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Electro-stimulation of S. cerevisiae wine yeasts by pulsed electric field and its effects on fermentation capacity J. Mattara,b*, M. Turkc, M. Nonusa, N.I. Lebovkaa,d, H. El Zakhemb, E. Vorobieva

a Équipe TAI Laboratoire TIMR 4297, Université de Technologie de Compiègne, Centre de Recherche de Royallieu BP 20529 - 60205 Compiègne cedex, France

bDépartement de Génie Chimique, Université de Balamand, BP 33 Amioun, Liban cÉquipe TAI Laboratoire TIMR 4297, Ecole Supérieure de Chimie Organique et

Minérale, Centre de Recherche de Royallieu BP 20529 - 60205 Compiègne cedex, France dDeparment of Physical Chemistry of Disperse Minerals, Institute of Biocolloidal

Chemistry named after F.D. Ovcharenko, NAS of Ukraine, 42, blvr. Vernadskogo, Kyiv 03142, Ukraine, lebovka@gmail.com

Abstract

The batch fermentation process, inoculated by pulsed electric field (PEF) treated wine yeasts (S. cerevisiae Actiflore F33) , was studied. PEF treatment was applied to the aqueous yeast suspensions (0.12 % wt.) at the electric field strengths of E=100 and 6000 V/cm using the same pulse protocol (number of pulses of n=1000, pulse duration of ti=100 µs, and pulse repetition time of Δt=100 ms). Electro-stimulation was confirmed by the observed growth of electrical conductivity of suspensions. The fermentation was running at 30ºC for 150 hours in an incubator with synchronic agitation. The obtained results clearly evidence the positive impact of PEF treatment on the batch fermentation process. Electro-stimulation resulted in improvement of such process characteristics as mass losses, consumption of soluble matter content (°Brix) and synthesis of proteins. It also resulted in a noticeable acceleration of consumption of sugars at the initial stage of fermentation in the lag phase. At the end of the lag phase (t=40 hours), consumption of fructose in samples with electrically activated inoculum exceeded fructose consumption in samples with control inoculums by ≈2.33 times when it was activated at E=100 V/cm and by ≈3.98 times after treatment at E=6000 V/cm. At the end of the log phase (120 hours of fermentation), ≈30% mass reduction was reached in samples withPEF-treated inoculums (E=6000 V/cm), whereas the same mass reduction of the control sample required approximately, 20 hours of extra fermentation. The possible mechanisms of electro-stimulation are also discussed in details.

Key words: S. cerevisiae; Pulsed electric fields; Electro-stimulation; Biomass;

Fermentation 1. Introduction Yeasts can convert sugar to ethanol under anaerobic conditions. This process is referred

to as fermentation. Industrially important is stabilization of the yeast multiplication and enhancement of the process productivity and fermentation yield (Ribéreau-Gayon et al., 2006).

*Corresponding author at: Équipe TAI Laboratoire TIMR 4297, Université de Technologie de Compiègne, Centre de Recherche de

Royallieu, BP 20529 - 60205 Compiègne cedex, France. Tel.: +33648110611. E-mail address: jessy.mattar@utc.fr (Jessy Mattar).

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Recently, various emerging technologies were demonstrated to have a stimulating effect on the microbial flora multiplication process. E.g., it was shown that continuously supplied low-power ultrasonic in the range of 20-30 kHz could enhance ethanol production via stimulation of S. cerevisiae M30 performance (Klomklieng and Prateepasen, 2011). The optimum ultrasonic treatment at the frequency of 25 kHz resulted in 15.6% ethanol concentration that was higher than in the control system (≈ 12.0%). The positive influence of static magnetic fields (SMF) on the growth of S. cerevisiae biomass was also demonstrated (Muniz et al., 2007). The biomass (g/L) increment in eight samples, exposed to SMF treatment, after their 24 h fermentation at 23ºC, was 2.5 times greater as compared with control cultures. The differential biomass growth rate (135%) in SMF-treated samples was slightly higher than glucose consumption rate (130 %), which indicates an increase in biomass production of the magnetized cells.

