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Diauxic growth of the fission yeast Schizosaccharomyces pombe in mixtures

of D-glucoseand ethanol or acetate1


C. STANT S A I ~AND ANTONIOJ . AVELEDO
Department of Chemistry, Carleton University, Ottawa, Ont., Canada KIS 5B6
IANJ . MCDONALDA N D BYRONF . JOHNSON
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada KIA OR6

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Received July 2, 1986


Accepted March 5 , 1987
TSAI,C. S., AVELEDO,A. J., MCDONALD,I. J., and JOHNSON,B. F. 1987. Diauxic growth of the fission yeast Schizosaccharomyces pombe in mixtures of D-glucose and ethanol or acetate. Can. J. Microbiol. 33: 593-597.
The fission yeast Schizosaccharomyces pombe was unable to utilize ethanol or acetate as its sole carbon source for growth.
However, ethanol and acetate were utilized in the presence of D-glucose during diauxic growth. No mutants capable of utilizing
ethanol or acetate as sole carbon source were isolated from cultures grown in glucose together with ethanol or acetate. Low
concentrations of acetate facilitated growth with glucose, whereas high concentrations of ethanol or acetate were inhibitory.
The exogenous [1-I4c]acetate was initially incorporated into
Growing cells readily took up [I -I4c]ethanol and
biomacromolecules which were subsequently catabolized.

acetat acetate.

TSAI, C. S., AVELEDO,A. J., MCDONALD,I. J., and JOHNSON,B. F. 1987. Diauxic growth of the fission yeast Schizosaccharomyces pombe in mixtures of D-glucose and ethanol or acetate. Can. J. Microbiol. 33 : 593-597.
La levure Schizosaccharomyces pombe est incapable d'utiliser l'ethanol ou l'acetate comme seule source de carbone pour la
croissance. Cependant, en presence de D-glucose, l'ethanol et l'acetate se sont averes tous les deux utilisables au cours de la
croissance diauxique. Aucun mutant capable d'utiliser .l'Cthanol ou l'acetate comme seule source de carbone n'a pu Ctre isole
a partir de cultures croissant sur milieu glucosC en prisence d'ethanol ou d'acitate. De faibles concentrations en acetate
facilitent la croissance en presence de glucose, alors que des concentrations Clevees sont inhibitrices. Les cellules en croissance
absorbent rapidement 1'Cthanol [14c-1] et l'acitate [I4c-11. L'acitate [14c-11 exogkne est initialement incorpork a des
biomacromol~culesqui sont, subsequemment, catabolisees.
[Traduit par la revue]

Introduction
Saccharomyces (S.) cerevisiae and Schizosaccharomyces
(Sch.) pombe are t w o o f the most thoroughly studied model
microorganisms. Both yeasts utilize D-glucose which, a t high
concentrations, represses respiration ( D e Deken 1966). Thus,
they are capable of aerobic alcoholic fermentation provided that
the glucose concentration is in excess. In a batch culture, S .
cerevisiae utilizes D-glucose in diauxic growth, whereas the
growth o f Sch. p o m b e is reported t o b e monoauxic (Fiechter e t
al. 1981).
T h e rates of growth and fermentaion of S . cerevisiae are
profoundly affected by ethanol and acetate either produced in the
batch fermentation o r added from a n external source (Huiikova
and Fencl 1978; Moulin e t a l . 1980; Le3o and van Uden 1982).
After an adaptation period, S . cerevisiae utilizes ethanol and
acetate (Eaton and Klein 1954; Gosling and Duggen 197 1).
Schizosaccharomyces p o m b e , o n the other hand, has been
considered incapable o f utilizing ethanol and acetate (Barnett e t
a l . 1983; Mitchison 1970). In view o f great similarities between
the two yeasts, the utilization of these t w o carbon substrates by
Sch. pombe requires reinvestigation. Furthermore, organic acid
metabolism of fission yeasts might have s o m e commercial
application (Yang 1973; Gallander 1977; S n o w and Gallander
1979), thus more detailed examination o n acetate utilization
was in order. T h e present work demonstrates that ethanol and
acetate are utilized during diauxic growth o f Sch. p o m b e in the
presence of D-glucose .

Materials and methods


Materials
Reagent-grade chemicals were obtained from a variety of commersources. D - ~ ~ - ~ 4 ~ ~ g l(u2 c. 2oms ~e i / m m o l ; 1 c i = 37 GBq),

'NRCC no. 27666.


