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The influence of pressure and temperature of compressed CO2 on the survival of yeast cells
journal
of
biotechnology Journal
of Biotechnology
39 (1995) 229-237
The influence of pressure and temperature of compressed CO, on the survival of yeast cells A. Isenschmid, I.W. Marison, U. von Stockar
*
Swiss Federal Institute of Technology, CH-1015 Lazmanne, Switzerland Received
24 August
1994; accepted
17 January
1995
Abstract
In order to study the potential use of supercritical CO, extraction for the recovery of products from yeast cell cultures, the effects of CO, on different yeast strains over a range of pressures and temperatures have been examined. Viability was shown to be dependent on temperature and dissolved CO, concentration, and can be described by a sigmoidal (S-shaped) curve. Cell death was mainly due to an ‘anaesthesia effect’ rather than cell rupture. Important differences in sensitivity were observed for the strains studied, with the following order of resistance: Kluyveromyces fragilis > Saccharomyces cerevisiae > Candida utilis. Keywords: Supercritical
CO,; Extraction; Yeast; Viability; Pressure sensitivity
1. Introduction Supercritical extraction has become an established process, with some industrial and many lab- and pilot-scale applications (Hoyer, 1985). The most important supercritical solvent, especially for food and pharmaceutical applications, is CO,. The characteristics of supercritical carbon dioxide extraction fit well with biotechnological production conditions: mild temperatures, lack of toxicity, good selectivity and ultra-pure products (no solvent residues). Very often product inhibition lowers the efficiency of an industrial bioprocess and to overcome this problem an in situ extraction without cell separation would be desir-
characteristics
* Corresponding author. 016%1656/95/$09.50 0 1995 Elsevier SSDZ 0168-1656(95)00018-6
able. For primary metabolites and biomass production, however, the extraction fluid has to be ‘biocompatible’. In the literature two different opinions exist concerning the biocompatability of compressed carbon dioxide. Some authors proclaim good survival (van Eijs et al., 1988; Thibault et al., 1987; L’Italien et al., 1989), while others speak of a rapid loss of viability in such a solvent (Rau, 1985; Kamihira et al., 1987; Haas et al., 1989; Wei et al., 1991; Lin et al., 1991,1992,1993). To clarify the situation a detailed study of the behaviour of yeast viability in compressed carbon dioxide has been undertaken. We have focused mainly on the parameters of temperature and pressure, since they greatly influence the solvent
Science
B.V. All rights reserved
of the fluid.
A. Isenschmid et al. /Journal
230
2. Materials
2.2. Procedure and analyses
and methods
2.1. High-pressure installation
A pressurizable reactor system has been constructed (Fig. 1) in which the pressure within the reactor is continuously measured by quartz pressure transducer coupled to a Mac IIci computer operating with LabView acquisition/control software. A control algorithm enabled careful control of the compression, holding and decompression time as well as ensuring reproducibility between experiments. The compression rate was 51 bar min-’ and the decompression rate 73 bar min- ‘. The autoclave used to contact the cell suspension with the compressed carbon dioxide consisted of a cylindrical vessel with a volume of 80.9 ml (internal diameter: 22 mm) and a magnetic stirrer. The reactor was made of stainless steel (316 L-steel) which was immersed in a water bath to control the temperature (for further details see Isenschmid, 1994). For all experiments presented here, the CO,inlet was at the base of the vessel in order to sparge the gas through the suspension during compression. 17
of Biotechnology 39 (1995) 229-237
16
15
Three yeast strains, Candida utilis NCYC 9950, Kluyveromyces fragilis NRRL 1109 and Saccharomyces cerevisiae CBS 426, were used as test organisms. Each strain was grown in a 3-l bioreactor under batch conditions as previously described (Isenschmid, 1994). Cells were harvested after the exponential growth phase, washed and re-suspended in fresh medium without glucose and incubated at - 70” C for 24 h, followed by storage in liquid nitrogen (for further details see Isenschmid, 1994). To expose them to compressed carbon dioxide the cells were thawed and split into a control sample of 2 ml and a pressure sample of 2 ml. The control sample received the same treatment as the pressure sample except that it was kept at atmospheric pressure. The viability ratio between treated and untreated cells was called ‘% viable cells’ and the cell density ratio ‘% intact cells’. The viability was determined by counting the number of colony-forming units (CFU) obtained after plating of suitably diluted culture samples on a medium composed (l- ‘) of 10 g yeast extract, Oxoid L21; 20 g Bacto Peptone, Oxoid L37; 20 g glucose, Fluka 49150 and 16 g agar, Oxoid Ll 1. The cell density as well as the cell size distribution were determined with a Coulter counter (Coultronics Model ZM). The number of cells larger than 4.75 pm (5.6 X lo-” ml) was also determined because of a shrinking effect observed after the treatment.
3. Results and discussion
Fig. 1. Schematic representation of the pressurizable reactor configuration. 0, balance (level control); 1, pressurized CO,; 2, thermostat: 5” C; 3, precooling coil; 4, HPLC pump, cooled; 5, valve (computer directed); 6, thermostat of the pressure vessel; 7, preheating coil; 8, choice of compression: sparged or surface gassing; 9, pressure vessel (80.9 ml, stainless steel); 10, magnetic stirrer; 11, temperature controller 5” C; 12, temperature controller (pressure vessel); 13, pressure controller (computer directed); 14, pH probe; 15, pressure reduction valve; 16, decompression valves (computer directed); 17, outlet.