Exposure of microorganisms to pulsed electric field (PEF) allows their electro-permeabilization or killing depending on parameters of the applied field (Sale and Hamilton, 1967). PEF treatment was demonstrated to be a promising food industry technique for both acceleration of extraction from cells and inactivation of microorganisms (Barbosa-Cánovas et al., 2001; Heinz et al., 2003; Vorobiev and Lebovka, 2006). Thus, dramatic increase in the permeability of cell membrane was observed when the trans-membrane potential exceeded a certain critical value (of the order of 0.5-1 V) (Weaver and Chizmadzhev, 1996). However, PEF application to microorganisms, such as bacteria and yeasts, may cause a lethal action at sufficiently high values of applied electric field strength or at long times of treatment. For example, the PEF treatment at optimal values of energy consumption (186 kJ/kg) and electric field strength (29 kV/cm) permitted reduction of the population of spoilage flora in must and wine up to 99.9% (Puertolas et al., 2009). In contrast to other methods, employed for microbial cell inactivation, PEF treatment selectively removes the membrane barriers in cells and does not cause any visible damage of cells (El Zakhem et al., 2006a, 2006b; Sale and Hamilton, 1967; Somolinoset al., 2008). It was shown that PEF treatment of S. cerevisiae improved accumulation of zinc and magnesium in the yeast biomass (Pankiewicz and Jamroz, 2010, 2011). PEF treatment allowed selective release of different components (ionic, proteins and nucleic acids) from the yeast cells (Liuet et al., 2013; Ohshima et al., 1995; Shynkaryk et al., 2009). E.g., PEF treatment of the aqueous suspension of wine yeasts (S. cerevisiae bayanus, strain DV10) at E=10 kV/cm allowed high extraction of ionic components and small extraction of high molecular weight components (Shynkaryk et al., 2009). Application of PEF-treatment at E=40 kV/cm to the aqueous suspension of the same wine yeasts allowed extraction of 70% of ionic substances, 1% of proteins and 16% of nucleic acids (Liu et al., 2013).

Application of PEF with restrictions of electric field strength and time of treatment within certain reasonable ranges allowed preservation of functionality of cell membranes (Fologea et al., 2004). PEF technique allowed facilitation of the efficient transformation of cells (Costaglioli et al., 1994; Shen et al., 2013; Teissié et al., 2002) and fusion of cell (electrofusion) (Hu et al., 2013). The stimulation of activity of the living cells under the impact of PEF treatment has also attracted a great attention. A stress response analysis of S. cerevisiae has shown that PEF-induced expression of the oxidation genes and glutathione played an important role in the stress resistance toward PEF (Taninoet al., 2012). The electrical stimulation was verified to alter the S. cerevisiae culture cycles and to promote synchrony in cell division (Araújo et al., 2004). Moreover, electrical stimulation resulted in smaller size of bacterial populations. Application of continuous direct current (DC) or alternating current (AC) treatments to a culture broth after inoculation by yeast suspension allowed significant increase in the cell growth and alcohol

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production rates (Nakanishi et al., 1998). The positive role of electrical current (alternating current or pulsed direct current) as a tool for stimulation of microbe reactions (i.e., fermentation) was also reported (Carlin, 1981). However, the effects of PEF treatment on microbial activity, metabolism and microbe reactions, practically, were not yet studied.

The main purpose of the present work was to investigate PEF-induced effects on S. cerevisiae growth in synthetic media. PEF treatment was applied to the aqueous suspensions of wine yeast cells S. cerevisiae in distilled water. The electric field strength was E=100 V cm-1 and 6000 V cm-1, the pulse duration was ti=100 µs, and the number of pulses was n=1000. The conditions of inoculation, incubation and harvesting of cell populations were carefully kept constant in order to obtain identical fermentation properties. The comparative studies of the kinetics of fermentation were carried out in order to characterize the PEF-induced stress responses of S. cerevisiae.

