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Yeast cell inactivation related to local heating induced by low-intensity electric fields with long-duration pulses
International Journal of Food Microbiology 113 (2007) 180 – 188 www.elsevier.com/locate/ijfoodmicro
Yeast cell inactivation related to local heating induced by low-intensity electric fields with long-duration pulses Stéphane Guyot, Eric Ferret, Jean-Baptiste Boehm, Patrick Gervais ⁎ GPAB Laboratory, ENSBANA, 1 Esplanade Erasme, F-21000 DIJON, France Received 11 August 2005; received in revised form 8 February 2006; accepted 13 June 2006
Abstract The effects of electric field (EF) treatments on Saccharomyces cerevisiae viability were investigated using a PG200 electroporator (Hoefer Scientific Instrument, San Fransisco, CA, USA) with specific attention to induced thermal effects on cell death. Lethal electric fields (1.5 kV cm− 1 for 5 s) were shown to cause heat variations in the cell suspension medium (water + glycerol), while corresponding classical thermal treatments at equivalent temperatures had no effect on the cells viability. Variations of the electrical conductivity of the intra- and extracellular matrix caused by ions and solutes transfer across the membrane were shown to be involved in the observed heating. The results permitted to build a theoretical model for the temperature variations induced by electric fields. Using this model and the electrical conductivity of the different media, a plausible explanation of the cell death induced by low-intensity electric fields with long-duration pulses has been proposed. Indeed, cell mortality could in part be caused by direct and indirect effects of electric fields. Direct effects are related to well known electromechanical phenomena, whereas indirect effects are related to secondary thermal stress caused by plasma membrane thermoporation. This thermoporation was attributed to electrical conductivity variations and the corresponding intracellular heating. © 2006 Elsevier B.V. All rights reserved. Keywords: Electric field; Cell inactivation; Temperature; S. cerevisiae; Conductivity; Modeling
1. Introduction Pulsed electric fields (PEF) applications are considered as a nonthermal decontamination process that involves the maintenance of biological samples between electrodes, and their subjection to high-intensity, short-duration electric fields. Some studies reported that electrical treatments could affect cell physiology directly by intracellular disorganization (Aronsson et al., 2001), by DNA and RNA decomposition (Takayuki et al., 1999) or by inactivation of enzymes (Van Loey et al., 2002). Alternatively, they could act indirectly by lethal molecule generation from the suspension medium (Reyns et al., 2004). Thus, the mechanisms by which microorganisms subjected to such physical treatments are inactivated remain unclear. The
⁎ Corresponding author. Tel.: +33 3 80 39 66 54; fax: +33 3 80 39 68 98. E-mail address: [email protected] (P. Gervais). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.06.036
most commonly accepted theory is that electric fields cause the polarization of cells, which generates electromechanical compression of the plasma membrane and subsequent permeabilization (Zimmermann, 1986; Bryant and Wolfe, 1987; Tsong, 1989; Qin et al., 1996). The membrane can be considered as a capacitor and exposure to an external electric field leads to a build-up of the membrane potential difference, V, caused by a charge separation across the membrane. V is proportional to the field strength, E, and to the radius of the cell. An increase in the membrane potential leads to a reduction in membrane thickness. Breakdown occurs if the critical breakdown voltage, Vc (about 1 V), is reached with a further increase in the external field strength. It is assumed that breakdown causes the formation of transmembrane pores, which leads to the depolarization of the cell and subsequent decompression of the membrane. Breakdown is reversible if the pores are few and small with regard to the total membrane surface. Alternatively, permeabilization may lead to irreversible breakdown associated with the mechanical
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destruction of the cell (Zimmermann, 1986). All these events depend on various factors such as the cell size, the conductivity of the medium, and the field characteristics (Weaver, 1993; Jayaram et al., 1992; Zhang et al., 1994). However, although the application of the laws of electrical forces to cells is accepted by all authors, some physical laws applying to the cells have been neglected. Thus, the previous theoretical approach does not recognize the increase in temperature arising from the electrical discharge across the cell suspension. Because PEF treatments do not cause significant increases in the mean temperature of the liquid medium (Qin et al., 1996; Harrison et al., 1997), the mechanisms of cell death are often explained with no reference to the thermal aspects. Nevertheless, some have reported an important contribution to the destruction of the cells by the thermal effects naturally associated with electric fields. Indeed, Shillcock and Seifert (1998), Winterhalter (1997), and Qin et al. (1996) proposed that the induction of pore formation was a consequence of the thermal fluctuations of the lipid elements. PEF treatments of 20–50 kV cm− 1 increase the temperature of the treated medium by about 8–12 °C (Qin et al., 1996; Harrison et al., 1997; Sale and Hamilton, 1967), and Vega-Mercado et al. (1997) demonstrated the thermal aspects of the PEF process, and its requirement for cooling. Furthermore, Pliquett and Gusbeth (2000) explained the breakdown of the plasma membrane by the subsequent heating of the PEF, which reached the temperature of the lipid phase transition. These results suggest the relative importance of the temperature variations that occur when an electric field is applied to a solution. Other examples show that the effects of electrical and heat treatments on cells do not differ greatly. Gervais and Martinez de Marañon (1995) demonstrated that a sudden rise in temperature, from 25 °C to 50 °C, was followed by a significant expulsion of the intracellular medium, which could be the basis of cell destruction. Therefore, it is possible to compare classical heat treatments and PEF treatments in terms of membrane behavior. Thus, it appears that PEF treatment of a cell suspension medium, could induce heat variation which could be a key factor in cell death. To understand better the mechanisms involved in this type of mortality, this paper deals with the heat dissipation caused by exposure of yeast cell suspensions to direct current (DC). Low-intensity (1.5 kV cm− 1) and longduration pulses (0.5–5.0 s) were applied in a static treatment chamber. Specific experiments were undertaken to distinguish between heating variations caused by the cells presence and the suspension medium, and to estimate the heat dissipation in both. First, heat variations induced by electric fields were compared in different types of high conductivity solutions with and without yeast cells. Taking into account the variation of the electrical conductivity of the solutions caused by increasing temperature, a model was proposed to estimate the heat variation with regard to electric field duration. Second, we compared the effect of electric fields to heat treatments on yeast viability. These results lead us to propose a hypothesis that relates electric field-induced cell death to electrical conductivity and heat variations.
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2. Materials and methods 2.1. Microorganisms and culture conditions Yeast cells (Saccharomyces cerevisiae strain CBS 1171) were grown in 100 mL of Malt Wickerham (MW) medium for 60 h at 25 °C on a continuously agitated (250 rpm), temperaturecontrolled platform shaker (New Brunswick Scientific, Edison, USA). This medium is composed of 10 g glucose (Sigma, Saint Quentin Fallavier, France), 3 g pancreatic peptone (Sigma), 3 g yeast extract (Sigma), and 1.5 g Na2HPO4 (Prolabo, Paris, France) in 1 L distilled water. Cells were harvested at the stationary phase. Because conductivity is an important parameter in electrical treatments, and to establish conductivity close to the water activity (aw) of the culture medium (0.992), before stressing, all samples were washed twice in a binary water–glycerol (Sigma) medium corresponding to an aw of 0.992. 2.2. Electric field The electroporation chamber has been previously described by Ferret et al. (1999). Two parallel platinum electrodes were inserted into the static treatment chamber, and connected to the generator (Progenetor 200; Hoefer Scientific Instruments, San Fransisco, CA, USA). The space between the two electrodes had a volume of 1.2 mL. Direct electric fields of 1.5 kV cm− 1, ranging in duration (di) from 0.5 to 5 s, were applied to the samples. To simplify calculations, electric field intensity is approximated by Eq. (1), where E is the electric field intensity (V cm− 1), U0 is the applied voltage (V), and d is the distance between the two electrodes (0.3 cm). E ¼ U0 =d
ð1Þ
For all experiments, electric fields were applied to samples at room temperature (measured between 23 and 25 °C). Because such treatment could induce a temperature increase, samples were cooled to room temperature at the rate of 1.25 °C min− 1 before viability measurements by maintaining samples at room temperature. Temperature decrease was controlled using a Kthermocouple (TCSA, Dardilly, France). 2.3. Heat treatments Different heat treatments were applied for comparison with electric field treatments. Experiments were performed at either 50 up to 70 °C, in a heating bath. 2 mL of washed cells, at an initial concentration of 1.5 × 108 cells mL− 1 (measured using a Malassez cell, VWR International, Limonest, France), were injected into glass flasks containing 18 mL of binary medium that had been equilibrated to the chosen temperature. So, washed cells were diluted 10 folds prewarmed binary medium and final cell concentration was equal to 1.5 × 107 cells mL− 1. The heat shocks were maintained for 10 s, to approximate the corresponding electrical treatment times. As in the case of electric field
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treatments, samples were cooled to room temperature at the rate of 1.25 °C min− 1 before measuring cell viability by maintaining samples at room temperature. Temperature decrease was controlled using a K-thermocouple (TCSA). 2.4. Conductivity and temperature measurements Electrical conductivity was measured with a conductimeter (LF 323-B, Wissenchaft-Technische Werkstätten, Weilheim, Germany). Furthermore, measurements of the temperature during the heat treatment or after the electrical stress were made using a K-thermocouple (TCSA). The different temperature variations induced by the application of electrical stresses were evaluated 10 s after the end of the treatment, which corresponds to the time required to remove electrodes and to introduce the thermocouple into the chamber. The measures were repeated three to six times and the 95% confidence intervals were calculated. 2.5. Cell enumerations Cell enumerations, after both heat and electrical treatments followed by a cooling period, were made using the plating method, by plating cells in Petri dishes containing MW medium supplemented with 15 g L− 1 Pastagar A (Biokar Diagnostics, Beauvais, France). Each sample was decimally diluted in binary water–glycerol solution and, 0.3 mL of appropriate dilutions was spread on MW-Pastagar A. Petri dishes were then incubated for two days at 25 °C. Viability was determined by counting Colonies-Forming Unit (CFU) and related with the viability of controls (unstressed samples) using previous dilution coefficients. Each treatment was repeated on at least three independent cultures. 3. Results and discussion Because this method of study focused on the dynamic aspects of electric field effects, the chosen field characteristics must produce a low-intensity and long-duration pulse. Most of the fields previously used in food-processing pilot plants have been of much higher intensity, between 10 and 50 kV cm− 1 (Qin et al., 1996; Vega-Mercado et al., 1997). Preliminary viability experiments on S. cerevisiae suspensions allowed the definition of an electrical treatment duration range involving a cell death ratio around 50%; we chose a low-intensity electric field of 1.5 kV cm− 1 over a long period ranging from 0.5 to 5 s. 3.1. Electric field-induced temperature variations 3.1.1. Temperature measurements of cell suspensions Fig. 1 shows the temperature variations caused by the application of the electric field for different times to different cell concentrations. In all cases, a significant temperature increase was observed for pulses of longer duration. Such thermal variations are well described in the literature in the case of pulsed electric fields (Fiala et al., 2001; Fleischman et al., 2004; Sepulveda et al., 2005).