Samples of Kluyveromyces fragilis, Saccharomyces cerevisiae and Candida utilis were exposed for 5 min to compressed carbon dioxide at 33” C and different pressures (Fig. 2). All three yeast strains showed the same tendency: with increasing CO, pressure the viability decreased following a typical S-shaped curve. Near-critical CO, is less harmful than supercritical CO, to all of the cells tested. However, this borderline from harmless to harmful - or the inflexion point of
A. Isenschmid et al. /Journal
this curve - does not necessarily occur at the critical pressure, but is strain dependent. The inflexion points were 53 bar for C. utilis, 61 bar for S. cerevisiae and 67 bar for K. fragilis. Similar tendencies were also found for different temperatures. Fig. 3 shows the results for four different temperatures exhibiting similar Sshaped curves. 3.1. Macroscopic effect of compressed carbon dioxide (solvent effect)
A solvent effect of compressed carbon dioxide - which would be a macroscopic effect - can occur when there is a two-phase system with the cells suspended in an aqueous phase and the CO, as the extracting phase (either liquid or fluid). The decrease in cell density (see Fig. 3, halfsquares) at each of the four temperatures is small and cannot account for the observed loss of viability. Even at 13” C, where the greatest effect of cell lysis was observed, just 45% of the cells lysed, whereas the viability dropped by over 99%. Thus this shows that cell death is not necessarily accompanied by cell lysis. This is particularly apparent at temperatures higher than 27”C, where essentially no cell lysis was observed; however, viability dropped to about 1%. The solvent characteristics of compressed CO, depend strongly on its density. A phase change therefore involves a large change in dissolving
40 pressure
60 (bar)
Fig. 2. Influence of CO, pressure on different yeast strains, suspended in buffered, sugar-free medium. Legend: o, Muyveromyces fragilis; l, Saccharomyces cerevisiae; q , Candida utilis. Conditions: cell type, see legend; pressure, variable; temperature, 33” C, volume, 2 ml; agitation, 1500 U min-‘; time of contact, 5 min.
of Biotechnology 39 (1995) 229-237
231
power of the fluid. Consequently, a possible solvent effect would clearly show up at the phase change and, therefore, at the saturation pressure of the fluid. This seems to be the case at 8°C (saturation pressure of pure CO, = 43 bar) and 13” C (saturation pressure = 48 bar) because the major viability drop occurs at the same pressure as the phase change occurs. However, at 28°C (saturation pressure = 69 bar) the major drop of viability occurs at pressures which are 14 bar less than the saturation pressure (Fig. 3). Therefore at 8” C and 13” C a solvent effect must be taken in account as a possible reason for the loss of viability. However, this cannot be the case for temperatures above 18” C where this loss of viability occurred at pressures considerably lower than the saturation pressure and where the CO, is gaseous and thus acts as a very poor extracting phase. Interesting effects are observed for the cell size distribution (see Fig. 4). Gaseous CO, (65 bar, 33°C) shows hardly any effect on the distribution, whereas an important effect of supercritical CO, (85 bar, 33” C) is observed. Although the total cell number remains constant, a significant decrease of large cells and a parallel increase in small cells is observed. One could interpret this phenomena as a simple cell shrinking effect, however, another explanation is also possible. The number of budding cells (G2) of the samples is rather high. Such cells have a diameter of 5 to 5.1 pm (Munch, 1992). Single cells (Gl) are smaller and show a diameter between 3 and 4.5 pm. It could well be that supercritical carbon dioxide provokes a cell separation between daughter and parent cell and increases (in the best case: doubles) the cell number. This would explain, for instance, the measurements that indicated cell numbers higher than 100% (results not shown), which would be difficult to explain by cell shrinking. To quantify this phenomena, the number of cells having a volume larger than 5.6 x lo-” ml, which, assuming spherical shape, corresponds to a diameter of 4.75 pm, were also measured. Fig. 3 shows the data evaluated in this way (full squares). For higher temperatures (> 27” C) there is a huge difference between the total cell number and the number of cells larger than 5.6 x 10-i’ ml. The constant cell density at these tem-
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peratures is a rather surprising effect. The extraction power of CO, to extract, for instance lipids, usually increases when the temperature exceeds the critical temperature of 3P C (Wong and Johnston, 1986; Hammam and Sivik, 1991). However, invoking the explanation of forced cell separation, 100% cell densities would mean a cell loss of up to 50%, which would be a much more reasonable result. Furthermore, the G2 peak has indeed disappeared. It is also difficult to imagine that a cell which has suffered a forced separation could still resist the supercritical solvent. Fig. 4 shows clearly that this is not the case. The viability drop parallels the decrease in the number of
281 K
0
of Biotechnology
cells having a volume larger than 5.6 x lo-” ml or occurs at lower pressures (Fig. 5). For temperatures higher than 27” C cell death occurs at lower pressures (Fig. 3) which must be the result of some other effect. Until now all tentative explanations have been focused on the solvent effect of CO,. However, cell death may also result from CO, acting at the molecular level as dissolved CO,. 3.2. Microscopic effect of compressed carbon dioxide
Solubilization of CO, in a water phase depends on both pressure and temperature. The
286K
(WC)
0
50 100 pressure(bar)
301 K
39 (1995) 229-237
80
50 100 pressure(bar)
316 K
(28°C)
(13°C)
(43°C)
80 $ 60 % zj 40 ._
60 40 20 0 0
50 100 pressure(bar)
0
50 100 pressure(bar)
Fig. 3. Influence of pressure and temperature on the viability and the cell density of Candida utilk after a 5 min exposure to compressed carbon dioxide. Legend: n , % intact cells; 9, % intact cells > 4.75 Km; 0, % viable cells; + , phase change of pure CO,; -, data fitting; -+ , largest gradient of density of supercritical CO,. Conditions: cell type, C. utih; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min-‘; time of contact, 5 min.
A. Isenschmid et al. /Journal of Biotechnology 39 (1995) 229-237 2 5Et6
7E r .Z t g
(a)
20E+6
15E+6 1.OEc6
= t
50Ec5
O.OE+O OE+OO
ZE-11
4E-11
6E-11 cell volume
6E+6
BE-II
IE-10
1Z’E-IO
1
,
5E+6
7 -E a
.Z
14E-10
(ml)
09
4Ec6 t 3E+6
: d
2E+6
t u
1E+6 OE+O OE+OO
ZE-11
4E-11
6E-11 cell volume
BE-1
1
IE-10
WE-10
1.4E-IO
(ml)
Fig. 4. Cell size distribution of treated and untreated cells, which were harvested in (a) the growth phase (8.6 h) and (b) the late growth phase of a batch culture (10.8 h). The number of budding cells (G2) is high compared to the number of single cells (Gl). 5 min of exposure to gaseous CO, has neither an influence on the size distribution nor the cell density. However, supercritical CO2 has a large influence on the size distribution. The cells either shrink or the budding cells get separated from the mother cell (forced cell separation). Legend: 0, freshly harvested, untreated; 0, treated with 65 bar; 0, treated with 85 bar. Arrows indicate the limit of 5.6X lo-” ml for the cell volume. Conditions: cell type, C. utilis; pressure, 65 bar, 85 bar; temperature, 33°C; volume, 5 ml; agitation, 250 U min-‘; time of contact, 5 min.
