Effects of ethanol and acetate on glucose-limited chemostat cultures of

Schizosaccharomyces pombe, a fission yeast1

I A NJ . MCDONALD,TEENA
WALKER,A N D BYRONF. JOHNSON
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada KIA OR6
ANTONIOJ . AVELEDOA N D C . STANT S A I ~
Department of Chemistry, Carleton Universio, Ottawa, Ont., Canada KlS 5B6

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Received July 2 1, 1986

Accepted March 5, 1987
MCDONALD,
I. J., WALKER,
T., JOHNSON,
B. F., AVELEDO,
A. J., and TSAI,C. S. 1987. Effects of ethanol and acetate on
glucose-limited chemostat cultures of Schizosaccharomyces pombe, a fission yeast. Can. J. Microbiol. 33: 598-601.
Glucose-limited chemostat cultures of Schizosaccharomyces pombe were studied to assess metabolic changes induced by the
presence of ethanol and acetate. Both ethanol and acetate were utilized by the chemostat cultures. Ethanol increased the
maintenance rate of glucose assimilation. Acetate reduced the maintenance rate of glucose consumption and cell counts without
significantly affecting the glucose requirements for growth and cell yield. Ethanol accumulation in the chemostat cultures was
facilitated by high dilution rates and noninhibitory concentrations of acetate.
MCDONALD,
I. J., WALKER,
T . , JOHNSON,
B. F., AVELEDO,
A. J., et TSAI,C. S. 1987. Effects of ethanol and acetate on
glucose-limited chemostat cultures of Schizosaccharomyces pombe, a fission yeast. Can. J. Microbiol. 33 : 598-601.
Des cultures en chkmostat de Schizosaccharomyces pombe ont etk limitees en glucose en vue d'inventorier les changements
mktaboliques induits par la presence d'kthanol et d'acktate. L'kthanol ainsi que l'acktate ont kt6 utilisks par les cultures. L'kthanol
a fait croitre le taux du maintien de l'assimilation du glucose. L'acktate a reduit le taux du maintien de la consommation du
glucose et les comptes cellulaires, mais sans affecter de faqon significative les exigences en glucose pour la croissance et le
rendement des cellules. L'accumulation d'kthanol dans les cultures a kt6 facilitk par des taux de dilution klevks et par des
concentrations en acktate qui n'ktaient pas inhibitrices.
[Traduit par la revue]

Introduction
Although the fission yeast ~chizosaccharomyces
pornbe is
unable to metabolize ethanol o r acetate a s the sole carbon source
for growth, diauxic growth occurred in the presence of
D-glucose with added ethanol o r acetate (Tsai et a/. 1987).
Because ethanol and acetate are products of D-glucose metabolism, and because ethanol is a useful fermentation product whose
production might be limited in the presence of acetate,
knowledge of their interactive utilization and production by
Sch. pombe would b e desirable.
Glucose-limited chemostat cultures of Sch. pombe were
studied to
the metabolism when the carbon source was

glucose
Or when
Or acetate' The

was added together with

conditions of continuous

(Tempest 1970; Tempest and Wouters 1981) are particularly

suited for such studies because growth rates and concentrations
of limiting nutrients for the growth of microorganisms can be
easily controlled. Earlier studies on continuous cultures of Sch.
pombe were concerned only with its growth o n D-glucose
(Kothari et al. 1972; McDonald et al. 1982; Vrana, 1983). This
report describes the utilization of D-glucose in relation to
utilization and production of its metabolic products, ethanol and
acetate.

Materials and methods

Yeast strain
A strain of Schizosaccharomyces pombe, NCYC 132 Sz-2
(McDonald et al. 1982), was used throughout. Cultures were maintained by periodic transfers in 2% (w/v) malt extract broth (Oxoid)
incubated at 30C.
Chemostat cultures
Chemostat cultures of Sch. pombe were grown in modified EMM2

'NRCC no. 27674.