Fig. 1. Measurements of temperature variations at different cell concentrations subjected to a 1.5 kV cm− 1 electric field for different pulse durations (0.05–5 s). Bars represent 95% confidence intervals. Highly concentrated cell suspension: (•) 1.5 × 108 cell mL− 1; slightly concentrated cell suspensions: (♦) 1.5 × 107 cell mL− 1; (n) 1.5 × 103 cell mL− 1; (○) water–glycerol (aw = 0.992) without cells.
Nevertheless, when electrical treatments were performed on highly concentrated cell suspensions (about 1.5 × 108 cells mL− 1), the temperature variation increase was higher than in slightly concentrated cell suspensions (1.5 × 103 to 1.5 × 107 cells mL− 1). Concerning highly concentrated cell suspensions, temperature variations presented a sharp increase up to 2 s (about 25 °C s− 1) and then remained stable. In the case of the slightly concentrated cell suspensions, all the temperature curves presented the same profile and the same variations as the control sample containing the water–glycerol medium without cells. The temperature variations increase linearly at the rate of about 5 °C s− 1. To understand better the difference in the temperature variation between highly concentrated and slightly concentrated cell suspensions, we focused our interest on the involvement of electrical conductivity (σ) in such thermal variations. Indeed, as shown by Fiala et al. (2001) and by Hayashi (2004) the electrical conductivity of a solution, which is a key factor in electric field treatments, increases with temperature increase. Measurements of σ as a function of cell concentrations (Fig. 2), showed that the electrical conductivity of slightly concentrated cell suspensions (2.31 × 103 to 1.50 × 107 yeasts mL− 1) was the same as a water–glycerol solution without yeast: about 8.5 μS cm− 1 . Electrical conductivity of more concentrated yeast suspensions (2.31 × 107 to 2.31 × 108 yeasts mL− 1) significantly increased with cell concentration. Such an observation shows that the heat variations induced by low-intensity electric fields with long-duration pulses could be related to yeast concentration and to electrical conductivity. The more highly concentrated suspensions were characterized by higher temperature variations and higher electrical conductivity values than those of slightly concentrated suspensions (Table 1). To quantify this, we performed experiments on the electrical conductivity of nutritive medium (MW lacking cells). 3.1.2. Temperature measurements in nutritive medium (MW) This conductive medium was used to approximate the intracellular medium, which presents an electrical conductivity of 2.3–5 mS cm− 1 (Harris and Kell, 1983; Hölzel, 1992) 1000 folds higher than the suspension medium (in the case of a
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Fig. 2. Measurements of electrical conductivity as a function of yeast cell concentration at 25 °C. Cell suspensions were prepared in a binary water–glycerol medium (aw = 0.992) as described in the Materials and methods section. Bars represent 95% confidence intervals.
water–glycerol solution) because of charged components present in the cell cytoplasm (i.e., proteins, nuclear acids, ions, etc.). Indeed, MW is composed in part of yeast extract (3 g L− 1), as explained in the Materials and methods section, and presents similar water activity than yeast cytoplasm (about 0.992). Moreover, this nutritive medium presents an electrical conductivity, about 1.5 mS cm− 1, which is relatively close to the yeast intracellular medium one. If we consider a cell suspension undergoing an electric field treatment and if both cell concentration and poration state allow an electric field pathway, it is reasonable to infer that the cell cytoplasm will be subjected to a significant increase in temperature. Tο study the involvement of the electrical conductivity in electric fields-induced temperature of the intracellular medium, different electrical conductivities of MW solutions were measured realized by adjusting the MW concentration using serial decimal dilutions of water–glycerol mixes. Measurements of the temperature according to electric field duration, and of the electrical conductivity with regard to temperature for different concentrations of MW, are presented in Figs. 3 and 4 respectively. As in the presence of yeast cells (Fig. 1), Fig. 3 shows that the temperature increased with pulse duration and with the increase in MW concentration. For the 10-fold dilution,
the temperature variations increased linearly up to about 40 °C for a 1 s pulse (at the rate of 40 °C s− 1) and then reached a plateau. For the 100-fold dilution, the temperature increased linearly up to 36 °C for a 4 s pulse duration (about 9 °C s− 1) and then remained stable. However, for the 1000-fold dilution and the water–glycerol medium, the temperature variation increased
Table 1 Main characteristics of the different media subjected to electric fields Medium
Concentration (dilution Linear slope σ25 (μS cm− 1) in water–glycerol) (°C s− 1)
Highly concentrated 1.5 × 108 cells mL− 1 cell suspension Slightly concentrated cell 1.5 × 107 cells mL− 1 suspensions 1.5 × 103 cells mL− 1 MW without cells 10− 1 10− 2 10− 3 Water–glycerol without cells
25
17.2
5 5 40 9 5 5
9.3 8.0 151.7 22.2 9.0 7.9
Linear slopes of the temperature variations according to pulse duration are shown in Figs. 1 and 3 and electrical conductivities at 25 °C (σ25) are shown in Figs. 2 and 4.
Fig. 3. Comparisons of experimental and modeled temperature variations according to time for different decimal dilutions of MW in water–glycerol. Experimental data (bars represent 95% confidence intervals) are compared with the theoretical curves deduced from Eq. (2). A: (♦) 10-fold dilution; (○) water– glycerol (aw = 0.992), without cells. B: (n) 100-fold diluted sample; (▴) 1000fold diluted sample.
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Fig. 4. Measurements of electrical conductivity according to temperature for different decimal dilutions of MW in water–glycerol. (♦) 10-fold dilution; (n) 100-fold diluted sample; (▴) 1000-fold diluted sample; (○) water–glycerol (aw = 0.992), without cells. (Dotted lines): linear regression.