data of Fig. 3 can be transformed to indicate the dependency on dissolved carbon dioxide and the possible influence of molecular carbon dioxide (Fig. 6). The data obtained once again display typical S-shaped curves. With these graphs socalled LB,, values of dissolved CO, were determined, corresponding to 50% cell survival. The same was done for the density of cells larger than 5.6 X 10-l’ ml and the results plotted in Fig. 7. Fig. 8 shows the LD,, values for the viability and compares it with the ‘LDSO pressures’ from Fig. 3, where 50% of the cells survive. The LB,,-pressure curve shows a very strange behaviour and culminates in a maximum at 28” C. Obviously two different influences concur here to reduce cell
233
viability. Fig. 8 illustrates these two influences: the temperature dependence of the solubility of the carbon dioxide (open squares) and the temperature dependence of the resistance of the yeast (open circles). The combination of these two results is the LD,,-pressure curve (solid squares). Thus, dissolved carbon dioxide seems to be responsible for cell death. At a high temperature ( - 40” C> the threshold value of dissolved carbon dioxide to kill a yeast cell is very low, at a low temperature (- 10” C) the threshold value can be more than twice as high. A possible explanation for the influence of temperature on the cell inactivation could be a purely chemical one, with the rate of inactivation increasing with temperature. With higher temperatures the inactivation reactions get accelerated. However, this hypothesis is difficult to defend by looking at Fig. 9 which shows the temperature dependence of the viablility drawn for three different and constant dissolved CO, concentrations. Furthermore, it was shown by a kinetic study that the viability is not influenced by a simple Arrhenius dependency (results shown in Isenschmid, 1994). In Fig. 9, three curves can be observed for each dissolved-CO, concentration each with a typical inflexion temperature.
E ZL Z d
8
100 80
I
20 f; 8.8 ! ‘. J 0 0
17% ;
20
40
60
80
I 100
% viable cells Fig. 5. Parity plot of the viable cells vs. cell density of cells larger than 4.75 pm after different CO, treatments. The correlation is good, the viability loss however is higher than the cell density loss. Conditions: cell type, C. utilis; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min-‘; time of contact, 5 min.
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20
60
40
of Biotechnology
39 (1995) 229-237
NJ
.
04
dissolved carbon dioxide (g I-‘)
270
280
300
290 temperature
;s
dissolved carbon dioxide (g 1.‘)
Fig. 6. Influence of dissolved CO, on (a) the viability and (b) the density of cells larger than 4.75 pm. For each temperature a ‘threshold solubility’ was found, where the viability and cell density loss increases significantly. Legend: A, 8” C + 13” C; A, 18°C; l , 23’C; W, 28°C; 0, 33°C; 0, 38°C; o, 43°C. Conditions: cell type, C. utilis; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min- ‘; time of contact, 5 min.
60
i c” 2 ) 0 0 B ;u
T
70
-_
60
--
(K)
The viability of yeast cells thus depends on three main parameters, (a) the dissolved carbon dioxide concentration, (b) temperature and (c) the strain used. With respect to the dissolved CO,, as well as to the temperature, a sudden mechanism must be responsible for cell death. With constant temperature a ‘threshold value of dissolved carbon dioxide’ exists where cell death
80
50-40
320
Fig. 8. LD,, pressures for Candida utilis for different temperatures where the viability loss is 50% within 5 min of exposure (solid squares). Open symbols: explanation for the existence of the optimum temperature for yeast resistance. The negative slope of the LD,,-CO, curve (open circles) is less steep than the slope of the Henry coefficient curve (open squares) until 301 K (28°C). Therefore, the LD,, pressure increases until this temperature. Legend: n , LD,, pressure; 0, LD,, doses of dissolved CO,; 0, coefficient of Henry. Conditions: cell type, C. utilk; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min-I; time of contact, 5 min.
7i,
I
CO 310
--
70
30 --
8
20
-_
4A
IO
--
v
280
290 temperature
300
6o 50
a E .9
40
I 320
c’
20
viability
0, 270
In 7
310
(K)
Fig. 7. Cell survival as a function of dissolved CO, and temperature. ‘LD,,-carbon dioxide dissolved’ means the amount of dissolved CO, that provokes the death of 50% of the cells or a cell density drop of 50%, respectively. Legend: 0, LD,, doses of dissolved CO, to cause 50% decrease in viability; 0, LD,, doses of dissolved CO, to cause 50% decrease in density of cells > 4.75 pm. Conditions: cell type, C. utilk; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min -I; time of contact, 5 min.
30
10 0 275
285
295
305
315
temperature(K)
Fig. 9. Influence of temperature on the inactivation of Candida utilis at constant concentration of dissolved carbon dioxide. Legend: 0, 60 g I-’ dissolved CO,; 0, 50 g 1-l dissolved CO,; q , 40 g 1-l dissolved CO,. Conditions: cell type, C. utif~; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min -‘; time of contact, 5 min.
A. Isenschmid et al. /Journal
of Biotechnology 39 (1995) 229-237
Fig. 10. Electron micrograph showing the cell membranes of two different cells of Saccharomyces cereui.siue, both treated fc with 98 bar CO, at 33” C. The upper membrane shows no perturbation, while the lower shows shrinking and aggregation of de xes.
235
min Jtein
236
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suddenly increases. The same was observed for constant concentration of dissolved carbon dioxide, where a ‘threshold temperature’ was found. Thus, it may be asked how molecular CO, reacts with the cell and what kind of inactivation mechanism is involved. 3.3. Tentative inactivation mechanism Carbon dioxide has some unique properties, such as its lipo- and hydro-phility. CO, is relatively soluble in water but is even more soluble in organic solvents such as ethanol (4.5times better) or acetone (9.9-times better). This is the reason why CO, diffuses so easily through, and into, the plasma membrane of micro-organisms and allows them to ‘expire’ the produced carbon dioxide. Clearly the opposite, rapid diffusion into the cell, can also occur. It was shown that the pH decrease of the bulk does not provoke this viability loss (result in Isenschmid, 1994). However, it could be that too much molecular CO, passes through the membrane and lowers the internal pH by exceeding the buffer capacity of the cell pool. The formed bicarbonate as well as molecular CO, can interfere in the cell metabolism (Jones and Greenfield, 1982; Oura et al., 1980; Shkidchenko, 1976). Molecular carbon dioxide can also diffuse into the cell membrane and accumulate there, since the inner layer is lipophilic. The fluidity of such a membrane increases because of the disturbing effect of the foreign molecules and the order loss of the lipid chains (‘anaesthesia effect’). With the fluidity the permeability increases, the membrane fusion characteristics alter (important for cell budding!) and destructuring of essential membrane domains occurs (Miller, 1968; Esplin et al., 1973; Lenaz et al., 1975; Poole-Wilson, 1978; Syapin and Noble, 1979; Pitchard, 1979). Temperature also affects the fluidity of the lipids and causes similar effects to dissolved CO, in the membrane, which could explain the parallelity of the two parameters. The differences in membrane composition (Kotyk and Janacek, 1977) could explain the different behaviour of the three strains tested. The hypothesis that molecular CO, attacks the viability of the cell is supported by the
of Biotechnology 39 (1995) 229-237
electron micrographs (Fig. 10). Approx. 25% of the cells show intact plasma membranes (no shrinking, no aggregation of protein complexes), whereas viability is as low as 2.2%. This suggests that cells which have apparently intact membranes have lost their viability. This is more likely to occur with a microscopic inactivation mechanism than with a solvent effect.
