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Sterilization by H2O2 droplets under corona discharge
Journal of Electrostatics 56 (2002) 173–187
Sterilization by H2O2 droplets under corona discharge Mitsuo Yamamoto*, Masateru Nishioka, Masayoshi Sadakata Department of Chemical System Engineering, University of Tokyo, Tokyo 113-8656, Japan Received 14 April 2001; received in revised form 8 September 2001; accepted 13 October 2001
Abstract A new sterilization method that combines an injection of hydrogen peroxide droplets (30 wt%) by spraying and a corona discharge is very effective for high-speed sterilization. Ultraviolet light, gaseous species and fine H2O2 droplets were considered to be important factors of this sterilization method. In order to investigate the sterilization mechanism, these three factors were examined. It was shown that ultraviolet light was not effective for sterilization. Among the gaseous species investigated, positive ions were proved to be effective through experiments using electric fields. A quadrupole mass spectrometer was used to measure positive ions. It was confirmed that H2O2 was decomposed and the number of H2O2 droplets was reduced. H2O2 droplet size was measured by a laser scattering method, CNC (condensation nuclei counter) and DMA (differential mobility analyzer). It was found that droplets larger than 3 nm still existed even if the droplet size was reduced. It was concluded that both fine H2O2 droplets and positive ions had important effects on sterilization. r 2002 Elsevier Science B.V. All rights reserved. Keywords: Sterilization; Hydrogen peroxide; Discharge; Ion; Droplet
1. Introduction In the food industry and the medical field, sterilization is one of the most important processes for guaranteeing the safety of foods or medicines. Many sterilization methods have been investigated so far [1], including autoclaving, chemical treatment with ethylene oxide gas ((CH2)2O) [2], hydrogen peroxide [3,4] and ozone gas treatment, radiation by ultraviolet light [5], gamma ray and electron *Corresponding author. Tel./fax: +81-3-5841-7363. E-mail address: [email protected] (M. Yamamoto). 0304-3886/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 8 8 6 ( 0 1 ) 0 0 1 9 5 - 4
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beam. However, each method has advantages and disadvantages. In the autoclaving method, a long sterilization time is required because it is a batch process. In the chemical methods, residual chemical substances have adverse effects in application, so their amounts should be reduced as much as possible. Even though the sterilization ability of ultraviolet radiation is very strong, heavy instruments are required as the shielding system. Hence, a new high-speed and secure sterilization process is urgently required. In previous works, many discharge methods were examined for use in sterilization. Glow discharge plasma at atmospheric pressure was used for the sterilization of contaminated matter [6]. On the other hand, a pulsed corona discharge was utilized for the sterilization of microorganisms in water [7,8]. In those methods, the impact of the discharge and the chemical species produced by it were thought to be the main causes of the high sterilization rate. The active species produced by the pulsed corona discharge were determined by optical emission spectral analysis [9] to be O, H and OH radicals. A new method of dry-type sterilization, aiming at high-speed and secure sterilization, was examined [10]. This sterilization method utilized hydrogen peroxide and a pulsed corona discharge under atmospheric pressure. In this study, a sterilization method using a corona discharge with H2O2 droplets was studied and the effective species in this process were examined by various methods. This sterilization method is expected to be applied to the food packaging field. At present, plastic films that are used for packaging are sterilized by soaking in hydrogen peroxide. Thus, it takes a long time to sterilize all bacteria and remove residual H2O2 by washing with water. If a sterilization method using a corona discharge with H2O2 droplets were established, the sterilization time would be reduced. The main objective of this study is to identify the effective species for sterilization in order to confirm the safety of this sterilization method.
2. Experimental apparatus and method The experimental apparatus is shown in Fig. 1. This apparatus consists of a discharge chamber and a sterilization chamber for optical measurement. Both chambers are made of acrylic resin. The discharge chamber is shown in Fig. 2. This chamber consists of coaxial cylindrical electrodes where a 1/4 inch stainless-steel pipe is installed at the center of the quartz tube as a high-voltage electrode and aluminum foils are wound outside the quartz tube as an earth electrode. The inside diameter of the quartz tube is 17 mm. The length of the earth electrode is 120 mm. The H2O2 droplets are supplied together with argon gas by a nebulizer (Kinoshita J-753) and a corona discharge is formed in the discharge chamber. The concentration of H2O2 droplets is 30 wt%. The inside diameter of the sterilization chamber is 110 mm and the height is 300 mm. A bio-indicator is installed at the bottom of the sterilization chamber. A distance from the output port of the corona discharge chamber to the bio-indicator is fixed at 200 mm, so the residence time is 1.4 min.
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Ar, H2O2 Discharge chamber
300mm
Gas Bio-indicator
Sterilization chamber
Exhaust gas 110mm Fig. 1. Schematic diagram of the sterilization chamber.
Ar, H 2 O2
Tubular electrode (stainless-steel pipe)
Glass tube
120 mm
Cylindrical electrode (aluminum foil)
17 mm Fig. 2. Schematic diagram of the discharge chamber.
Chemical species produced in the discharge chamber flow into the sterilization chamber and reach the bio-indicator. Under the standard experimental conditions, gas flow rate is 2 Nl/min, the flow rate of H2O2 droplets supplied by the nebulizer is 38.7 ml/min and the discharge type is AC corona whose voltage is 12 kV (50 Hz, 0-peak).
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Escherichia coliphage (E.coli, JM109 TAKARA) is used as the bio-indicator. E. coli are supplied on the glass plate (diameter: 15 mm), whose surface is dry. The initial number of E. coli on the plate is approximately 24,000. The incubation procedure is as follows: 1. E. coli on the glass plate are used in the sterilization experiment. 2. After the experiment, residual E. coli are transferred to water and sprinkled on the agar in the Petri dish. 3. E. coli is incubated for 24 h at 371C. 4. The number of colonies is counted with a colony counting pen. 5. Two or three samples are examined under the same experimental conditions. The average number is used in the calculation of survival ratio. Survival ratio Z is defined as Z ¼ NðtÞ=N0 ; where NðtÞ is the number of E. coli cells when the sterilization time is t [min]. N0 is the initial number of E. coli cells (sterilization time is 0 min).