linearly (at the rate of about 5 °C s− 1) according to pulse duration and electrical conductivity at 25 °C. To understand better the influence of MW or yeast cell concentration on the temperature increase, we measured changes in the electrical conductivity of different decimal dilutions of MW in water–glycerol mixes according to temperature changes. Results are presented in Fig. 4 and show that, whatever the MW concentration, the electrical conductivity increased linearly with the temperature. For 10-fold dilutions, the electrical conductivity increased from about 150 μS cm− 1 at 21 °C to about 350 μS cm− 1 at 77 °C. However, for higher dilutions, electrical conductivity variations according to the temperature were less important, and for the 100-fold diluted solutions they varied from about 21 μS cm− 1 at 21 °C to about 55 μS cm− 1 at 77 °C. The 1000-fold diluted solutions showed a slight increase of the electrical conductivity from about 7.9 μS cm− 1 at 21 °C to about 19 μS cm− 1 at 77 °C. These results are in accordance with the model proposed by Sorensen and Glass (1987). Moreover, from the results presented in Figs. 3 and 4, it is evident that the electrical responses of MW solutions depended on the dilution rate and the electrical conductivity at 25 °C (σ25) (see also Table 1). These data underline the role of electrical conductivity in the rate of temperature increase according to pulse duration. Nevertheless, in some cases (Figs. 1 and 3) a plateau appeared, indicating that the maximum temperature variation had been reached. We assume that the temperature reached 100 °C and then during the 10 s needed to remove electrodes and introduce the thermocouple in the chamber cooled to about 70 °C in the case of MW solutions (ΔT ≈ 45 °C) or to greater than ≈ 75 °C in the case of cell suspensions. To verify this hypothesis and to understand better the causes of the thermal effects induced by electric field application, we established a mathematical model that includes electrical conductivity as discussed in the next section. 3.2. Model of heat dissipation To estimate the electric field-induced temperature, our model outlines the temperature evolution of the MW medium
subjected to an electric field (Fig. 3). The model does not deal with cells exposed to electric fields, because there are no data on the kinetics of ion release from yeast cells caused by electroporation. The theoretical expression of the temperature variation of a suspension subjected to an electric field is well described in the literature (Jayaram et al., 1992; Castro et al., 1993; Lebovka et al., 2000). However the novelty of the proposed model is to highlight the temperature decrease between the end of the electrical treatment and the instance of temperature measurement. The temperature variations induced by electric fields and consecutive cooling are given by Eq. (2), below. In this, Ti and To are the temperatures (°C) of the inner and outer chamber respectively; t is the time (s), E is the electric field strength (1.5 kV cm− 1); σ is the electrical conductivity (μS cm− 1), which depends on both T and the concentration of MW; h is an estimated parameter defined as the convective coefficient of cooling between the inner and outer chambers; A is the exchange chamber surface (4 cm2); ρ is the solution density (1 g cm− 3); Cp is the specific heat of the solution (4.18 J °C− 1 g− 1) and V is the sample volume (1 cm3). It should be noted that this equation comprises two parts: first, the heating effects induced by the electric fields, which depend mainly on E and σ, and second, the cooling effects induced by thermal exchanges between inside and outside of the chamber. dTi E2 :rðTi ðtÞÞ h:A:ðTi ðtÞ To Þ ¼ q:Cp q:Cp :V dt |fflfflfflfflfflfflffl{zfflfflfflfflfflfflffl} |fflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflffl} Heating
ð2Þ
Cooling
As explained in the Materials and methods section, (i) E = 1.5 kV cm− 1 when 0 b t ≤ di, and E = 0 kV cm− 1 when t N di (duration) and the experimental temperature variations presented in Fig. 3 were measured 10 s after the electrical stresses (the time required to remove electrodes and to introduce the thermocouple in the chamber). Thus, during the electric treatment (i.e., until t ≤ di) the MW solution was submitted to heating effects induced by electric fields and to cooling effects caused by temperature exchanges between inside and outside of the chamber. During the time required to remove the electrodes and to introduce the thermocouple (10 s and E = 0 kV cm− 1), heating ceased and only cooling occurred. The model shows that, ignoring the last cooling period at the end of the electric treatment, in the cases of 10-fold and 100-fold MW dilutions, the temperature reaches a maximum (ebullition point) and then remains stable (data not shown). During this cooling period (10 s), the temperature curves present the same profile as previously described (a slope followed by a plateau), but the maximum ΔT reached is about 40 °C. As shown in Fig. 3, this model is in accordance with the experimental data. Moreover, it permits us to understand better the difference in temperature variations between yeast cell suspensions (Fig. 1) and MW solutions (Fig. 3). Indeed, the electrical conductivity of yeast cytoplasm is higher than that of MW solutions. Therefore, temperature increases are more important in the presence of yeast cells than in their absence. This can be extrapolated to the behavior of the intracellular medium. According to the literature
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(Harris and Kell, 1983; Hölzel, 1992) and our own estimates, intracellular conductivity is between 2.3 and 5.5 mS cm− 1, or about 1000 times higher than that for a water–glycerol mix (about 12 μS cm− 1). The temperature increase will thus be higher, as has been corroborated by others authors. Pliquett and Gusbeth (2000) observed local transport regions (LTR) within a skin sample subjected to an electric field. The temperature of the center of these areas was estimated to be below 70 °C, whereas the theoretical calculation of the temperature gradient showed a maximum of about 100 °C. Heat transfer again explained the differences between experimental data and theoretical calculations. Although for MW solutions electrical conductivity is the main factor involved in temperature variations induced by electric fields, it seems that other factors could be involved in the case of cell suspensions. Indeed, as shown in Table 1, for the same σ25 level (i.e., between 17 and 22 μS cm− 1), highly concentrated cell suspensions showed greater temperature variations than the 100-fold MW dilution. We assume that intracellular-ion release, induced by plasma membrane electroor thermoporation, increases the electrical conductivity of the extracellular matrix, leading to a higher temperature. Nevertheless, in the case of low σ25 levels (about 5 μS cm− 1), the temperature variations were identical with and without yeast cells. We therefore compared the effects of thermal stresses with those of electrical stresses on yeast physiology to understand better the thermal mechanisms involved in this type of cell death. 3.3. Compared effects of electrical and thermal treatments on yeast physiology 3.3.1. Cell enumerations We measured the viability measurements of electric and heat-shocked yeast cells to evaluate the thermal effects of electric treatment. A total of 1.5 × 107 cells mL- 1 was exposed to a low-intensity electric field of 1.5 kV cm− 1 over a long period ranging from 0.5 to 5 s and to different levels of heat shock ranging from 50 to 70 °C for 10 s. The viability of electric-shocked cells is presented in Table 2. The results were similar to those of other authors (Qin et al., 1995), and they indicate an increase in microbial inactivation after longer pulses. The shorter pulses were associated with a lower level of inactivation of the yeast cells. Viabilities after pulses of 0.5 s, 3 s and 5 s were close to 50%, 15% and 0.7%, respectively. These fields allowed the observation of biophysical phenomena occurring just before cell death. Indeed, these kinds of electrical treatments are known to cause permeabilization of the plasma membrane, and Ferret et al. (1999) demonstrated that human K562 cells show reductions in volume in response to low-intensity electric fields.
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Table 3 Viability of Saccharomyces cerevisiae CBS 1171 exposed to different levels of heat shock Temperature variations, ΔT, (°C) Viability (%)
0
25
30
35
40
45
100.± 0
100.± 0
87.8 ± 8.0
6.7 ± 2.8
1.4 ± 1.4
0.± 0
Heat treatments were carried out in a binary water–glycerol medium (aw = 0.992) for 10 s. Results are shown as means and 95% confidence intervals.
To evaluate the thermal effects of electrical treatment, heat shocks were applied for a similar time (10 s) in a heat bath, with similar cooling conditions as explained in the Materials and methods section. Results are presented in Table 3. Heating from 25 to 50 °C (ΔT = 25 °C) produced no loss in viability, whereas the equivalent electrical treatment, resulting in a final measured temperature of 51 °C (ΔT = 26 °C; Fig. 1), produced a viability of only 0.7% (Table 2). Thus, in the case of a final measured temperature of 51 °C (ΔT = 26 °C; Fig. 1) induced by the electric treatment, this lethality cannot be only explained by the rise in temperature of the suspension medium. Nevertheless, in the case of highly concentrated cell suspensions, temperature variations caused by electric treatment could be involved in cell death because, as shown in Fig. 1, they are greater than 75 °C (ΔT = 50 °C). It is noteworthy that such temperature variations could play an important role in this type of cell death as shown in Table 3. This study thus confirms numerous previous works (Bryant and Wolfe, 1987; Weaver, 1993; Qin et al., 1996) about the nonthermal aspects of pulsed electric fields. Nevertheless, a recent study using high pulsed electric fields (in the range of 31–40 kV cm− 1) showed that the temperature achieved by the electrical treatment is a very important parameter in microbial inactivation and is independent of the electric field strength (Sepulveda et al., 2005). This observation leads us to understand better the role played by this parameter in this type of cell death. 3.3.2. Relationship between electrical conductivity of the medium and heating As previously explained, electrical conductivity is one of the main factors involved in electric field-induced temperature increase. Thus, immediately after a heat shock at 50 °C for 2 min, there is a significant leakage of ions (Na+, K+ and Ca2+) from yeast cells to the suspension medium (Martinez de Marañon et al., 1999). Thus, the temperature variations caused by electric fields application could alter plasma membrane integrity by thermoporation. As recently suggested by Sepulveda et al. (2005), in the case of pulsed electric fields, such thermal variations (giving a final temperature of 55 °C) would increase the killing effect of the electrical treatments. The
Table 2 Mean viabilities and 90% confidence intervals for S. cerevisiae subjected to a 1.5 kV cm− 1 electric field for different pulse durations Pulse durations (s) Viability (%)
0.5 55.1 ± 20.2
1 41.0 ± 11.8
Cell concentration was adjusted to about 1.5 × 107 cells mL− 1.
