Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci., Matica Srpska Novi Sad,
¥ 113, 271—284, 2007
UDC 663.14.039.3
S t e l i o s L o g o t h e t i s1
G r a e m e W a l k e r1
E l i a s T. N e r a n t z i s2 *
1
University of Abertay Dundee, School of Contemporary Sciences,
Bell Street DD1 HG1 Dundee, Scotland
2 TEI of Athens Department of Oenology and Spirit Technology,
Biotechnology & Industrial Fermentations Lab Agiou Spiridonos
12210 Egaleo, Athens, Greece E-mail: [email protected]
* Author to whom all correspondence should be addressed
EFFECT OF SALT HYPEROSMOTIC STRESS
ON YEAST CELL VIABILITY
ABSTRACT: During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low
temperature; and later, increased ethanol concentrations. Such conditions trigger a series of
biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene
expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations
under osmotic stress conditions. Specifically, the research work focused on the evaluation
of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13),
particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful
conditions. Experiments were designed by pretreating cells with different sodium chloride
concentrations (NaCl: 4%, 6% and 10% w/v) growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v)
using salt-adapted cells as inocula. We present evidence that osmostress induced by mild
salt pre-treatments resulted in beneficial influences on both cell viability and fermentation
performance of an industrial wine yeast strain.
KEY WORDS: Saccharomyces cerevisiae, wine yeast, salt stress, cell growth, cell
viability
INTRODUCTION
During industrial fermentations that exploit yeast, cells are confronted
with a multitude of chemical, physical and biological stresses that may impair
cell function and thus fermentation progress. Cells adapt to such stresses by
eliciting responses designed to maintain cell growth and survival. Yeast stress
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responses can be distinguished by different stages: cellular changes that occur
immediately as direct consequences of the physico-chemical forces; activation
of the primary defense processes and changes in cell homeostasis. Concerning
osmotic stress, a number of physiological changes take place, including: efflux
of intracellular H2O, rapid reduction in total cell volume, including the vacuole
(1); transient increases in glycolytic intermediates (2); accumulation of cytosolic glycerol; and triggering of the HOG (Hyper Osmotic Glycerol) signaling
pathway (3).
Microorganisms such as the yeast, Saccharomyces cerevisiae develop
systems to counteract the effect of osmotic stress such as salt stress (NaCl).
Specifically, salt-induced stress results in two different phenomena: ion toxicity and osmotic stress. Defense responses to salt stress are based on osmotic
adjustments by osmolyte synthesis and cation transport systems for sodium
exclusion. In osmostressed S. cerevisiae, polyols (glycerol in particular) are the
major osmolytes produced accumulated by cells (4, 5, 6). Other products
synthesized by yeast during stress conditions are trehalose and glycogen that
may collectively represent 25% of the dry cell mass depending on the environmental conditions. The disaccharide trehalose accumulates not only during salt
adaptation (7, 8), but also in response to a number of other stress conditions,
and has been shown to protect cells against high temperature by stabilizing
proteins and maintaining membrane integrity (9, 10, 11).
Exposing yeast cells in a hyper osmotic environment leads to a rapid initial efflux of water into the medium, which, in other words, is what is meant
as cell dehydration. Dehydration is a rapid process mediated solely by water
efflux through the lipid bilayer. Intracellular water is recruited from the vacuole into the cytoplasm thus partially compensating for the sudden increase in
macromolecular concentration. Additionally, the cytoskeleton collapses leading
to depolarization of actin patches. This cell dehydration leads to growth arrest.
Under these conditions, cellular reprogramming or “adaptation" represents major defenses including accumulation of compatible solutes to balance the intracellular osmotic pressure with the external environment. The compatible solutes can be: glycerol, trehalose, amino acids, and fatty acids in cell membranes.