3. Results and discussion Fig. 3 shows the relationship between survival ratio and sterilization time. The experimental conditions are as follows. The voltage of AC corona discharge was 12 kV. Gas flow rate is 2 Nl/min and the surface of the bio-indicator is always dry. The survival ratio (t ¼ 0:5 min) in the case of a corona discharge with H2O2 droplets is about 1000 times lower than that in the case of supplying H2O2 droplets without discharge. This result indicates that the sterilization effect is improved by using a 0
10
-1
Survival ratio [-]
10
Only H2 O2 -2
10
-3
10
H2 O2 + discharge
-4
10
0
1 Time [min]
2
Fig. 3. Relationship between sterilization time and survival ratio (AC corona discharge: 12 kV (50 Hz, 0-peak), distance: 200 mm, flow rate: 2 Nl/min).
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corona discharge, as reported in a previous work [10], and chemical species with a long lifetime are considered effective for sterilization, because the residence time is longer than 1 min in this case. From Fig. 3, it was predicted that not only H2O2 droplets but also other factors, such as the chemical species produced by the corona discharge, were effective for sterilization because the difference in survival ratio between the two conditions was significant. Therefore, factors contributing to a high sterilization rate are as follows: 1. Ultraviolet light produced by a corona discharge. 2. Chemical species in the gas phase produced by a corona discharge. 3. Residual H2O2 droplets. These three factors were investigated to find the most effective species in this study.
4. Examination of effective species In this section, a different type of bio-indicator was used to simplify the evaluation of the sterilization effect. The bio-indicator used here was in the wet condition. E. coli cells (JM109, TAKARA), the number of which was controlled at 1000, were sprinkled on the agar in a Petri dish (diameter: 86 mm). This bio-indicator was then placed at the bottom of the sterilization chamber. The chamber used here is the same as the chamber used for sterilization shown in Fig. 1. After the experiment, residual E. coli in the bio-indicator was incubated for 24 h at 371C. Then, the number of colonies was counted and the difference in survival ratio was evaluated. 4.1. Ultraviolet light Ultraviolet light has been utilized for sterilization [5] in various fields, such as medicine, air and water purification. Two experiments were carried out in order to investigate the effect of ultraviolet light. First, four initial gas conditions were examined in the experiments as follows: pure argon gas, H2O droplets supplied by a nebulizer with argon gas, 25 vol% O2 gas with argon and 30 wt% H2O2 droplets with argon gas. In the case of pure Ar gas, UV light was not produced by a corona discharge. However, in the other three cases, UV light was produced as confirmed by emission spectrum measurement. In this experiment, AC corona discharge (11 kV) at a gas flow rate of 2 Nl/min was utilized. The difference in survival ratio among four conditions is shown in Fig. 4. The survival ratio of H2O2 corona discharge is more than 100 times lower than that of Ar corona discharge. In the case of H2O corona discharge, the survival ratio is also high and almost the same as that of Ar corona discharge. This result indicates that ultraviolet light is not effective for sterilization. Although the survival ratio in O2 corona discharge is slightly lower than that in Ar corona discharge, the difference between O2 discharge and H2O2 discharge is much larger. Since ozone may be produced in O2 discharge, it is proved that O3 is not the main cause of the sterilization.
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Survival ratio [-]
10
Ar + discharge H 2O + discharge O 2 + discharge H 2O 2 + discharge
10-1
-2
10
0
1
2
3
4
5
Time [min] Fig. 4. Dependence of initial gas conditions on sterilization (AC corona: 11 kV, distance: 200 mm, flow rate: 2 Nl/min).
Gas
Quartz glass Petri dish
Fig. 5. Experimental apparatus for investigation of UV effect.
The effect of ultraviolet light on sterilization was investigated further. An experiment was carried out using quartz glass as shown in Fig. 5. The UV transmittance (200–400 nm) of the quartz glass is higher than 90%. In this setup, since the quartz glass was used to cover the Petri dish, only ultraviolet light without chemical species affects the sterilization. However, the survival ratio is almost the same as that of Ar discharge in Fig. 4 which means that almost no bacteria are sterilized. According to these experimental results, it can be concluded that ultraviolet light is not the main cause of the sterilization effect. Fig. 4 shows that H2O2 droplets themselves or chemical species produced from the corona discharge may be the main causes of the sterilization.
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4.2. Chemical species in gas phase In order to investigate the effect of chemical species in the gas phase, the size distribution of residual H2O2 droplets was measured to evaluate whether all of the H2O2 droplets were decomposed or not. If no H2O2 droplets are observed, only the chemical species in the gas phase are considered to be effective for sterilization. The size distribution of H2O2 droplets produced by the nebulizer is shown in Fig. 6. The average radius is approximately 5:5 mm: This distribution was measured by the laser diffraction method (He–Ne laser, 1 mW, 632.8 nm) using LDSA-1400A (Tounichi Computer Applications). In this apparatus, the identification limit of droplet size was 1:4 mm: Next, the size distribution in the case of AC corona discharge application (13 kV, 50 Hz) was measured. No droplets could be detected, which meant that almost all H2O2 droplets disintegrated, although there was a possibility that fine droplets less than 1:4 mm in size still existed as the identification limit of droplet size was 1:4 mm: As almost all H2O2 droplets were decomposed, it was predicted that chemical species in the gas phase was one of the major factors effecting sterilization. Therefore, the determination of the effective species in the gas phase was carried out. The polarity of the gas species was investigated at first. Two electrodes were installed as shown in Fig. 7 and electric field (E ¼ 8 V/m) was applied in the vertical direction. In this setup, three kinds of species (positive, negative and neutral) could be controlled depending on the condition of the electric field. Four conditions were examined, as shown in Fig. 8, where each kind of species was expected to behave in a different way according to the direction of the electric field. The direction of the electric field from the top to the bottom is defined as the positive direction [A]. It is expected that negative ions cannot reach the bio-indicator under this condition. On the other hand, positive ions cannot reach the bio-indicator in the case of the negative direction [B]. If both electrodes are connected to the ground and no voltage is applied [C], all species can reach the bio-indicator. However, if neither electrode is 15
100
10 60 40
5
Cumulation [%]
Frequency [%]
80
20 0 1
0 5 10 Diameter [m]
50
Fig. 6. Size distribution of H2O2 droplets in the case of no discharge.