2 32.1 ± 17.3
3 16.1 ± 17.4
4 4.5 ± 6.5
5 0.7 ± 1.6
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Table 4 Electrical conductivity variations of a cell suspension exposed to different level of heat shocks Temperature variations, ΔT (°C) Electrical conductivity variations, Δσ (μS cm− 1)
25 1.9 ± 0.3
30 6.4 ± 1.3
35 6.6 ± 1.9
Heat treatments were applied for 10 s. Cell concentration was adjusted to about 1.5 × 107 cells mL− 1. Results are shown and confidence intervals were calculated at 95%.
authors attributed this effect to temperature-related phase transition of phospholipids in the cell membranes. Indeed, phospholipid fluidity could change according to the temperature, reducing the thickness and the mechanical resistance of the plasma membrane. As is well described in the literature, such changes to phase transition could alter plasma membrane permeability (Block et al., 1975; Beney and Gervais, 2001). To evaluate the effect of such thermoporation on ion leakage and so with environmental electrical conductivity, we measured electrical conductivity variations of cell suspensions submitted to different levels of heat shocks for 10 s (Table 4). The
electrical conductivities increased with temperature variations and could therefore damage plasma membranes and lead to intracellular-ion release from yeast cytoplasm to suspension medium. This increases the electrical conductivity of the suspension medium and increases temperature variations. Indeed, as shown in Fig. 4, such thermal variations increase electrical conductivity, which is a predominant factor in the temperature variations induced by electric fields (Eq. (2)). Therefore, for electric treatments, electrical conductivity and thermal variations are inversely related. More precisely, we assume that released ions could be localized first in a thin peripheral cellular sublayer where turbulence is absent, unlike the water–glycerol matrix which is turbulent. With ionic diffusion from the intracellular matrix to the peripheral sublayer and then from the sublayer to the extracellular matrix, ionic concentrations in the sublayer develop to form an ionic gradient between the intracellular matrix and the sublayer and between the sublayer and the extracellular matrix. Therefore, according to the temperature-induced electrical conductivity variations previously measured (Fig. 4), this sublayer is strongly exposed to time-dependent thermal variations. Such high
Fig. 5. Proposed biophysical events involved in cell death related to electric field treatment. Native cells (1) are exposed to an electric field causing plasma membrane electroporation (2). This could induce (3) the formation of an ionic sublayer via intracellular-ion leakage (which induces increases in sublayer-electrical conductivity and temperature). (4) The final step involves by ionic migration from the sublayer to the environmental matrix. This increases the electrical conductivity and temperature of the matrix. This could explain cell death induced by an electric treatment. Note that sublayer temperature increases could enhance intracellular-ion leakage via plasma membrane thermoporation. Symbols: (●) represents ions, (ΔTx): temperature variations of the x medium, ( ): induced event, and (!): ion migration.
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temperature shifts localized at a cellular level could play a role in yeast death by initiating cell thermoporation. This localized high temperature increase corresponds to a low amount of heat transfer in the medium caused by the small size of the cell and so the corresponding mean increase in temperature stays low. Each cell constitutes a thermal source that slightly influences the temperature of the extracellular matrix. To appreciate better the killing effects of such thermal variations, we propose a model that includes parameters such as the heat transfer by conduction between sublayers and the environmental matrix. 3.4. Proposition of a comprehensive model of cell death This study underlines the involvement of two elements in cell death induced by low-intensity electric fields with long-duration pulses: first the variations in electrical conductivity and temperature; second the electro- and thermo-mechanical phenomena (electroporation and thermoporation) involved in intracellular medium leakage. Interestingly, this paper and other authors such as Kotnick and Milklavčič (2000) demonstrate the plausible role of the electric field-induced temperature in the behavior of cells subjected to such an electric stress. Our study which describes mainly the cause–effect relations between electrical conductivity and temperature variations, leads us to propose a possible scenario for the cellular response to a lowintensity electric field with long-duration pulses (Fig. 5). On the one hand the electric fields affect the yeast cells mechanically (Table 2) and induce plasma membrane poration (Zimmermann, 1986), which leads to both intracellular material leakage (such as ions) and an increase of the extracellular electrical conductivity, and to the transit of the electric field into yeast cells. Such an event, because of the high intracellular electrical conductivity, leads to an increase in inner cell temperature. On the other hand, electric fields affect the extracellular matrix directly. Indeed, electrical treatment increases the extracellular temperature (Figs. 1 and 3) and then the electrical conductivity which could also increase via the leakage of the intracellular medium. As previously explained, ions could first diffuse from the cytoplasm to a peripheral cellular sublayer, thereby increasing the electrical conductivity of this layer. Therefore, transient temperature variations induced by the electrical treatment would be localized at a cellular level. Ions could then diffuse from the sublayer to the environmental matrix (i.e., the suspending medium). Such events imply that whatever the studied medium (intra- or extracellular), an electrical conductivity variation would induce a rapid temperature shift (as with a heat shock treatment), which leads to cell injury (Table 3). Such an association between extracellular conductivity medium variations caused by cell permeabilization and temperature medium variations has been recently confirmed by Pavlin et al. (2005). Interestingly, cell death could be induced directly by the electric field itself, with electroporation followed by a leakage of the intracellular components (Aronsson et al., 2005) and indirectly by a high- and short-time heat shock induced in the cell or in the sublayer surrounding the cell (i.e. thermoporation) caused by an increase in electrical conductivity.
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4. Conclusions Experimental and theoretical considerations lead us to conclude that the mechanisms implicated in the observed death of cells exposed to low-intensity electric fields with long-duration pulses, which can be explained by membrane polarization phenomena, can also be explained by thermal phenomena. Such suggested mechanisms must be confirmed in the case of cells exposed to high-intensity electric fields with short-duration pulses. This paper emphasizes the relation between extra- and intracellular temperature and electrical conductivity variations. A comprehensive model and experimental data suggest that yeast cell death can be induced by both direct and indirect effects of such treatment. We suggest that membrane polarization mechanisms are certainly associated with thermal effects, mainly in the permeabilization of the cell membrane and the subsequent increase of extracellular conductivity. However, if heat and mass transfers are taken into account, the theoretical temperature increase in the intracellular medium or in the cell's vicinity is much higher than that of the extracellular medium, and could explain the destruction of the cell. Further investigations are needed for a full understanding of the behavior of cells subjected to PEF treatment. References Aronsson, K., Lindgren, M., Johansson, B.R., Rönner, U., 2001. Inactivation of microorganisms using pulsed electric fields: the influence of process parameters on Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae. Innovative Food Science and Emerging Technologies 2, 41–54. Aronsson, K., Rönner, U., Borch, E., 2005. Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae in relation to membrane permeabilization and subsequent leakage of intracellular compounds due to pulsed electric field processing. International Journal of Food Microbiology 99, 19–32. Beney, L., Gervais, P., 2001. Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses. Applied Microbiology and Biotechnology 57, 34–42. Block, M.C., Van Der Neut-Kok, E.C.M., Van Deenen, L.L.M., De Gier, J., 1975. The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes. Biochimica and Biophysica Acta 406, 187–196. Bryant, G., Wolfe, J., 1987. Electromechanical stress produced in the plasma membrane of suspended cells by applied electric fields. Journal of Membrane Biology 96, 129–139. Castro, A.J., Barbosa-Cánovas, G.V., Swanson, B.G., 1993. Microbial inactivation of foods by pulsed electric fields. Journal of Food Processing and Preservation 17, 47–73. Ferret, E., Evrard, C., Foucal, A., Gervais, P., 1999. Volume changes of isolated human K562 leukemia cells induced by electric pulse. Biotechnology and Bioengineering 67, 520–528. Fiala, A., Wouters, P.C., Van Der Bosch, E., Creyghton, Y.L.M., 2001. Coupled electrical-fluid model of pulsed electric field treatment in a model food system. Innovative Food Science and Emerging Technologies 2, 229–238. Fleischman, G.J., Ravishankar, S., Balasubramaniam, V.M., 2004. The inactivation of Listeria monocytogenes by pulsed electric fields treatment in a static chamber. Food Microbiology 21, 91–95. Gervais, P., Martinez de Marañon, I., 1995. Effect of kinetics of temperature variation on Saccharomyces cerevisiae viability and permeability. Biochimica and Biophysica Acta 1235, 52–56. Harrison, S.L., Barbosa-Cánovas, G.V., Swanson, B.G., 1997. Saccharomyces cerevisiae structural changes induced by pulsed electric field treatment.
