751—Electrochemical sterilization of microbial cells

393

Bioelectrochemistry and Bioenergetics, 13 (1984) 393-400 A section of J. Eleetroanal. Chem., and constituting Vol. 174 (1984) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

751-ELECTROCHEMICAL

STERILIZATION

OF MICROBIAL

CELLS

TADASHI MATSUNAGA, YOICHI NAMBA and TOSHIAKI NAKAJIMA Department of Applied Chemistry for Resources, Tokyo University of Agriculture & Technology, Koganei, Tokyo 184 (Japan) (Revised manuscript received January 18th 1985)

SUMMARY The respiratory activity of microbial cells on the surface of a graphite electrode was inhibited by applying a constant potential to the graphite electrode. The respiratory activity of whole cells of S. cerevisiae decreased to 25% of the initial activity after 7 min when the electrode potential was controlled at +0.74 V versus s.c.e. The loss of respiratory activity was also observed for B. subtilis and E. coli. CoA existing in the cell wall was electrochemically oxidized to dimeric CoA and, as a result, the respiration of cells was inhibited. Inhibition of the respiratory activity resulted in the death of the microbial cells.

INTRODUCTION

Sterilization of microbial cells is important in the medical and bioindustrial field. Antibiotics, metabolic inhibitors and bactericides have been employed for this purpose. Microbial cells were also physically sterilized by heat and U.V. irradiation. However, a more effective, selective and safe method is still desired. Various electrochemical methods have been developed for the sterilization of microbial cells. The passage of alternating current through a cell suspension causes inhibition of cell division or results in bacterial death. Toxic substances such as free chlorine [l], Pt complex [2] and H,O, (31 generated by electrolysis were shown to be responsible for the killing effect. The experimental results show that the current affects the cell’s viability indirectly. Recently, the present authors found that electrons are directly transferred from microbial cells to a graphite electrode by applying cyclic voltammetry and differential pulse voltammetry [4,5]. The electron transfer was mediated by CoA in the cell wall. In this paper, electrochemical sterilization of microbial cells based on direct electron transfer between cells and an electrode was attempted. Whole cells of Saccharomyces cerecisiae were attached to the surface of a graphite electrode, and respiratory activity and viability of the microbial cells were controlled by applying a constant potential to the graphite electrode. The mechanism of sterilization. was also studied. 0302-4598/84/$03.00

0 1984 Elsevier Sequoia S.A.

394 EXPERIMENTAL

Materials

Yeast extract was purchased from Difco Laboratories (Detroit, MI) and polypeptone from Kyokuto Pharmaceutical Co. (Tokyo, Japan). Phosphotransacetylase (EC2,3,1,8) was obtained from P.L. Biochemicals (Milwaukee, WI). Coenzyme A was purchased from Sigma Chem. Co. (Saint Louis, MO) and acetylphosphate from Boehringer Mannheim (Mannheim, F.R.G.). Microbial cells Saccharomyces cerevisiae was cultured aerobically at 30°C for 12 h in 100 cm3 of a medium (pH 7.0) containing 4 g of glucose, 1 g of polypeptone, 0.5 g of KH,PO,, and 0.2 g of MgSO, - 7 H,O. Escherichia coli K12 was incubated aerobically at 37°C for 12 h in 100 cm3 of a medium (pH 7.2) containing 0.1 g of glucose, 1 g of bacto-tryptone, 0.5 g of yeast extract and 0.5 g of NaCl. Bacillus subtilis MI112 was cultured aerobically in 100 cm3 of a medium. Apparatus

Figure 1 shows the schematic diagram of the experimental system for the electrochemical regulation of microbial cells. The electrode system consists of a basal-plane pyrolytic graphite electrode (surface area, 0.17 cm*), a counter electrode (platinum wire) and a membrane filter for retaining microbial cells. A triangular potential sweep and constant potential were applied using a potentiostat (Hokuto Denko, Model HA301) and a function generator (Hokuto Denko, Model HB104). The graphite electrode was polished with emery paper (No. 2000) before use in order to remove surface-adsorbed species. The experimental cell was of an all-glass construction, approximately 25 cm3 in volume and incorporated a conventional three-electrode system. The reference electrode was the saturated calomel electrode. The reference electrode was separated from the main cell compartment by immersion in a glass tube ending in a sintered glass frit. Procedure