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

medium (Mitchison 1970) containing D-glucose (2.0 mg mL- ') and

or ethanol (0-10 mg mL-').
either sodium acetate (0-1 a75 mg
Unless otherwise noted, the temperature of growth was 30C. Cultures
were maintained at pH 5.0 by means of an automatic pH controller
(Radiometer pH 28 and Titrator 11, Radiometer, Copenhagen) through
the addition of sterile 1 N HCl or 2 N KOH. Culture medium was
supplied to the fermentor by means of a peristaltic pump (model 1203,
Harvard Apparatus, Millis, MA) running at constant rates. Chemostat
cultures were sampled periodically after-a steady state (approximately
7td) was reached. 'The dilution rate (D) was calculated from the flow
rate (F ) and the culture volume ( V ) by D (h- I) = F (mL h-') / V (mL)
and related to the mass doubling time (td) by td (h) = ln 2 / (h-I).
~
Because the steady-state condition of chemostat cultures tends to
oscillate at low dilution rates (Furukawa et al. 1983), the steady-state
concentrations of fission yeast cells and metabolites were followed for a
pried longer than lotd after the steady-state
was established.
Analyses
To follow steady-state growth, the turbidity of chemostat cultures
was measured at 660 nm in a Beckman model DB spectrophotometer.
Cell numbers were counted microscopically in an Improved Neubauer
ultraplane chamber (A. H. Thomas Co.). Yeast cells from 10 mL of the
cultures were collected by centrifugation at 12 000 x g for 3 min and
washed three times with saline. They were transferred onto a
preweighed aluminum weighing dish (National Scientific Products
Corporation Ltd., Mississauga, Ont.), dried at 100C for 15- 18 h, and
weighed. The percent of conjugants and the division index (DI (5%))
were measured as previously described (McDonald et al. 1982;
Johnson et al. 1982). To screen for mutants capable of utilizing ethanol
or acetate as sole carbon source, washed cells were plated on modified
EMM2 agar containing either ethanol or acetate as the sole carbon
source.
Glucose, ethanol, and acetate concentrations of the cell-free culture
medium were determined as described (Tsai et al. 1987).

Results
Changes in cell counts, steady-state concentrations of glucose, ethanol, and acetate in a D-glucose-limited chemostat

599

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McDONALD ET AL.

DILUTION RATE ( h - ' 1

DILUTION RATE ( h - ' 1

FIG. 1. D-Glucose limited chemostat culture of Schizosaccharomycespombe. A continuous culture with 2.0 mg D-glucosemL-' as the
carbon source was maintained at 30C, pH 5 .O. Steady-state concentrations of cells (@), D-glucose ( 0 ) , ethanol (O), and acetate ( A ) were
analyzed.

FIG.3. Chemostat culture of Schizosaccharomyces pombe with

2.0 mg D-glucose mL- and 1.0 mg acetate mL- as the carbon source
at 30C, pH 5.0. Steady-state concentrations of cells (@), D-glucose
(O), ethanol (O), and acetate ( A ) were analyzed.

'

DILUTION RATE ( h - ' I

FIG. 2. Chemostat culture of Schizosaccharomyces pombe with

2.0 mg D-glucose mL-' and 0.30 mg ethanol mL-' as the carbon
source at 30C, pH 5.0. Steady-state concentrations of cells (@),
D-glucose ( 0 ) , ethanol (O), and acetate ( A ) were analyzed.

culture of Schizosaccharomyces pombe were followed with

respect to changes in dilution rate (Fig. 1). At very low dilution
rates, D-glucose was nearly completely utilized. As dilution
rates were increased, glucose consumption and cell density
decreased, while ethanol production increased. Acetate was
formed only as dilution rates approached washout (the point at
which addition of medium is faster than the growth rate of the
cells).
In the presence of ethanol, both D-glucose and ethanol were
metabolized by the growing yeast at steady state (Fig. 2). The
ethanol concentration of the chemostat culture was lower than
the feed concentration at low dilution rates, although it
accumulated at high dilution rates. Acetate was not detected at
any dilution rates between 0.05 to 0.25 h- I. Schizosaccharomyces pombe was capable of concurrently utilizing acetate and
D-glucose (Fig. 3). While the steady-state acetate concentration
stayed below the feed concentration, the cell density remained
relatively constant, glucose utilization was marginally reduced,
and ethanol production was facilitated. In the chemostat
cultures of Sch. pombe when the growth is glucose limited, both
D-glucose and acetate or D-glucose and ethanol in mixtures of
two carbon sources are simultaneously utilized.
In the glucose-limited chemostat cultures, a relationship
between dilution rates ( D ) and rates of substrate utilization (V,)
can be often described by V, = m + Y .D where m and Y are the

'

DILUTION RATE ( h - ' 1

FIG.4. Rates of glucose consumption as the function of dilution

rates. Chemostat cultures of Schizosaccharomyces pombe were maintained at 30C, pH 5.0, with 2.0 mg D-glucose mL-' alone ( 0 ) or with
2.0 mg D-glucose mL- in the presence of 0.30 mg ethanol mL- ( 0 )
or 1.0 mg acetate mL- (A).

'
'

'

maintenance coefficient and the substrate requirement for the

growth or yield coefficient, respectively (Pirt 1965; Abbott and
Clamen 1973). Thus, plots of V, (glucose consumption) versus
D ( D 1 0.075 h- I) are linear (Fig. 4). The linear relationships
were also observed for rates of increasing cell concentration
versus D ( D 2 0.140 h-I). Values of m and Y obtained by linear
regression analysis (Table 1) show that acetate (1.0 mg mL-I)
reduced the maintenance rates of glucose assimilation and cell
concentration without significantly affecting the glucose requirement for growth and cell yield. This high tolerance of Sch. pombe
for acetate may arise from its ability for deacidification (Ethiraj
and Suresh 1978). Ethanol (0.30 mg m ~ - ' )raised the maintenance rates of glucose assimilation and cell concentration but
sharply reduced the yield coefficient of cell concentration.
Yeasts are known to produce and oxidize ethanol concurrently (Maleszka and Schneider 1982). However, rates of
ethanol production became appreciable in the chemostat cultures of Sch. pombe at dilution rates of 0.10-0.20 h-' (Fig. 3).
Ethanol at 0.30 mg mL- inhibited the cellular effectiveness of
ethanol production, whereas acetate at 1.O mg mL-' was
stimulatory, presumably by sparing the consumption of ethanol
(Fig. 5).
The division index (DI; the percentage of cells in a recognizable stage of division) indicates the proportion of the cell cycle