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Lebensmittel-Wissenschaft und-Technologie—Food Science and Technology 30, 236–240. Hayashi, M., 2004. Temperature–electrical conductivity relation of water for environmental monitoring and geophysical data inversion. Environmental Monitoring and Assessment 96, 119–128. Harris, C.M., Kell, D.B., 1983. The radio-frequency dielectric properties of yeast cells measured with a rapid, automated, frequency-domain dielectric spectrometer. Bioelectrochemistry and Bioenergetics 11, 15–28. Hölzel, R., 1992. Non-invasive determination of bacterial single cell properties by electroporation. Biochimica and Biophysica Acta 1450, 53–60. Jayaram, S., Castle, G.S.P., Margaritis, A., 1992. Kinetics of sterilization of Lactobacillus brevis cells by the application of high voltage pulses. Biotechnology and Bioengineering 40, 1414–1420. Kotnick, T., Milklavčič, D., 2000. Theoretical evaluation of the distributed power dissipation in biological cells exposed to electric fields. Bioelectromagnetics 21, 385–394. Lebovka, N.I., Bazhal, M.I., Vorobiev, E., 2000. Simulation and experimental investigation of food material breakage using pulsed electric field treatment. Journal of Food Engineering 44, 213–223. Martinez de Marañon, I., Chaudanson, N., Joly, N., Gervais, P., 1999. Slow heat rate increases yeast thermotolerance by maintaining plasma membrane integrity. Biotechnology and Bioengineering 65, 177–181. Pavlin, M., Kandušer, M., Reberšek, M., Pucihar, G., Hart, F.X., Magjareviæ, R., Miklavæiæ, D., 2005. Effect of cell electroporation on the conductivity of a cell suspension. Biophysical Journal 88, 4378–4390. Pliquett, U.F., Gusbeth, C.A., 2000. Perturbation of human skin due to application of high voltage. Bioelectrochemistry 51, 41–51. Qin, B.L., Chang, F.J., Barbosa-Cánovas, G.V., Swanson, B.G., 1995. Nonthermal inactivation of Saccharomyces cerevisiae in apple juice using pulsed electric fields. Lebensmittel-Wissenschaft und-Technologie—Food Science and Technology 28, 564–568. Qin, B.L., Pothakamury, U.R., Barbosa-Cánovas, G.V., Swanson, B.G., 1996. Nonthermal pasteurization of liquid foods using high intensity pulsed electric fields. Critical Review in Food Science and Nutrition 36, 603–627. Reyns, K.M.F.A., Diels, A.M.J., Michiels, C.W., 2004. Generation of bactericidal and mutagenic components by pulsed electric field treatment. International Journal of Food Microbiology 93, 165–173.
Sale, A.J.H., Hamilton, W.A., 1967. Effects of high electric fields on microorganisms: killing of bacteria and yeasts. Biochimica Biophysica Acta 148, 781–788. Sepulveda, D.R., Góngora-Nieto, M.M., San-Martin, M.F., Barbosa-Cánovas, G.V., 2005. Influence of treatment temperature on the inactivation of Listeria innocua by pulsed electric fields. Lebensmittel-Wissenschaft und-Technologie—Food Science and Technology 38, 167–172. Shillcock, J.C., Seifert, U., 1998. Thermally induced proliferation of pores in a model fluid membrane. Biophysical Journal 74, 1754–1766. Sorensen, J.A., Glass, G.E., 1987. Ion and temperature dependence of electrical conductance for natural waters. Analytical Chemistry 59, 1594–1597. Takayuki, O., Takahiro, O., Masayuki, S., 1999. Decomposition of nucleic acid molecules in pulsed electric field and its release from recombinant Escherichia coli. Journal of Electrostatics 46, 163–170. Tsong, T.Y., 1989. Electroporation of cells membranes: mechanisms and applications. In: Neumann, E., Sowers, A.E., Jordan, C.A. (Eds.), Electroporation and electrofusion in cell biology. Plenum Press, New York, pp. 149–164. Van Loey, A., Verachtert, B., Hendrickx, M., 2002. Effects of high electric field pulses on enzymes. Trends in Food Science and Technology 12, 94–102. Vega-Mercado, H., Martin-Belloso, O., Qin, B.L., Chang, F.J., Gongora-Nieto, M.M., Barbosa-Cánovas, G.V., Swanson, B.G., 1997. Non thermal food preservation: pulsed electric fields. Trends in Food Science and Technology 8, 151–157. Weaver, J.C., 1993. Electroporation: a general phenomenon for manipulating cells and tissues. Journal of Cellular Biochemistry 51, 425–435. Winterhalter, M., 1997. On the defect growth after short electric field pulses in planar lipid bilayers. Colloids and Surfaces A-Physicochemical and Engineering Aspects 149, 161–169. Zhang, Q., Monsalve-Gonzales, A., Barbosa-Cánovas, G.V., Swanson, B.G., 1994. Inactivation of Saccharomyces cerevisiae in apple juice by square wave and exponential decay pulsed electric fields. Journal of Food Process Engineering 17, 469–478. Zimmermann, U., 1986. Electrical breakdown, electropermeabilization and electrofusion. Reviews of Physiology Biochemistry and Pharmacology 105, 176–256.
Yeast cell inactivation related to local heating induced by low-intensity electric fields with long-duration pulses Stéphane Guyot, Eric Ferret, Jean-Baptiste Boehm, Patrick Gervais ⁎ GPAB Laboratory, ENSBANA, 1 Esplanade Erasme, F-21000 DIJON, France Received 11 August 2005; received in revised form 8 February 2006; accepted 13 June 2006
Abstract The effects of electric field (EF) treatments on Saccharomyces cerevisiae viability were investigated using a PG200 electroporator (Hoefer Scientific Instrument, San Fransisco, CA, USA) with specific attention to induced thermal effects on cell death. Lethal electric fields (1.5 kV cm− 1 for 5 s) were shown to cause heat variations in the cell suspension medium (water + glycerol), while corresponding classical thermal treatments at equivalent temperatures had no effect on the cells viability. Variations of the electrical conductivity of the intra- and extracellular matrix caused by ions and solutes transfer across the membrane were shown to be involved in the observed heating. The results permitted to build a theoretical model for the temperature variations induced by electric fields. Using this model and the electrical conductivity of the different media, a plausible explanation of the cell death induced by low-intensity electric fields with long-duration pulses has been proposed. Indeed, cell mortality could in part be caused by direct and indirect effects of electric fields. Direct effects are related to well known electromechanical phenomena, whereas indirect effects are related to secondary thermal stress caused by plasma membrane thermoporation. This thermoporation was attributed to electrical conductivity variations and the corresponding intracellular heating. © 2006 Elsevier B.V. All rights reserved. Keywords: Electric field; Cell inactivation; Temperature; S. cerevisiae; Conductivity; Modeling
1. Introduction Pulsed electric fields (PEF) applications are considered as a nonthermal decontamination process that involves the maintenance of biological samples between electrodes, and their subjection to high-intensity, short-duration electric fields. Some studies reported that electrical treatments could affect cell physiology directly by intracellular disorganization (Aronsson et al., 2001), by DNA and RNA decomposition (Takayuki et al., 1999) or by inactivation of enzymes (Van Loey et al., 2002). Alternatively, they could act indirectly by lethal molecule generation from the suspension medium (Reyns et al., 2004). Thus, the mechanisms by which microorganisms subjected to such physical treatments are inactivated remain unclear. The
⁎ Corresponding author. Tel.: +33 3 80 39 66 54; fax: +33 3 80 39 68 98. E-mail address: [email protected] (P. Gervais). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.06.036
most commonly accepted theory is that electric fields cause the polarization of cells, which generates electromechanical compression of the plasma membrane and subsequent permeabilization (Zimmermann, 1986; Bryant and Wolfe, 1987; Tsong, 1989; Qin et al., 1996). The membrane can be considered as a capacitor and exposure to an external electric field leads to a build-up of the membrane potential difference, V, caused by a charge separation across the membrane. V is proportional to the field strength, E, and to the radius of the cell. An increase in the membrane potential leads to a reduction in membrane thickness. Breakdown occurs if the critical breakdown voltage, Vc (about 1 V), is reached with a further increase in the external field strength. It is assumed that breakdown causes the formation of transmembrane pores, which leads to the depolarization of the cell and subsequent decompression of the membrane. Breakdown is reversible if the pores are few and small with regard to the total membrane surface. Alternatively, permeabilization may lead to irreversible breakdown associated with the mechanical
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destruction of the cell (Zimmermann, 1986). All these events depend on various factors such as the cell size, the conductivity of the medium, and the field characteristics (Weaver, 1993; Jayaram et al., 1992; Zhang et al., 1994). However, although the application of the laws of electrical forces to cells is accepted by all authors, some physical laws applying to the cells have been neglected. Thus, the previous theoretical approach does not recognize the increase in temperature arising from the electrical discharge across the cell suspension. Because PEF treatments do not cause significant increases in the mean temperature of the liquid medium (Qin et al., 1996; Harrison et al., 1997), the mechanisms of cell death are often explained with no reference to the thermal aspects. Nevertheless, some have reported an important contribution to the destruction of the cells by the thermal effects naturally associated with electric fields. Indeed, Shillcock and Seifert (1998), Winterhalter (1997), and Qin et al. (1996) proposed that the induction of pore formation was a consequence of the thermal fluctuations of the lipid elements. PEF treatments of 20–50 kV cm− 1 increase the temperature of the treated medium by about 8–12 °C (Qin et al., 1996; Harrison et al., 1997; Sale and Hamilton, 1967), and Vega-Mercado et al. (1997) demonstrated the thermal aspects of the PEF process, and its requirement for cooling. Furthermore, Pliquett and Gusbeth (2000) explained the breakdown of the plasma membrane by the subsequent heating of the PEF, which reached the temperature of the lipid phase transition. These results suggest the relative importance of the temperature variations that occur when an electric field is applied to a solution. Other examples show that the effects of electrical and heat treatments on cells do not differ greatly. Gervais and Martinez de Marañon (1995) demonstrated that a sudden rise in temperature, from 25 °C to 50 °C, was followed by a significant expulsion of the intracellular medium, which could be the basis of cell destruction. Therefore, it is possible to compare classical heat treatments and PEF treatments in terms of membrane behavior. Thus, it appears that PEF treatment of a cell suspension medium, could induce heat variation which could be a key factor in cell death. To understand better the mechanisms involved in this type of mortality, this paper deals with the heat dissipation caused by exposure of yeast cell suspensions to direct current (DC). Low-intensity (1.5 kV cm− 1) and longduration pulses (0.5–5.0 s) were applied in a static treatment chamber. Specific experiments were undertaken to distinguish between heating variations caused by the cells presence and the suspension medium, and to estimate the heat dissipation in both. First, heat variations induced by electric fields were compared in different types of high conductivity solutions with and without yeast cells. Taking into account the variation of the electrical conductivity of the solutions caused by increasing temperature, a model was proposed to estimate the heat variation with regard to electric field duration. Second, we compared the effect of electric fields to heat treatments on yeast viability. These results lead us to propose a hypothesis that relates electric field-induced cell death to electrical conductivity and heat variations.
