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Indian Journal of Science and Technology Vol. 4 No. 1 (Jan 2011) ISSN: 0974- 6846

Role of insoluble glycogen in ethanol adaptation mechanism of Saccharomyces italicus

M. S. Dake, M. B. Khetmalas and S. V. Amarapurkar

Dr. D. Y. Patil Biotechnology & Bioinformatics Institute, Survey No. 87/88, Mumbai–Pune Bypass Express Highway,
Tathawade, Pune-411033, Maharashtra, India
man1d_2@rediffmail.com

Abstract

Saccharomyces italicus has two pools of glycogen–one is the soluble pool and the remainder water insoluble linked
with cell wall or β-glucans. Soluble pool of glycogen present is assigned to produce energy required for metabolic
activities but the insoluble pool in the cell wall plays a special physiological role in providing the osmotolerance under
ethanol stress conditions. S. italicus grown in the presence of 2-8% v/v ethanol exhibits exponential increase in
insoluble glycogen content as a protective measure against the stress created across the plasma membrane by the
action of ethanol. But ethanol concentration above 8%v/v reduces hydrophobic interactions thereby decreasing the
insoluble glycogen content with increase in membrane permeability.

Keywords: Saccharomyces, glycogen, osmotolerance, ethanol, yeast cell wall.

Introduction cell wall β-glucan (Smith et al., 1977). Due to different

Yeast cell wall is a major structure, which plays an
important role in transport of nutrients and in maintaining
osmotolerance. Cell wall is also involved in flocculation,
conjugation and cell-cell interaction process.
Macromolecular constituents of yeast comprise proteins,
glycoproteins, polysaccharides, polyphosphates, lipids
and nucleic acids. The wall of a yeast cell contains some
15 to 25% of the dry mass of the cell. Major structural
constituents of the cell wall are polysaccharides (80-
90%), mainly glucans and mannans with a minor
percentage of chitin. Carbohydrate portion of yeast cell
wall consist of β-glucans, mannans and chitin. β-glucan,
the insoluble component present in yeast cell wall is
composed of branched β(1-3)-linked polymer along with
β(1-3) and β(1-6) interchain linkages forming a major
fraction whereas, β(1-6) linked polymer represents a
minor fraction (Manners et al., 1973). These major and
minor fractions are also termed as alkali insoluble and
acid soluble fractions respectively. β-1,3 glucan forms
amorphous components as well as a fibrous network of
the inner surface of yeast cell wall (Kopecka et al.,1974).
Mannans present as cell surface mannoproteins
consisting of short chains of α-(1-2) and α-(1-3) linked
mannose residues (Ballou, 1975). Yeast wall
mannoproteins are highly glycosylated polypeptides,
often 50 to 95% carbohydrate by weight (Vaart et al.,
1995; Orlean, 1997). Chitin, localized mainly in the bud
scar region is an unbranched polysaccharide containing
β-(1-4) linked N-acetylglucosamine units. The β-1,3
glucan-chitin complex is the major constituent of the inner
wall (Lipke & Ovalle, 1998). Apart from these cell wall
components, presence of two pools of glycogen has also
been reported in yeast as one intracellular cytoplasmic
soluble pool and the other cell wall bound glycogen
rendered water insoluble owing to its covalent linkage to
Research article
©Indian Society for Education and Environment (iSee)
physiological roles, the metabolic regulation of these two
pools of glycogen is independent. There is also presence
of glycogen in the periplasmic space of yeast cell (Smith
& Smith, 1974). Metabolism and storage level of glycogen
in yeast is affected by physiological, environmental and
nutritional stress conditions. Depletion of carbon, nitrogen
or sulfur sources induce glycogen accumulation in yeast
(Lillie & Pringle, 1980). A secondary metabolite produced
by yeast cell is the ethanol, a stress factor affecting cell
growth, viability (Thomas et al., 1978), intracellular
proteins and glycolytic enzymes (Nagodawithana et al.,
1977).
The fermenting microorganism, usually
Saccharomyces cerevisiae cannot tolerate more than
about 10-12% by volume of ethanol (Taylor et al., 1995).
An important correlation between insoluble glycogen
content and ethanol yield has already been established.
This implies the certain role of cell wall bound insoluble
glycogen and ethanol as indicators of stress created in
yeast by external factors. Here especially the effect of
externally added ethanol on the yeast glycogen content is
an important aspect for such study. Flocculation, a
physiological process exhibited by yeast showing
aggregation of cells is a cell surface phenomenon. The
content of acid soluble glycogen is correlated with the
process of flocculation in yeast (Patel & Ingledew, 1975).
So, study regarding the role of glycogen in flocculation
process will conclude their correlation with ethanol.
Presence of third pool of glycogen in the form of α-
glucans at cell surface level of yeast is confirmed by
previous studies (Arvindekar, 1995). Effect of ethanol on
cell weight, total cell count and glycogen content in
Saccharomyces sp. is studied previously. In accordance
to this the effect of variable concentrations of ethanol on
the corresponding level of cell surface α-glucans as well
“Ethanol tolerance in yeast” Dake et al.
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 53