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Ar, H2O2
Grid electrode Bio-indicator
E = 8 V/cm
Fig. 7. Schematic diagram of the sterilization chamber.
Fig. 8. Expected behavior of chemical species under each condition.
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Table 1 Effect of electric field on survival ratio (E ¼ 8 V/cm, t ¼ 1 min)
Survival ratio
Only H2O2
[A]
[B]
[C]
[D]
0.12
0.019
0.029
0.017
0.047
connected to the ground [D], the upper electrode is predicted to charge up and only neutral species can reach the bio-indicator. Table 1 shows the survival ratio under each condition when the sterilization time was 1 min. Comparing [D] with [C], the survival ratio of [C] is lower than that of [D], which means that ions are effective for sterilization. The survival ratio of [A] is lower than that of [B], which indicates that positive ions are more effective than negative ones. In order to confirm the effect of the polarity of ions, an electric field was applied in the horizontal direction, as shown in Fig. 9. The supplied electric field was from 6.4 to 35 V/cm, where the electric field was enough to move ions. In this setup, positive ions are attracted to the cathode side, while negative ions are attracted to the anode. Thus, it was possible to compare the survival ratio of the cathode side with that of the anode side. The result indicated that the survival ratio of the positive ion attracted side (cathode side) is lower than that of the negative ion attracted side (Fig. 10). These results indicate that positive ions are more effective for sterilization than negative ones. In the next step, the identification of positive ions was carried out. First, the emission spectrum of AC corona discharge was measured. In this measurement, the gas flow rate was 2 Nl/min and the voltage of the discharge was 13 kV. Fig. 11 shows the emission spectrum where many active species such as OH, O and Ar radicals could be confirmed. With respect to positive ions, O2+ and O+ were detected. There was a possibility that neutral radicals, such as OH and O, were effective for sterilization. However, O and OH radicals are quenched by recombination or collision with other species because the residence time of this experiment is 1.4 min. Thus, O and OH radicals are not the effective factors for sterilization. The determination of positive ions using a quadrupole mass spectrometer (Q-MS, HIDEN ANALYTICAL, 3F/PIC series EPIC) was also carried out. As it was difficult to detect ions under atmospheric pressure with Q-MS, the detection was
Cathode (-)
Positive ion
Negative ion
Anode (+)
Petri dish
Fig. 9. Experimental setup in the case of a horizontal electric field.
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Fig. 10. Difference in colony distribution between cathode side and anode side.
Ar
Ar+
+
Ar
O
OH
O
H
O
+
O O
O2+ O
OH
0.5
H2
Ar
1 OH
Intensity [-]
Ar Ar
Ar
×105 ] 1.5
0 300
400
500 600 700 Wavelength [nm]
800
Fig. 11. Emission spectrum of AC corona discharge (13 kV, 50 Hz).
carried out under reduced pressure. Although this condition is different from actual sterilization conditions, some important information is expected to be obtained. The discharge chamber connected to Q-MS is shown in Fig. 12. Pressure was 1.3 Torr; the voltage of the discharge of a positive corona was only 650 V. Fig. 13 shows the difference in neutral species profile between no discharge and discharge. The intensity of H2O2 (mass number: 34) was reduced by the discharge. This result indicates that H2O2 was decomposed and ions were produced. This result is also one proof that ions are effective species for sterilization. In the case of neutral species, it is possible to measure the species under atmospheric pressure. Thus, the
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183
Fig. 12. Discharge chamber connected to the quadrupole mass spectrometer.
10000 no discharge discharge
Intensity [c/s]
8000 6000 4000 2000 0 28
30
32 34 Mass number
36
38
Fig. 13. Difference in neutral species profile between no discharge and discharge.
measurement was also carried out under atmospheric pressure. The result was the same as that under reduced pressure and H2O2 decomposition was confirmed. Positive ions under reduced pressure were measured and shown in Fig. 14. As O+, OH+, H2O+ and O+ 2 were observed in this measurement, these species were considered to be effective for sterilization. No large ions or clusters were detected.
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40000
Intensity [c/s]
+
H 2O
30000 OH 20000
+
Ar
+
+
+
O
O2
10
20 30 40 Mass number
10000
0
50
60
Fig. 14. Positive ion spectrum under reduced pressure (pressure: 1.3 Torr, voltage: 650 V).
It was concluded that positive ions such as O+, OH+ and O+ 2 are effective for sterilization.
