JOURNAL 01~ I~RMJBNTATION AND B I O ~ N O ~ O Vol. 75, NO. 2, 99-102. 1993

Resistance of Yeast and Bacterial Spores to High Voltage Electric Pulses YOSHIMASA YONEMOTO, l TETSUO YAMASHITA, 1 MASAFUMI MURAJI, 2 WATARU TATEBE, ~-

HIROSHI OOSHIMA, 3 JYOJI KATO, 3 AKIRA KIMURA, 4 AND KOUSAKU MURATA 4.

Division of Food & Beverage Research, Otsuka Chemical Co., Ltd., Kawauchi, Tokushima 771-01,1 Department of Electrical Engineering, 2 Department of Bioapplied Chemistry, 3 Osaka City University, Sugimoto,

Sumiyoshi-ku, Osaka 558, and Research Institute for Food Science, Kyoto University, Ufi, Kyoto 611, 4 Japan

Received 8 September 1992/Accepted 19 November 1992

Spores of a yeast, Saccharomyces cerevisiae, and a bacterium, Bacillus subtUis, were exposed to high voltage electric pulses. The vinbillties of spores and vegetative cells of the yeast were significantly decreased after the electric pulse treatment, and some of the spores and almost all of the cells were stained red with an agent, phlox- ine B. On the other hand, (endo) spores of the bacterium were highly resistant to the electric pulses and little decrease in viability was observed, although the viability of vegetative cells was sharply lowered. The results revealed marked structural and/or biochemical differences between eukaryotic and prokaryotic spores.

As described previously (1), microbial spores are excep- tionally resistant to extreme environments such as high tem- perature, high osmotic pressure, high and low pHs, toxic compounds and mechanical shocks compared with vegeta- tive cells, although they have proteins (enzymes), nucleic acids, membranes and structures basically similar to those of vegetative cells. Our recent studies also indicated that yeast spores contain all of the enzymes present in vegeta- tive cells at almost the same activity levels (2), and that bacterial (endo)spores are highly resistant to lytic en- zymes produced by microbes unless they are physically damaged (1).

These structural and biochemical properties of micro- bial spores suggest that they can be used as a biocatalyst in place of vegetative cells, and in fact we have already used yeast spores as a biocatalyst for the continuous dephos- phorylation of p-nitrophenylphosphate in a bioreactor system (3). In order to use the various enzymes in micro- bial spores, efficient methods of inducing enzyme activ- ities in the spores must be developed. A few reports on the action of electric pulses on the viability and perme- ability of vegetative ceils of yeast and bacteria have been published (4, 5). An analysis of the effect of electric pulses revealed that they caused reversible loss of permeability (5, 6); this phenomenon has been utilized for the intro- duction of genetic materials into microbial cells (7, 8). However, the effect of electric pulses on microbial spores has not been investigated thus far. As a first step towards the preparation of enzymatically active microbial spores, we investigated the effect of electric pulses on the structure and viability of yeast and bacterial spores.

MATERIALS AND METHODS

Slmres and vegetative cells Cells of a yeast, Saccha- romyces cerevisiae 4011, and a bacterium, Bacillus subtilis no. 1 (1), were aerobically grown at 30°C for 20h in 100 ml of YPD (2.0% glucose, 1.0% bactopeptone, 0.5% yeast extracts: pH 5.0) and NS (0.5% meat extracts, 1.0% peptone, 0.5% NaCI: pH 7.2) media, respectively. Bac- topeptone and yeast extracts were purchased from Difco

* Corresponding author.

Laboratories, Detroit, Michigan, USA. The cells (vegeta- tive cells) were collected, washed once in chilled 0.85% NaC1 and then suspended in the same solution to make a concentration of 3-5 x l0 s cells/ml. Spores of the two strains were prepared by the same procedures as those described previously (1, 3). Spores were also suspended in 0.85% NaCI to give a concentration of approximately l0 s spores/ml. Yeast spores form aggregates in aqueous con- ditions; the yeast spore suspension was thus vigorously stirred before use.

Electric pulses The electric circuit used for the pulse treatment of vegetative cells and spores is illustrated in Fig. 1. The cell or spore suspension (2.0 ml) was placed between the two electrodes in the chamber and pulsed 3 or 10 times for 90 ps each time with an interval of 30 s between pulses. These conditions are nearly the same as those used for the transformation of yeast cells (8). The detailed pulse conditions are described under Results and Discussion.