4. Conclusions Cell lysis cannot be the reason for the viability loss of yeast cells in compressed carbon dioxide, since decreases in cell density are never as high as decreases in viability. A solvent effect might be the reason for the viability and cell density loss at low temperatures. At higher temperatures (> 18°C) the influence of the concentration of dissolved CO, appears to be the key parameter, in addition to temperature and the type of microorganism. Among the three yeast tested distinct differences were found. The interesting dependency on temperature and dissolved carbon dioxide, the density loss of cells larger than 5.6 x lo-” ml and the electron micrographs which show membrane perturbations, indicate an ‘anaesthesia effect’ as the main reason for the viability loss. The results also indicate that compressed carbon dioxide can force a budding cell to separate which would explain why cell densities are frequently difficult to interpret. The decrease in the number of cells having a volume larger than 5.6 x lo-” ml may be due to a solvent effect of the compressed CO, at low temperature or due to an ‘anaesthesia effect’ which provokes separation of budding cells.
References Esplin, D.W., Capek, R. and Esplin, B.A. (1973) An intracellular study of the actions of carbon dioxide on the spinal monosynaptic pathway. Can. J. Physiol. Pharmacol. 51, 424436. Haas, G.J., Prescott, H.E., Jr., Dudley, E., Dik, R., Hintlian, C. and Keane, L. (1989) Inactivation of microorganisms by carbon dioxide under pressure. J. Food Safety 9, 253-265. Hammam, H. and Sivik, B. (1991) Fractionation of gluten
A. Isenschmid et al. /Journal
lipids with supercritical carbon dioxide. Fat. Sci. Technol. 93(3), 104-108. Hoyer, G.G. (1985) Extraction with supercritical fluids: Why, how, and so what? Chemtechnology 15440-448. Isenschmid, A. (1994) Effekt von iiberkritischem und unterkritischem Kohlendioxid auf die Viabilitlt von Mikroorganismen. PhD Thesis No. 1257, ETH, Lausanne. Jones, R.P. and Greenfield, P.F. (1982) Effect of carbon dioxide on yeast growth and fermentation. Enyme Microb. Technol. 4, 201-223. Kamihira, M., Taniguchi, M. and Kobayashi, T. (1987) Sterilization of microorganisms with supercritical carbon dioxide. Agric. Biol. Chem. 51, 407-412. Kotyk, A. and Janacek, K. (1977) Biomembranes, Volume 9. Plenum Press, New York, edn. 1, pp. 37 and 43. Lenaz, G., Curatola, G. and Masotti, L. (1975) Perturbation of membrane fluidity. J. Bioenerg. Biomembr. 7, 223-299. Lin, H., Chan, E., Chen, C. and Chen, L. (1991) Disintegration of yeast cells by pressurized carbon dioxide. Biotechnol. Prog. 7, 201-204. Lin, H., Yang, Z. and Chen, L. (1992) An improved method for disruption of microbial cells with pressurized carbon dioxide. Biotechnol. Prog. 8, 165-166. Lin, H., Yang, Z. and Chen, L. (19931 Inactivation of leuconostoc dextranicum with carbon dioxide under pressure. Chem. Eng. J. 52, B29-B34. L’ltalien, Y., Thibault, J. and Leduy, A. (1989) Improvement of ethanol fermentation under hyperbaric conditions. Biotechnol. Bioeng. 33(l), 471-476. Miller, S.L. (1968) Effects of anesthetics on water structure. Fed. Proc. 27(3), 879-883. Munch, T. (1992) Zellzyklusdynamik von Saccharomyces cereuisiae in Bioprozessen. PhD Thesis, ETH, Ziirich.
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Oura, E., Haarasilta, S. and Londonsborough, J. (1980) Carbon dioxde fixation by baker’s yeast in a variety of growth conditions. J. Gen. Microbial., 118, 51-58. Pitchard, J.B. (1979) Toxic substances and cell membrane function. Fed. Proc. 38, 2220-2225. Poole-Wilson, P.A. (1978) Inhibition of calcium uptake by acidosis in the myocardium of the rabbit. Proc. Physiol. Sot. 277, 79P-93P. Rau, G. (1985) Die Anwendung von verdichtetem Kohlendioxid zur Qualitltsverbesserung von Drogen. PhD Thesis, Univ. Saarland, Saarbriicken, Germany. Shkidchenko, A.N. (1976) Effect of carbon dioxide on growth of Candida utills during continuous chemostat cultivation. Mikrobiologiya 45, 67-72. Syapin, P.J. and Noble, E.P. (1979) Studies on ethanol’s effects on cells in culture. In: Majchrowicz, E. and Noble, E.P. (Eds.), Biochemistry and Pharmacology of Ethanol, Plenum Publishing Co., New York, Vol. 1, pp. 521-540. Thibault, J., Leduy, A. and Cot’e, F. (1987) Production of ethanol by Saccharomyces cerevisiae under high pressure conditions. Biotechnol. Bioeng. 30, 74-80. van Eijs, A.M.M., Wokke, J.M.P., Ten Brink, B. and Dekker, K.A. (1988) Downstream processing of fermentation broths with supercritical carbon dioxide. Nice, France, International Symposium on Supercritical Fluids, 17-19.10.1988, pp. 799-805. Wei, C.I., Balaban, M.O., Fernando, S.Y. and Peplow, A.J. (19911 Bacterial effect of high pressure CO, treatment on foods spiked with Listeria or Salmonella. J. Food Protect. 54, 189-193. Wong, J.M. and Johnston, K.P. (1986) Solubilisation of biomolecules in carbon dioxide based supercritical fluids. Biotechnol. Progress 2, 29-39.