2 ~ u t h oto
r whom reprint requests should be addressed.

ethanol (20 mCi/mmol), and [l-14~]sodiumacetate (50 mCi/


mrnol) were purchased from NEN DuPont Canada Inc. and Amersham
Corp., respectively.
Cultures
The fission yeast Schizosaccharomyces pombe, NCYC 132 S2-2
(McDonald et al. 1982), was maintained by periodic transfers to 2%
(w/v) malt extract (Oxoid) broth at 30C.
Batch cultures were grown in EMM2, a minimal salts medium
described by Mitchison (1970) and modified to contain D-glucose
(0-80mg m ~ - ' ) and either ethanol (0-lOmg mL-l) or acetate
(0-2.0 mg d - I ) as the carbon sources. Batch cultures were grown in
Erlenmeyer flasks (10 mL medium in 50-mL flask, 20 in 125, 100 in
500, or 250 in 1000) with constant shaking (200 rpm) at 30C in a G24
environment shaker or a Gyrotherm bath, both manufactured by New
Bmnswick Scientific Co., New Brunswick, NJ.
Radiolabelled experiments
For uptake experiments, the ethanol-EMM2 medium contained
2 m g D-glucose d-'and 5 mg [ l - 1 4 ~ ] e t h a n omL-I
l
(2 nCi/mg),
while the acetate-EMM2 medium contained 2 mg D-glucose m ~ - and
'
2 mg [ I -14c]acetate mL-' (10 nCi/mg). Erlenmeyer flasks (125 mL)
containing 10 mL of the 14C-labelled medium were inoculated and
shaken at 200rpm in a G75 shaker bath (New Brunswick Scientific
Co.) at 30C. At intervals, 1.O-mL samples of culture were removed
and centrifuged (Sorvall RC2-B , Sorvall Inc .) at 12 000 X g for 3 min.
Sedimented cells were washed twice with 0.9% NaCl solution and then
suspended in 1.0 mL of 0.9% NaCl. The cell suspension was filtered
through a Millipore filter (0.2 pm) to collect cells for 14ccounting.
To follow I4cO2 production, cultures were set up as described for
the uptake experiments. Erlenmeyer flasks were fitted with an inlet
sidearm (with a 0.2-pm filter) through which water-saturated air was
delivered by a pump (Hagen Elite 802) at a rate of 10 mL min- I, and an
outlet sidearm through which the respired air was bubbled through
lOmL of 4.0 N KOH. Samples of KOH were withdrawn at time
determination.
intervals for I4co2
For chemical extraction of cellular compounds, the yeast was grown
for 24 h (G75 shaker as described above) in (i) lOOmL of EMM2

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594

CAN. 1. MICROBIOL. VOL. 3 3 , 1987

containing 2 mg ~ - [ ~ - ' ~ C ] ~ l umc o~s-e(25


' nCi m g I ) , (ii) 100 mL of
acetate-EMM2 containing 2 mg D - [ u - ' ~ c ] ~ ~ u cmL-'
o s ~ (24 nCi
mg-I) and 2 mg acetate m ~ - ' , or (iii) lOOmL acetate-EMM2
containing 2 mg D-glucose m I - ' and 2 mg [~-'~C]acetate
mI-'
(25 nCi mg-I). Cells were collected, washed, and extracted by the
following procedure (Sutherland and Wilkinson 1971). An initial
extract and a washing with cold 10% aqueous trichloroaceticacid were
pooled (small molecular weight fraction). The nucleic acid was
extracted by heating (90C) with 5% aqueous trichloroacetic acid for
30 min. After centrifugation, the cell pellet was suspended in distilled
water and warmed to 65C. An equal volume of 90% aqueous phenol
was added and mixed vigorously for 5 min. The aqueous layer was
collected as a polysaccharide fraction and the phenol layer was pooled
as a protein fraction. The residue was referred to as cell wall debris.
Each extracted liquid fraction was assayed for 14Cincorporation. The
residue fraction was transferred onto a Whatman 3MM paper disc for
Time (days)
counting. The radioactivity of samples derived from D-[~-'~C]gluFIG. 1. Growth curves of Schizosaccharomyces pombe in batch
cose, [ I-14c]ethanol, or [I -14c]acetate in 5 mL of aquasol (NEN) was
culture of EMM2 medium supplied with 2.0 mg D-glucose mL-' and
measured with a Beckman LS 120 scintillation counter.
10 mg ethanol mL- at pH 5 .O. The cultures were incubated aerobically
Analysis
with a constant shaking (200 rpm) in a G-24 environmental shaker at
The turbidity of batch cultures was measured at 660nm in a
30C as described in the text. a, Total cell count; 0, D-glucose
Beckman model DB spectrophotometer. The cell number was counted
concentration; 0, ethanol concentration; A, acetate concentration.
microscopically in an Improved Neubauer ultraplane chamber (A. H.
Thomas Co.). To determine cell mass, samples of cultures (10 mL)
were filtered through preweighed nitrocellulose filters (0.45 km,
Schleicher & Schuell Inc.). Collected cells were washed with distilled
water, dried in an oven at 100C for 16- 18 h, and weighed. Glucose
concentration of the cell-free culture medium was determined by a
glucose oxidase and peroxidase-coupled colorimetric method (Spiro
1966) using the Sigma Chemical Co. glucose assay kit (no. 510).
Ethanol and acetate concentrations were determined by the use of a
GOW/MAX gas chromatograph (series 750; equipped with a HewlettPackard 3390 integrator) employing a coiled column (6 ft. X 0.25 in.;
1 ft. = 0.3048 m, 1 in. = 25.4 mm) packed with 0.10% free fatty acid
packing and 1.0% H3PO4on 100-200 mesh chromosorb W, A/W, at a
column temperature of 150C (Ackman 1972). The column was
calibrated with standard ethanol and acetate solutions using i-pentanol
(0.10%) as the internal reference.
0
1
2
3
4
5

