ISSN 00181439, High Energy Chemistry, 2012, Vol. 46, No. 3, pp. 212–215. © Pleiades Publishing, Ltd., 2012. Original Russian Text © A.I. Maksimov, I.K. Naumova, A.V. Khlyustova, 2012, published in Khimiya Vysokikh Energii, 2012, Vol. 46, No. 3, pp. 259–262.

PLASMA CHEMISTRY

Sterilization of Solutions by Underwater Electric Discharges A. I. Maksimova, I. K. Naumovab, and A. V. Khlyustovaa a

Institute of Chemistry of Solutions, Russian Academy of Sciences, Akademicheskaya ul. 1, Ivanovo, 153045 Russia Email: kav@iscras.ru b Ivanovo State Academy of Agriculture, ul. M. Ryabininoi 45, Ivanovo, 153012 Russia Received October 17, 2011; in final form, November 16, 2011

Abstract—A possibility of using lowvoltage underwater pinhole discharge for sterilization of aqueous solu tions has been considered. It was experimentally established that the time of complete sterilization of a solu tion is determined by the type of bacterial culture and the number of diaphragms. The posteffect phenome non has been revealed. The sterilizing property of the solution persists for a long time. An analysis of the results has allowed for identification of the main sterilizing factors, which include chemically active species, dischargeinduced shock and sound waves, and UV radiation. DOI: 10.1134/S0018143912030058

Sterilization of water, aqueous solutions, and med ical tools placed in solution is an important factor in human and veterinary medicine. Cold gas plasma has unique characteristics, since it contains a variety of biochemical active agents, such as UV radiation, OH radicals, and oxygen atoms. However, the plasma–liq uid system is neither widespread and nor wellunder stood as compared with classical lowpressure cold plasma with metal electrodes [1]. A number of studies on the use of cold plasma in medicine have been reported [2–8]. Pulsed spark dis charge in water was used for sterilization of aqueous solutions in [2, 3]. It was shown that as short as 30s gas discharge treatment renders the solutions sterile. KamgangYoubi et al. [4] discussed the use of gliding arc in humid air as a means of sterilizing aqueous solu tions. They noted the socalled posteffect, the dura tion of which varied from 20 to 30 min depending on the time of plasma contact with water (2 to 10 min). A wealth of studies [5–8] concerned the effect of dielec tric barrier discharge on various objects. Studies showed that the main sterilizing factors are charged particles, electric field, the microscale thermal effect, and vacuum ultraviolet radiation generated by this type of discharge. In this paper, we present the results of investigation of the sterilizing effect of lowvoltage underwater dis charge.

shown that the generation of diaphragm discharge is accompanied by partial destruction of the electrodes and the transition of ions into solution. It is known that the presence of certain ions in solution can pro mote both growth and death of bacteria. For example, the sterilizing effect of hydrogen peroxide and active oxygen radicals is enhanced in the presence of Ag, Cu, and Zn ions and declines when Fe and Mn are added. Mo, Ni, and Al ions have an additive effect with H2O2. For this reason, graphite rods of 5 mm in diameter were selected as working electrodes. The test materials were Escherichia coli M17 (E. coli) and Staphylococcus aureus strains. The bacte rial culture concentration varied from 104 to

EXPERIMENTAL Lowvoltage ac discharge of the diaphragm type, which is excited in an electrolyte solution, was used in experiments (Fig. 1). The discharge current was varied in the range of 20–120 mA, and the operating voltage did not exceed 1500 V. The gasdischarge treatment time varied from 1 to 10 min. Earlier experiments have 212

4

3 2

7 5

6

4

1

Fig. 1. Schematic of the laboratory sterilization cell: (1) working solution, (2) cover, (3) thermometer, (4) elec trodes, (5) quartz ampule, (6) stirrer, and (7) pinholes.

STERILIZATION OF SOLUTIONS BY UNDERWATER ELECTRIC DISCHARGES t, min 17 16 15 14 Staph. 13 12 11 10 9 8 7 6 5 E. coli 4 3 2 1 0 104 Colonies/mL

213

C, Colonies/mL

1000

100 2 10

1

1 0

Fig. 2. Time of complete sterilization of solution for differ ent bacterial cultures.