of
biotechnology Journal
of Biotechnology
39 (1995) 229-237
The influence of pressure and temperature of compressed CO, on the survival of yeast cells A. Isenschmid, I.W. Marison, U. von Stockar
*
Swiss Federal Institute of Technology, CH-1015 Lazmanne, Switzerland Received
24 August
1994; accepted
17 January
1995
Abstract
In order to study the potential use of supercritical CO, extraction for the recovery of products from yeast cell cultures, the effects of CO, on different yeast strains over a range of pressures and temperatures have been examined. Viability was shown to be dependent on temperature and dissolved CO, concentration, and can be described by a sigmoidal (S-shaped) curve. Cell death was mainly due to an ‘anaesthesia effect’ rather than cell rupture. Important differences in sensitivity were observed for the strains studied, with the following order of resistance: Kluyveromyces fragilis > Saccharomyces cerevisiae > Candida utilis. Keywords: Supercritical
CO,; Extraction; Yeast; Viability; Pressure sensitivity
1. Introduction Supercritical extraction has become an established process, with some industrial and many lab- and pilot-scale applications (Hoyer, 1985). The most important supercritical solvent, especially for food and pharmaceutical applications, is CO,. The characteristics of supercritical carbon dioxide extraction fit well with biotechnological production conditions: mild temperatures, lack of toxicity, good selectivity and ultra-pure products (no solvent residues). Very often product inhibition lowers the efficiency of an industrial bioprocess and to overcome this problem an in situ extraction without cell separation would be desir-
characteristics
* Corresponding author. 016%1656/95/$09.50 0 1995 Elsevier SSDZ 0168-1656(95)00018-6
able. For primary metabolites and biomass production, however, the extraction fluid has to be ‘biocompatible’. In the literature two different opinions exist concerning the biocompatability of compressed carbon dioxide. Some authors proclaim good survival (van Eijs et al., 1988; Thibault et al., 1987; L’Italien et al., 1989), while others speak of a rapid loss of viability in such a solvent (Rau, 1985; Kamihira et al., 1987; Haas et al., 1989; Wei et al., 1991; Lin et al., 1991,1992,1993). To clarify the situation a detailed study of the behaviour of yeast viability in compressed carbon dioxide has been undertaken. We have focused mainly on the parameters of temperature and pressure, since they greatly influence the solvent
Science
B.V. All rights reserved
of the fluid.
A. Isenschmid et al. /Journal
230
2. Materials
2.2. Procedure and analyses
and methods
2.1. High-pressure installation
A pressurizable reactor system has been constructed (Fig. 1) in which the pressure within the reactor is continuously measured by quartz pressure transducer coupled to a Mac IIci computer operating with LabView acquisition/control software. A control algorithm enabled careful control of the compression, holding and decompression time as well as ensuring reproducibility between experiments. The compression rate was 51 bar min-’ and the decompression rate 73 bar min- ‘. The autoclave used to contact the cell suspension with the compressed carbon dioxide consisted of a cylindrical vessel with a volume of 80.9 ml (internal diameter: 22 mm) and a magnetic stirrer. The reactor was made of stainless steel (316 L-steel) which was immersed in a water bath to control the temperature (for further details see Isenschmid, 1994). For all experiments presented here, the CO,inlet was at the base of the vessel in order to sparge the gas through the suspension during compression. 17
of Biotechnology 39 (1995) 229-237
16
15
Three yeast strains, Candida utilis NCYC 9950, Kluyveromyces fragilis NRRL 1109 and Saccharomyces cerevisiae CBS 426, were used as test organisms. Each strain was grown in a 3-l bioreactor under batch conditions as previously described (Isenschmid, 1994). Cells were harvested after the exponential growth phase, washed and re-suspended in fresh medium without glucose and incubated at - 70” C for 24 h, followed by storage in liquid nitrogen (for further details see Isenschmid, 1994). To expose them to compressed carbon dioxide the cells were thawed and split into a control sample of 2 ml and a pressure sample of 2 ml. The control sample received the same treatment as the pressure sample except that it was kept at atmospheric pressure. The viability ratio between treated and untreated cells was called ‘% viable cells’ and the cell density ratio ‘% intact cells’. The viability was determined by counting the number of colony-forming units (CFU) obtained after plating of suitably diluted culture samples on a medium composed (l- ‘) of 10 g yeast extract, Oxoid L21; 20 g Bacto Peptone, Oxoid L37; 20 g glucose, Fluka 49150 and 16 g agar, Oxoid Ll 1. The cell density as well as the cell size distribution were determined with a Coulter counter (Coultronics Model ZM). The number of cells larger than 4.75 pm (5.6 X lo-” ml) was also determined because of a shrinking effect observed after the treatment.
3. Results and discussion
Fig. 1. Schematic representation of the pressurizable reactor configuration. 0, balance (level control); 1, pressurized CO,; 2, thermostat: 5” C; 3, precooling coil; 4, HPLC pump, cooled; 5, valve (computer directed); 6, thermostat of the pressure vessel; 7, preheating coil; 8, choice of compression: sparged or surface gassing; 9, pressure vessel (80.9 ml, stainless steel); 10, magnetic stirrer; 11, temperature controller 5” C; 12, temperature controller (pressure vessel); 13, pressure controller (computer directed); 14, pH probe; 15, pressure reduction valve; 16, decompression valves (computer directed); 17, outlet.
