Journal of Applied Microbiology 2001, 91, 306311

The inuence of dissolved CO2 concentration on the death kinetics of Saccharomyces cerevisiae
M. Shimoda1, J. Cocunubo-Castellanos1, H. Kago1, M. Miyake2, Y. Osajima3 and I. Hayakawa1
1

Department of Bioscience and Biotechnology, Kyushu University, 2Shimadzu Co., and 3School of Bioresources, Hiroshima Prefectural University, Japan

614/10/00: received 27 October 2000, revised 28 February 2001 and accepted 15 March 2001

M. SHIMODA, J. COCUNUBO-CASTELLANOS, H. KAGO, M. MIYAKE, Y . O S A J I M A A N D I . H A Y A K A W A . 2001.

Aims: The effects of temperature and concentration of dissolved CO2 on the inactivation of Saccharomyces cerevisiae were investigated using a plug-ow system. Methods and Results: Several combinations of pressure (4, 6, 8, 10 mega-Pa (MPa)) and temperature (30, 34, 36, 38C) were used. The D-values obtained were 014 min at 8 MPa and 38C, and 015 min at 10 MPa and 36C. The log D-values were related linearly to the treatment temperature and to the dissolved CO2 concentration. The thermal resistance constant (zCO2(T)) was 95C in the media, including signicant levels of CO2, and the CO2 resistance constant was ztemp.(c) 72 c. Conclusions: This work has shown that inactivation followed rst-order death kinetics, and the effects of temperature and CO2 concentration were consistent through the critical temperature and pressure of CO2. Therefore, it is feasible to estimate D-values at any temperature and any CO2 concentration. Signicance and Impact of the Study: Non-thermal inactivation of micro-organisms in acidic beverages could be realized by the present technique.
INTRODUCTION The thermal inactivation process of micro-organisms has been widely used for killing vegetative cells in foods. It is, however, well known that their avours essentially deteriorate during the thermal process. One of the possible methods for reducing the inactivation temperature is the addition of antimicrobial agents, but this is highly restricted. Carbon dioxide (CO2) can be used as an antimicrobial agent because it is an ultra-pure product, free from toxicity, which can be easily removed from food products. Extensive studies on the microbial inhibition by CO2 near atmospheric pressure, and changes in the function of the cell membrane, have been reviewed (Jones and Greeneld 1982; Eklund 1984; Dixon and Kell 1989). A further consideration is that CO2 may exert its inuence on cells by affecting the rate at which particular reactions proceed via induction or represCorrespondence to: M. Shimoda, Department of Bioscience and Biotechnology, Kyushu University, 6101 Hakozaki, Higashi-ku, Fukuoka 8128581, Japan (e-mail: mshimoda@agr.kyushu-u.ac.jp).

