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Inactivation characteristics of Bacillus thuringiensis spore in liquid using atmospheric torch plasma using oxygen
Vacuum 88 (2013) 173e176
Contents lists available at SciVerse ScienceDirect
Vacuum journal homepage: www.elsevier.com/locate/vacuum
Inactivation characteristics of Bacillus thuringiensis spore in liquid using atmospheric torch plasma using oxygen Nobuya Hayashi a, *, Yusuke Akiyoshi b, Yasuyo Kobayashi c, Kouzo Kanda c, Kazusato Ohshima c, Masaaki Goto d a
Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, 6-1 Kasuga-kouen, Kasuga-shi, Fukuoka 816-8580, Japan Faculty of Science and Engineering, Saga University, 1 Honjo-machi, Saga-shi, Saga 840-8502, Japan Faculty of Agriculture, Saga University, 1 Honjo-machi, Saga-shi, Saga 840-8502, Japan d Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga-shi, Saga 849-8501, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2011 Received in revised form 8 March 2012 Accepted 8 March 2012
The atmospheric plasma torch produced by a barrier discharge using air and oxygen as working gases is adopted to inactivate microorganisms in a liquid. The inactivation characteristics of bacillus spore are investigated and compared with the heat sterilization method, and sterilization factors are active species with short-lifetimes produced in water. The population of Bacillus thuringiensis spore in pure water decreases 103 times within 20 min, and the obtained result is equivalent to the heat sterilization of 95 C. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Atmospheric torch plasma Oxygen radicals Low temperature treatment Bacillus inactivation Spores in liquid
1. Introduction Disinfections of microorganisms in liquid is an important procedure in medicine [1] and the food Industry [2]. The unique method that has been adopted to inactivate the microorganisms in a liquid is the heat pulse method [3], which applies heat to the liquid for a short period of several minutes. The temperature of the pressurized water increases to 130 C at 200 kPa, and microorganisms including bacillus spores are inactivated. However, any liquid to be sterilized tends to suffer modification in taste and aroma, in spite of the short period of heat application. Another certain application of the low temperature sterilization of a liquid is for body fluids on wounds. The expected volume of the liquid to be sterilized in the medical treatment is the order of ml or ml. Recently, atmospheric barrier discharge plasma using He gas [4e7] has been adopted to inactivate microorganisms in water [8,9]. This method offers non-heat sterilization of a liquid. Since the cost of He gas used in the method is significantly high, the He torch plasma sterilization method is difficult to utilize practically. The torch plasma produced by the surface dielectric barrier discharge (SDBD) has been adopted for sterilization of water. There are three
* Corresponding author. Tel./fax: þ81 92 583 7649. E-mail address: [email protected] (N. Hayashi). 0042-207X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vacuum.2012.03.023
advantages of the torch type SDBD for practical use: (i) The SDBD torch plasma is a kind of remote plasma where the treatment region separates from the discharge region. Therefore, low temperature treatment is realized and only energetic neutral particles and radicals with long lifetime are obtained at treatment region. (ii) The cost of torch electrodes and power supply is low because precise manufacturing of electrodes is not necessary. (iii) The discharge gap of SDBD is controlled spontaneously depending on circumstances such as material gas species, gas flow rate, humidity and temperature. Therefore, the SDBD is a significantly robust discharge and suitable for practical uses. In this study, the atmospheric plasma torch produced by the oxygen gas SDBD is studied to inactivate microorganisms in liquid. The inactivation characteristics of bacilli in spore and vegetative form in a liquid are investigated comparing the conventional heat sterilization method, and the sterilization factors are specified to be oxygen radicals with short lifetime. 2. Experimental procedure Fig. 1(a) shows the SDBD plasma torch used in this experiment. The copper thin sheet as a grounded electrode is wrapped around an alumina tube of the length of 100 mm and the inner diameter of 3 mm. The wound tungsten wire of diameter 0.23 mm is set in the inner surface of the alumina tube as a cathode. When a high voltage
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N. Hayashi et al. / Vacuum 88 (2013) 173e176
with high frequency of 10 kHz is applied to the wire cathode electrode inside the alumina tube, the discharge occurs around the surface of the wire electrode. The pure oxygen, air, and Ar gases are used as the material gases of the plasmas. As the gas flows through the alumina tube, the ions and radicals are ejected from the opening edge of the alumina tube. The distance between the opening edge of the torch tube and the liquid surface is 15 mm, in order to prevent evaporation of the liquid by the gas flow. The applied voltage and the gas flow rate are kept constant at 6 kV and 1 slm, respectively. The bacilli utilized as sample microorganisms in the pure water are in spore and vegetative form of the Bacillus thuringiensis that is tolerant against heat and UV light. The spore with the population of order of 107 is dispersed in the water, and the water is pasteurized for the inactivation of unexpected microorganisms using a water bath for 20 min at 60 C. The torch plasma irradiates the surface of the water in the small petri dish. The amount and depth of the water are 20 ml and 3 mm, respectively. The treatment period (that is the plasma irradiation period) is 10, 20, or, 30 min. After the plasma irradiation, the bacilli in liquid are put on the agar in petri dishes and cultivate for 48 h. The number of surviving bacilli in petri dishes is determined using the colony count method.
