Biotechnology Letters 23: 677–681, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Stress effect of ethanol on fermentation kinetics by stationary-phase cells of Saccharomyces cerevisiae Virginie Ansanay-Galeote∗ , Bruno Blondin, Sylvie Dequin & Jean-Marie Sablayrolles INRA, Institut des produits de la vigne, Laboratoire de Microbiologie et Technologie des Fermentations, Montpellier, France ∗ Author for correspondence (Fax: +33 4 99 61 28 57; E-mail:
[email protected]) Received 29 November 2000; Revisions requested 18 December 2000; Revisions received 23 February 2001; Accepted 26 February 2001
Key words: alcoholic fermentation, ethanol stress, ethanol tolerance, stationary phase, wine yeast
Abstract When 4% (v/v) ethanol was added progressively to two strains exhibiting different fermentative abilities, K1 (a commercial wine strain) and V5 (a strain derived of a wine yeast), the fermentation rate correlated directly to the ethanol concentration for both strains. In contrast, the effect of sudden addition of 2%, 4% or 6% (v/v) ethanol was different depending on the strain. While the same effect was observed for K1 whatever the way of ethanol addition, V5 required an adaptation period after the shock addition of ethanol.
Introduction The inhibitory effect of ethanol to Saccharomyces cerevisiae produced during fermentation is complex and is the main reason for slow and incomplete enological fermentations. Therefore, a better knowledge of the ethanol effect on yeasts would be of interest to improve wine-making fermentations. Ethanol retards the growth rate of yeasts (Jones & Greenfield 1985, Kalmokoff & Ingledew 1985, Mota et al. 1984), their viability (Brown et al. 1981, Kalmokoff & Ingledew 1985), and their fermentative ability (Casey et al. 1984, Holzberg et al. 1967). Ethanol also modifies plasma membrane fluidity (Alexandre et al. 1994, D’Amore et al. 1990, Thomas et al. 1978), stimulates the activity of plasma membrane H+-ATPase (Alexandre et al. 1993, Monteiro & Sa-Correia 1998) and inhibits glucose transport (Leão & Van Uden 1982, Salmon et al. 1993). Ethanol also triggers a stress response with Saccharomyces cerevisiae (Piper et al. 1994). Indeed, ethanol between 4% and 6% (v/v) induces formation of heat shock proteins in yeast cultures. However, less information is available for enological conditions, which are characterized by high initial sugar concentrations (150–260 g l−1 ), and thus high final ethanol con-
centrations (11–15% v/v), low pH (2.9–3.6) and low nitrogen availability. The main feature of this type of fermentation is that most of the ethanol is produced by stationary-phase cells. During the stationary phase, the fermentation rate decreases until complete sugar exhaustion. The impact of ethanol on the fermentation rate decrease has never been precisely addressed. The purpose of this paper is to assess the effect of ethanol on yeast stationary-phase cells during wine-making conditions. To this end, a specific experimental set-up was designed and two strains were studied, a commercial wine strain (K1) and a strain derived from a wine yeast (V5). The effect of progressive additions of ethanol, which mimic ethanol formation during fermentation, was compared with the effects of ethanol-induced shock, which were used in most of the previous ethanol stress studies. In order to detect minor differences in the kinetics, the fermentation rate was precisely measured by using on-line monitoring of the CO2 production.
678 Materials and methods
Addition of ethanol
Yeast strains and culture conditions
Ethanol was added at the beginning of the stationary phase, when 2% (v/v) ethanol was produced, in two different ways, (i) additions of 2%, 4% or 6% (v/v) of ethanol and (ii) perfusion of 50 ml ethanol solution (corresponding to a 4% (v/v) ethanol addition), over 22 h. The control fermentation was perfused in the same way with water.
Saccharomyces cerevisiae strains used in this study were V5 (ScV5M, MATa, ura3) derived from a Champagne wine strain and the industrial strain K1 ICVINRA. Batch fermentation experiments were carried out in synthetic medium simulating the composition of a grape must, as described by Bely et al. (1990), except the concentration of assimilable nitrogen was 70 mg l−1 and sugar concentrations were 150 to 250 g glucose l−1 . Fermentations were inoculated with 106 cells ml−1 in fermenters (1.1 l working volume) equipped with fermentation locks. They were carried out at 24 ◦ C with continuous stirring (500 rpm). The cells had been previously pre-cultured on synthetic medium at 28 ◦ C in 50 ml flasks for 24 h (industrial strain) and 36 h (V5 strain). Monitoring and control of fermentations The amount of CO2 release was determined by automatic measurements of fermenter weight loss every 20 min (Sablayrolles et al. 1987). The CO2 production rate was calculated by polynomial smoothing of the last ten evolved CO2 values. The frequent acquisitions of CO2 release and the precision of the weighing (0.1 g or 1 mg) allowed calculation of the CO2 production rate with good precision and reproducibility: variation coefficient of (dCO2 /dt)max = 0.8% (Bely et al. 1990). Analytical methods Biomass Yeast cells were counted using an electronic particle counter (ZM, Coultronics). Viability measurements Yeast viability was determined by an epifluorescence method using the magnesium salt of 1-anilino-8naphthalene sulfonic acid (King et al. 1981). Ethanol production The ethanol concentration, E (% v/v), was estimated from the release of CO2 (g l−1 ) with the following relation: E = (0.94 · CO2 + 2.7)/7.89
(El Haloui et al. 1988).