2. Material and Methods

2.1. Preparation of yeast suspensions

The wine yeasts, S. cerevisiae, strain Actiflore F33 (Laffort, Bordeaux, France) was used throughout this study. The industrial dry powder (rod-shaped particles (El Zakhem et al., 2006a)) was mixed with distilled water with electrical conductivity of 4 µS/cm at 20°C, the concentration of yeasts was 0.12 % wt. The yeast suspension was submitted to vortexing (during 2 minutes, rotation speed 150 rpm and amplitude 4.5 mm) using Top Mix (Bioblock Scientific, Germany). Note that more vigorous mixing may lead to a drop in viability of cells due to their breakage, so, it was avoided. The initial suspension conductivity after the vortexing was σi≈18±3 µS/cm. Then the suspension was gently agitated (100 rpm) at 30°C using magnetic agitator. The swelling process was monitored by means of conductivity measurements (Inolab Level 1, Germany) at the frequency of 50 Hz. In PEF treatment experiments, the suspension was initially agitated for 15 min, treated by PEF (total time of keeping suspension in PEF treatment chamber was 100 s) and agitated again. The total time of agitation of PEF-treated and untreated suspensions was the same (tin=30 min). After agitation, the (treated and untreated yeast suspensions were immediately inoculated into fermentation substrate.

2.2. Fermentation substrate

The fermentation process may reflect the composition of the reaction broth medium, which is not stable for a natural product containing a multitude of components (e.g., grape juice). In order to make comparison of the fermentation effects for untreated and PEF treated inoculums, the synthetic fermentation medium was used in this work.

The composition of the fermentation medium was the following: 5 g/L yeast extract (Sigma Aldrich Steinheim), 6.36 g/L of ammonium sulfate (as a source of mineral nitrogen), 6.36 g/L of monoammonium phosphate (as a source of phosphoric content), and 142 g/L of sugar (as source of carbohydrates), including 68 g/L of glucose, 73.6 g/L of fructose and 0.4 g/L of sucrose(Ribéreau-Gayon et al., 2006). For sterilization purposes, all reagents and apparatus were treated for 15 min at 121°C in autoclave (Lequeux, Paris, France).

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2.3. PEF-treatment

The PEF generator, Hazemeyer 5kV- 1kA (Hazemeyer, Saint Quentin, France), providing bipolar pulses of near-rectangular shape, was used in this work. The treatment chamber consisted of a propylene container, into which 2 stainless electrodes (≈143 cm2) separated by 7 mm gap, were inserted. The volume of the treatment chamber was 100 mL. The PEF experiments were carried out at two different electric field strengths of E=100 and 6000 V/cm using the same protocol of pulses: number of pulses n=1000, pulse duration ti=100 µs, and pulse repetition time Δt=100 ms. The time of PEF treatment was 0.1 s and total time of keeping suspension in PEF treatment chamber was 100 s. The current and voltage data were measured and then collected using a data logger and specific software, adapted by Service Electronique UTC, Compiègne, France. The temperatures before and after PEF treatment were controlled by a thermocouple. The initial temperature was 30°C and the temperature elevation resulting from PEF treatment was less than 5°C. The chamber was disinfected before and after each treatment with an 80% ethanol solution.

2.4. Batch fermentation

Batch fermentation experiments were initiated by transferring 10 mL of 0.12 % wt. yeast suspension to 300 mL of sterilized synthetic medium. The final concentration of yeasts in the fermentation substrate was ≈0.0039 % wt. The small-scale fermentations were carried out in six different vessels (4 were PEF treated and 2 were the control ones) for 150 hours under the controlled temperature (30°C) with synchronic agitation at 150 rpm (HT Inforsag Bottmingen).

2.5. Analytical methods

Fermentation performance was estimated throughout the fermentation period using the periodic (each six hours) measurements of mass of the fermentation substrate m and soluble matter content (°Brix). The value of °Brix was measured using a digital refractometer AR 200 (Leica Microsystems Inc., Buffalo, USA). The initial values of mass mi and °Brixi were 130±0.1 g and 13.0±0.1, respectively.