4.3. Residual H2O2 droplets The effect of residual H2O2 droplets less than 1 mm in size was examined. In Table 1, the survival ratio under condition [D] is lower than that under the condition of only H2O2 droplets without corona discharge. It was considered that neutral gaseous species and fine residual droplets were effective for sterilization under condition [D]. However, neutral active species in the gas phase cannot reach the bioindicator because the residence time is longer than 1 min. Thus, there is a possibility that fine droplets are effective for sterilization. It is necessary to confirm whether H2O2 droplets less than 1 mm in size exist or not. The concentration of fine H2O2 droplets was measured by CNC (condensation nuclei counter, KANOMAX). The objective of this measurement was to confirm the existence of H2O2 droplets less than 1 mm in size. The measurement range of particle size using this CNC is from 3 nm to 1 mm: Ar gas was supplied under no corona discharge in the first condition. H2O2 droplets with Ar gas were supplied under no discharge in the second condition, and H2O2 droplets were supplied under AC corona discharge (12 kV, 50 Hz) in the third condition (H2O2 discharge). Table 2 shows the concentrations of particles under these three conditions. The number concentration of H2O2 with a corona discharge was one-tenth of that of H2O2 without a corona discharge and was more than one hundred times higher than that of pure Ar. These results indicate that the number concentration of particles decreases to less than one-tenth of the initial one and droplets with size greater than 3 nm are residual in the sterilization chamber. However, there is a possibility that almost all of the less than 1 mm in size droplets are entrained with the air flow. In order to confirm that the ultrafine droplets are
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Table 2 Number concentration of particles under each condition Condition
Concentration [cm3]
Ar gas+no discharge Ar gas+H2O2+no discharge Ar gas+H2O2+discharge
6.3 19,000 1300
attached to the surface, H2O2 determination was carried out using the colorimetric method [11] according to the potassium iodide reaction 2KI þ H2 O2 -2KOH þ I2 : In this reaction, I 3 ion is oxidized and the absorbance at 350 nm is measured with a spectrophotometer (Hitachi U-400). The procedure for H2O2 determination is as follows: 1. A Petri dish containing 20 ml water instead of a bio-indicator is set. 2. Corona discharge is applied for 10 min at 12 kV with H2O2 droplets (the same conditions as those for sterilization). 3. After the corona discharge, the solution in the Petri dish is taken out and mixed with the solution containing potassium iodide. 4. The mixed solution is set in a spectrophotometer and absorbance at 350 nm is measured. Fig. 15 shows the absorption spectra under two different conditions. The first condition is that H2O2 droplets are supplied to the discharge chamber and a positive corona discharge is applied (12 kV, 50 Hz). The second condition is that H2O2 is supplied by the bubbling method with no corona discharge. The solid line in Fig. 15 indicates the absorption spectrum of H2O2 droplets with the corona discharge and we confirmed the absorption at 350 nm. This result indicated that fine H2O2 droplets remain in the sterilization chamber and are attached to the surface of the bioindicator. However, there is a possibility that gaseous H2O2 dissolves in water. The dashed line in Fig. 15 indicates the absorption spectrum of H2O2 produced from saturated gaseous H2O2. The absorbance of H2O2 droplets with the corona discharge is larger than that of saturated H2O2 without the corona discharge. This result indicates that H2O2 droplets also attach to the surface of the bio-indicator. According to these results, it was proved that H2O2 droplets less than 1 mm in size were effective for sterilization. Finally, the size distribution of particles was measured by DMA (differential mobility analyzer, Wyckoff). The purpose of this detection is to determine the size distribution of residual H2O2 droplets. In this measurement, the size distribution under two conditions was determined. One is to supply H2O2 droplets with no corona discharge, and the other is to supply H2O2 droplets with AC corona discharge (12 kV, 50 Hz). Although measurement of the size distribution from 0.5 nm to 1 mm is desirable, the range from 0.5 to 200 nm was examined because of the
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Absorbance [-]
6 H2O2 droplets (discharge)
4 H2O2 bubbling (no discharge)
2 0
350
400 Wavelength [nm]
450
Fig. 15. Difference in H2O2 concentration according to the different supply methods, namely, nebulizer and bubbling.
-3
Concentration [cc ]
H2O2 H2O2 + discharge
10
10
4
3
1
10 Diameter [nm]
10 2
Fig. 16. Size distribution of particles generated by AC corona discharge (positive ions).
measurement limit of this DMA. The result is shown in Fig. 16. It was predicted that the average diameter of the droplets decreased by using the corona discharge. However, the average diameter did not change in this range. On the other hand, the number concentration had decreased. In CNC measurement, the number concentration of H2O2 droplets decreased to one-tenth of the initial number concentration due to the discharge. However, in Fig. 16, the number concentration had decreased to only one-fourth of the initial number concentration. It means that most H2O2 droplets greater than 200 nm in size had decreased and only fine particles were left. Thus, there is a possibility that droplets 10–100 nm in size are effective for sterilization even though the number concentration is decreased. It can be considered that the properties of the fine droplets change after a corona discharge. The reasons as to why the fine droplets are much more effective for sterilization are as follows. First, the concentration of H2O2 in the droplets increases. Since the boiling point of
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H2O2 is higher than that of H2O, H2O is vaporized faster than H2O2. Thus, it is likely that the H2O2 concentration in each droplet rises gradually as the diameter becomes small by the discharge. At the entrance of the discharge chamber, the average radius of H2O2 droplets is 5:5 mm: After the discharge, there are no droplets greater than 1 mm in size and only fine droplets remain. It is predicted that these fine droplets whose H2O2 concentration becomes higher are effective for sterilization even if the number concentration of the droplets is decreased. In addition, it is possible that droplets charged by the corona discharge more easily diffuse into the E. coli cells than non-charged droplets, or that H2O2 droplets are decomposed into more active species by the corona discharge. 5. Conclusions The effect of the sterilization method using a corona discharge with H2O2 droplets was proved to be more than 100 times higher than that of the sterilization method without a discharge. In this sterilization method, both positive ions and fine droplets (10–100 nm) are effective for sterilization. References [1] D. Dempsey, R. Thirucote, Sterilization of medical devices: a review, J. Biomater. Appl. 3 (1989) 454–523. [2] S. Kercher, V. Mortimer, Before and after: an evaluation of engineering controls foar ethylene oxide sterilization in hospitals, Appl. Ind. Hyg. 2 (7) (1987) 7–12. [3] W. Freeze, Room sterilization using vaporous hydrogen peroxide, Bioprocess Eng. 27 (1993) 37. [4] G. Cerny, Testing of aseptic machines for efficiency of sterilization of packaging materials by means of hydrogen peroxide, Packag. Technol. Sci. 5 (1992) 77–81. [5] Y. Yamagoshi, E. Ishiyama, The applicability of the flow UVFsterilizer to the secondary effluent of sewage treatment plant, Study Ultraviolet Rays 17 (1998) 27–32. [6] M. Laroussi, Sterilization of contaminated matter with an atmospheric pressure plasma, IEEE Trans. Plasma Sci. 24 (1996) 1188–1191. [7] M. Sato, K. Tokita, M. Sadakata, T. Sakai, K. Nakanishi, Sterilization of microorganisms by a highvoltage, pulsed discharge under water, Int. Chem. Eng. 30 (4) (1990) 695–698. [8] M. Sato, T. Ohgiyama, J. Clements, Formation of chemical species and their effects on microorganisms using a pulsed high-voltage discharge in water, IEEE Trans. Ind. Appl. 32 (1) (1996) 106–112. [9] B. Sun, M. Sato, Optical study of active species produced by a pulsed streamer corona discharge in water, J. Electrostatics 39 (1997) 189–202. [10] A. Mizuno, M. Kurahashi, S. Imano, T. Ishida, M. Nagata, Sterilization using OH radicals produced by pulsed discharge plasma in atmospheric pressure, IEEE Industry Application Society Annual Meeting, 1997, pp. 2032–2036. [11] C. Hochanadel, Effects of cobalt g radiation on water and aqueous solutions, J. Chem. Phys. 56 (1952) 587–595.