106 Colonies/mL. Normal saline (0.9% NaCl) was used as the working electrolyte. After discharge treatment, the active medium of 0.1 mL volume was inoculated from the containers on the surface of Endo’s medium in accordance with aseptic rules, being distributed evenly over the entire surface with a sterile spatula. Then, the cultures were incubated in a thermostat at 37°С. Colonyforming units were counted after 24 h on the basis of cultural properties characteristic of Escherichia coli and Sta phylococcus aureus colonies on Endo’s medium. RESULTS Figure 2 illustrates the influence of the nature of the bacterial culture on the time of complete steriliza tion of infected solution by diaphragm discharge treat ment for initial inoculum concentrations. The effi ciency of gas discharge treatment of staphylococcus containing solutions is well below (by a factor of 3–4) that in the case of E. coli. The diagram in Fig. 2 shows that 3–6min treatment completely suppress the growth of viable cells in the case of E. coli with a con centration of 104–105 Colonies/mL, whereas this effect using Staphylococcus aureus as a bacterial cul ture is achieved only after 12–16 min of treatment (for its concentration in the solution of 104–105 Colo nies/mL, respectively). The presumable reason for this phenomenon is a difference in cell wall structure between the test microbial cells. Thus, the wall thickness of staphylo coccus is 15–20 µm and that of E. coli is 10 µm. Bac Vol. 46

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40

60 80 t, min

100

120

Fig. 3. Discharge treatment posteffect on the amount of (1) E. coli and (2) Staphylococcus aureus bacterial cultures in the solution.

105 Colonies/mL

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terial walls have highly individual chemical properties. For example, the E. coli cell wall has the lipoprotein nature, it includes a large amount of aromatic amino acids, arginine, and proline, whereas the staphylococ cus “shell” is composed of a glycerophosphate proteid complex, which is more stable. The posteffect, the existence of a sterilizing activity after turning off the discharge, was observed experi mentally. The behavior of Escherichia coli and Staphy lococcus aureus cells in the saline after its shortterm diaphragm discharge treatment was investigated. The treatment time was chosen in such a way that full ster ilization of the solution was not achieved during this period of time, so that a certain amount of viable bac teria remained in its volume. Figure 3 depicts cell sur vival curves. From these data it is seen that within a few minutes after cessation of gas discharge treatment, the concentration of bacterial cells in the solution decreased by about half. This suggests that after switching off the discharge, changes that we qualify as posteffect continue in the solution. The figure also shows that after a sharp decrease in the amount of bac terial cultures in the solution within a short time, the death process slows down over 30–40 min for both types of bacteria. This “bacteriostatic effect” is explained in terms of the fact that enhancement of destructive factors increases the immunity of bacteria. The sterilizing activity of the solution persists for 7 days. The number of pinholes affects the time taken to achieve complete sterility of the solution (Table). As can be seen from the data in the table, an increase in the number of pinholes reduces the time of treatment to achieve complete sterility of the solution.

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Influence of the number of pinholes on the sterilizing properties of discharge and the level of sterility of the solution Number of pinholes, pc

Treatment time, min

1 1 50 50

10 5 10 5

It is known that excitation of electric discharge generates a large amount of chemically active species in solution and in the liquid phase. We performed a set of experiments to elucidate the mechanism of action of underwater discharge on a bacterial culture. Patho genic and nonpathogenic bacterial cultures were placed in the working solution pretreated with dia phragm discharge (1–3 min) (Fig. 4). The results show that in the case of the nonpathogenic bacteria E. coli, the solution acquired sterility within 1 h after placing the bacteria in the solution. In the case of pathogenic Staphylococcus aureus bacteria, the effect of complete sterilization was observed only after 2 h. Thus, it can be concluded that destruction of the body of nonpatho genic bacteria is due to the interaction of chemically active species present in the solution. This is insuffi cient for breaking or destroying the body of pathogenic bacteria because of thicker walls of the bacteria. DISCUSSION Definitely, one of the sterilizing factors of dia phragm discharge is chemically active species gener ated in the solution by the action of electrical dis C, Colonies/mL 10000 1000 100 1 10 1 2

0.1 0.01 1E–3 0

20

40

60 80 t, min

100

120

Fig. 4. Effect of preliminary gas discharge treatment on the survival of (1) Staphylococcus aureus and (2) Escherichia coli M17.