'

Results
Schizosaccharomyces pombe would not grow in batch
cultures supplied with ethanol or acetate as the sole carbon
source. This result supports earlier observations made by
Mitchison (1970) and Barnett et al. (1983). However, the
sequential utilization of ethanol in the presence of D-glucose
was characterized by diauxic growth of Sch. pombe (Fig. 1).
Diauxic growth was also observed for the fission yeast grown
with a mixture of D-glucose and acetate (Fig. 2). Slight albeit
opposite effects on cell concentration at the stationary phase of
cultures were observed for acetate versus ethanol. We have
estimated the cell density from linear relationships between cell
mass and cell concentration of exponentially growing cells.
Acetate increases while ethanol decreases the cell density
(Table 1).
Schizosaccharomyces pombe has not been noted to have
diauxic growth in batch culture with glucose as the sole carbon
source. However, on reexamination of growth with glucose as
sole carbon source, the sequential utilization of glucose,
ethanol, and acetate was found (Fig. 3a). Ethanol production
was concurrent with D-glucose utilization, but upon exhaustion
of D-glucose, ethanol was utilized with the subsequent accumulation of acetate. This resulted in a short but discernible
interlogarithmic phase characteristic of diauxic growth, which
is apparent on average but not seen in every experiment: diauxie
in 7 of 12 repetitions (Fig. 3b).
Although D-glucose is the preferred substrate, the rate of

Time (days)

FIG. 2. Growth curves of Schizosaccharomyces pombe in batch


culture containing 2.0 mg D-glucosemL-I and 2.0 mg acetate mL-'.
See Fig. 1 for symbol legends.
TABLE1. Growth parameters for batch cultures of Schizosaccharomyces pombe at pH 5.0, 30C

Medium

Specific
rate of D-glucose
consumption
(h- '1

Cell
density
(pglcell)

D-Glucose, 0.2Oh
D-Glucose,0.2%, + ethanol, 1 .O%
D-Glucose,0.296, + acetate, 0.2%

0.133
0.079
0.108

22.9
15.7
25.6

NOTE:Growth parameters of the exponential phase are given.

D-glucose depletion in the tropophase was slightly slower in the


presence of ethanol or acetate (Figs. 1-3). Ethanol followed its
general transitory pattern in concert with glucose utilization and
acetate accumulation (Figs. 1-3). At the end of diauxic growth,
the cultures were plated on EMM2 agar with either ethanol or
acetate as the sole carbon source and examined for mutant cells
capable of growth. No such mutants were detected.
The inability of Sch. pombe to grow in ethanol or acetate was
not due to a failure in the uptake of ethanol or acetate, because
concentrations of exogenous ethanol and acetate in the batch

TSAI ET AL.

TABLE2. Incorporation of D-[u-14C]glucoseand [1-I4c]acetateinto biomacromolecules of Schizosaccharomyces pombe grown for 24 h at pH 5.0, 30C

Fraction

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Small mol. wt ."


~iomacromolecules~
Cell wall debrisc

D- [u-14C]glucose

~-[u-'~~]~lucose
+ acetate
[ I-I4C]acetate

33.3
52.2
34.4

NOTE:Data are given as nmol 14C-labelledsubstrate incorporated per 10 mL culture.


"Small mol.wt. fraction refers to cold 10% TCA extract.
bBiomacromolecules include nucleic acid, polysaccharide, and protein fractions as referred to in text.
'Cell wall debris is residue after extractions.