Sterilization by H2O2 droplets under corona discharge Mitsuo Yamamoto*, Masateru Nishioka, Masayoshi Sadakata Department of Chemical System Engineering, University of Tokyo, Tokyo 113-8656, Japan Received 14 April 2001; received in revised form 8 September 2001; accepted 13 October 2001
Abstract A new sterilization method that combines an injection of hydrogen peroxide droplets (30 wt%) by spraying and a corona discharge is very effective for high-speed sterilization. Ultraviolet light, gaseous species and fine H2O2 droplets were considered to be important factors of this sterilization method. In order to investigate the sterilization mechanism, these three factors were examined. It was shown that ultraviolet light was not effective for sterilization. Among the gaseous species investigated, positive ions were proved to be effective through experiments using electric fields. A quadrupole mass spectrometer was used to measure positive ions. It was confirmed that H2O2 was decomposed and the number of H2O2 droplets was reduced. H2O2 droplet size was measured by a laser scattering method, CNC (condensation nuclei counter) and DMA (differential mobility analyzer). It was found that droplets larger than 3 nm still existed even if the droplet size was reduced. It was concluded that both fine H2O2 droplets and positive ions had important effects on sterilization. r 2002 Elsevier Science B.V. All rights reserved. Keywords: Sterilization; Hydrogen peroxide; Discharge; Ion; Droplet
1. Introduction In the food industry and the medical field, sterilization is one of the most important processes for guaranteeing the safety of foods or medicines. Many sterilization methods have been investigated so far [1], including autoclaving, chemical treatment with ethylene oxide gas ((CH2)2O) [2], hydrogen peroxide [3,4] and ozone gas treatment, radiation by ultraviolet light [5], gamma ray and electron *Corresponding author. Tel./fax: +81-3-5841-7363. E-mail address: [email protected] (M. Yamamoto). 0304-3886/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 8 8 6 ( 0 1 ) 0 0 1 9 5 - 4
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beam. However, each method has advantages and disadvantages. In the autoclaving method, a long sterilization time is required because it is a batch process. In the chemical methods, residual chemical substances have adverse effects in application, so their amounts should be reduced as much as possible. Even though the sterilization ability of ultraviolet radiation is very strong, heavy instruments are required as the shielding system. Hence, a new high-speed and secure sterilization process is urgently required. In previous works, many discharge methods were examined for use in sterilization. Glow discharge plasma at atmospheric pressure was used for the sterilization of contaminated matter [6]. On the other hand, a pulsed corona discharge was utilized for the sterilization of microorganisms in water [7,8]. In those methods, the impact of the discharge and the chemical species produced by it were thought to be the main causes of the high sterilization rate. The active species produced by the pulsed corona discharge were determined by optical emission spectral analysis [9] to be O, H and OH radicals. A new method of dry-type sterilization, aiming at high-speed and secure sterilization, was examined [10]. This sterilization method utilized hydrogen peroxide and a pulsed corona discharge under atmospheric pressure. In this study, a sterilization method using a corona discharge with H2O2 droplets was studied and the effective species in this process were examined by various methods. This sterilization method is expected to be applied to the food packaging field. At present, plastic films that are used for packaging are sterilized by soaking in hydrogen peroxide. Thus, it takes a long time to sterilize all bacteria and remove residual H2O2 by washing with water. If a sterilization method using a corona discharge with H2O2 droplets were established, the sterilization time would be reduced. The main objective of this study is to identify the effective species for sterilization in order to confirm the safety of this sterilization method.
2. Experimental apparatus and method The experimental apparatus is shown in Fig. 1. This apparatus consists of a discharge chamber and a sterilization chamber for optical measurement. Both chambers are made of acrylic resin. The discharge chamber is shown in Fig. 2. This chamber consists of coaxial cylindrical electrodes where a 1/4 inch stainless-steel pipe is installed at the center of the quartz tube as a high-voltage electrode and aluminum foils are wound outside the quartz tube as an earth electrode. The inside diameter of the quartz tube is 17 mm. The length of the earth electrode is 120 mm. The H2O2 droplets are supplied together with argon gas by a nebulizer (Kinoshita J-753) and a corona discharge is formed in the discharge chamber. The concentration of H2O2 droplets is 30 wt%. The inside diameter of the sterilization chamber is 110 mm and the height is 300 mm. A bio-indicator is installed at the bottom of the sterilization chamber. A distance from the output port of the corona discharge chamber to the bio-indicator is fixed at 200 mm, so the residence time is 1.4 min.
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175
Ar, H2O2 Discharge chamber
300mm
Gas Bio-indicator
Sterilization chamber
Exhaust gas 110mm Fig. 1. Schematic diagram of the sterilization chamber.
Ar, H 2 O2
Tubular electrode (stainless-steel pipe)
Glass tube
120 mm
Cylindrical electrode (aluminum foil)
17 mm Fig. 2. Schematic diagram of the discharge chamber.
Chemical species produced in the discharge chamber flow into the sterilization chamber and reach the bio-indicator. Under the standard experimental conditions, gas flow rate is 2 Nl/min, the flow rate of H2O2 droplets supplied by the nebulizer is 38.7 ml/min and the discharge type is AC corona whose voltage is 12 kV (50 Hz, 0-peak).
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Escherichia coliphage (E.coli, JM109 TAKARA) is used as the bio-indicator. E. coli are supplied on the glass plate (diameter: 15 mm), whose surface is dry. The initial number of E. coli on the plate is approximately 24,000. The incubation procedure is as follows: 1. E. coli on the glass plate are used in the sterilization experiment. 2. After the experiment, residual E. coli are transferred to water and sprinkled on the agar in the Petri dish. 3. E. coli is incubated for 24 h at 371C. 4. The number of colonies is counted with a colony counting pen. 5. Two or three samples are examined under the same experimental conditions. The average number is used in the calculation of survival ratio. Survival ratio Z is defined as Z ¼ NðtÞ=N0 ; where NðtÞ is the number of E. coli cells when the sterilization time is t [min]. N0 is the initial number of E. coli cells (sterilization time is 0 min).