Samples were also harvested for offline chemical analysis. UV absorption spectra were measured in the spectral range of 190–900 nm by UV spectrophotometer (Spectronic 20 Genesys, Spectronic Instruments, Rochester, NY). The path length of the Suprasil quartz cuvette was 10 mm (Hellma, Mullheim,Germany). The concentration of sugars was determined enzymatically using glucose and fructose analysis kit (Enzytec fluid Glucose/Fructose, R-Biopharm). The concentration of proteins P(μg/ml) was determined using the Bradford procedure (Bradford, 1976). The protein content calibrations were done using measurements of absorbance at 595 nm with Bovine serum albumin (BSA) (Sigma A7030) as reference substance (Bradford, 1976).

2.7. Statistical analysis

All the experiments and respective analysis were done, at least, in triplicate. Means and standard deviations of data were calculated. One-way analysis of variance was used for statistical analysis of the data using the Statgraphics plus (version 5.1, Statpoint TechnologiesInc., Warrenton, VA, USA). For each analysis, significance level of 5 % was assumed. The error bars presented on the figures correspond to the standard deviations.

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3. Results and discussion

During the swelling the electrical conductivity σ of concentrated inoculums (0.12 % wt. aqueous suspensions of S. cerevisiae yeasts) grew with time and reached the stable value of 25±5 µS/cm, approximately, 60 minutes of agitation (Fig.1). To investigate the effect of PEF treatment on fermentation of the synthetic media, the same concentrated inoculums (0.12 % wt. ) were treated at electric field strength E=100 V/cm and E=6000 V/cm. These values of electric field strength E corresponded to the relatively small fields that can induce only at the early stages of yeast cells damages (El Zakhem et al., 2006b).

Fig. 1. Electrical conductivity ratio σ/σi versus time t during the swelling for PEF treated and untreated (control) suspensions. Here, σi≈15 µS/cm is the initial suspension conductivity. The concentration of yeasts was 0.12 % wt. and temperature was 20°C. Gray shading corresponds to the period of PEF treatment. Symbols: (■) control, (▲) PEF at E=100V/cm, (●) PEF at E=6000V/cm.

The commonly reported critical field strength, needed for high degree of disintegration of the S. cerevisiae cells, was rather high (>7.5 kV/cm) (Cserhalmi et al., 2002; Schrive et al., 2006; Zhang et al., 1994). However, it was expected that fields below this critical value can cause noticeable electrical stimulation of S. cerevisiae cultures.

During the PEF treatment, electrical conductivity jumped to some extent (see, gray shading in Fig. 1), and continued to increase after the PEF treatment (t>16.5 min). It evidently reflected the leakage of intracellular ionic components, caused by electroporation of S. cerevisiae cells during and after the PEF treatment (El Zakhem et al., 2006a, 2006b; Ganeva et al., 2003; Ohshima et al., 1995; Ohshima and Sato, 2004).

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Fig. 2. Time dependences of relative weight m/mi (a) and relative soluble matter content °Brix/°Brixi (b) during the process of fermentation (t=0-140 hours) in samples with untreated and PEF-treated inoculums. Symbols: (■) control, (▲) PEF at E=100V/cm, (●) PEF at E=6000V/cm. The time periods corresponding to the different growth phases (lag, log and declined) are shown.

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Noticeable changes of σ were observed after PEF treatment of the sample with smaller electric field strength (E=100 V/cm). However, they were rather small after treatment at E=6000 V/cm and electrical conductivity reached saturation at t> 30 min. The time evolutions of relative weight m/mi and relative soluble matter content °Brix/°Brixi during the process of fermentation (t=0-140 hours) are presented in Fig. 2a and Fig. 2b, respectively. During the lag phase, individual S. cerevisiae cells are maturing and not yet able to divide.

This process is accompanied with decrease of the relative weight m/mi (Fig. 2a), whereas °Brix/°Brixi passes through minor maximum (Fig. 2b). At this stage, the difference between PEF-treated and control inoculums was unessential; however it was increasing with time.