The cultured cells were suspended in 0.1 M phosphate buffer, and diluted to a final cell concentration of 7.1 X lo6 cells/cm3. One cm3 of cell suspension was dropped on a membrane filter (Toyo membrane filter, Type TM-2, nitrocellulose, 0.45 pm pore size, 25 mm diameter) with slight suction. The microbial cells were retained on the surface of the membrane filter. The membrane filter containing the microbial cells (6.0 X lo5 cells/cm*) was cut to the size of the graphite electrode. Then, the graphite electrode was inserted into the reaction cell filled with 10 cm3 of

395

-0 -0

X-Y recorder 9

Cell Membranefilter

Fig. 1. Schematic diagram of the experimental system for the electrochemical regulation of microbial cells. The microbial cells (6.0 X 10’ cells) were attached to the surface of the graphite electrode. (W) Working electrode; (C) counter electrode; (R) reference electrode.

0.1 M phosphate buffer (pH 7.0), and a constant potential or a sweeping potential was applied to the electrode. Measurement of respiratory activity

The respiratory activity of the microbial cells was determined by the microbial electrode system [6]. After the direct electrochemical reaction of microbial cells, the membrane filter retaining microbial cells was attached to the Teflon membrane of an oxygen electrode (Ishikawa Seisakujo Co., Model A, diameter 1.7 cm, height 7.2 cm, PVC casing). The oxygen electrode was immersed in 50 cm3 of 0.1 M phosphate buffer saturated with oxygen. When the current of the electrode became constant, 0.5 cm3 of 50 mM glucose was added to the buffer solution The relative respiratory activity of the microbial cells could be calculated from the current decrease, a linear relationship between the total respiratory activity of microbial cells and the current decrease. Determination of viable cell numbers

The number of viable cells on the membrane filter was determined by plating suitably diluted samples of a culture and counting the colonies that appeared after 24 h of incubation at 37°C.

0

0.5

1.0

Fig. 2. Cyclic voltammograms of whole cells of S. cereuisiae in 0.1 M phosphate buffer (pH 7.0). The potential was sweeped in the range of 0 to + 1 V. Scan rate was 10 mV s-‘. The potential was cycled two times. Cell concentration was 1.28 X lo8 cells/cm3.

Assay of CoA and dimeric CoA

CoA was determined by the phosphotransacetylase method of Stadtmann et al. [7]. CoA and dimeric CoA were separated by thin-layer chromatography on silica gel using acetic acid-methanol (1: 10) as the developing solvent, and detected by U.V. light. RESULTS AND DISCUSSION

Cyclic voltammograms of microbial cells

Figure 2 shows the cyclic voltammograms of whole cells of S. cerevisiae in the range of 0 to + 1.0 V versus s.c.e. An anodic peak current appeared at + 0.74 V versus s.c.e. in the first scan in the positive direction. Upon scan reversal, no corresponding reduction peak was obtained. The peak currents decrease when the scan was repeated. Therefore, the respiratory activity of the microbial cells was measured by the oxygen electrode after each scan. The respiratory activity of the microbial cells decreased with increasing cycle number, and only 30% of the initial respiratory activity was observed after 5 cycles. The respiratory activity decreased to 30-35% when the potential was cycled 5 times in the range over 0 to +0.6 V, 0 to +0.7 V and 0 to +0.8 V. On the other hand, 85% of the initial respiratory activity was retained after 3 cycles of the potential sweep in the range of 0 to +0.5 V versus

397 100 ,

0

I 3

t (min) 5

I 7

10

Fig. 3. Time-course of respiratory activity of S. cereuisiae during controlled-potential V wsw s.c.e.) in 0.1 M phosphate buffer (pH 7.0).

electrolysis (+ 0.74

s.c.e. The result indicates that the respiratory activity also depends on the potential of the graphite electrode. Inhibition of respiratory activity of microbial cells The respiratory activity of whole cells of S. cerevisiae was directly controlled by the constant-potential electrolysis. Figure 3 shows the time-course of the respiratory activity of cells on the membrane filter when the electrode potential was controlled at +0.74 V versus s.c.e. The respiratory activity decreased and only 25% of the initial activity was observed after 7 min. Therefore, the electrolysis of microbial cells was carried out for 10 min at various potentials. Figure 4 shows the relationship between the respiratory activity and the controlled potential. The respiratory activity decreased with increasing potential. The minimum respiratory activity of whole cells of S. cerevisiae was obtained at + 0.74 V versus s.c.e. However, the activity increased again at more than + 0.80 V uersus s.c.e. Therefore, controlled-potential electrolysis at +0.74 V uersus s.c.e. is suitable for S. cereuisiae. The direct electrolysis was applied to other microbial cells. Table 1 shows the respiratory activity when gram-positive bacteria (B. subtilis) and gram-negative bacteria (E. coli) on the membrane filter were electrolyzed at various potentials. The maximum decrease of respiratory activity was observed at + 0.68 V versus s.c.e. for B. subtilis and + 0.72 V versus s.c.e. for E. coli. Figure 5 shows the relationship between the number of viable cells on the membrane filter and the controlled potentials. T’he viability of the cells was correlated with the respiratory activity. The number of viable cells decreased with increasing potential. The number of viable cells also reached a minimum at + 0.74 V uersus s.c.e. and increased again at more than + 0.80 V versus s.c.e.

uvs.5.c.e.