3. Results and discussion Fig. 3 shows the relationship between survival ratio and sterilization time. The experimental conditions are as follows. The voltage of AC corona discharge was 12 kV. Gas flow rate is 2 Nl/min and the surface of the bio-indicator is always dry. The survival ratio (t ¼ 0:5 min) in the case of a corona discharge with H2O2 droplets is about 1000 times lower than that in the case of supplying H2O2 droplets without discharge. This result indicates that the sterilization effect is improved by using a 0
10
-1
Survival ratio [-]
10
Only H2 O2 -2
10
-3
10
H2 O2 + discharge
-4
10
0
1 Time [min]
2
Fig. 3. Relationship between sterilization time and survival ratio (AC corona discharge: 12 kV (50 Hz, 0-peak), distance: 200 mm, flow rate: 2 Nl/min).
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corona discharge, as reported in a previous work [10], and chemical species with a long lifetime are considered effective for sterilization, because the residence time is longer than 1 min in this case. From Fig. 3, it was predicted that not only H2O2 droplets but also other factors, such as the chemical species produced by the corona discharge, were effective for sterilization because the difference in survival ratio between the two conditions was significant. Therefore, factors contributing to a high sterilization rate are as follows: 1. Ultraviolet light produced by a corona discharge. 2. Chemical species in the gas phase produced by a corona discharge. 3. Residual H2O2 droplets. These three factors were investigated to find the most effective species in this study.
4. Examination of effective species In this section, a different type of bio-indicator was used to simplify the evaluation of the sterilization effect. The bio-indicator used here was in the wet condition. E. coli cells (JM109, TAKARA), the number of which was controlled at 1000, were sprinkled on the agar in a Petri dish (diameter: 86 mm). This bio-indicator was then placed at the bottom of the sterilization chamber. The chamber used here is the same as the chamber used for sterilization shown in Fig. 1. After the experiment, residual E. coli in the bio-indicator was incubated for 24 h at 371C. Then, the number of colonies was counted and the difference in survival ratio was evaluated. 4.1. Ultraviolet light Ultraviolet light has been utilized for sterilization [5] in various fields, such as medicine, air and water purification. Two experiments were carried out in order to investigate the effect of ultraviolet light. First, four initial gas conditions were examined in the experiments as follows: pure argon gas, H2O droplets supplied by a nebulizer with argon gas, 25 vol% O2 gas with argon and 30 wt% H2O2 droplets with argon gas. In the case of pure Ar gas, UV light was not produced by a corona discharge. However, in the other three cases, UV light was produced as confirmed by emission spectrum measurement. In this experiment, AC corona discharge (11 kV) at a gas flow rate of 2 Nl/min was utilized. The difference in survival ratio among four conditions is shown in Fig. 4. The survival ratio of H2O2 corona discharge is more than 100 times lower than that of Ar corona discharge. In the case of H2O corona discharge, the survival ratio is also high and almost the same as that of Ar corona discharge. This result indicates that ultraviolet light is not effective for sterilization. Although the survival ratio in O2 corona discharge is slightly lower than that in Ar corona discharge, the difference between O2 discharge and H2O2 discharge is much larger. Since ozone may be produced in O2 discharge, it is proved that O3 is not the main cause of the sterilization.
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Survival ratio [-]
10
Ar + discharge H 2O + discharge O 2 + discharge H 2O 2 + discharge
10-1
-2
10
0
1
2
3
4
5
Time [min] Fig. 4. Dependence of initial gas conditions on sterilization (AC corona: 11 kV, distance: 200 mm, flow rate: 2 Nl/min).
Gas
Quartz glass Petri dish
Fig. 5. Experimental apparatus for investigation of UV effect.
The effect of ultraviolet light on sterilization was investigated further. An experiment was carried out using quartz glass as shown in Fig. 5. The UV transmittance (200–400 nm) of the quartz glass is higher than 90%. In this setup, since the quartz glass was used to cover the Petri dish, only ultraviolet light without chemical species affects the sterilization. However, the survival ratio is almost the same as that of Ar discharge in Fig. 4 which means that almost no bacteria are sterilized. According to these experimental results, it can be concluded that ultraviolet light is not the main cause of the sterilization effect. Fig. 4 shows that H2O2 droplets themselves or chemical species produced from the corona discharge may be the main causes of the sterilization.
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4.2. Chemical species in gas phase In order to investigate the effect of chemical species in the gas phase, the size distribution of residual H2O2 droplets was measured to evaluate whether all of the H2O2 droplets were decomposed or not. If no H2O2 droplets are observed, only the chemical species in the gas phase are considered to be effective for sterilization. The size distribution of H2O2 droplets produced by the nebulizer is shown in Fig. 6. The average radius is approximately 5:5 mm: This distribution was measured by the laser diffraction method (He–Ne laser, 1 mW, 632.8 nm) using LDSA-1400A (Tounichi Computer Applications). In this apparatus, the identification limit of droplet size was 1:4 mm: Next, the size distribution in the case of AC corona discharge application (13 kV, 50 Hz) was measured. No droplets could be detected, which meant that almost all H2O2 droplets disintegrated, although there was a possibility that fine droplets less than 1:4 mm in size still existed as the identification limit of droplet size was 1:4 mm: As almost all H2O2 droplets were decomposed, it was predicted that chemical species in the gas phase was one of the major factors effecting sterilization. Therefore, the determination of the effective species in the gas phase was carried out. The polarity of the gas species was investigated at first. Two electrodes were installed as shown in Fig. 7 and electric field (E ¼ 8 V/m) was applied in the vertical direction. In this setup, three kinds of species (positive, negative and neutral) could be controlled depending on the condition of the electric field. Four conditions were examined, as shown in Fig. 8, where each kind of species was expected to behave in a different way according to the direction of the electric field. The direction of the electric field from the top to the bottom is defined as the positive direction [A]. It is expected that negative ions cannot reach the bio-indicator under this condition. On the other hand, positive ions cannot reach the bio-indicator in the case of the negative direction [B]. If both electrodes are connected to the ground and no voltage is applied [C], all species can reach the bio-indicator. However, if neither electrode is 15
100
10 60 40
5
Cumulation [%]
Frequency [%]
80
20 0 1
0 5 10 Diameter [m]
50
Fig. 6. Size distribution of H2O2 droplets in the case of no discharge.