TlME (days)

FIG. 5. Uptake of ~l-l~C]acetate


by batch cultures (2.0 mg D-glucose rnL- and 2.0 mg acetate mL- ) of Schizosaccharomyces pombe.
0,
D-Glucose concentration; A , acetate concentration in the culture;
A , [1-'4~]acetateincorporated per lo7 cells;
I4co2production rate
per lo6 cells.

'

TlME (days)

FIG. 3. Growth curves of Schizosaccharomyces pombe in batch


cultures containing 2.0 mg D-glucose mL-' . (a) 0 , Total cell count, 12
experiments; 0 , D-glucose concentration, 3 experiments; , ethanol
concentration, 3 experiments; and A , acetate concentration, 3 experiments. (b) 0 , Total cell count, 7 experiments, all showing diauxie.

'

+,

TABLE3. Effect of ethanol concentrations on growth of Schizosaccharomyces pombe


Initial concn. ethanol
(mg m ~ - ' )

Cell concn.
(cells m I - ')

Ethanol consumed

(%I

NOTE:Batch cultures of Sch. pombe were grown aerobically in modified EMM2 medium
containing 2 . 0 m g D-glucose mL-' and 0-lOmg ethanol mL-' in Erlenmeyer flasks
(125 mL) which were placed in a (3-24 environmental shaker at 30C, pH 5 . 0 , for 5 days.

TlME (days)

FIG. 4. Uptake of [ I-14c]ethanol by batch cultures (2.0 mg D-glucose mL-' and 5.0 mg ethanol mL-') of Schizosaccharomycespombe.
0 , D-Glucose concentration; 13,ethanol concentration in the culture;
W, [ I-'4C]ethanol incorporated per lo7 cells.

cultures (Figs. 1 and 2) fluctuated in consonance with changes in


the endogenous ethanol and acetate concentrations of the batch
culture of which D-glucose was the sole carbon source (Fig. 3).
Furthermore, the exogeneous [ 1- 14c]ethanol (Fig. 4) and
[1-14c]acetate (Fig. 5) readily entered the growing cells. A
rapid uptake of [ 1 - 14C]acetateduring the initial 24 h growth was

followed by a decrease in the cellular radiolabel upon the


depletion of culture ethanol (Figs. 2 and 5).
Since ethanol is metabolized via acetate, cells utilizing
ethanol are likely to have problems identical with the cells
utilizing acetate. Therefore, the fate of [1-14c]acetate that
entered the fission yeast cells was traced. The 14c0,production from [1-14c]acetate followed two discrete stages (Fig.
5). Schizosaccharomyces pombe cells grown for 24 h in D-[U14~]glucoseand [1-14c]acetate were extracted into different
was
chemical fractions. As shown in Table 2, D-[U-14~]glucose
incorporated into every biomolecular fraction. [I - l 4 c ] ~ c e t a t e
was found mainly in cell wall debris and biomacromolecular
fractions. The effects of acetate on D-glucose incorporation
suggest a synergistic utilization of D-glucose and acetate in
syntheses of cell wall and biomacromolecular fractions.
Addition of ethanol or acetate to the batch culture affects the
total growth of the fission yeast cells (Tables 3 and 4). Thus,

596

CAN. J. MICROBIOL. VOL. 33, 1987

TABLE
4. Effect of acetate concentrations on growth of Schizosaccharomyces pombe
Initial concn. acetate
(mg m ~ - ' )

Cell concn.
(cells mL- ')

Acetate consumed

(%I

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NOTE: Batch cultures of Sch, pombe were grown at 30C, pH 5.0, for 8 days as
described in Table 3 except that acetate (0-4.0 mg rnL-') replaced ethanol.

high concentrations of ethanol are inhibitory, as observed


earlier for Saccharomyces and Candida (Moulin et al. 1980;
Novak et al. 1981; KO and Edward 1975; Huiikova and Fencl
1977). Indeed, the cell counts declined with the amounts of
added ethanol (Table 3). However, the addition of acetate elicits
a different response, for subinhibitory concentrations of acetate
stimulate increased production of cells (Table 4).