A commonly used osmolyte in experiments to cause hyperosmotic stress
is sodium chloride. It has been established that the intracellular concentration
of glycerol increases in parallel to the external concentration of sodium chloride. In general, an increase in the intracellular concentration of glycerol can
be the result of increased glycerol production, increased retention by cytoplasmic membranes, or decreased dissimilation or uptake of glycerol from the medium. Glycerol is produced during glycolysis by reduction of dihydroxyacetone phosphate to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase
(GPD). (12, 13, 14). Under osmotic stress, increased levels of glycerol take
place due to the increase of the activity of GPD. Glycerol formation requires
an equimolar amount of cytoplasmatic NADH and when cells are osmotically
stressed, there is a decreased reduction of acetaldehyde to ethanol and an increased oxidation to acetate. The observed decrease in the synthesis of alcohol
dehydrogenase as well as the increase of the aldehyde dehydrogenase could
account for this alteration in flux. The present work was based on the hypo272
thesis that mild osmotic stress caused by NaCl would improve wine yeast fermentation performance due to accumulation of cellular protecting molecules.
The results obtained with a wine yeast indicate that salt adaptation or “preconditioning" may have potential benefits for industrial fermentation processes.
MATERIALS AND METHODS
Microorganism: Saccharomyces cerevisiae (strain Vin 13 from Anchor,
S. Africa) and was grown in glucose-based defined medium, without shaking
at 25°C.
Medium: 200 gr glucose/lit, 1 gr of K2HPO4, 1 gr of K2H2PO4, 0.2 gr of
ZnSO4, 0.2 gr of MgSO4, 2 gr of yeast extract, 2 gr of NH4SO4.
Yeast count: Yeast cell number was determined using haemocytometer
(Thoma type). Glucose measurement: Glucose was determined using the DNS
method (15)
Yeast cell viability: 1 ml of yeast taken from Erlenmeyer flask diluted in
9 ml of deionized water and then 1ml of this solution dissolved with 1ml of
10% methylene blue solution. After 10 min 0.01 ml added to a haemocytometer (Thoma type). Continuously yeast total cell number and yeast living cell
number took place by optical microscope (Olympus).
Fermentations: 250 ml volume conical Erlenmeyer flasks without shaking used in a temperature of 25°C.
Repeat fermentations: During the second part of the experiments repeat
fermentations have been performed. After 400 hours a new medium containing
200 gr lit of D-glucose inoculated by 5x103 living cells preconditioned in 6%
and 10% of NaCl. After the end of the fermentation new medium containing
300 gr lit of D-glucose inoculated by 5x103 living cells. Finally after the end
of the fermentation new medium containing 400 gr lit of D-glucose inoculated
by 5x103 living cells. This experiment was to observe the tolerance of the preconditioned cells to high sugar concentrations.
RESULTS AND DISCUSSION
The overall aim of this work was to gain further understanding of yeast
fermentation performance during osmotic stress conditions. Specifically, work
was focused on the evaluation of NaCl-induced osmotic stress responses on industrial strains of the yeast, Saccharomyces cerevisiae.
Figure 1 shows that osmotic stress had a minor effect on yeast cell
growth while sodium chloride concentrations increased. During the first 16
hours fermentation with glucose, no differences were observed in yeast growth
rate. After this period, differences between stressed and unstressed cells became apparent, especially around 90 hours. The lag phases of the cultures treated with 4% and 6% w/v NaCl were protracted by 24 hours. For the 4% and
6% NaCl concentration, the total cell number measurements occurred to have
a difference from the beginning of the fermentation compared to that of the
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untreated cells. As far as the 10% sodium chloride concentration was concerned, the growth rate had shown minimum changes.
Fig. 1 — Yeast growth under NaCl stress
Growth of S. cerevisiae (VIN 13) was monitored in the presence of 0%
4%, 6%, 10% w/v NaCl in glucose-based defined medium without shaking at
25°C
In contrast to the total cell number and yeast growth rate, it was surprising that cells under the higher osmotic stress conditions (especially 10%
NaCl) maintained a high viability over time compared with that of unstressed
cells (Figure 2). After prolonged fermentation times, such differences became
Fig. 2 — Viability under NaCl stress
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more pronounced. Under physiological conditions, without sodium chloride,
yeast cell viability fell to around 50%. In contrast, viability remained at high
levels for salt-stressed cells, even after 180 hours of fermentation. These data
clearly show that at 10%w/v NaCl concentration, whilst yeasts retained their
viability, their growth rate was adversely affected.