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Ar, H2O2
Grid electrode Bio-indicator
E = 8 V/cm
Fig. 7. Schematic diagram of the sterilization chamber.
Fig. 8. Expected behavior of chemical species under each condition.
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Table 1 Effect of electric field on survival ratio (E ¼ 8 V/cm, t ¼ 1 min)
Survival ratio
Only H2O2
[A]
[B]
[C]
[D]
0.12
0.019
0.029
0.017
0.047
connected to the ground [D], the upper electrode is predicted to charge up and only neutral species can reach the bio-indicator. Table 1 shows the survival ratio under each condition when the sterilization time was 1 min. Comparing [D] with [C], the survival ratio of [C] is lower than that of [D], which means that ions are effective for sterilization. The survival ratio of [A] is lower than that of [B], which indicates that positive ions are more effective than negative ones. In order to confirm the effect of the polarity of ions, an electric field was applied in the horizontal direction, as shown in Fig. 9. The supplied electric field was from 6.4 to 35 V/cm, where the electric field was enough to move ions. In this setup, positive ions are attracted to the cathode side, while negative ions are attracted to the anode. Thus, it was possible to compare the survival ratio of the cathode side with that of the anode side. The result indicated that the survival ratio of the positive ion attracted side (cathode side) is lower than that of the negative ion attracted side (Fig. 10). These results indicate that positive ions are more effective for sterilization than negative ones. In the next step, the identification of positive ions was carried out. First, the emission spectrum of AC corona discharge was measured. In this measurement, the gas flow rate was 2 Nl/min and the voltage of the discharge was 13 kV. Fig. 11 shows the emission spectrum where many active species such as OH, O and Ar radicals could be confirmed. With respect to positive ions, O2+ and O+ were detected. There was a possibility that neutral radicals, such as OH and O, were effective for sterilization. However, O and OH radicals are quenched by recombination or collision with other species because the residence time of this experiment is 1.4 min. Thus, O and OH radicals are not the effective factors for sterilization. The determination of positive ions using a quadrupole mass spectrometer (Q-MS, HIDEN ANALYTICAL, 3F/PIC series EPIC) was also carried out. As it was difficult to detect ions under atmospheric pressure with Q-MS, the detection was
Cathode (-)
Positive ion
Negative ion
Anode (+)
Petri dish
Fig. 9. Experimental setup in the case of a horizontal electric field.
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Fig. 10. Difference in colony distribution between cathode side and anode side.
Ar
Ar+
+
Ar
O
OH
O
H
O
+
O O
O2+ O
OH
0.5
H2
Ar
1 OH
Intensity [-]
Ar Ar
Ar
×105 ] 1.5
0 300
400
500 600 700 Wavelength [nm]
800
Fig. 11. Emission spectrum of AC corona discharge (13 kV, 50 Hz).
carried out under reduced pressure. Although this condition is different from actual sterilization conditions, some important information is expected to be obtained. The discharge chamber connected to Q-MS is shown in Fig. 12. Pressure was 1.3 Torr; the voltage of the discharge of a positive corona was only 650 V. Fig. 13 shows the difference in neutral species profile between no discharge and discharge. The intensity of H2O2 (mass number: 34) was reduced by the discharge. This result indicates that H2O2 was decomposed and ions were produced. This result is also one proof that ions are effective species for sterilization. In the case of neutral species, it is possible to measure the species under atmospheric pressure. Thus, the
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Fig. 12. Discharge chamber connected to the quadrupole mass spectrometer.
10000 no discharge discharge
Intensity [c/s]
8000 6000 4000 2000 0 28
30
32 34 Mass number
36
38
Fig. 13. Difference in neutral species profile between no discharge and discharge.
measurement was also carried out under atmospheric pressure. The result was the same as that under reduced pressure and H2O2 decomposition was confirmed. Positive ions under reduced pressure were measured and shown in Fig. 14. As O+, OH+, H2O+ and O+ 2 were observed in this measurement, these species were considered to be effective for sterilization. No large ions or clusters were detected.
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40000
Intensity [c/s]
+
H 2O
30000 OH 20000
+
Ar
+
+
+
O
O2
10
20 30 40 Mass number
10000
0
50
60
Fig. 14. Positive ion spectrum under reduced pressure (pressure: 1.3 Torr, voltage: 650 V).
It was concluded that positive ions such as O+, OH+ and O+ 2 are effective for sterilization.
4.3. Residual H2O2 droplets The effect of residual H2O2 droplets less than 1 mm in size was examined. In Table 1, the survival ratio under condition [D] is lower than that under the condition of only H2O2 droplets without corona discharge. It was considered that neutral gaseous species and fine residual droplets were effective for sterilization under condition [D]. However, neutral active species in the gas phase cannot reach the bioindicator because the residence time is longer than 1 min. Thus, there is a possibility that fine droplets are effective for sterilization. It is necessary to confirm whether H2O2 droplets less than 1 mm in size exist or not. The concentration of fine H2O2 droplets was measured by CNC (condensation nuclei counter, KANOMAX). The objective of this measurement was to confirm the existence of H2O2 droplets less than 1 mm in size. The measurement range of particle size using this CNC is from 3 nm to 1 mm: Ar gas was supplied under no corona discharge in the first condition. H2O2 droplets with Ar gas were supplied under no discharge in the second condition, and H2O2 droplets were supplied under AC corona discharge (12 kV, 50 Hz) in the third condition (H2O2 discharge). Table 2 shows the concentrations of particles under these three conditions. The number concentration of H2O2 with a corona discharge was one-tenth of that of H2O2 without a corona discharge and was more than one hundred times higher than that of pure Ar. These results indicate that the number concentration of particles decreases to less than one-tenth of the initial one and droplets with size greater than 3 nm are residual in the sterilization chamber. However, there is a possibility that almost all of the less than 1 mm in size droplets are entrained with the air flow. In order to confirm that the ultrafine droplets are
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Table 2 Number concentration of particles under each condition Condition
Concentration [cm3]
Ar gas+no discharge Ar gas+H2O2+no discharge Ar gas+H2O2+discharge