During log (logarithmic or exponential) phase, S. cerevisiae cells become doubling with intensive depletion of nutrients. During this phase, the difference between behavior of m/mi (Fig. 2a) and °Brix/°Brixi (Fig. 2b) in samples with PEF-treated and control inoculums became noticeable. E.g., the mass losses were essentially smaller in samples with inoculums treated at E=6000 V/cm that in samples treated at E=100 V/cm, or in untreated inoculums (Fig. 2a). From the other side, kinetics of °Brix values was approximately the same in samples with inoculums, treated by PEF at E=100 V/cm and at E=6000 V/cm and noticeably faster than in samples with control inoculums (Fig. 2b). So, the process of fermentation in the log phase was significantly affected by PEF treatment.

Fig. 3. Absorbance A versus wavelength λ for culture solutions inoculated by untreated and PEF-treated yeasts. The time of fermentation was t=40 hours. Insert shows the values of absorbance measured at λ =550 nm for control and PEF treated inoculums at different times of fermentation t. Symbols: (■) control, (▲) PEF at E=100V/cm, (●) PEF at E=6000V/cm.

At the beginning of declined (stationary) phase (t≈120 hours), depletion of essential nutrients and formation of the inhibitory products (e.g., organic acids, etc.) resulted in restriction of fermentation. At this time (120 hours of fermentation), ≈30% mass reduction was attained in

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samples with PEF-treated (E=6000 V/cm) inoculums, whereas the same reduction in control sample required approximately, 20 hours of extra fermentation. In general, it may be concluded that faster kinetics of fermentation in samples with PEF-activated inoculums was accompanied by high consumption of carbon sources in the culture medium.

Figure 3 presents examples of UV absorption spectra for culture solutions inoculated by untreated and PEF-treated yeasts at the beginning of log phase (t=40 hours). The spectra have the peaks at λ≈ 260 nm that are generally attributed to the absorption of nucleic acids (Rosenheck and Doty, 1961), presumably of RNA (Liu et al., 2013). The PEF treatment of inoculums resulted in a noticeable increase of spectral line intensities, and approximately the same spectra were observed after PEF treatment at E=100 V/cm and E=6000 V/cm. Absorbance value A at λ≈ 550 nm may be used for estimation of bacterial counts(Arbianti et al., 2012). Insert in Fig. 3 shows that values of absorbance were systematically higher for culture solutions, inoculated by PEF-treated S. cerevisiae yeasts. It evidenced in favor of more accelerated growth of biomass in samples with PEF-treated inoculums. The similar effects were observed in the previous study of the effect of static magnetic field on S. cerevisiae biomass growth (Muniz et al., 2007), where magnetized yeasts showed accelerated kinetics of fermentation and higher biomass growth rate.

Fig. 4. Time dependences of protein concentration P during the fermentation (t=0-140 hours) in samples with untreated and PEF-treated inoculums. Symbols: (■) control, (▲) PEF at E=100V/cm, (●) PEF at E=6000V/cm.

PEF treatment of inoculums also affected the process of protein synthesis. Figure 4 shows time dependences of the concentration of proteins P during the process of fermentation (t=0-140 hours) in samples with untreated and PEF-treated inoculums. During the initial log phase (t<40 hours), the value of P was nearly constant for untreated or PEF-treated by weak field inoculums at E=100 V/cm, however, it noticeably decreased for inoculums PEF-treated at E=100 V/cm. During the lag phase (t=40-100 hours), the concentration of produced proteins continuously

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increased and PEF treatment resulted in increase of P value. Consumption of sugars (glucose and fructose) during the fermentation (t=0-140 hours) in samples with untreated and PEF-treated inoculums is presented in Fig. 5. Said sugars are important nutrients, related to the growth of S. cerevisiae. The faster kinetics of consumption was observed for glucose than for fructose. It is typical for fermentation with participation of S. cerevisiae and can be explained by higher affinity of hexose transporters (which translocate each sugar into the cell) to glucose than fructose (Ribéreau-Gayon et al., 2006; Tronchoni et al., 2009). In our experiments, the fermentation processes were beginning at approximately equal concentrations of both sugars (Ci=68 g/L for glucose and Ci=73.6g/L for fructose), however, at the end of t=100 hours, practically all glucose was consumed and the yeasts continued to consume only fructose in the following fermentation (Fig. 5).