Results Effect of ethanol on K1 strain Shock additions of 2% (v/v), 4% (v/v) and 6% (v/v) ethanol in the early stationary phase had an immediate effect on the fermentation rate which was instantaneously reduced by about 10%, 20%, and 40%, respectively (Figure 1A). However the fermentation profile is not significantly altered after the ethanol shock. (Note that the total fermentation duration cannot be compared since different initial sugar concentrations were used to obtain the same final ethanol concentration.) The final viability was at least 85% in all cases indicating that the decrease in the fermentation rate in our experiments was not due to cell mortality (data not shown). When the progressive addition of ethanol was carried out, the fermentation rate decreased progressively during the addition (Figure 1B). After the addition, the CO2 production rate was the same as when adding 4% (v/v) ethanol in the shock experiment. The rate of fermentation was plotted against the ethanol concentration in the medium for both types of experiments. As shown in Figure 2, all the curves are superimposable pointing out a direct correlation between fermentation rate and ethanol concentration. This result indicates that, in these conditions, the ethanol concentration is the key parameter that significantly influences the fermentation rate. Therefore a given increase in ethanol concentration has the same effect on the fermentation rate over a wide range (2 to 14% v/v) of ethanol concentration (Figure 2). Effect of ethanol on V5 strain As observed with the strain K1, the addition of ethanol led to a decrease of fermentative activity (Figure 3), without any consequences on the final viability of the cells which were at least 80% (data not shown).
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Fig. 2. Rate of fermentation versus the % ethanol of the medium (v/v) for the Saccharomyces cerevisiae K1 strain on MS70 medium containing different concentrations of glucose: N, 250 g glucose l−1 without ethanol added; O, 216 g glucose l−1 and 2% (v/v) ethanol added; ◦, 182 g glucose l−1 and 4% (v/v) ethanol added continuously; •, 182 g glucose l−1 and 4% (v/v) ethanol added; , 149 g glucose l−1 and 6% (v/v) ethanol added.
Fig. 1. Evolution of the CO2 production rate of the K1 strain on MS70 medium with different concentrations of glucose and different ethanol additions: (A) N, 250 g glucose l−1 without ethanol added; O, 216 g glucose l−1 and 2% (v/v) ethanol added; •, 182 g glucose l−1 and 4% (v/v) ethanol added; , 149 g glucose l−1 and 6% (v/v) ethanol added. (B) N, 250 g glucose l−1 without ethanol added; ◦, 182 g glucose l−1 and 4% (v/v) ethanol added continuously, •, 182 g glucose l−1 and 4% (v/v) ethanol added.
The fermentation rate was decreased by about 30%, 40% and 55% as a result of addition of 4% in progressive addition, 4% in shock addition or 6% (shock addition) respectively. Similarly to K1, when ethanol is added progressively, a direct correlation was observed between the fermentation rate and the amount of ethanol added (Figure 4). In contrast, the decrease in fermentation rate triggered by shock addition is more pronounced than the decrease associated to its natural formation or progressive addition. How-
Fig. 3. Evolution of the CO2 production rate of the V5 strain on MS70 medium with different concentrations of glucose and different ethanol additions: N, 250 g glucose l−1 without ethanol added; ◦, 182 g glucose l−1 and 4% (v/v) ethanol added continuously; •, 182 g glucose l−1 and 4% (v/v) ethanol added; , 149 g glucose l−1 and 6% (v/v) ethanol added.
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Fig. 4. Rate of fermentation versus the % ethanol of the medium (v/v) for the Saccharomyces cerevisiae V5 strain on MS70 medium containing different concentrations of glucose: N, 250 g glucose l−1 without ethanol added; ◦, 182 g glucose l−1 with 4% (v/v) ethanol added continuously; •, 182 g glucose l−1 with 4% (v/v) ethanol added; , 149 g glucose l−1 with 6% ethanol added.
ever, at the end of fermentation, the fermentation rates were similar whatever method had been used to increase the ethanol concentration. This indicates that after an ethanol shock, the cells become less sensitive to a further increase in ethanol concentration.
& Salmon 1992, Salmon 1989, Salmon et al. 1993). In addition, it has been demonstrated that ethanol inhibits glucose transport (Leão & Van Uden 1982, Salmon et al. 1993). The observation that ethanol affects the fermentation rate in a linear way (Figure 2; Figure 4, progressive addition) suggests that the target which limits the fermentation rate is a single component. Thus, we favour the hypothesis that the decrease of fermentation rate observed in this work point to an inhibition of the hexose transporters by ethanol. After shock addition of ethanol more complex mechanisms may be involved and the two strains analysed exhibited different responses to such shocks. Indeed, V5 strain was more sensitive to an abrupt increase of ethanol than strain K1 and the fermentation rate was not directly correlated to the amount of ethanol added when shock addition was performed. However, the ethanol shock seems to render the yeast cells less sensitive to a further increase in ethanol concentration. Theses observations point to a possible mechanism of cellular adaptation to ethanol stress. Moreover, the experimental model presented here could be an excellent system to assess the gene response specific to ethanol stress under enological conditions.
References Discussion Alcoholic fermentation in winemaking conditions occurs mainly with yeasts in stationary phase. During this phase, the fermentation rate decreases progressively as the fermentation progresses. The factors which govern this decrease in fermentation rate are not well defined. In this study, we observed that ethanol plays a major role in the control of the fermentation rate. When simulating a natural ethanol production by a progressive addition, a direct correlation was observed between the rate of fermentation and the concentration of ethanol in the medium, with the two strains used in this study. The major target of ethanol is the plasma membrane. Ethanol stress alters the membrane organization and permeability (Alexandre et al. 1994, D’Amore et al. 1990, Ingram & Buttke 1984, Thomas et al. 1978) perturbing transport systems of the cell: inhibition of uptake of hexose, ammonium and amino acids. Under enological conditions, characterized by stationary-phase cells, sugar transport was shown to be the limiting factor of fermentation rate (Mauricio
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