sion of cytoplasmic enzyme synthesis (for examples, see Bowien and Leadbeater 1984; Dixon et al. 1988). As the microbial inhibitory activity of CO2 near atmospheric pressure is gentle, CO2 gas has been applied mainly for the preservation of foodstuffs. On the other hand, the antimicrobial activity of pressurized CO2 is remarkable and hence, the application of pressurized CO2 for sterilization of foods is expected to minimize the degradation of heatsensitive substances. However, there are great differences in the microbial inhibitory activity of pressurized CO2. Some authors have observed poor activities (Thibault et al. 1987; L'Italien et al. 1989) while others have reported good activity (Kamihira et al. 1987; Haas et al. 1989; Lin et al. 1991, 1992, 1993; Wei et al. 1991; Isenschmid et al. 1995; Ishikawa et al. 1995). Signicant differences in the inhibitory activity were thought to result from differences in the concentration of dissolved CO2 in the cell suspension. It has been reported that microbubbling of pressurized CO2 facilitated the dissolution of CO2 molecules in the sample solution and hence, enabled effective inactivation of micro-organisms (Ishikawa et al.
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1995). For the development of this technique for the food industry, a continuous processor using microbubbles of pressurized CO2 was built. The continuous processor demonstrated extremely high performance on the inactivation of micro-organisms, but it was impossible to analyse the inactivation process kinetically (Shimoda et al. 1998). In this paper, the effects of temperature and concentration of dissolved CO2 was investigated, using a plug-ow treatment system, on the inactivation of S. cerevisiae. MATERIALS AND METHODS Microbial culture Saccharomyces cerevisiae (ATCC 18824) was used as a test micro-organism. A starter culture was initiated by inoculating an individual test tube containing 5 mg YM agar (Difco). After multiplication to approximately 109 cfu ml1, the culture was transferred to 250 ml asks containing 100 ml YM medium and maintained at 35C for 24 h. The micro-organisms were collected by centrifugation and resuspended in physiological saline at about 108109 cfu ml1. Continuous ow system For continuous treatment of aqueous samples, an original instrument (Fig. 1) was constructed. Liquid CO2 and physiological saline were simultaneously pumped through a CO2 dissolving vessel (28 cm i.d. 35 cm high; 215 ml volume) with respective ow rates of 1016 g min1 for CO2 and 2038 ml min1 for physiological saline. Liquid CO2 changed to the gaseous or supercritical state through an evaporator. The evaporated CO2 uid was dispersed into the

saline solution from a stainless steel mesh lter attached to the bottom of the dissolving vessel. The average pore size of the lter was 10 lm, which gave the highest CO2 concentration (Ishikawa et al. 1995). The microbubbles of pressured CO2 migrated upwards while dissolving CO2 in the saline. The saline saturated with CO2 was heated to the experimental temperature by an electrical heater. A suspension (108109 cfu ml1) of S. cerevisiae was introduced at a ow rate of 10 ml min1 into the ow of saline. Residence coils of different volumes (1887 ml) were replaced to change the residence time or inactivation time. After the physiological saline containing cells of S. cerevisiae had owed through the residence coil, it was withdrawn via a pressure control valve (II), keeping the pressure at 01 MPa. The temperature of the residence coil was maintained within 02C. It is assumed that the CO2 molecules permeating into the cells expand explosively in the outlet of the pressure control valve (II) to burst the cells. The treatment conditions were as follows: treatment pressure, 4, 6, 8, 10 MPa; temperature, 30, 34, 36, 38C; ow rate of physiological saline, 2233 ml min1; ow rate of CO2, 1017 g min1; feeding rate of microbial suspension, 1 ml min1. All experiments were carried out over 95% saturation with CO2. Enumeration of survivors Samples were serially diluted in sterile physiological saline. Initial cell counts and survivors were determined by plating 01 ml of diluted or non-diluted samples on triplicate plates of standard plate count agar (Difco). Colonies were counted after incubation at 37C for 48 h.

Fig. 1 Schematic diagram of continuousow system for microbial inactivation with microbubbles of pressurized CO2
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Kinetic parameters for inactivation of microbial cells in media including signicant levels of CO2 The approach to describing changes in microbial populations as a function of time uses the survivor curve equation:
1 1 log [N N 0 t D

where N microbial population at any time, t; N0 initial microbial population; D decimal reduction time, or time required for a 1-log cycle reduction in the microbial population. The inuence of temperature on the CO2-inactivation rates in a medium including signicant levels of CO2 was expressed in terms of the thermal resistance constant (zCO2 (T)) using the following model:
1 1 log [D D T0 T T0 zCO2 (T)

The thermal resistance constant zCO2(T) is the temperature increase needed to accomplish a 1-log cycle reduction in the D-value at a given CO2 concentration. The reference decimal reduction time (DT0) is the magnitude at a reference temperature (T0). The inuence of dissolved CO2 concentration on CO2 inactivation rates was expressed in terms of the CO2 resistance constant (ztemp. (c)) using the following model:
1 1 Log [D D c0 c c0 ztemp: c