Indian Journal of Science and Technology Vol. 4 No. 1 (Jan 2011) ISSN: 0974- 6846

as on cell wall bound insoluble glycogen is studied in study variations in glycogen content resulting from
detail. The study will reflect the associated correlation ethanol stress generated in S. cerevisiae.
between ethanol and glycogen content in S. italicus.
Since the prime target for the action of ethanol is the cell Media for set A: Increasing sugar concentration (Flasks:
wall and plasma membrane of yeast. The components of a, b, c, d & e) 2, 4, 6, 8 and 10% glucose; 0.5% peptone
yeast cell wall i.e. cell wall bound insoluble glycogen and and 0.3% yeast extract in a set of 5 flasks each.
cell surface α-glucans are significant factors which can
contribute towards the osmotolerance of cell against Media for set B: Increasing ethanol concentration with
higher ethanol concentrations. constant concentration of carbohydrate (Flasks: f, g, h, i, j
& k). 8% glucose; 0.5% peptone, 0.3% yeast extract and
Materials and methods 2, 4, 6, 8, 10, 12% (v/v) ethanol in a set of 6 flasks each.
Organisms & culture conditions
Bottom flocculating Saccharomyces italicus NCIM– Media for set C: Increasing sugar concentration and
3230 was obtained from National Chemical Laboratory, constant ethanol concentration (Flasks: l, m, n, o, p & q)
Pune and is routinely maintained on YEPD agar at 40C. 2, 4, 6, 8, 10 and 12% glucose, 0.5% peptone, 03% yeast
Peptone, yeast extract and malt extract were from Difco extract and 8 % (v/v) ethanol in a set of 6 flasks.
Laboratory, Detroit, Mich. Amyloglucosidase was All the flasks from Set A, B and C were inoculated
purchased from Sigma Chemical Company, USA and and fermentation was carried out at 25 ± 2°C temperature
Glucose oxidase kit was from Biolab. Viablity of cells was for 48 h. Then yeast cells were harvested from each flask,
accessed using methylene blue stain. centrifuged under cold conditions (+4°C) at 7000 rpm for
Glucose was measured using glucose oxidase 10 min, washed repeatedly with chilled distilled water.
peroxidase method (Lloyd & Whelan, 1969) and total Cells were subjected to alkali digestion process to
carbohydrate was estimated by phenol sulfuric acid determine the amount of soluble and insoluble glycogen
method (Dubois et al., 1956). Proteins were analyzed by (Arvindekar & Patil, 2002).
Folin Lowry method (Lowry et al., 1951).
Media with variable concentrations of glucose as a Isolation of glycogen
carbon source as well as ethanol were used to study their Glycogen was isolated from yeast cells by alkali
effect on insoluble pool of glycogen and mechanism of digestion method (Smith et al., 1977). The resultant alkali
flocculation. Different media compositions were used to digest obtained as reddish brown coloured liquid was