Initial concentration, Colonies/mL

E. coli content after treatment

106 104 106 104

Traces 0 0 0

charge, such as the H and OH radicals, the solvated electron, and H2O2. In the case of underwater electri cal discharge, the active zone in which the generation of active species takes place is a thin layer of solution in contact with the plasma bubble. We believe that the most chemically reactive species is the hydroxyl radi cal. Assuming that the primary active particles convert into OH radicals according to the following mecha nism [9]: H2O

OH + H,

H2O* + e H2O* + e

O– + OH,

H– + OH + e OH*,

H2O+ + H2O

H3O+ + OH,

we suggest that hydroxyl radicals can react with neigh boring (adjacent) organic molecules, leading to the chain process and, thereby, destroying bacterial DNA molecules, depolarizing nucleic acids, and breaking other biologically active substances. Hydrogen perox ide, the combination product of OH radicals, can readily penetrate across the cell membrane, causing the death of the bacteria [10]. During operation of diaphragm discharge, instabil ity due to overheating and breakdown appear in the pinhole area, wherein the breakdown is accompanied by a high shock pressure (about 1000 atm) and emer gence of electric field with a high strength (~105 V/cm) and a high local temperature (about 105 K in the pinhole area). According to published data [11, 12], a pressure drop greater than 1.7 MPa leads to a local increase in temperature to 100°С, whereas the temperature of the surrounding solution increases only by 10°С. Mechanical rupture of a bac terial cell requires a pressure of about 0.6–1.3 MPa. Thus, the shock wave induced by the collapse of the plasma bubble leads to mechanical destruction of bac teria. In addition to the factors discussed above, we find that the discharge radiation, namely, its UV part, also contributes to the sterilization process. Primarily, the emission due to OH radicals in the range of 306– 309 nm is meant. The depth of penetration of UV radiation of this range to water is 25 cm [13]. Thus, the cumulative effect of all the sterilizing fac tors of diaphragm discharge results in deactivation of HIGH ENERGY CHEMISTRY

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STERILIZATION OF SOLUTIONS BY UNDERWATER ELECTRIC DISCHARGES

both types of bacterial cultures and in complete steril ization of the solution. CONCLUSIONS Based on our experimental results and published data, we can conclude that the sterilizing factors of diaphragm discharge have different effects on different types of bacterial cultures. Chemically active species are mainly responsible for the damage to a nonpatho genic microbial cell. In the case of pathogenic bacte ria, shock and sound waves and UV radiation have the crucial effect, leading to a mechanical rupture of the bacterial cell. REFERENCES 1. Kutepov, A.M., Zakharov, A.G., and Maksimov, A.I., VacuumPlasma and Plasma–Solution Modification of Polymer Materials, Tsivadze, A.Yu., Ed., Moscow: Nauka, 2004, p. 496. 2. Takai, O., Pure Appl. Chem., 2008, vol. 80, no. 9, p. 2003. 3. Akishev, Yu., Grushin, M., Karalnik, V., Trushkin, N., Kholodenko, A., Chugunov, A., Kobzev, E., Zhirkova, N., Irkhina, I., and Kireev, G., Pure Appl. Chem., 2008, vol. 80, no. 9, p. 1953.

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4. KamgangYoubi, G., Herry, J.M., BellonFontaine, M.N., Brisset, JL., Doubla, A., and Naitali, M., Appl. Envi ron. Microbiol., 2007, vol. 73, no. 15, p. 4791. 5. Garamoon, A.A., Elkashar, F.F., Nossair, A.M., and Kotb, E.F., Plasma Source Sci. Technol., 2002, vol. 11, no. 3, p. 254. 6. Morgan, N.N., Elsabbagh, M.A., Desoky, S., and Gar amoon, A.A., Eur. Phys. J.: Appl. Phys., 2009, vol. 46, no. 3, p. 31001. 7. Fridman, G., Brooks, A.D., Balasubramanian, M., Fridman, A., Gustol, A., Vasilets, V.N., Ayan, H., and Friedman, G., Plasma Process. Polym., 2007, vol. 4, no. 4, p. 370. 8. Cooper, M., Fridman, G., Staak, D., Gustol, A., Vasi lets, V.N., Anandan, S., Cho, Y.I., Fridman, A., and Tsapin, A., IEEE Trans. Plasma Sci., 2009, vol. 37, no. 6(1), p. 866. 9. Pikaev, A.K., Kabakchi, S.A., and Makarov, I.E., High Temperature Radiolysis of Water and Aqueous Solutions, Moscow: Energoatomizdat, 1988. 10. Dobrynin, D., Fridman, G., Friedman, G., and Frid man, A., New J. Phys., 2009, vol. 11, no. 11, p. 115020. 11. Vilkov, K.V. and Nagel’, Yu.A., Pis’ma Zh. Tekh. Fiz., 2004, vol. 30, no. 5, p. 88. 12. Vilkov, K.V., Grigor’ev, A.L., Nagel’, Yu.A., and Uvarova, I.V., Pis’ma Zh. Tekh. Fiz., 2004, vol. 30, no. 7, p. 48. 13. Zolotarev, V.M., Morozov, V.N., and Smirnova, E.V., Handbook of Optical Constants of Natural and Technical Media, Leningrad: Khimiya, 1984.