Fig. 2 Levels of dissolved CO2 as related to CO2 feeding ratio. a Kuenen's gas absorption coefcient. CO2 feeding ratio was dened as the ratio of CO2 ow rate (g min1) to sample ow rate (g min1). (*), saturated solubility at 35C and 10 MPa

The CO2 resistance constant ztemp.(c) is the increase in the concentration of dissolved CO2 needed to accomplish a 1-log cycle reduction in the D-value at a given temperature. The reference decimal reduction time (Dc0) is the magnitude at a reference CO2 concentration (c0). Replication and statistical treatment All experiments were done in triplicate. The data presented are the means and standard deviations of three replicate experiments.

feeding ratio higher than 035 to ensure the saturated solubility of CO2. First-order death kinetics were known with heat-treated vegetative cells, but the kinetics on microbial inactivation with pressurized CO2 were unknown. As shown in Fig. 3, a straight relationship was observed between the values of log [N No1] and treatment temperature. This indicated rstorder death kinetics based on CO2 inactivation. From the

The concentration of dissolved CO2 in the outlet of the CO2 dissolving vessel was measured volumetrically. The concentration of dissolved CO2 was estimated as a Kuenen's gas absorption coefcient (c), which is dened as the volume under normal conditions of the amount of gas dissolved in a unit volume of solvent. Figure 2 shows the concentration of dissolved CO2 as related to the CO2 feeding ratio, which was dened as the ratio of the ow rate of CO2 and that of physiological saline. It was shown that when the CO2 feeding ratio was higher than 025 at 10 MPa and 35C, it was possible to dissolve CO2 molecules to the almost saturated level. All experiments were carried out at a CO2

log (N No1)

RESULTS

Treatment time (min)

Fig. 3 First-order death of Saccharomyces cerevisiae in saline saturated with pressurized CO2. (s), Treatment at 8 MPa and 38 C; (d), treatment at 10 MPa and 36C

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slopes of straight lines, the decimal reduction times (D-values) were calculated. Under the condition of CO2 saturation, the D-values were 014 min for the treatments at 8 MPa and 38C, and 015 min at 10 MPa and 36C. Despite the treatment temperature being the optimum for multiplication in a nutrient broth, the treatment was very effective for the inactivation of S. cerevisiae. On the other hand, the D-value of the present strain was 035 min at 60C under atmospheric conditions. The effect of treatment pressure on the inactivation of S. cerevisiae is shown in Fig. 4. The inactivation power in the treatment at 35C was relatively weak below the critical pressure of CO2 (73 MPa), i.e. the D-values were 41 min at 4 MPa and 11 min at 6 MPa. The D-value, however, decreased steeply with increases in the treatment pressure, i.e. the D-values were 033 min at 8 MPa and 02 min at 10 MPa. It is assumed that higher pressure could result in higher inactivation power. The effect of treatment temperature on the inactivation of S. cerevisiae in saline including signicant levels of CO2 are shown in Fig. 5. The plots of the log D-values against treatment temperature gave straight lines. The thermal resistance constant of S. cerevisiae in the saline saturated with the pressurized CO2, which was estimated from the

Fig. 5 Effect of treatment temperature on D-value of Saccharomyces cerevisiae in saline saturated with pressurized CO2. (s), Treatment at 8 MPa; (d), treatment at 10 MPa

slope of straight line, was 86C at 10 MPa and 70C at 8 MPa. DISCUSSION In order to elucidate the effect of treatment pressure on the inactivation of S. cerevisiae, log D-values were plotted against the pressure of CO2 (the gure is not shown). The plots, however, did not give a straight relationship. Then, the log D-values were plotted against the concentration of dissolved CO2 in the sample solution during the treatment. The CO2 concentration was estimated by interpolating the data on CO2 solubility under pressurized conditions (Seidell and Linke 1958). As shown in Fig. 6, the log D-value was clearly related to the concentration of CO2. This indicated that the inactivation of S. cerevisiae was directly correlated with the concentration of dissolved CO2. It was also found that the cells were inactivated by the same mechanism through the critical pressure of CO2. The CO2 resistance constant, ztemp. (c), which was estimated from the slope of the straight line in Fig. 6, was 72 c with respect to the treatment at 35C. As shown in Fig. 4, the plots of the log D-values against treatment temperature gave straight lines, but it was still necessary to take into account the change in the CO2 solubility accompanied by the change in treatment temperature. The effect of temperature on the solubility of CO2 was also estimated by interpolating the data of Seidell and Linke (1958). With the purpose of clarifying the net effect of treatment temperature at a xed CO2 concentration and at