Viability Vegetative cells and spores before the after treatment with electric pulses were diluted with 0.85% NaC1 and spread on agar (1.5%) plates of YPD (for the

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FIG. 1. Electric circuit for electroporation. The conditions for the pulse experiments are described in the text. S1, Switch for charg- ing condenser; $2, switch for discharge to chamber.

100 YONEMOTO ET AL. J. FEINT. BXO~G.,

yeast) and NS (for the bacterium) media. Plates were incu- bated at 30°C and colonies on the plates were counted.

Phloxine B uptake In order to discern any changes in the permeabilities of vegetative cells and spores of the yeast after the electric pulse, 20/~1 of 10 mg/ml phloxine B (Sigma Chemical Co., St. Louis, MO, USA) dissolved in 0.85% NaCI was added to 0.2 ml of spore or vegetative cell suspension before and exactly 1 rain after the electric pulse treatment (8). After incubation at room temperature for 2 min, the number of spores or cells stained with the red dye agent was counted by using an optical microscope.

M i c r o g r a p h s Yeast spores were treated with 2.0% glutaraldehyde for 1 h at room temperature, dried on a crit- ical dryer (Hitachi CTD-1) and then examined by scanning electron microscope (Hitachi S-450). Yeast spores after in- cubation with phloxine B were directly examined as above. Bacterial spores were negatively stained with phospho- tungstic acid in 2% KOH, and an electron micrograph was taken by a transmission electron microscope (Hitachi H-700).

RESULTS AND DISCUSSION

Electric pulse treatment The electric circuit for the pulse experiments is shown in Fig. 1. Using electrical energy from a dc power source the condenser (capacitance, C = 8 pF) was first charged by closing S1. The energy was then discharged to the vegetative cell or spore suspension, which was placed between the two electrodes in the cham- ber, by opening S1 and closing $2. The waveforms of the voltage between the electrodes (Fig. 2A) and the current through the suspension (Fig. 2B) were observed on a dig- ital storage oscilloscope (DCS-9300 Kenwood). The initial field strength was calculated to be 5,400 V/cm, since the distance between the two electrodes was 0.5 cm (Fig. 1). I f a suspension contains components of resistance R and the circuit is ideally regarded as an RC series circuit, the volt- age V between the two electrodes after the circuit is closed is expressed as:

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V= Vo exp ( - t /r) , Where r is the time constant (r =RC) that indicates the period of an electric pulse applied to the suspension. The time constant r was determined to be 90 ps by approximat- ing the voltage waveform with the least square method to an ideal exponential damped wave through Vo at t=0 . In order to elucidate the behaviour of the yeast and bacterial spores toward high voltage electric pulses, 90 ps pulses with voltage and current waveforms as shown in Fig. 2 were applied to the spore and vegetative suspensions, with an interval of 30 s between pulses.

E f f e c t o f e l e c t r i c p u l s e o n y e a s t s p o r e s Spores and vegetative cells of S. cerevisiae 4011 were highly suscepti- ble to the high voltage electric pulse treatment, their viabil- ities being greatly reduced (more than 90°/~) after pulses were repeated ten times (Fig. 3A). The spores before and after electric pulse treatment were examined by scanning electron microscope (Figs. 4A-1 and A-2). Little structural change was observed between the two spore preparations. However, spores with many small holes on their surfaces were found only in the spore population after the electric pulse treatment (Fig. 4A-2, indicated by an arrow); the fre- quency of appearance of such spores was approximately 0.1% under the conditions employed. Although no direct

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FIG. 3. Effect of electric pulses on vegetative cells and spores. (A) Effect on S. cereviaiae. Vegetative cells and spores were dectri- caliy pulsed by the method described in text. Viability and phloxine B uptake are expressed as a function of the number of times electric pulses applied to the suspension. Phloxine B uptake represents the percentage of spores or vegetative cells stained with the agent. ) , Viability of spores; O, viability of vegetative cells; m, phloxine B up- take by spores; ~3, phloxine B uptake by vegetative cells. (B) Effect on B. subtilis. Viability of vegetative cells and spores after electric pulses was determined and expressed as above. ) , Viability of spores; o, viability of vegetative cells.

VoL. 75, 1993 RESISTANCE OF MICROBIAL SPORES TO ELECTRIC PULSES 101

A 1 2

B

FIG. 4. Effect of electric pulses on structure and uptake of phloxine B of yeast spores. (A) Scanning electron micrographs of yeast spores before [1] and after [2] treatment with electric pulses three times. A beehive-like spore is indicated by an arrow. Bars represent 1.0 pm in length. (B) Photographs of spores stained with phloxine B before [1] and after [2] treatment with electric pulses three times. Arrows in B2 indicate spores stained red by phloxine B. Bars represent 6.0/an in length.

evidence was obtained, such beehive-like spores seemed to be one of the products of the high voltage electric pulse treatment.