6.3 19,000 1300
attached to the surface, H2O2 determination was carried out using the colorimetric method [11] according to the potassium iodide reaction 2KI þ H2 O2 -2KOH þ I2 : In this reaction, I 3 ion is oxidized and the absorbance at 350 nm is measured with a spectrophotometer (Hitachi U-400). The procedure for H2O2 determination is as follows: 1. A Petri dish containing 20 ml water instead of a bio-indicator is set. 2. Corona discharge is applied for 10 min at 12 kV with H2O2 droplets (the same conditions as those for sterilization). 3. After the corona discharge, the solution in the Petri dish is taken out and mixed with the solution containing potassium iodide. 4. The mixed solution is set in a spectrophotometer and absorbance at 350 nm is measured. Fig. 15 shows the absorption spectra under two different conditions. The first condition is that H2O2 droplets are supplied to the discharge chamber and a positive corona discharge is applied (12 kV, 50 Hz). The second condition is that H2O2 is supplied by the bubbling method with no corona discharge. The solid line in Fig. 15 indicates the absorption spectrum of H2O2 droplets with the corona discharge and we confirmed the absorption at 350 nm. This result indicated that fine H2O2 droplets remain in the sterilization chamber and are attached to the surface of the bioindicator. However, there is a possibility that gaseous H2O2 dissolves in water. The dashed line in Fig. 15 indicates the absorption spectrum of H2O2 produced from saturated gaseous H2O2. The absorbance of H2O2 droplets with the corona discharge is larger than that of saturated H2O2 without the corona discharge. This result indicates that H2O2 droplets also attach to the surface of the bio-indicator. According to these results, it was proved that H2O2 droplets less than 1 mm in size were effective for sterilization. Finally, the size distribution of particles was measured by DMA (differential mobility analyzer, Wyckoff). The purpose of this detection is to determine the size distribution of residual H2O2 droplets. In this measurement, the size distribution under two conditions was determined. One is to supply H2O2 droplets with no corona discharge, and the other is to supply H2O2 droplets with AC corona discharge (12 kV, 50 Hz). Although measurement of the size distribution from 0.5 nm to 1 mm is desirable, the range from 0.5 to 200 nm was examined because of the
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Absorbance [-]
6 H2O2 droplets (discharge)
4 H2O2 bubbling (no discharge)
2 0
350
400 Wavelength [nm]
450
Fig. 15. Difference in H2O2 concentration according to the different supply methods, namely, nebulizer and bubbling.
-3
Concentration [cc ]
H2O2 H2O2 + discharge
10
10
4
3
1
10 Diameter [nm]
10 2
Fig. 16. Size distribution of particles generated by AC corona discharge (positive ions).
measurement limit of this DMA. The result is shown in Fig. 16. It was predicted that the average diameter of the droplets decreased by using the corona discharge. However, the average diameter did not change in this range. On the other hand, the number concentration had decreased. In CNC measurement, the number concentration of H2O2 droplets decreased to one-tenth of the initial number concentration due to the discharge. However, in Fig. 16, the number concentration had decreased to only one-fourth of the initial number concentration. It means that most H2O2 droplets greater than 200 nm in size had decreased and only fine particles were left. Thus, there is a possibility that droplets 10–100 nm in size are effective for sterilization even though the number concentration is decreased. It can be considered that the properties of the fine droplets change after a corona discharge. The reasons as to why the fine droplets are much more effective for sterilization are as follows. First, the concentration of H2O2 in the droplets increases. Since the boiling point of
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H2O2 is higher than that of H2O, H2O is vaporized faster than H2O2. Thus, it is likely that the H2O2 concentration in each droplet rises gradually as the diameter becomes small by the discharge. At the entrance of the discharge chamber, the average radius of H2O2 droplets is 5:5 mm: After the discharge, there are no droplets greater than 1 mm in size and only fine droplets remain. It is predicted that these fine droplets whose H2O2 concentration becomes higher are effective for sterilization even if the number concentration of the droplets is decreased. In addition, it is possible that droplets charged by the corona discharge more easily diffuse into the E. coli cells than non-charged droplets, or that H2O2 droplets are decomposed into more active species by the corona discharge. 5. Conclusions The effect of the sterilization method using a corona discharge with H2O2 droplets was proved to be more than 100 times higher than that of the sterilization method without a discharge. In this sterilization method, both positive ions and fine droplets (10–100 nm) are effective for sterilization. References [1] D. Dempsey, R. Thirucote, Sterilization of medical devices: a review, J. Biomater. Appl. 3 (1989) 454–523. [2] S. Kercher, V. Mortimer, Before and after: an evaluation of engineering controls foar ethylene oxide sterilization in hospitals, Appl. Ind. Hyg. 2 (7) (1987) 7–12. [3] W. Freeze, Room sterilization using vaporous hydrogen peroxide, Bioprocess Eng. 27 (1993) 37. [4] G. Cerny, Testing of aseptic machines for efficiency of sterilization of packaging materials by means of hydrogen peroxide, Packag. Technol. Sci. 5 (1992) 77–81. [5] Y. Yamagoshi, E. Ishiyama, The applicability of the flow UVFsterilizer to the secondary effluent of sewage treatment plant, Study Ultraviolet Rays 17 (1998) 27–32. [6] M. Laroussi, Sterilization of contaminated matter with an atmospheric pressure plasma, IEEE Trans. Plasma Sci. 24 (1996) 1188–1191. [7] M. Sato, K. Tokita, M. Sadakata, T. Sakai, K. Nakanishi, Sterilization of microorganisms by a highvoltage, pulsed discharge under water, Int. Chem. Eng. 30 (4) (1990) 695–698. [8] M. Sato, T. Ohgiyama, J. Clements, Formation of chemical species and their effects on microorganisms using a pulsed high-voltage discharge in water, IEEE Trans. Ind. Appl. 32 (1) (1996) 106–112. [9] B. Sun, M. Sato, Optical study of active species produced by a pulsed streamer corona discharge in water, J. Electrostatics 39 (1997) 189–202. [10] A. Mizuno, M. Kurahashi, S. Imano, T. Ishida, M. Nagata, Sterilization using OH radicals produced by pulsed discharge plasma in atmospheric pressure, IEEE Industry Application Society Annual Meeting, 1997, pp. 2032–2036. [11] C. Hochanadel, Effects of cobalt g radiation on water and aqueous solutions, J. Chem. Phys. 56 (1952) 587–595.