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2. Materials and methods 2.1. Microorganisms and culture conditions Yeast cells (Saccharomyces cerevisiae strain CBS 1171) were grown in 100 mL of Malt Wickerham (MW) medium for 60 h at 25 °C on a continuously agitated (250 rpm), temperaturecontrolled platform shaker (New Brunswick Scientific, Edison, USA). This medium is composed of 10 g glucose (Sigma, Saint Quentin Fallavier, France), 3 g pancreatic peptone (Sigma), 3 g yeast extract (Sigma), and 1.5 g Na2HPO4 (Prolabo, Paris, France) in 1 L distilled water. Cells were harvested at the stationary phase. Because conductivity is an important parameter in electrical treatments, and to establish conductivity close to the water activity (aw) of the culture medium (0.992), before stressing, all samples were washed twice in a binary water–glycerol (Sigma) medium corresponding to an aw of 0.992. 2.2. Electric field The electroporation chamber has been previously described by Ferret et al. (1999). Two parallel platinum electrodes were inserted into the static treatment chamber, and connected to the generator (Progenetor 200; Hoefer Scientific Instruments, San Fransisco, CA, USA). The space between the two electrodes had a volume of 1.2 mL. Direct electric fields of 1.5 kV cm− 1, ranging in duration (di) from 0.5 to 5 s, were applied to the samples. To simplify calculations, electric field intensity is approximated by Eq. (1), where E is the electric field intensity (V cm− 1), U0 is the applied voltage (V), and d is the distance between the two electrodes (0.3 cm). E ¼ U0 =d
ð1Þ
For all experiments, electric fields were applied to samples at room temperature (measured between 23 and 25 °C). Because such treatment could induce a temperature increase, samples were cooled to room temperature at the rate of 1.25 °C min− 1 before viability measurements by maintaining samples at room temperature. Temperature decrease was controlled using a Kthermocouple (TCSA, Dardilly, France). 2.3. Heat treatments Different heat treatments were applied for comparison with electric field treatments. Experiments were performed at either 50 up to 70 °C, in a heating bath. 2 mL of washed cells, at an initial concentration of 1.5 × 108 cells mL− 1 (measured using a Malassez cell, VWR International, Limonest, France), were injected into glass flasks containing 18 mL of binary medium that had been equilibrated to the chosen temperature. So, washed cells were diluted 10 folds prewarmed binary medium and final cell concentration was equal to 1.5 × 107 cells mL− 1. The heat shocks were maintained for 10 s, to approximate the corresponding electrical treatment times. As in the case of electric field
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treatments, samples were cooled to room temperature at the rate of 1.25 °C min− 1 before measuring cell viability by maintaining samples at room temperature. Temperature decrease was controlled using a K-thermocouple (TCSA). 2.4. Conductivity and temperature measurements Electrical conductivity was measured with a conductimeter (LF 323-B, Wissenchaft-Technische Werkstätten, Weilheim, Germany). Furthermore, measurements of the temperature during the heat treatment or after the electrical stress were made using a K-thermocouple (TCSA). The different temperature variations induced by the application of electrical stresses were evaluated 10 s after the end of the treatment, which corresponds to the time required to remove electrodes and to introduce the thermocouple into the chamber. The measures were repeated three to six times and the 95% confidence intervals were calculated. 2.5. Cell enumerations Cell enumerations, after both heat and electrical treatments followed by a cooling period, were made using the plating method, by plating cells in Petri dishes containing MW medium supplemented with 15 g L− 1 Pastagar A (Biokar Diagnostics, Beauvais, France). Each sample was decimally diluted in binary water–glycerol solution and, 0.3 mL of appropriate dilutions was spread on MW-Pastagar A. Petri dishes were then incubated for two days at 25 °C. Viability was determined by counting Colonies-Forming Unit (CFU) and related with the viability of controls (unstressed samples) using previous dilution coefficients. Each treatment was repeated on at least three independent cultures. 3. Results and discussion Because this method of study focused on the dynamic aspects of electric field effects, the chosen field characteristics must produce a low-intensity and long-duration pulse. Most of the fields previously used in food-processing pilot plants have been of much higher intensity, between 10 and 50 kV cm− 1 (Qin et al., 1996; Vega-Mercado et al., 1997). Preliminary viability experiments on S. cerevisiae suspensions allowed the definition of an electrical treatment duration range involving a cell death ratio around 50%; we chose a low-intensity electric field of 1.5 kV cm− 1 over a long period ranging from 0.5 to 5 s. 3.1. Electric field-induced temperature variations 3.1.1. Temperature measurements of cell suspensions Fig. 1 shows the temperature variations caused by the application of the electric field for different times to different cell concentrations. In all cases, a significant temperature increase was observed for pulses of longer duration. Such thermal variations are well described in the literature in the case of pulsed electric fields (Fiala et al., 2001; Fleischman et al., 2004; Sepulveda et al., 2005).
Fig. 1. Measurements of temperature variations at different cell concentrations subjected to a 1.5 kV cm− 1 electric field for different pulse durations (0.05–5 s). Bars represent 95% confidence intervals. Highly concentrated cell suspension: (•) 1.5 × 108 cell mL− 1; slightly concentrated cell suspensions: (♦) 1.5 × 107 cell mL− 1; (n) 1.5 × 103 cell mL− 1; (○) water–glycerol (aw = 0.992) without cells.
Nevertheless, when electrical treatments were performed on highly concentrated cell suspensions (about 1.5 × 108 cells mL− 1), the temperature variation increase was higher than in slightly concentrated cell suspensions (1.5 × 103 to 1.5 × 107 cells mL− 1). Concerning highly concentrated cell suspensions, temperature variations presented a sharp increase up to 2 s (about 25 °C s− 1) and then remained stable. In the case of the slightly concentrated cell suspensions, all the temperature curves presented the same profile and the same variations as the control sample containing the water–glycerol medium without cells. The temperature variations increase linearly at the rate of about 5 °C s− 1. To understand better the difference in the temperature variation between highly concentrated and slightly concentrated cell suspensions, we focused our interest on the involvement of electrical conductivity (σ) in such thermal variations. Indeed, as shown by Fiala et al. (2001) and by Hayashi (2004) the electrical conductivity of a solution, which is a key factor in electric field treatments, increases with temperature increase. Measurements of σ as a function of cell concentrations (Fig. 2), showed that the electrical conductivity of slightly concentrated cell suspensions (2.31 × 103 to 1.50 × 107 yeasts mL− 1) was the same as a water–glycerol solution without yeast: about 8.5 μS cm− 1 . Electrical conductivity of more concentrated yeast suspensions (2.31 × 107 to 2.31 × 108 yeasts mL− 1) significantly increased with cell concentration. Such an observation shows that the heat variations induced by low-intensity electric fields with long-duration pulses could be related to yeast concentration and to electrical conductivity. The more highly concentrated suspensions were characterized by higher temperature variations and higher electrical conductivity values than those of slightly concentrated suspensions (Table 1). To quantify this, we performed experiments on the electrical conductivity of nutritive medium (MW lacking cells). 3.1.2. Temperature measurements in nutritive medium (MW) This conductive medium was used to approximate the intracellular medium, which presents an electrical conductivity of 2.3–5 mS cm− 1 (Harris and Kell, 1983; Hölzel, 1992) 1000 folds higher than the suspension medium (in the case of a
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Fig. 2. Measurements of electrical conductivity as a function of yeast cell concentration at 25 °C. Cell suspensions were prepared in a binary water–glycerol medium (aw = 0.992) as described in the Materials and methods section. Bars represent 95% confidence intervals.