600

CAN. J. MICROBIOL. VOL. 33, 1987

TABLE1. Effects of ethanol and acetate on rates of glucose assimilation and increasing cell concentration in chemostat cultures of Schizosaccharomyces pombe

TABLE
3. Temperature effect on growth behavior of Schizosaccharomyces pombe in chemostat cultures

- -

Glucose

Ethanol
and glucose

Acetate
and glucose
T("C)

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Glucose assimilation
m (mM h-')
Y (mM)
n
r

NOTE:The feed concentrations of glucose, ethanol, and acetate were 2 . 0 , 0.30, and
1.0mg d-I
respectively.
,
rn, maintenance rate; Y, glucose requirement for growth or
yield coefficient of cell density; n, number of observations analyzed; r , correlation
coefficient.

DILUTION RATE ( h - ' 1

FIG.5. Rates of ethanol production as a function of dilution rates.

Experimental details and legends as indicated in Fig. 4.
TABLE
2. Effects of dilution rates and acetate on division index and
conjugants in chemostat cultures of Schizosaccharomyces pombe
Glucose
DI

(%I

Cell mass
(mgm~-')

DI

Conjugant

(%)

(%I

NOTE:The chemostat cultures ( 2 . 0 mg D-glucose d '

and 1 .O mg acetate dl)
were
maintained at pH 5 . 0 , D = 0.055 2 0.001 h-I.

Cell concentration
m (pg mL1' h- ')
Y(pg mL '1
n
r

D (h-')

Concn. of
glucose
consumed
( m mL-')
~

Conjugant

(%I

Glucose and acetate

DI

Conjugant

(%)

(%)

NOTE:See Table 1 . DI (division index), the percentage of cells in a recognizable state

of division; conjugant, a recognizable sexual cell.

devoted to an essential function: the higher the division index,

the more slowly division proceeds (Johnson et al. 1982). The
division index was not markedly affected by varying the dilution
rate (Table 2), but the frequency of conjugants rose at low D, as
shown earlier by McDonald et al. (1 982). The division index
was lower in the presence of acetate (Table 2), suggesting that
acetate enhances the cellular effectiveness of division processes
as well as ethanol production. At lower temperatures, the cell
mass, the rate of passage through cell division, and the frequency
of conjugants were all reduced (Table 3).

Discussion
Studies on chemostat cultures of Sch. pombe confirm the
observations made from batch culture (Tsai et al. 1987) that the
fission yeast is capable of metabolizing ethanol or acetate in the
presence of D-glucose. The exogenous ethanol or acetate is
metabolized concomitantly with D-glucose at low dilution rates.
Glucose at high concentrations represses yeast respiration by
suppressing activities of several enzymes from the TCA cycle,
glyoxalate bypass, and electron-transfer chain (Fiechter et al.
1981). Because the dilution rate of feed medium regulates the
growth rate which in turn controls the glycolytic rate of
D-glucose consumption, a change in dilution rates shifts the
state of D-glucose repression. The shift occurs at a dilution rate
which is characterized by changes in biomass, D-glucose, and
ethanol concentrations of the chemostat cultures. The sensitivity of Sch. pombe to D-glucose repression is manifested by a
lack of ethanol accumulation in the derepressed state at low
dilution rates and the aerobic ethanol production in the repressed
state at dilution rates higher than the critical value.
The conversion of D-glucose into ethanol under the aerobic
glucose repressed state is as effective as that of the anaerobic
fermentation (Fiechter et al. 1981) . Furthermore, continuous
cultures allow operators to choose an appropriate dilution rate
for the optimal ethanol production (Leuenberger 1972; Tempest
and Wouters 1981). Ethanol production by yeasts is of
biotechnological interest (Ghose and Tyagi 1979; Jin et al.
1981; Miitze and Wandrey 1983), thus the aerobic fermentation
by continuous cultures of D-glucose sensitive yeasts shows
considerable application potential. Acetate, below inhibitory
concentrations, facilitates ethanol production. Acetate is a
frequent contaminant of commercially available carbon sources
for ethanol fermentation. Consequently, the enhancement of
ethanol production of the fission yeast by noninhibitory concentrations of acetate may have some interesting ramifications for
alcohol industries.
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