Fig. 4 Effect of treatment pressure on the inactivation of Saccharomyces cerevisiae in saline saturated with pressurized CO2. The treatment temperature was 35C

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Fig. 6 Relationship between log D-values and dissolved CO2 concentration. Saccharomyces cerevisiae in saline including signicant levels of CO2 was treated at 35C

Fig. 7 Modifying the D-values of Saccharomyces cerevisiae by CO2 solubility as related to treatment temperature. (s), Treatment at 8 MPa; (d), treatment at 10 MPa. The dashed lines indicate the modied relationship. It was supposed that cells were inactivated in the saline including CO2 saturation level at 35C

saturated level at 35C, modication was carried out by using the CO2 resistance constant, ztemp. (c)-value (Fig. 7). The net thermal resistance constant, zCO2(T)-value, was 95C for both 8 and 10 MPa treatments. The zCO2(T)values were comparable with the thermal resistance constant under atmospheric conditions. On the other hand, it should be noted that the plot of log D-value at 10 MPa and 30C followed a straight line. This indicated that cells are inactivated by the same mechanism through the critical temperature of CO2 (311C). Furthermore, the thermal resistance constant (70C for 8 MPa and 86C for 10 MPa) calculated from the slope of straight lines in Fig. 4 could be dened as an overall thermal resistance constant (zsat. CO2(T)-value) under CO2 saturation. Comparing Fig. 6 with Fig. 7, the effect of dissolved CO2 on the inactivation was found to be equivalent to the effect of treatment temperature. This suggests that molecular CO2 permeated into cell membranes and accumulated there, increasing the uidity of membranes by the disturbing effect of foreign molecules, indicating that temperature increase promotes uidity. Moreover, the fact that the thermal resistance constant (zCO2(T)) for inactivation was independent of the dissolved CO2 concentration, demonstrated that the treatment temperature and the CO2 concentration contributed independently to the inactivation. Isenschmid et al. (1995) termed the promotion of membrane uidity due to CO2 accumulation an ``anaesthesia

effect''. In batch treatments, which were characterized by gentle expansion of CO2, it was considered that cell death in a medium including a signicant level of CO2 was mainly due to the anaesthesia effect, rather than to cell burst. On the other hand, as the present continuous treatment was accompanied by explosive expansion of CO2 due to continuous pressure release, cell death was considered to be due not only to the anaesthesia effect but also, to cell burst. The mechanical impact on the cells in the anaesthesia state prompted cell rupture and hence, the explosive expansion of CO2 was very important for the improvement of inactivation power (Shimoda et al. 1998). In conclusion, the inactivation of S. cerevisiae in saline including signicant levels of CO2 followed rst-order death kinetics. The effects of temperature and dissolved CO2 concentration on the inactivation were consistent through critical temperature and pressure of CO2. The D-values were 014 min at 8 MPa and 38C, and 015 min at 10 MPa and 36C. The log D-values were related linearly not only to treatment temperature but also to dissolved CO2 concentration. The thermal resistance constant (ZCO2(T)) was 95C in the saline including signicant levels of CO2, and the CO2 resistance constant was Ztemp. (c) 72 c. These results permitted an estimation of the D-value for the inactivation of S. cerevisiae at any temperature and any CO2 concentration, as the temperature and CO2 concentration contributed independently to the inactivation.

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2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 306311