Samples of Kluyveromyces fragilis, Saccharomyces cerevisiae and Candida utilis were exposed for 5 min to compressed carbon dioxide at 33” C and different pressures (Fig. 2). All three yeast strains showed the same tendency: with increasing CO, pressure the viability decreased following a typical S-shaped curve. Near-critical CO, is less harmful than supercritical CO, to all of the cells tested. However, this borderline from harmless to harmful - or the inflexion point of
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this curve - does not necessarily occur at the critical pressure, but is strain dependent. The inflexion points were 53 bar for C. utilis, 61 bar for S. cerevisiae and 67 bar for K. fragilis. Similar tendencies were also found for different temperatures. Fig. 3 shows the results for four different temperatures exhibiting similar Sshaped curves. 3.1. Macroscopic effect of compressed carbon dioxide (solvent effect)
A solvent effect of compressed carbon dioxide - which would be a macroscopic effect - can occur when there is a two-phase system with the cells suspended in an aqueous phase and the CO, as the extracting phase (either liquid or fluid). The decrease in cell density (see Fig. 3, halfsquares) at each of the four temperatures is small and cannot account for the observed loss of viability. Even at 13” C, where the greatest effect of cell lysis was observed, just 45% of the cells lysed, whereas the viability dropped by over 99%. Thus this shows that cell death is not necessarily accompanied by cell lysis. This is particularly apparent at temperatures higher than 27”C, where essentially no cell lysis was observed; however, viability dropped to about 1%. The solvent characteristics of compressed CO, depend strongly on its density. A phase change therefore involves a large change in dissolving
40 pressure
60 (bar)
Fig. 2. Influence of CO, pressure on different yeast strains, suspended in buffered, sugar-free medium. Legend: o, Muyveromyces fragilis; l, Saccharomyces cerevisiae; q , Candida utilis. Conditions: cell type, see legend; pressure, variable; temperature, 33” C, volume, 2 ml; agitation, 1500 U min-‘; time of contact, 5 min.
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power of the fluid. Consequently, a possible solvent effect would clearly show up at the phase change and, therefore, at the saturation pressure of the fluid. This seems to be the case at 8°C (saturation pressure of pure CO, = 43 bar) and 13” C (saturation pressure = 48 bar) because the major viability drop occurs at the same pressure as the phase change occurs. However, at 28°C (saturation pressure = 69 bar) the major drop of viability occurs at pressures which are 14 bar less than the saturation pressure (Fig. 3). Therefore at 8” C and 13” C a solvent effect must be taken in account as a possible reason for the loss of viability. However, this cannot be the case for temperatures above 18” C where this loss of viability occurred at pressures considerably lower than the saturation pressure and where the CO, is gaseous and thus acts as a very poor extracting phase. Interesting effects are observed for the cell size distribution (see Fig. 4). Gaseous CO, (65 bar, 33°C) shows hardly any effect on the distribution, whereas an important effect of supercritical CO, (85 bar, 33” C) is observed. Although the total cell number remains constant, a significant decrease of large cells and a parallel increase in small cells is observed. One could interpret this phenomena as a simple cell shrinking effect, however, another explanation is also possible. The number of budding cells (G2) of the samples is rather high. Such cells have a diameter of 5 to 5.1 pm (Munch, 1992). Single cells (Gl) are smaller and show a diameter between 3 and 4.5 pm. It could well be that supercritical carbon dioxide provokes a cell separation between daughter and parent cell and increases (in the best case: doubles) the cell number. This would explain, for instance, the measurements that indicated cell numbers higher than 100% (results not shown), which would be difficult to explain by cell shrinking. To quantify this phenomena, the number of cells having a volume larger than 5.6 x lo-” ml, which, assuming spherical shape, corresponds to a diameter of 4.75 pm, were also measured. Fig. 3 shows the data evaluated in this way (full squares). For higher temperatures (> 27” C) there is a huge difference between the total cell number and the number of cells larger than 5.6 x 10-i’ ml. The constant cell density at these tem-
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peratures is a rather surprising effect. The extraction power of CO, to extract, for instance lipids, usually increases when the temperature exceeds the critical temperature of 3P C (Wong and Johnston, 1986; Hammam and Sivik, 1991). However, invoking the explanation of forced cell separation, 100% cell densities would mean a cell loss of up to 50%, which would be a much more reasonable result. Furthermore, the G2 peak has indeed disappeared. It is also difficult to imagine that a cell which has suffered a forced separation could still resist the supercritical solvent. Fig. 4 shows clearly that this is not the case. The viability drop parallels the decrease in the number of
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0
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cells having a volume larger than 5.6 x lo-” ml or occurs at lower pressures (Fig. 5). For temperatures higher than 27” C cell death occurs at lower pressures (Fig. 3) which must be the result of some other effect. Until now all tentative explanations have been focused on the solvent effect of CO,. However, cell death may also result from CO, acting at the molecular level as dissolved CO,. 3.2. Microscopic effect of compressed carbon dioxide
Solubilization of CO, in a water phase depends on both pressure and temperature. The
286K
(WC)
0
50 100 pressure(bar)
301 K
39 (1995) 229-237
80
50 100 pressure(bar)
316 K
(28°C)
(13°C)
(43°C)
80 $ 60 % zj 40 ._
60 40 20 0 0
50 100 pressure(bar)
0
50 100 pressure(bar)
Fig. 3. Influence of pressure and temperature on the viability and the cell density of Candida utilk after a 5 min exposure to compressed carbon dioxide. Legend: n , % intact cells; 9, % intact cells > 4.75 Km; 0, % viable cells; + , phase change of pure CO,; -, data fitting; -+ , largest gradient of density of supercritical CO,. Conditions: cell type, C. utih; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min-‘; time of contact, 5 min.
A. Isenschmid et al. /Journal of Biotechnology 39 (1995) 229-237 2 5Et6
7E r .Z t g
(a)
20E+6
15E+6 1.OEc6
= t
50Ec5
O.OE+O OE+OO
ZE-11
4E-11
6E-11 cell volume
6E+6
BE-II
IE-10
1Z’E-IO
1
,
5E+6
7 -E a
.Z
14E-10
(ml)
09
4Ec6 t 3E+6
: d
2E+6
t u
1E+6 OE+O OE+OO
ZE-11
4E-11
6E-11 cell volume
BE-1
1
IE-10
WE-10
1.4E-IO
(ml)
Fig. 4. Cell size distribution of treated and untreated cells, which were harvested in (a) the growth phase (8.6 h) and (b) the late growth phase of a batch culture (10.8 h). The number of budding cells (G2) is high compared to the number of single cells (Gl). 5 min of exposure to gaseous CO, has neither an influence on the size distribution nor the cell density. However, supercritical CO2 has a large influence on the size distribution. The cells either shrink or the budding cells get separated from the mother cell (forced cell separation). Legend: 0, freshly harvested, untreated; 0, treated with 65 bar; 0, treated with 85 bar. Arrows indicate the limit of 5.6X lo-” ml for the cell volume. Conditions: cell type, C. utilis; pressure, 65 bar, 85 bar; temperature, 33°C; volume, 5 ml; agitation, 250 U min-‘; time of contact, 5 min.