centrifuged at 10,000 rpm for 20 min at room
Table 1. Fermentation carried out using three sets of media. temperature (20°C-25°C) and separated into
Flask Glycogen mg/g cells Carbohydrate mg/g cells supernatant and residual fractions. The supernatant
Set
No. Soluble Insoluble Soluble Insoluble obtained contained soluble glycogen and other
a 4.15±0.01 3.13±0.03 16.44±0.04 11.02±0.02 components. The residual gelly like mass representing
b 5.25±0.03 5.21±0.01 22.13±0.03 16.23±0.02 insoluble glycogen was washed with distilled water
Set
A
c 5.32±0.02 6.01±0.01 25.41±0.01 21.41±0.01 repeatedly till the supernatant gives negative phenol
d 5.67±0.01 7.42±0.02 25.50 23.21±0.01 sulphuric acid test.
e 4.24±0.01 9.23±0.03 29.02±.02 27.02±0.02
f 2.02±0.02 8.03±0.03 26.22±0.02 25.31±0.01 Estimation of soluble & insoluble glycogen content
g 2.62±0.02 10.51±0.01 28.09±0.01 28.03±0.03 Degradation of glycogen was carried out specifically
Set h 3.22±0.02 13.12±0.02 29.03±0.03 31.21±0.01 by the action of amyloglucosidase (2 IU) and fungal α-
B i 3.61±0.01 15.02±0.02 30.40 33.40 amylase mixture (1 IU) (Smith et al., 1977). For routine
j 4.02±0.02 11.41±0.01 21.39±0.01 24.59±0.01 estimation 1 ml yeast digest as supernatant and
k 4.21±0.01 6.12±0.02 11.02±0.02 13.61±0.01 residual suspension was treated with the enzyme
l 0.61±0.01 2.60 19.02±0.02 18.03±0.03 mixture prepared in sodium acetate buffer pH 4.2 at
m 1.79±0.01 4.51±0.01 23.01±0.01 25.22±0.02 37°C for 1 h. The reaction was terminated by keeping
Set n 2.99±0.01 10.02±0.02 25.01±0.03 27.05 the tubes in boiling water bath for 5 min. The reaction
C o 3.41±0.01 15.12±0.02 28.16±0.04 31.03±0.03 mixture was centrifuged and librated glucose was
p 4.10 10.37±0.03 19.02±0.02 22.41±0.01 estimated by glucose oxidase peroxidase method. The
q 4.30 4.34±0.04 12.52±0.02 10.02±0.02 amount of glucose liberated in supernatant and residual
Set A: Variation in glycogen and carbohydrate with increase in fractions represents soluble and insoluble glycogen
sugar concentration (2-10% w/v); Set B: Variation in glycogen and content respectively.
carbohydrate with increase in ethanol content (2–10% v/v). Sugar
concentration kept constant (8% w/v); Set C: Variation in glycogen Results and discussion
and carbohydrate with increase in sugar concentration (2–12% In yeast the presence of two pools of glycogen is
w/v). Ethanol concentration kept constant (10% v/v) (The values established on the basis of results obtained from
are expressed as mg/g wet weight of yeast cells. The data
chemical and enzymatic analysis. Both pools of
represents mean ± S D of three sets of observations).
Research article “Ethanol tolerance in yeast” Dake et al.
©Indian Society for Education and Environment (iSee) http://www.indjst.org Indian J.Sci.Technol.