Viability of S. cerevisiae (VIN 13) was monitored in the presence of 0%
4%, 6%, 10% w/v NaCl culture in glucose-based defined medium without shaking at 25°C.
Fig. 3 — Yeast cell viability over an extended period of 192 hours after fermentation for cells
with 0% 4%, 6%, 10% w/v NaCl culture in defined medium without shaking at 25°C
Fig. 4 — Cell viability over an extended time period of 400 hours including fermentation for
cells with 0%, 4%, 6%, 10% w/v NaCl stress culture in defined medium without shaking at 25°C
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Figures 3 and 4 clearly show that sodium chloride had a positive effect in
maintaining yeast cell viability especially for extended fermentation periods of
time. After 400 hours, cells treated with 6% and 10% NaCl had the highest
viability. It is possible that maintenance of high yeast viability in presence of
high salt was due to the synthesis of stress responsive compounds including
trehalose and glycerol.
Continuous fermentations at increasing glucose concentrations were performed. Pre-conditioned stressed cells at 6% and 10% w/v/ NaCl used as inoculum for fermentations media containing 20, 30, and 40% of D-glucose in the
absence of NaCl stress. These experiments were designed to observe the
fermentability, cell growth and viability under non-stress conditions. After 400
hours, media containing 20% w/v D-glucose inoculated by 5x103 living cells
preconditioned in 6% and 10% w/v NaCl. Subsequently, media containing 30,
then 40% w/v D-glucose were inoculated in a similar manner. Yeast cell
growth and viability were monitored. These experiments aimed to observe the
capability of salt-preconditioned cells to grow and ferment high sugar concentrations.
Figures 5, 6, 7 and 8 show that for 40% of D-glucose, the fermentation
time was 116 hours longer and the total cell number was almost the half comparing to the other two fermentations of 20 and 30% w/v of D-glucose.
Growth rate in media with 20% and 30% of D-glucose was similar. The most
important observation was that for higher glucose concentrations (30% and
40%) the total cell number was close to 5x107/ml for the fermentation of 40%
D-glucose and close to 10 x107 for the fermentation of 30% D-glucose.
Fig. 5 — Growth for pre-conditioning cells in 6% and 10% of NaCl cultured
in define medium containing 20% of D-Glucose
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Fig. 6 — Yeast growth for pre-conditioning cells in 6% and 10% of NaCl cultured
in define medium containing 30% of D-Glucose
Fig. 7 — Yeast growth for pre-conditioning cells in 6% and 10% of NaCl cultured
in define medium containing 40% of D-Glucose
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Fig. 8 — Yeast growth for pre-conditioning cells in 6% and 10% of NaCl cultured
in define medium containing 20, 30 and 40% of D-Glucose
Figures 9, 10 and 11 show that for 20% D-glucose consumption was almost linear and the amount for residual sugars was the same (26 gr lit). For
the fermentation in media with 30% D-glucose the sugar consumption occurred to be significant when the inoculum cells pre-treated with 10% NaCl. At
very high glucose levels (40% w/v), a rapid decrease in sugar consumption
was observed between 48 and 96 hours from 395 gr lit to 64 gr lit of glucose
and then remains constant.
Fig. 9 — Glucose consumption in wine yeast cells pre-conditioned in 6% and
10% w/v NaCl in a medium containing 20% w/v D-glucose
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Fig. 10 — Glucose consumption for pre-conditioning cells in 6% and 10% of
NaCl cultured in define medium containing 30% of D-Glucose
Fig. 11 — Glucose consumption for pre-conditioning yeast cells in 6% and 10% of NaCl
cultured in define medium containing 40% of D-Glucose
Figures 12, 13, 14 and 15 show that yeast cell viability decreased when
the sugar levels increased. For example, for 20% D-glucose yeast viability remained high even towards the end of the fermentation. However, at 30% and
40% D-glucose yeast viability remained low.