3. Results and discussion 3.1. Production of active species The applied cathode voltage and the discharge current waveforms of the SDBD are shown in Fig. 1(b). The pulse-like waveforms indicate the typical barrier discharge, and the maximum discharge current is 23 mA. Typical power consumption of the torch obtained from the Lissajous figure is approximately 5 W in this experimental condition. The light emission of the air and oxygen torch plasmas is observed below the opening edge of the plasma torch with a length of approximately 5 mm, which originates mainly from the excited nitrogen. Fig. 2(a) and (b) illustrates the light emission spectra of the plasma torch using air and oxygen, respectively, measured horizontally using a multi-channel spectrometer. Fig. 2(a) indicates that the nitrogen second positive band is significant in the spectrum of the air plasma. The nitrogen metastable particles would affect the bacilli in the water. In the case of the oxygen plasma, the peak at 777 nm is significant in the light emission spectra as shown in Fig. 2(b), which is assigned as the triplet oxygen atom O(3P). In the atmospheric discharge generating high-energy electrons, the ground state O2 molecule is excited to B3S u , and then the reaction below occurs [10]:
B3
a
Gas
H.V. power supply
Tungsten wire
a Intensity (arb units)
b
u
40
6
30
0 0 -10 -2 -0.05
-20 0.00
0.05
0.10
0.15
Time (ms) Fig. 1. (a) Schematic diagram of atmospheric pressure plasma torch apparatus, and (b) typical cathode voltage and discharge current waveforms.
N2 2nd positive
6000 5000 4000
N2 1st negative
3000 2000 1000 300
400
b Intensity (arb units)
Applied voltage (kV)
10
Discharge current (mA)
20
7000
0 200
4
2
/Oð3 PÞ þ Oð1 DÞ
The O(3P) is excited and emits light of wavelength 777 nm. On the other hand, the light emission of O(1D) is significantly small due to its long lifetime. Therefore, the light emission of O(3P) is the index of the excited singlet oxygen atom O(1D). Fig. 2(b) suggests that the O(1D) is the major active species in the oxygen torch plasma. The O(1D) is a candidate for the inactivation factor of bacilli, because the lifetime of O(1D) is the order of milliseconds and significantly larger than that of other active oxygen species such as OH with a lifetime of ms. The chemical indicator which is specialized for oxygen radical detection shows the generation of active oxygen
Alumina tube
Copper sheet
X
500
600
700
v800
Wavelength [nm] 160 140 120 100 80 60 40 20 0 200
3
O(P)
1
O( D)
300
400
500
600
700
800
Wavelength [nm] Fig. 2. Typical light emission spectra of (a) air plasma, and (b) oxygen plasma generated inside the plasma torch.
N. Hayashi et al. / Vacuum 88 (2013) 173e176
175
Fig. 3. Oxygen radical detection in gas phase and H2O2, NO 2 , O3 measurement in liquid.