Discussion
We have demonstrated that the growth of Schizosaccharomyces pombe in low D-glucose concentrations is likely diauxic.
Ethanol and acetate are sequentially produced from glucose,
and are utilized in sequence. However, sequential utilization of
the three compounds does not lead to triauxie. Diauxic growth is
also observed for batch cultures containing mixtures of D-glucose and ethanol or acetate, both of which prolong the
interlogarithmic phase. Although it is not a classical diauxie
(Fiechter et al. 1981), the sum of the evidence indicates a
diauxic metabolism. Schizosaccharomycespombe is reported to
be D-glucose sensitive, yet to utilize the sugar in monauxic
growth (Fiechter et al. 1981). Furthermore, the fission yeast is
considered to be unable to grow on either ethanol or acetate as
sole C source (Mitchison 1970), observations we have confirmed (data not shown).
The major route of D-glucose catabolism in yeasts is the
glycolytic pathway leading to the formation of pyruvate (Barnett
1976). Facultative yeasts such as Sch. pombe and S. cerevisiae
are capable of aerobically metabolizing pyruvate via alcohol
fermentation and respiration (Fiechter et al. 1981). Changes in
D-glucose, ethanol, and acetate concentrations of the batch
cultures of Sch. pombe are consistent with the known metabolic
sequences. Schizosaccharomyces pombe does not grow in
cultures with ethanol or acetate as the sole carbon source,
although the fission yeast utilizes both ethanol and acetate in the
presence of D-glucose. At high D-glucose concentrations, the
repression of respiration (De Deken 1966) of Saccharomyces
results in aerobic ethanol production. The depletion of D-glucose derepresses the respiratory metabolism allowing the oxidation of ethanol with a concurrent accumulation of acetate.
Ethanol equilibrates readily across yeast membranes (Guijmo
and Lagunas 1984; Dasari et al. 1985) and is oxidized to acetate
(Sols et al. 1971). Uptake of exogenous acetate occurs during
the period when D-glucose and ethanol are metabolized. This
concurs with the incorporation of
into cellular
compounds which are subsequently utilized in respiration. It is
noted that [14c]acetate accumulation decreases as soon as
glucose and ethanol are exhausted. Furthermore, the decline
persists while acetate is being actively metabolized. This
observation suggests a possible switch in the role played by the
acetate carbon.

acetate

The cells grew during the initial stages of these experiments,


while the glucose and ethanol were being consumed, and while
acetate was being accumulated or stored. Upon the exhaustion
of glucose and ethanol, acetate was used as the carbon source.
Only when acetate was added at high concentration at inoculation did we see growth while the acetate concentration declined.
Hence, the obvious deduction is that acetate was not ordinarily
utilized for growth. Nevertheless, we note that noninhibitory
concentrations of acetate, added at inoculation, enhanced the
final number of cells per millilitre. Thus, the presence of
acetate, at this initial concentration, did foster growth. This is
not the only enigma, for the evidence suggests that glucose must
be present before either ethanol or acetate supports growth, yet
the acetate concentration in the medium remained at high levels
until the glucose and ethanol had been consumed. Only after the
glucose and ethanol ostensibly disappeared did the cells
consume the acetate. Although one expects ethanol and acetate
to share metabolic pathways and fates, it is clear that "ethanol
utilization" and "acetate utilization" have different physiological meanings. There's nothing in the yeast literature to indicate
the nature of these differences.
The incorporation of [1-14c]acetate into proteins in conjunction with the increased cell density and cell concentration of the
fission yeast grown in a mixture of D-glucose and acetate
suggests an anabolic utilization of acetate by Sch. pombe. Two
anaplerotic systems, the pyruvate carboxylase bypass and the
glyoxalate cycle, have been demonstrated in S. cerevisiae
(Haarasilta and Oura 1975). For the anabolic utilization of
two-carbon compounds such as ethanol and acetate, the
glyoxalate cycle, which provides a bypass to the decarboxylating
steps of the TCA cycle, functions anaplerotically to replenish a
four-carbon intermediate (Kornberg and Elsden 1961). Isocitrate lyase and malate synthetase are the two key enzymes of
the glyoxalate cycle (Kornberg and Elsden 1961). However, in
Sch. pombe, we have not been able to detect these enzymic
activities in cell extracts which is in agreement with earlier work
(Fiechter et al. 1981). Work is in progress to elucidate the
anaplerotic pathway for ethanol and acetate anabolisms in Sch.
pombe.
In this paper we demonstrate that either ethanol or acetate can
be metabolized by Sch. pombe in the presence of D-glucose,
while neither substrate can serve as the sole carbon source for
growth. We conclude that glucose assimilation is required for
initiating either one of the ethanol or acetate anabolic systems. It
is clear that the concomitant metabolisms of D-glucose and
ethanol or acetate by fission yeast cells are interrelated in complex ways that merit further study.

Acknowledgements
We thank Teena Walker and Ken Mitton for assistance with
portions of the experimental work.
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