data of Fig. 3 can be transformed to indicate the dependency on dissolved carbon dioxide and the possible influence of molecular carbon dioxide (Fig. 6). The data obtained once again display typical S-shaped curves. With these graphs socalled LB,, values of dissolved CO, were determined, corresponding to 50% cell survival. The same was done for the density of cells larger than 5.6 X 10-l’ ml and the results plotted in Fig. 7. Fig. 8 shows the LD,, values for the viability and compares it with the ‘LDSO pressures’ from Fig. 3, where 50% of the cells survive. The LB,,-pressure curve shows a very strange behaviour and culminates in a maximum at 28” C. Obviously two different influences concur here to reduce cell
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viability. Fig. 8 illustrates these two influences: the temperature dependence of the solubility of the carbon dioxide (open squares) and the temperature dependence of the resistance of the yeast (open circles). The combination of these two results is the LD,,-pressure curve (solid squares). Thus, dissolved carbon dioxide seems to be responsible for cell death. At a high temperature ( - 40” C> the threshold value of dissolved carbon dioxide to kill a yeast cell is very low, at a low temperature (- 10” C) the threshold value can be more than twice as high. A possible explanation for the influence of temperature on the cell inactivation could be a purely chemical one, with the rate of inactivation increasing with temperature. With higher temperatures the inactivation reactions get accelerated. However, this hypothesis is difficult to defend by looking at Fig. 9 which shows the temperature dependence of the viablility drawn for three different and constant dissolved CO, concentrations. Furthermore, it was shown by a kinetic study that the viability is not influenced by a simple Arrhenius dependency (results shown in Isenschmid, 1994). In Fig. 9, three curves can be observed for each dissolved-CO, concentration each with a typical inflexion temperature.
E ZL Z d
8
100 80
I
20 f; 8.8 ! ‘. J 0 0
17% ;
20
40
60
80
I 100
% viable cells Fig. 5. Parity plot of the viable cells vs. cell density of cells larger than 4.75 pm after different CO, treatments. The correlation is good, the viability loss however is higher than the cell density loss. Conditions: cell type, C. utilis; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min-‘; time of contact, 5 min.
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20
60
40
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NJ
.
04
dissolved carbon dioxide (g I-‘)
270
280
300
290 temperature
;s
dissolved carbon dioxide (g 1.‘)
Fig. 6. Influence of dissolved CO, on (a) the viability and (b) the density of cells larger than 4.75 pm. For each temperature a ‘threshold solubility’ was found, where the viability and cell density loss increases significantly. Legend: A, 8” C + 13” C; A, 18°C; l , 23’C; W, 28°C; 0, 33°C; 0, 38°C; o, 43°C. Conditions: cell type, C. utilis; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min- ‘; time of contact, 5 min.
60
i c” 2 ) 0 0 B ;u
T
70
-_
60
--
(K)
The viability of yeast cells thus depends on three main parameters, (a) the dissolved carbon dioxide concentration, (b) temperature and (c) the strain used. With respect to the dissolved CO,, as well as to the temperature, a sudden mechanism must be responsible for cell death. With constant temperature a ‘threshold value of dissolved carbon dioxide’ exists where cell death
80
50-40
320
Fig. 8. LD,, pressures for Candida utilis for different temperatures where the viability loss is 50% within 5 min of exposure (solid squares). Open symbols: explanation for the existence of the optimum temperature for yeast resistance. The negative slope of the LD,,-CO, curve (open circles) is less steep than the slope of the Henry coefficient curve (open squares) until 301 K (28°C). Therefore, the LD,, pressure increases until this temperature. Legend: n , LD,, pressure; 0, LD,, doses of dissolved CO,; 0, coefficient of Henry. Conditions: cell type, C. utilk; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min-I; time of contact, 5 min.
7i,
I
CO 310
--
70
30 --
8
20
-_
4A
IO
--
v
280
290 temperature
300
6o 50
a E .9
40
I 320
c’
20
viability
0, 270
In 7
310
(K)
Fig. 7. Cell survival as a function of dissolved CO, and temperature. ‘LD,,-carbon dioxide dissolved’ means the amount of dissolved CO, that provokes the death of 50% of the cells or a cell density drop of 50%, respectively. Legend: 0, LD,, doses of dissolved CO, to cause 50% decrease in viability; 0, LD,, doses of dissolved CO, to cause 50% decrease in density of cells > 4.75 pm. Conditions: cell type, C. utilk; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min -I; time of contact, 5 min.
30
10 0 275
285
295
305
315
temperature(K)
Fig. 9. Influence of temperature on the inactivation of Candida utilis at constant concentration of dissolved carbon dioxide. Legend: 0, 60 g I-’ dissolved CO,; 0, 50 g 1-l dissolved CO,; q , 40 g 1-l dissolved CO,. Conditions: cell type, C. utif~; pressure, variable; temperature, variable; volume, 2 ml; agitation, 1500 U min -‘; time of contact, 5 min.
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Fig. 10. Electron micrograph showing the cell membranes of two different cells of Saccharomyces cereui.siue, both treated fc with 98 bar CO, at 33” C. The upper membrane shows no perturbation, while the lower shows shrinking and aggregation of de xes.
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min Jtein
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suddenly increases. The same was observed for constant concentration of dissolved carbon dioxide, where a ‘threshold temperature’ was found. Thus, it may be asked how molecular CO, reacts with the cell and what kind of inactivation mechanism is involved. 3.3. Tentative inactivation mechanism Carbon dioxide has some unique properties, such as its lipo- and hydro-phility. CO, is relatively soluble in water but is even more soluble in organic solvents such as ethanol (4.5times better) or acetone (9.9-times better). This is the reason why CO, diffuses so easily through, and into, the plasma membrane of micro-organisms and allows them to ‘expire’ the produced carbon dioxide. Clearly the opposite, rapid diffusion into the cell, can also occur. It was shown that the pH decrease of the bulk does not provoke this viability loss (result in Isenschmid, 1994). However, it could be that too much molecular CO, passes through the membrane and lowers the internal pH by exceeding the buffer capacity of the cell pool. The formed bicarbonate as well as molecular CO, can interfere in the cell metabolism (Jones and Greenfield, 1982; Oura et al., 1980; Shkidchenko, 1976). Molecular carbon dioxide can also diffuse into the cell membrane and accumulate there, since the inner layer is lipophilic. The fluidity of such a membrane increases because of the disturbing effect of the foreign molecules and the order loss of the lipid chains (‘anaesthesia effect’). With the fluidity the permeability increases, the membrane fusion characteristics alter (important for cell budding!) and destructuring of essential membrane domains occurs (Miller, 1968; Esplin et al., 1973; Lenaz et al., 1975; Poole-Wilson, 1978; Syapin and Noble, 1979; Pitchard, 1979). Temperature also affects the fluidity of the lipids and causes similar effects to dissolved CO, in the membrane, which could explain the parallelity of the two parameters. The differences in membrane composition (Kotyk and Janacek, 1977) could explain the different behaviour of the three strains tested. The hypothesis that molecular CO, attacks the viability of the cell is supported by the
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electron micrographs (Fig. 10). Approx. 25% of the cells show intact plasma membranes (no shrinking, no aggregation of protein complexes), whereas viability is as low as 2.2%. This suggests that cells which have apparently intact membranes have lost their viability. This is more likely to occur with a microscopic inactivation mechanism than with a solvent effect.