Fig. 5. Consumption of sugars (glucose and fructose) during the fermentation (t=0-140 hours) in samples with untreated and PEF-treated inoculums. The data presented as the relative concentration C/Ci, where Ci is the initial concentration. Symbols: (■) control, (▲) PEF at E=100V/cm, (●) PEF at E=6000V/cm.

The fermentation-stimulation effects of PEF treatment resulted in more accelerated sugar depletion, and the most evident differences were observed at the end of the lag phase (at t≈40 hours). E.g., by this moment, consumption of fructose in samples with electrically activated inoculums exceeded that in the samples with control inoculums by ≈2.33 times for E=100 V/cm and by ≈3.98 times for E=6000 V/cm. The most essential differences between fermentation activity of electrically activated and control inoculums were observed during the log (exponential) phase. At the end of this phase (120 hours of fermentation), ≈30% mass reduction was attained in samples with PEF-treated inoculums (E=6000 V/cm), whereas the same mass reduction in the control sample required 20 hours of extra fermentation.

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4. Concluding remarks

The obtained results clearly evidence the positive impact of PEF treatment of wine S. cerevisiae yeast inoculums on the batch fermentation process. PEF treatment was applied to yeast suspensions (0.12 % wt.) and electro-stimulation was confirmed by the observed grown of electrical conductivity of suspensions. It evidently reflected the leakage of intracellular ionic components, caused by electroporation of S. cerevisiae cells. The electrical stimulation of S. cerevisiae cultures resulted in a noticeable enhancement of the fermentation kinetics that was seen from time dependencies of mass losses, soluble matter content °Brix, consumption of sugars and synthesis of proteins. It is remarkable that noticeable acceleration of sugar consumption was observed at the initial stage of fermentation in the lag phase. E.g., at the end of this phase (t=40 hours), consumption of fructose in samples with electrically activated inoculums exceeded that in samples with for control inoculums by ≈2.33 times at E=100 V/cm and ≈3.98 times at E=6000 V/cm. At the end of the log phase (120 hours of fermentation), ≈30% mass reduction was attained in samples with PEF treated inoculums (E=6000 V/cm), whereas the same mass reduction in control sample required approximately, 20 hours of extra fermentation.

The obtained results may reflect impact of PEF stimulation on the efficiency of synthesis of RNA, enzymes, frequency of cell division events, the probability of daughter cells survival, tolerance to ethanol, and fermentation capacity. Few possible mechanisms can be proposed for explanation of the observed phenomena. Application of rather small (E=100 V/cm and E=6000 V/cm) electric fields can induce only the early stages of yeast cell electroporation (El Zakhem et al., 2006b) and it is expected that killing efficiency of such electric fields is not high. In principle, the killing efficiency of electric field treatment depends on the diversity of cell size and their resistance to electrical stress (Weaver and Chizmadzhev, 1996). Owing to these effects, some portion of yeast subpopulations may be selectively killed and removed from the further fermentation process. It is reasonable to expect that killing effects are much less pronounced at E=100 V/cm than at E=6000 V/cm.

From the other side, the increase of E may result in stimulation of subpopulations of the yeast cells. Previous studies have shown that PEF treatment can influence the rate of survival of yeast the cells, and maximum of the rate of yeast cell survival was observed at optimal electric field 850 V/cm (Fologea et al., 1998). The mechanism of PEF-induced stimulation of yeasts is still unknown in all details. Note that phenomenon of temporary electroporation with following resealing (Angersbach et al., 2000) can result in modification of both the cell wall and plasma membrane. It is known that membrane proteins are electrogenic (e.g., they are membrane ion pumps) and external electric fields can induce protein activity (Serrano, 1991). The electric field can improve transport of useful nutrients through the modified cell membranes due to pore formation or activation of transport proteins (Castro et al., 2012). We can conclude that although the potential of the practical application of electrical stimulation for increasing performance of the yeast cells is high, the general mechanisms and optimal procedures of electrically-enhanced fermentation still require further detailed studies in the future.

Acknowledgements

The authors appreciate the financial support from the French Ministry of Research and Higher Education. Authors also thank Dr. N. S. Pivovarova for her help with preparation of the manuscript.

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