I

:‘I

0

0.3

0.4

0.5

006

(V) 007

I

0.8

0.9

Fig. 4. Relationship between the respiratory activity of S. cerevisiae and potential during controlledpotential electrolysis for 10 mm in 0.1 M phosphate buffer (pH 7.0). TABLE 1 Respiration activity of various microorganisms at controlled-potential

electrolysis

Microorganisms

Controlled potential (V vs. SCE)

Relative respiration activity (W

B. subtilis

0.68 0.72 0.74

34.0 44.1 58.8

E. Coli

0.68 0.72 0.74

70.7 47.1 49.4

Uvs: s.c.e. (V) 0

0.4

0.5

0.6

0.7

0.8

0.9

Fig. 5. Relationship between the number of viable cells of S. cereuisiue on the membrane filter and potential during controlled-potential electrolysis for 10 min in 0.1 M phosphate buffer (pH 7.0). Each unit represents 10’ cells.

399

Mechanism of sterilization

When the surface of the graphite electrode was covered with a dialysis membrane in order to prevent direct contact between the cells and the electrode, the respiratory activity of whole cells did not decrease. Therefore, the loss of respiratory activity seems to be due to a direct electron transfer between the microbial cells and the graphite electrode. The pH value of the solution around the graphite electrode did not change. Addition of catalase, albumin and cysteine did not change the respiratory activity of microbial cells. These results also deny any bactericidal effect of toxic substances such as H202 and free radicals formed by electrolysis. Recently, it was shown by the present authors that CoA mediates an electron transfer between cells and an electrode [4]. Therefore, the amount of CoA in the cell was enzymatically determined after the cells were electrolyzed for 10 min at + 0.74 V uersus s.c.e. As a result, the CoA content decreased from 45 nmol/lO* cells to 10 nmoles/108 cells. Since it has been reported that CoA is electrochemically oxidized to dimeric CoA with the hanging mercury electrode [8], the product of the electrochemical oxidation was analyzed by thin-layer chromatography. The chromatographic studies were performed on CoA, the purchased dimer CoA, and the electrolysis products of CoA and the exudate of whole cells. The exudate of whole cells was obtained by sonicating whole cells in the 0.1 M phosphate buffer solution. CoA in the cell wall and inside the cell was eluted in the buffer solution by sonication. The R, values were nearly identical (0.4) for the dimeric CoA and the electrolysis products of CoA and the exudate of whole cells. These results suggest that the electrolysis of whole cells at + 0.74 V uersus s.c.e. results in the formation of dimeric CoA in the cell. Next, protoplasts of S. cereuisiae were prepared by treatment Zymolyase-5000 (Kirin Brewery Co., Tokyo, Japan) [4]. During protoplast formation, the CoA content in the cell decreased from 45 nmoles/108 cells to 10 nmoles/lO’ cells. When a constant potential of +0.74 V uersm s.c.e. was applied to the graphite electrode, the protoplasts retained 90% of their initial respiratory activity after 10 min of electrolysis. The amount of CoA in the protoplasts after 10 min, was 8 nmoles/108 cells. The transport enzymes of CoA across the plasmalemma may be destroyed by the preparation of the protoplasts. Anyhow, the CoA remaining in the cell scarcely reacts with the electrode and the initial respiratory activity is almost retained. In conclusion, CoA depletion by electrochemical oxidation causes a decline in respiration, since this coenzyme is involved in the Krebs cycle and in fatty acid oxidation. Inhibition of the respiratory activity results in the death of microbial cells. Although in this paper the cells were attached to the surface of the graphite electrode so as to react efficiently with the electrode, electrochemical sterilization is possible in microbial suspension with the use of graphite or platinum electrodes (unpublished data, Matsunaga et al.). Further developmental studies in our laboratory are directed toward elucidating the exact mechanism of the electrochemical sterilization of microbial cells and applying the electrode system to sterilization of microbial cells in food.

400

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