular carbohydrate synthesis of about 100 com-

pared with the synthesis prev iously observed with this yeast

under anaerobic conditions (7), and an increase of about 40

compared with the synthesis observed in the shaker experi-

ments. In the shaker, there was a vigorous aerobic fermenta-

tion and the increase in the fat ty acid content of the cells was

more than 100 in excess of the fa tty acid synthesis observed in

the bubbler experiments. The synthesis was equivalent to the

conversion of 11 y0 of the added sugar to fat ty acids plus carbon

dioxide, if it is assumed that two-thirds of the glucose carbons

are converted to fa tty acid with an obligatory formation of car-

bon dioxide from one-third of the glucose carbons. The esti-

mated 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 shi fts 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 experi-

ments to be reported. The suspension was prepared as previ-

ously 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 ex-

periments, 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 capaci ty. Oxygen consumption and carbon di-

oxide 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 chem-

ical determinations, either separate Warburg vessels were pro-

vided 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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1256

Aerobic Assimilation of Glucose by Yeast Cells Vol. 235, No. 5

W

160

=

80

E

\ 40

6 70

L,

z 20

s

I6

I?

CARBON DIOXIDE

EXTRACELLULAR

CARBOHYDRATE

CARBOHYDRATE

.-

0 2

REACTJON TIME. HOURS

3

FI G. 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 fa tty acid and cellular carbohy-

drate 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 carbohy-

drate determinations was p< 0.02; for the 75-minute fatty acid

determina tions, p = 0.02; and for the other d etermination s, p <

0.01. The values recorded for the carbon dioxide, oxygen, and

extracellular carbohydrate were those obtained in single experi-

ments with each point being the average of duplicate determina-

tions.

glucose solution to a 500-ml Erlenmeyer flask.

When the Erlen-

meyer f lask 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 frit ted glass base was

used as the reaction vessel in the experiments in which air was

bubbled through the suspension.

The air was brought to the

temperature of the water bath and saturated with water before

it entered the reaction vessel . Air was passed through the sus-

pension at a rate of about 350 ml per minute. Yeast suspension

and glucose solution were added in the volumes of 80 and 20 ml,

respectively. Measurements of the fluid volume indicated that

there was no net evaporation or condensation during the course

of the experiments.

Also, there was no significant trapping of

the cells in the fritted glass base since a constant cell count was

observed. For each analysis, the bubbler and shaker experi-

ments 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 measure-

ments.

The total cellular and extracellular carbohydrate were deter-

mined 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 fa tty 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).

RESULTS

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

was a small increase in the glucose util ization 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 eff ect .

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 ob-

served 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 catab-

olism 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 glu-

cose concentration became limiting. A polarographic study of

the oxygen consumption rate of the yeast as affec ted by the con-

centration of ethanol, glucose, and pyruvate showed that the

same maximal oxygen consumption rate was obtained with the

three substrates, and indicated that at appropriate concentra-

tions they were all capable of saturating the same limiting reac-

tion 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

10”

M

for pyruvate. Thus, it is not surprising that the alcohol

successful ly competed as a substrate before the glucose concen-

tration 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 car-

bohydrate. However, the total carbon dioxide production for

the three hours was 13.7 y0 in excess of the total oxygen consump-

tion. This suggests that a portion of the alcohol initial ly 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 fa tty acid synthesis during the phase of al-

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May 1960

F. W. Fazes

1257

cohol oxidation, a period during which the rate of glycolys is was

minimal.

In contrast to the shaker experiments, no appreciable aerobic

fermentation was evident in the bubbler experiments. No alco-

hol 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 eff luent 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 apprecia-

ble accumulation of intermediates that could be rapidly oxidized.

Also, there was a marked reduction in the rate of fa tty acid syn-

thesis after the sugar became limiting in concentration.

DISCUSSION

The large differences in the metabolic pattern that were ob-

served 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 to-

gether with the decreased total oxygen consumption that was ob-

served in the bubbler experiments, suggests a higher over-all

effi ciency of oxidative phosphorylation at the higher oxygen ten-

sion. This increased effi ciency 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 o f the total oxygen consumption,

but in the bubbler experiments, oxidative fermentation was in-

hibited. The over-all effi ciency 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 carbo-

hydrate synthesis may be much higher than previously supposed.

Leloir and Carbib (10) have presented evidence that UDP-glu-

case 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

yeast phosphorylase reaction.

Since there is a considerable gly-

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 phos-

phorylation of the sugar. This proposed requirement of addi-

tional energy for cellular carbohydrate synthesis is helpful in

explaining the eff ect of azide on alcoholic fermentation. At an

appropriate azide concentration, the synthesis of cellular carbohy-

drate 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-requir-

ing step for the conversion of hexose phosphate to cellular carbo-

hydrate. However, if there were no energy-requiring step, then

the inhibition of the synthesis by the uncoupling of phosphoryla-

tion 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 tre-

halose and glycogen in the bubbler than in the shaker experi-

ments, 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 re-

quired reduced coenzyme may have been available for the fa tty

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 fa t synthesis. It is also possible

that the higher carbon dioxide tension in the shaker st imulated

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 rap-

idly 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 in-

hibition of aerobic fermentation, an increase in the synthesis of

cellular carbohydrate, and a decrease in the synthesis o f fat .

Thus both the catabolic and anabolic reactions were strongly

modified.

REFERENCES

1. MEYERHOF,

O., Bioche m. Z., 162, 43 1925).

2. WINZLER. R. J.. AND BAUMBERGER. J. P.. J. Cellular and ComD.

Physio i., 21,77 1943). ’ ’

3. GOTTSCHALK, A., Australian J. Exptl. Biol. Med. Sc i., 19, 211

1941).

4. STIER, T. J. B., Cold Spring Harbor sympo sia on quantitative

biology, Vo l. 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. Bacte rial., 69, 620 1955).

7. FALES, F. W., AND BAUMBERGER, J. P., J. Biol. Chem., 173,

1 1948).

8. BAUMBERGER, J. P., Cold Spring Harbor sympo sia on quanti-

tative 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.

Bioche m., 18, 409 1948).

15. CHANCE, B., J. Bio l. Chem., 217, 383 1955).

16. BRADY, R. O., Proc. Natl. Acad . Xci. U. S., 44, 993 1958).

17.

GIBSON,

D. M.,

TITCHENER,

E. B.,

AND WAKIL,

S. J., Biochim.

Biophys. Acta, 30 , 377 1958).

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Frank W. Fales

The Aerobic Assimilation of Glucose by Yeast Cells

1960, 235:1255-1257.J. Biol. Chem.

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