water–glycerol solution) because of charged components present in the cell cytoplasm (i.e., proteins, nuclear acids, ions, etc.). Indeed, MW is composed in part of yeast extract (3 g L− 1), as explained in the Materials and methods section, and presents similar water activity than yeast cytoplasm (about 0.992). Moreover, this nutritive medium presents an electrical conductivity, about 1.5 mS cm− 1, which is relatively close to the yeast intracellular medium one. If we consider a cell suspension undergoing an electric field treatment and if both cell concentration and poration state allow an electric field pathway, it is reasonable to infer that the cell cytoplasm will be subjected to a significant increase in temperature. Tο study the involvement of the electrical conductivity in electric fields-induced temperature of the intracellular medium, different electrical conductivities of MW solutions were measured realized by adjusting the MW concentration using serial decimal dilutions of water–glycerol mixes. Measurements of the temperature according to electric field duration, and of the electrical conductivity with regard to temperature for different concentrations of MW, are presented in Figs. 3 and 4 respectively. As in the presence of yeast cells (Fig. 1), Fig. 3 shows that the temperature increased with pulse duration and with the increase in MW concentration. For the 10-fold dilution,
the temperature variations increased linearly up to about 40 °C for a 1 s pulse (at the rate of 40 °C s− 1) and then reached a plateau. For the 100-fold dilution, the temperature increased linearly up to 36 °C for a 4 s pulse duration (about 9 °C s− 1) and then remained stable. However, for the 1000-fold dilution and the water–glycerol medium, the temperature variation increased
Table 1 Main characteristics of the different media subjected to electric fields Medium
Concentration (dilution Linear slope σ25 (μS cm− 1) in water–glycerol) (°C s− 1)
Highly concentrated 1.5 × 108 cells mL− 1 cell suspension Slightly concentrated cell 1.5 × 107 cells mL− 1 suspensions 1.5 × 103 cells mL− 1 MW without cells 10− 1 10− 2 10− 3 Water–glycerol without cells
25
17.2
5 5 40 9 5 5
9.3 8.0 151.7 22.2 9.0 7.9
Linear slopes of the temperature variations according to pulse duration are shown in Figs. 1 and 3 and electrical conductivities at 25 °C (σ25) are shown in Figs. 2 and 4.
Fig. 3. Comparisons of experimental and modeled temperature variations according to time for different decimal dilutions of MW in water–glycerol. Experimental data (bars represent 95% confidence intervals) are compared with the theoretical curves deduced from Eq. (2). A: (♦) 10-fold dilution; (○) water– glycerol (aw = 0.992), without cells. B: (n) 100-fold diluted sample; (▴) 1000fold diluted sample.
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Fig. 4. Measurements of electrical conductivity according to temperature for different decimal dilutions of MW in water–glycerol. (♦) 10-fold dilution; (n) 100-fold diluted sample; (▴) 1000-fold diluted sample; (○) water–glycerol (aw = 0.992), without cells. (Dotted lines): linear regression.
linearly (at the rate of about 5 °C s− 1) according to pulse duration and electrical conductivity at 25 °C. To understand better the influence of MW or yeast cell concentration on the temperature increase, we measured changes in the electrical conductivity of different decimal dilutions of MW in water–glycerol mixes according to temperature changes. Results are presented in Fig. 4 and show that, whatever the MW concentration, the electrical conductivity increased linearly with the temperature. For 10-fold dilutions, the electrical conductivity increased from about 150 μS cm− 1 at 21 °C to about 350 μS cm− 1 at 77 °C. However, for higher dilutions, electrical conductivity variations according to the temperature were less important, and for the 100-fold diluted solutions they varied from about 21 μS cm− 1 at 21 °C to about 55 μS cm− 1 at 77 °C. The 1000-fold diluted solutions showed a slight increase of the electrical conductivity from about 7.9 μS cm− 1 at 21 °C to about 19 μS cm− 1 at 77 °C. These results are in accordance with the model proposed by Sorensen and Glass (1987). Moreover, from the results presented in Figs. 3 and 4, it is evident that the electrical responses of MW solutions depended on the dilution rate and the electrical conductivity at 25 °C (σ25) (see also Table 1). These data underline the role of electrical conductivity in the rate of temperature increase according to pulse duration. Nevertheless, in some cases (Figs. 1 and 3) a plateau appeared, indicating that the maximum temperature variation had been reached. We assume that the temperature reached 100 °C and then during the 10 s needed to remove electrodes and introduce the thermocouple in the chamber cooled to about 70 °C in the case of MW solutions (ΔT ≈ 45 °C) or to greater than ≈ 75 °C in the case of cell suspensions. To verify this hypothesis and to understand better the causes of the thermal effects induced by electric field application, we established a mathematical model that includes electrical conductivity as discussed in the next section. 3.2. Model of heat dissipation To estimate the electric field-induced temperature, our model outlines the temperature evolution of the MW medium
subjected to an electric field (Fig. 3). The model does not deal with cells exposed to electric fields, because there are no data on the kinetics of ion release from yeast cells caused by electroporation. The theoretical expression of the temperature variation of a suspension subjected to an electric field is well described in the literature (Jayaram et al., 1992; Castro et al., 1993; Lebovka et al., 2000). However the novelty of the proposed model is to highlight the temperature decrease between the end of the electrical treatment and the instance of temperature measurement. The temperature variations induced by electric fields and consecutive cooling are given by Eq. (2), below. In this, Ti and To are the temperatures (°C) of the inner and outer chamber respectively; t is the time (s), E is the electric field strength (1.5 kV cm− 1); σ is the electrical conductivity (μS cm− 1), which depends on both T and the concentration of MW; h is an estimated parameter defined as the convective coefficient of cooling between the inner and outer chambers; A is the exchange chamber surface (4 cm2); ρ is the solution density (1 g cm− 3); Cp is the specific heat of the solution (4.18 J °C− 1 g− 1) and V is the sample volume (1 cm3). It should be noted that this equation comprises two parts: first, the heating effects induced by the electric fields, which depend mainly on E and σ, and second, the cooling effects induced by thermal exchanges between inside and outside of the chamber. dTi E2 :rðTi ðtÞÞ h:A:ðTi ðtÞ To Þ ¼ q:Cp q:Cp :V dt |fflfflfflfflfflfflffl{zfflfflfflfflfflfflffl} |fflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflffl} Heating
ð2Þ
Cooling
As explained in the Materials and methods section, (i) E = 1.5 kV cm− 1 when 0 b t ≤ di, and E = 0 kV cm− 1 when t N di (duration) and the experimental temperature variations presented in Fig. 3 were measured 10 s after the electrical stresses (the time required to remove electrodes and to introduce the thermocouple in the chamber). Thus, during the electric treatment (i.e., until t ≤ di) the MW solution was submitted to heating effects induced by electric fields and to cooling effects caused by temperature exchanges between inside and outside of the chamber. During the time required to remove the electrodes and to introduce the thermocouple (10 s and E = 0 kV cm− 1), heating ceased and only cooling occurred. The model shows that, ignoring the last cooling period at the end of the electric treatment, in the cases of 10-fold and 100-fold MW dilutions, the temperature reaches a maximum (ebullition point) and then remains stable (data not shown). During this cooling period (10 s), the temperature curves present the same profile as previously described (a slope followed by a plateau), but the maximum ΔT reached is about 40 °C. As shown in Fig. 3, this model is in accordance with the experimental data. Moreover, it permits us to understand better the difference in temperature variations between yeast cell suspensions (Fig. 1) and MW solutions (Fig. 3). Indeed, the electrical conductivity of yeast cytoplasm is higher than that of MW solutions. Therefore, temperature increases are more important in the presence of yeast cells than in their absence. This can be extrapolated to the behavior of the intracellular medium. According to the literature
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(Harris and Kell, 1983; Hölzel, 1992) and our own estimates, intracellular conductivity is between 2.3 and 5.5 mS cm− 1, or about 1000 times higher than that for a water–glycerol mix (about 12 μS cm− 1). The temperature increase will thus be higher, as has been corroborated by others authors. Pliquett and Gusbeth (2000) observed local transport regions (LTR) within a skin sample subjected to an electric field. The temperature of the center of these areas was estimated to be below 70 °C, whereas the theoretical calculation of the temperature gradient showed a maximum of about 100 °C. Heat transfer again explained the differences between experimental data and theoretical calculations. Although for MW solutions electrical conductivity is the main factor involved in temperature variations induced by electric fields, it seems that other factors could be involved in the case of cell suspensions. Indeed, as shown in Table 1, for the same σ25 level (i.e., between 17 and 22 μS cm− 1), highly concentrated cell suspensions showed greater temperature variations than the 100-fold MW dilution. We assume that intracellular-ion release, induced by plasma membrane electroor thermoporation, increases the electrical conductivity of the extracellular matrix, leading to a higher temperature. Nevertheless, in the case of low σ25 levels (about 5 μS cm− 1), the temperature variations were identical with and without yeast cells. We therefore compared the effects of thermal stresses with those of electrical stresses on yeast physiology to understand better the thermal mechanisms involved in this type of cell death. 3.3. Compared effects of electrical and thermal treatments on yeast physiology 3.3.1. Cell enumerations We measured the viability measurements of electric and heat-shocked yeast cells to evaluate the thermal effects of electric treatment. A total of 1.5 × 107 cells mL- 1 was exposed to a low-intensity electric field of 1.5 kV cm− 1 over a long period ranging from 0.5 to 5 s and to different levels of heat shock ranging from 50 to 70 °C for 10 s. The viability of electric-shocked cells is presented in Table 2. The results were similar to those of other authors (Qin et al., 1995), and they indicate an increase in microbial inactivation after longer pulses. The shorter pulses were associated with a lower level of inactivation of the yeast cells. Viabilities after pulses of 0.5 s, 3 s and 5 s were close to 50%, 15% and 0.7%, respectively. These fields allowed the observation of biophysical phenomena occurring just before cell death. Indeed, these kinds of electrical treatments are known to cause permeabilization of the plasma membrane, and Ferret et al. (1999) demonstrated that human K562 cells show reductions in volume in response to low-intensity electric fields.
