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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 .
594
'
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)
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
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
D- [u-14C]glucose
~-[u-'~~]~lucose
+ acetate
[ I-I4C]acetate
33.3
52.2
34.4
TlME (days)
'
TlME (days)
'
+,
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.
596
TABLE
4. Effect of acetate concentrations on growth of Schizosaccharomyces pombe
Initial concn. acetate
(mg m ~ - ' )
Cell concn.
(cells mL- ')
Acetate consumed
(%I
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.
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
Acknowledgements
We thank Teena Walker and Ken Mitton for assistance with
portions of the experimental work.
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TSAI ET AL.
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