Vegetative cells after the electric pulse treatment were efficiently stained with phloxine B, an agent often used for biostaining (8) (data not shown); the number of cells stained with the agent increased proportionally with the increase in the non-viable cell number (Fig. 3A), thus indi- caring that phloxine B could penetrate only into non- viable cells. Yeast spores after the electric pulse treatment were also found to be stained red in the presence of phlox- ine B (Fig. 4B-2). However, the number of spores stained with the agent was not proportional to that of non-viable spores (Fig. 3A). Judging from the size of spores presented in the scanning electron micrograph (Fig. 4A), the spore preparation used in this study contained no vegetative cells. Therefore, the non-proportional result observed on the numbers of non-viable and phloxine B-stained spores was presumably due to differential damage done to the spores by the electric pulses, since yeast spores have a ten- dency to form aggregates in aqueous solutions and despite vigorous stirring before use the aggregations were not eliminated, even after the electric pulse treatment (data not shown). The pldoxine B uptake results indicate that the electric pulse method can render spores, as well as vegetative cells, permeable to some chemicals and that

this method may be applicable to the preparation of enzy- matically active spores for use as a biocatalyst. The activa- tion of spores by this method and other physicochemical approaches will be reported elsewhere.

Effect of electric pulse on bacterial spores Spores and vegetative cells of B. subtilis no. 1 were also treated by electric pulses (Fig. 3B). Contrary to the case of the yeast S. cerevisiae (Fig. 3A), the bacterial spores were highly resistant to the treatment and Httle decrease in viability was observed, although vegetative cells sharply lowered their viability. Electron microscopic observation of spores before and after the electric pulse treatment revealed that they were structurally indistinguishable from each other. However, spores after the treatment had cracks on their surfaces (Fig. 5B-2, indicated by arrows) and black granules initially contained in the spores as 2-3 particles (Fig. 5A-2) were crushed, increasing in numbers. When the bacterial spores were ultrasonically treated, the granules were completely shattered into many small particles with a concomitant decrease in viability (1). Therefore, the repeated application of electric pulses may cause a de- crease in the viability of bacterial spores.

Thus, the high voltage electric pulse experiments indi- cated that bacterial (endo) spores are highly resistant to physical shocks and are structurally and/or biochemically different from yeast spores. We have obtained enzyme(s)

102 YONEMOTO ET AL. J. FERMENT. BIOENG.,

! 2

FIG. 5. Effect of electric pulses on structure of bacterial spores. Electron micrographs of spores were taken before (A) and after (B) treatment with electric pulse three times. One of the spores in AI and BI is magnified and shown in the right panel. Arrows in B2 indicate cracks formed after electric pulses. Bars represent 1.0/~m in length.

that induce lysis in bacterial spores (1). Recently, we have also found a bacterium producing enzyme(s) responsible for the lysis of yeast spores (unpublished data). The use of these enzymes that lyse yeast and bacterial spores may facil- itate a structural comparison of prokaryotic and eukary- otic spores.

REFERENCES

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2. Shigematsu, T., Matsutani, K., Fukuda, Y., Kimqra, A., and Mnrata, K.: Enzymes and germination of spores of a yeast Sac- charomyces cerevisiae. J. Ferment. Bioeng., (in press).

3. Shigematsu, T., Kimura, A., and Mnrata, K.: Use of yeast spores

as a biocatalyst. J. Ferment. Bioeng., 73, 467-470 (1992). 4. Sale, A. J. H. and Hamilton, W. A.: Effects of high electric fields

on microorganisms. I. Killing of bacteria and yeasts. Biochim. Biophys. Acta, 148, 781-788 (1967).

5. Jacob, H.-E., Fiirster, W., and Berg, H.: Microbiological impli- cations of electric field effects. II. Inactivation of yeast cells and repair of their cell envelope. Z. AUg. Mikrobiol., 21, 225-233 0981).

6. Kinoshita, K. Jr. and Tsong, T. Y.: Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature (London), 268, 438-441 (1977).

7. Shivarova, N., F6rster, W., Jacob, H.-E., and Grigorova, R.: Microbiological implications of electric field effects. VII. Stimula- tion of plasmid transformation of Bacillus cereus protoplasts by electric field pulses. Z. Allg. Mikrobiol., 23, 595-599 (1983).

8. Delorme, E.: Transformation of Saccharomyces cerevisiae by electroporation. Appl. Environ. Microbiol., 55, 2242-2246 (1989).