4. Conclusions Cell lysis cannot be the reason for the viability loss of yeast cells in compressed carbon dioxide, since decreases in cell density are never as high as decreases in viability. A solvent effect might be the reason for the viability and cell density loss at low temperatures. At higher temperatures (> 18°C) the influence of the concentration of dissolved CO, appears to be the key parameter, in addition to temperature and the type of microorganism. Among the three yeast tested distinct differences were found. The interesting dependency on temperature and dissolved carbon dioxide, the density loss of cells larger than 5.6 x lo-” ml and the electron micrographs which show membrane perturbations, indicate an ‘anaesthesia effect’ as the main reason for the viability loss. The results also indicate that compressed carbon dioxide can force a budding cell to separate which would explain why cell densities are frequently difficult to interpret. The decrease in the number of cells having a volume larger than 5.6 x lo-” ml may be due to a solvent effect of the compressed CO, at low temperature or due to an ‘anaesthesia effect’ which provokes separation of budding cells.
References Esplin, D.W., Capek, R. and Esplin, B.A. (1973) An intracellular study of the actions of carbon dioxide on the spinal monosynaptic pathway. Can. J. Physiol. Pharmacol. 51, 424436. Haas, G.J., Prescott, H.E., Jr., Dudley, E., Dik, R., Hintlian, C. and Keane, L. (1989) Inactivation of microorganisms by carbon dioxide under pressure. J. Food Safety 9, 253-265. Hammam, H. and Sivik, B. (1991) Fractionation of gluten
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lipids with supercritical carbon dioxide. Fat. Sci. Technol. 93(3), 104-108. Hoyer, G.G. (1985) Extraction with supercritical fluids: Why, how, and so what? Chemtechnology 15440-448. Isenschmid, A. (1994) Effekt von iiberkritischem und unterkritischem Kohlendioxid auf die Viabilitlt von Mikroorganismen. PhD Thesis No. 1257, ETH, Lausanne. Jones, R.P. and Greenfield, P.F. (1982) Effect of carbon dioxide on yeast growth and fermentation. Enyme Microb. Technol. 4, 201-223. Kamihira, M., Taniguchi, M. and Kobayashi, T. (1987) Sterilization of microorganisms with supercritical carbon dioxide. Agric. Biol. Chem. 51, 407-412. Kotyk, A. and Janacek, K. (1977) Biomembranes, Volume 9. Plenum Press, New York, edn. 1, pp. 37 and 43. Lenaz, G., Curatola, G. and Masotti, L. (1975) Perturbation of membrane fluidity. J. Bioenerg. Biomembr. 7, 223-299. Lin, H., Chan, E., Chen, C. and Chen, L. (1991) Disintegration of yeast cells by pressurized carbon dioxide. Biotechnol. Prog. 7, 201-204. Lin, H., Yang, Z. and Chen, L. (1992) An improved method for disruption of microbial cells with pressurized carbon dioxide. Biotechnol. Prog. 8, 165-166. Lin, H., Yang, Z. and Chen, L. (19931 Inactivation of leuconostoc dextranicum with carbon dioxide under pressure. Chem. Eng. J. 52, B29-B34. L’ltalien, Y., Thibault, J. and Leduy, A. (1989) Improvement of ethanol fermentation under hyperbaric conditions. Biotechnol. Bioeng. 33(l), 471-476. Miller, S.L. (1968) Effects of anesthetics on water structure. Fed. Proc. 27(3), 879-883. Munch, T. (1992) Zellzyklusdynamik von Saccharomyces cereuisiae in Bioprozessen. PhD Thesis, ETH, Ziirich.
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Oura, E., Haarasilta, S. and Londonsborough, J. (1980) Carbon dioxde fixation by baker’s yeast in a variety of growth conditions. J. Gen. Microbial., 118, 51-58. Pitchard, J.B. (1979) Toxic substances and cell membrane function. Fed. Proc. 38, 2220-2225. Poole-Wilson, P.A. (1978) Inhibition of calcium uptake by acidosis in the myocardium of the rabbit. Proc. Physiol. Sot. 277, 79P-93P. Rau, G. (1985) Die Anwendung von verdichtetem Kohlendioxid zur Qualitltsverbesserung von Drogen. PhD Thesis, Univ. Saarland, Saarbriicken, Germany. Shkidchenko, A.N. (1976) Effect of carbon dioxide on growth of Candida utills during continuous chemostat cultivation. Mikrobiologiya 45, 67-72. Syapin, P.J. and Noble, E.P. (1979) Studies on ethanol’s effects on cells in culture. In: Majchrowicz, E. and Noble, E.P. (Eds.), Biochemistry and Pharmacology of Ethanol, Plenum Publishing Co., New York, Vol. 1, pp. 521-540. Thibault, J., Leduy, A. and Cot’e, F. (1987) Production of ethanol by Saccharomyces cerevisiae under high pressure conditions. Biotechnol. Bioeng. 30, 74-80. van Eijs, A.M.M., Wokke, J.M.P., Ten Brink, B. and Dekker, K.A. (1988) Downstream processing of fermentation broths with supercritical carbon dioxide. Nice, France, International Symposium on Supercritical Fluids, 17-19.10.1988, pp. 799-805. Wei, C.I., Balaban, M.O., Fernando, S.Y. and Peplow, A.J. (19911 Bacterial effect of high pressure CO, treatment on foods spiked with Listeria or Salmonella. J. Food Protect. 54, 189-193. Wong, J.M. and Johnston, K.P. (1986) Solubilisation of biomolecules in carbon dioxide based supercritical fluids. Biotechnol. Progress 2, 29-39.