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Fig. 12 — Viability for pre-conditioning cells in 6% and 10% of NaCl cultured
in define medium containing 20% of D-Glucose
Fig. 13 — Viability for pre-conditioned cells in 6% and 10% of NaCl cultured
in define medium containing 30% of D-Glucose
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Fig. 14 — Viability for pre-conditioned cells in 6% and 10% of NaCl cultured
in define medium containing 40% of D-Glucose
Fig. 15 — Viability for pre-conditioned cells in 6% and 10% of NaCl cultured in define
medium containing 20, 30 and 40% of D-Glucose
CONCLUSIONS
Since the early seventies sodium chloride and the effects of saline stress
to yeast cells have been the subject of research in many research labs. Several
research studies have been contacted until today regarding the effects of salt
on yeast genes expression and transcriptional response (16, 17, and 18).
Scope of this paper was to observe the effect of high osmotic stress to
yeast growth and yeast viability. The main points derived from these results
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are the stress of yeast cells under 6% concentration NaCl and the high percentage of yeast cell viability for higher concentrations of 10% w/v NaCl. Growth
rate arrest under osmotic stress conditions was clearly showed in our experiments according to previous research work (18). Most important, was that
during the experiments, when salt concentration increased, the yeast growth
rate decreased almost proportionally.
The cell viability remained at high levels for all experiments series even
for those under extremely stress conditions such as 10% w/v of NaCl. For the
same levels of NaCl concentrations (6 and 10% w/v) it was shown that cell
viability remained almost constant and at high levels for more than 400 hours
of culture.
Repeated fermentations under increasing concentrations of glucose conclude that pre-conditioned yeast cells in 6 and 10% of NaCl remain viable for
a long period of time and exhibit high fermentability.
According to these results future work is needed using higher amounts of
sodium chloride (more than 10% w/v) in order to elucidate the effects of
salt-induced stress on yeast cell viability during alcoholic fermentation and the
ability of the cells to remain viable under different kind of stresses.
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UTICAJ HIPEROSMOTSKOG STRESA IZAZVANOG NaCl-om
NA VIJABILNOST ÃELIJA KVASCA
1
Stelios Logothetis1, Graeme Walker1, Elias T. Nerantzis2
University of Abertay Dundee, School of Contemporary Sciences,
Bell Street DD1 HG1 Dundee, Scotland
2 TEI of Athens Department of Oenology and Spirit Technology,
Biotechnology & Industrial Fermentations Lab Agiou Spiridonos
12210 Egaleo, Athens, Greece, e-mail: [email protected]
Rezime
U proizvodwi etanola kvasci su tokom fermentacije izloÿeni razliåitim vrstama fiziåko-hemijskog stresa kao što su: visoke poåetne koncentracije šeãera i niske temperature, a kasnije poveãane koncentracije etanola. Ovakvi uslovi su pokretaåi serije bioloških odgovora koji imaju za ciq odrÿavawe ãelijskog cikliånog razvoja i vijabilnosti ãelija kvasca. Što se tiåe
osmotskog stresa, mnoge studije su usmerene na transkripcionu aktivaciju i
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ekspresiju gena u laboratorijskim sojevima Sacchavomyces cerevisiae. Ciq ovog
rada je bio da se boqe sagledaju i performanse vinskog kvasca tokom fermentacije u uslovima osmotskog stresa. Istraÿivaåka aktivnost je posebno usmerena
na ispitivawe odziva industrijskih sojeva vinskog kvasca S. cerevisiae (VIN 13)
u uslovima stresa indukovanog NaCl-om, sa posebnim osvrtom na rast ãelija
kvasca i wihovu vijabilnost. Hipoteza je bila da se u uslovima osmotskog stresa energetski aktiviraju specifiåni geni koji omoguãuju preÿivqavawe kvasnih ãelija u datim uslovima. Ogled je izveden prethodnim tretirawem ãelija
rastvorima NaCl razliåitih koncentracija (NcAl: 4%, 6% i 10% m/v), korišãewem definisanih podloga sa D-glukozom i ispitivawem wihovog uticaja na
rast i vijabilnost ãelija. U kasnije sprovedenim ciklusima fermentacije poveãavane su koncentracije D-glukoze (20%, 30%, 40% m/v), a kao inokulum su se
koristile ãelije kvasca prethodno adaptirane na povišene koncentracije soli.
U radu je pokazano da osmotski stres indukovan blagim tretmanima u rastvorima soli pozitivno deluje i na vijabilnost ãelija i na fermentacione performanse industrijskih sojeva vinskog kvasca.
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