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Table 3 Viability of Saccharomyces cerevisiae CBS 1171 exposed to different levels of heat shock Temperature variations, ΔT, (°C) Viability (%)
0
25
30
35
40
45
100.± 0
100.± 0
87.8 ± 8.0
6.7 ± 2.8
1.4 ± 1.4
0.± 0
Heat treatments were carried out in a binary water–glycerol medium (aw = 0.992) for 10 s. Results are shown as means and 95% confidence intervals.
To evaluate the thermal effects of electrical treatment, heat shocks were applied for a similar time (10 s) in a heat bath, with similar cooling conditions as explained in the Materials and methods section. Results are presented in Table 3. Heating from 25 to 50 °C (ΔT = 25 °C) produced no loss in viability, whereas the equivalent electrical treatment, resulting in a final measured temperature of 51 °C (ΔT = 26 °C; Fig. 1), produced a viability of only 0.7% (Table 2). Thus, in the case of a final measured temperature of 51 °C (ΔT = 26 °C; Fig. 1) induced by the electric treatment, this lethality cannot be only explained by the rise in temperature of the suspension medium. Nevertheless, in the case of highly concentrated cell suspensions, temperature variations caused by electric treatment could be involved in cell death because, as shown in Fig. 1, they are greater than 75 °C (ΔT = 50 °C). It is noteworthy that such temperature variations could play an important role in this type of cell death as shown in Table 3. This study thus confirms numerous previous works (Bryant and Wolfe, 1987; Weaver, 1993; Qin et al., 1996) about the nonthermal aspects of pulsed electric fields. Nevertheless, a recent study using high pulsed electric fields (in the range of 31–40 kV cm− 1) showed that the temperature achieved by the electrical treatment is a very important parameter in microbial inactivation and is independent of the electric field strength (Sepulveda et al., 2005). This observation leads us to understand better the role played by this parameter in this type of cell death. 3.3.2. Relationship between electrical conductivity of the medium and heating As previously explained, electrical conductivity is one of the main factors involved in electric field-induced temperature increase. Thus, immediately after a heat shock at 50 °C for 2 min, there is a significant leakage of ions (Na+, K+ and Ca2+) from yeast cells to the suspension medium (Martinez de Marañon et al., 1999). Thus, the temperature variations caused by electric fields application could alter plasma membrane integrity by thermoporation. As recently suggested by Sepulveda et al. (2005), in the case of pulsed electric fields, such thermal variations (giving a final temperature of 55 °C) would increase the killing effect of the electrical treatments. The
Table 2 Mean viabilities and 90% confidence intervals for S. cerevisiae subjected to a 1.5 kV cm− 1 electric field for different pulse durations Pulse durations (s) Viability (%)
0.5 55.1 ± 20.2
1 41.0 ± 11.8
Cell concentration was adjusted to about 1.5 × 107 cells mL− 1.
2 32.1 ± 17.3
3 16.1 ± 17.4
4 4.5 ± 6.5
5 0.7 ± 1.6
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Table 4 Electrical conductivity variations of a cell suspension exposed to different level of heat shocks Temperature variations, ΔT (°C) Electrical conductivity variations, Δσ (μS cm− 1)
25 1.9 ± 0.3
30 6.4 ± 1.3
35 6.6 ± 1.9
Heat treatments were applied for 10 s. Cell concentration was adjusted to about 1.5 × 107 cells mL− 1. Results are shown and confidence intervals were calculated at 95%.
authors attributed this effect to temperature-related phase transition of phospholipids in the cell membranes. Indeed, phospholipid fluidity could change according to the temperature, reducing the thickness and the mechanical resistance of the plasma membrane. As is well described in the literature, such changes to phase transition could alter plasma membrane permeability (Block et al., 1975; Beney and Gervais, 2001). To evaluate the effect of such thermoporation on ion leakage and so with environmental electrical conductivity, we measured electrical conductivity variations of cell suspensions submitted to different levels of heat shocks for 10 s (Table 4). The
electrical conductivities increased with temperature variations and could therefore damage plasma membranes and lead to intracellular-ion release from yeast cytoplasm to suspension medium. This increases the electrical conductivity of the suspension medium and increases temperature variations. Indeed, as shown in Fig. 4, such thermal variations increase electrical conductivity, which is a predominant factor in the temperature variations induced by electric fields (Eq. (2)). Therefore, for electric treatments, electrical conductivity and thermal variations are inversely related. More precisely, we assume that released ions could be localized first in a thin peripheral cellular sublayer where turbulence is absent, unlike the water–glycerol matrix which is turbulent. With ionic diffusion from the intracellular matrix to the peripheral sublayer and then from the sublayer to the extracellular matrix, ionic concentrations in the sublayer develop to form an ionic gradient between the intracellular matrix and the sublayer and between the sublayer and the extracellular matrix. Therefore, according to the temperature-induced electrical conductivity variations previously measured (Fig. 4), this sublayer is strongly exposed to time-dependent thermal variations. Such high
Fig. 5. Proposed biophysical events involved in cell death related to electric field treatment. Native cells (1) are exposed to an electric field causing plasma membrane electroporation (2). This could induce (3) the formation of an ionic sublayer via intracellular-ion leakage (which induces increases in sublayer-electrical conductivity and temperature). (4) The final step involves by ionic migration from the sublayer to the environmental matrix. This increases the electrical conductivity and temperature of the matrix. This could explain cell death induced by an electric treatment. Note that sublayer temperature increases could enhance intracellular-ion leakage via plasma membrane thermoporation. Symbols: (●) represents ions, (ΔTx): temperature variations of the x medium, ( ): induced event, and (!): ion migration.
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temperature shifts localized at a cellular level could play a role in yeast death by initiating cell thermoporation. This localized high temperature increase corresponds to a low amount of heat transfer in the medium caused by the small size of the cell and so the corresponding mean increase in temperature stays low. Each cell constitutes a thermal source that slightly influences the temperature of the extracellular matrix. To appreciate better the killing effects of such thermal variations, we propose a model that includes parameters such as the heat transfer by conduction between sublayers and the environmental matrix. 3.4. Proposition of a comprehensive model of cell death This study underlines the involvement of two elements in cell death induced by low-intensity electric fields with long-duration pulses: first the variations in electrical conductivity and temperature; second the electro- and thermo-mechanical phenomena (electroporation and thermoporation) involved in intracellular medium leakage. Interestingly, this paper and other authors such as Kotnick and Milklavčič (2000) demonstrate the plausible role of the electric field-induced temperature in the behavior of cells subjected to such an electric stress. Our study which describes mainly the cause–effect relations between electrical conductivity and temperature variations, leads us to propose a possible scenario for the cellular response to a lowintensity electric field with long-duration pulses (Fig. 5). On the one hand the electric fields affect the yeast cells mechanically (Table 2) and induce plasma membrane poration (Zimmermann, 1986), which leads to both intracellular material leakage (such as ions) and an increase of the extracellular electrical conductivity, and to the transit of the electric field into yeast cells. Such an event, because of the high intracellular electrical conductivity, leads to an increase in inner cell temperature. On the other hand, electric fields affect the extracellular matrix directly. Indeed, electrical treatment increases the extracellular temperature (Figs. 1 and 3) and then the electrical conductivity which could also increase via the leakage of the intracellular medium. As previously explained, ions could first diffuse from the cytoplasm to a peripheral cellular sublayer, thereby increasing the electrical conductivity of this layer. Therefore, transient temperature variations induced by the electrical treatment would be localized at a cellular level. Ions could then diffuse from the sublayer to the environmental matrix (i.e., the suspending medium). Such events imply that whatever the studied medium (intra- or extracellular), an electrical conductivity variation would induce a rapid temperature shift (as with a heat shock treatment), which leads to cell injury (Table 3). Such an association between extracellular conductivity medium variations caused by cell permeabilization and temperature medium variations has been recently confirmed by Pavlin et al. (2005). Interestingly, cell death could be induced directly by the electric field itself, with electroporation followed by a leakage of the intracellular components (Aronsson et al., 2005) and indirectly by a high- and short-time heat shock induced in the cell or in the sublayer surrounding the cell (i.e. thermoporation) caused by an increase in electrical conductivity.
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