Indian Journal of Science and Technology
glycogen play an important role in yeast cell metabolism.
Since ethanol production by yeast cells was observed to
increase, there was a corresponding increase in insoluble
glycogen content. So, study regarding the correlation
between ethanol and insoluble glycogen in
Saccharomyces italicus was carried out in this work. The
results obtained display the effect of ethanol on the
insoluble pool of glycogen in S. italicus. The values of
soluble and insoluble glycogen as well as carbohydrate
are depicted in Table 1. From the experimental
observations it revealed that the content of soluble
glycogen did not exhibit any significant increase either
with increase in sugar concentrations (2-12% (w/v)) or
ethanol concentrations from 2-12% (v/v). The
corresponding values of soluble glycogen observed for
yeast cells varied from 0.6 mg to 5.6 mg/g wet weight of
yeast cells for all 3 experimental sets i.e. set A, set B and
set C. The insoluble glycogen content exhibit exponential
increase varying from 3 mg to 15 mg/g wet weight of
yeast cells with increase in ethanol concentration up to
8% (v/v) for both set B and C respectively. Total soluble
carbohydrate content of yeast cells consists of soluble
glycogen, α-glucan and mannan while total insoluble
carbohydrate comprised of insoluble glycogen, β-glucan
and chitin. So, carbohydrate content was estimated by
phenol sulphuric acid method. The amount of soluble and
insoluble carbohydrate showed parallel increase from 25
mg to 33 mg/g of weight of yeast cells grown with ethanol.
But cells grown with ethanol concentrations higher than
8% (v/v) displayed sharp decline in the values of
insoluble glycogen as well as carbohydrate from 15 mg to
Fig.1. Variation in wet weight of yeast cells in 3
different media compositions.
3
2.5
2
Cell weight in gms
1.5
1 Set C
0.5
0
2 4
Suga r concentra tion (% w/v) & Etha nol concentration (% v/v)
Research article
©Indian Society for Education and Environment (iSee)
54
Vol. 4 No. 1 (Jan 2011) ISSN: 0974- 6846
4 mg and 33 mg to 10 mg respectively. Thus cells grown
in media supplied with 2-8% (v/v) ethanol along with
sugar resulted in comparatively higher values of both
insoluble glycogen and carbohydrate than the cells grown
only with sugar. Fig. 1 indicates decrease in yeast cells
biomass grown in media containing ethanol higher than
8% (v/v). Thus higher ethanol concentrations above 8%
(v/v) adversely affects yeast cell metabolism resulting in
net depletion of cell biomass. The action of ethanol is
primarily on a membrane in agreement with the
conclusions of Leao and Uden (1984) for sugar transport
and membrane potential in yeast. Sudden increase in
insoluble glycogen content in yeast cells grown with
higher ethanol concentrations (up to 8% (v/v)) indicates
the major role of insoluble glycogen in ethanol tolerance
mechanism of yeast. Here ethanol acts by changing the
membrane permeability causing membrane leakage.
Interactions of ethanol with plasma membrane reduce
hydrophobic interactions thereby increasing its
permeability for free exchange of ethanol and other polar
solutes (Alexandre et al., 1993.)
Conclusion
When cells enter stationary growth phase, glycogen
accumulates in yeast batch cultures during transition from
exponential to stationary phase (Rothmans-Denes &
Cabib, 1970). Glycogen content in yeast cells undergoes
variations in response to changes in cultural conditions
like pH, temperature as well as percentage of carbon and
nitrogen source. Ethanol added to cultural medium of
yeast causes changes in cell membrane structure
Set A
Set B
6 8 10
“Ethanol tolerance in yeast” Dake et al.
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Indian Journal of Science and Technology
increasing its permeability and eventually leading to the
inactivation of cell growth and metabolism. The resulting
ethanol stress ultimately causes elevation in cell wall
insoluble glycogen content. Thus increased level of cell
wall glycogen in the presence of ethanol indicates the
role of glycogen in ethanol adaptation as well as ethanol
tolerance mechanism. Growth of yeast cells in the
presence of 2-8% (v/v) ethanol results in increasing the
insoluble glycogen as well as total carbohydrate content
as a protective measure towards osmostress. But higher
concentrations of ethanol above 8% v/v causes
dissolution of cell wall glycogen along with leakage of
membrane components showing net depletion of
glycogen & inhibition of cell growth.
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