species at the downstream of the torch plasma for air and oxygen plasmas, as shown in Fig. 3. When the SDBD torch plasma using air or Ar irradiates the water surface, hydrogen peroxide (H2O2) is generated in the water due to the dissociation of H2O by high-speed neutral particles. The concentration of H2O2 is measured using an H2O2 test strip (Merquant), as illustrated in Fig. 3. The H2O2 concentration increases monotonically with the discharge period, and reaches 5.0 mg/l after 20 min. Also nitric oxide is generated in the water with concentration of 25 mg/l by a discharge of 20 min. In the case of oxygen plasma, both particle density and energy of radicals ejected from the torch would be small and H2O2 would not be detected throughout the 20 min plasma irradiation using H2O2 test strips. Also, the dissolved nitric oxide is detected with concentration of 1 mg/l for both air and Ar discharges using a pack test, and is below the measurement limit (0.02 mg/l) in the water after the oxygen discharge for 20 min, as shown in Fig. 3. The high density Ar plasma induces the discharge of surrounding air and the produced nitric oxide is dissolved in the water. On the other hand, the density of oxygen ions and radicals generated by the oxygen discharge is relatively small when comparing air and Ar discharges. Therefore, generation and solution of nitric oxide in water would be significantly small. In the case of air and Ar discharges, the ozone generation in the gas phase is below approximately 50 ppm, whereas the oxygen discharge generates ozone with concentration over 600 ppm and ozone dissolved into water at a concentration of 2.0 mg/l. The ultra violet (UV) light emission from the SDBD torch plasma to the water is measured utilizing the UV detection label, which is located at the bottom of the water vessel. When the UV level is irradiated by the plasma torch, the UV light emission intensity from the plasma torch is negligibly smaller than that of the UV light tube for microorganism inactivation. The gas temperature in the plasma generation region is approximately 50 C, and the water surface is more than 10 cm apart from the plasma production region. The temperature on the surface of the water is measured using both the IR thermometer and the thermolabel. After the plasma irradiation for 30 min, the temperature on the water surface is below 40 C, which is approximately 15 C higher than the room temperature. This temperature is acceptable even for the vegetative state of B. thuringiensis. Therefore, this sterilization method is categorized as a low temperature sterilization. The above chemical species of H2O2, N2O, O3 in water have typical half-lives of at least several tens of minutes and they remain in the water. However, their concentration of below several ten mg/ l is too low to inactivate bacilli in water. Therefore, active species with short lifetime generated by the torch plasma, such as O and OH radical are candidates for causing the sterilization.
3.2. Inactivation of bacillus in liquid Fig. 4(a) illustrates the residual number of vegetative form of bacilli as a function of the oxygen plasma irradiation period, that is, the survival curves of vegetative state of B. thuringiensis with irradiation of oxygen torch plasma. Dashed lines indicate survival curves of heat inactivation. The effect of the plasma irradiation is equivalent to the heat inactivation with a temperature of 50 C in
Fig. 4. Survival curves of the vegetative state of Bacillus thuringiensis applying plasma and heat treatment, and (b) microscope images of Bacillus thuringiensis before (left) and after (right) the plasma irradiation.
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N. Hayashi et al. / Vacuum 88 (2013) 173e176
cases of the plasma irradiation with the oxygen gas flow rates of 1 l/ min and 3 l/min, the inactivation effect of the plasma irradiation is equivalent to the heat inactivation at a temperature of 95 C. Fig. 5(b) illustrates the microscope images of the spores of B. thuringiensis after the plasma irradiation. The significant variation of shape of the spore is not observed. Therefore, the decomposition of substances in a spore occurs by the oxygen plasma. All tendencies of the survival curves with the plasma treatments described above are similar to those with the heat treatment. This fact implies that the inactivation mechanism by the plasma irradiation would be same as the heat treatment that is modification and decomposition of proteins containing inside the cell wall. The expected volume of water to be sterilized by one SDBS plasma torch is typically several hundred ml/day. In order to apply the system to a beverage factory, the electrode array system should have hundreds of thousands of torches, which is difficult to realize under the present situation. If the torch electrode is improved and the number of torches can be reduced to 1/10, the SDBS plasma torch system could be adapted for use in a beverage factory. On the other hand, sterilization of body fluids on wounds is possible. The treatment of liquids of several ml is achieved by one plasma torch. 4. Conclusions
Fig. 5. (a) Survival curves of the spore of Bacillus thuringiensis using plasma and heat treatment, and (b) microscope image of the spore after the plasma irradiation.
The effects of the plasma irradiation are equivalent to heat inactivation at a temperature of 50 C and 95 C in the case of vegetative state and for spores of bacilli, respectively. All tendencies of the survival curves with the plasma treatments are similar to those with the heat treatment. This fact implies that the inactivation mechanism by the plasma irradiation would be same as the heat treatment, that is modification and decomposition of proteins contained inside the cell wall. References
the case of vegetative state and spore of bacilli. Fig. 4(b) shows the microscope images of the vegetative state of B. thuringiensis before (left photograph) and after (right photograph) the oxygen plasma irradiation, respectively. The length of the bacilli becomes shorter by plasma irradiation of the liquid surface. Therefore, oxygen radicals are dissolved in the liquid and decompose the chain structure of the bacilli. This phenomenon suggests that radicals in water damage the cell wall protein of the bacilli, and this is one of candidates for explaining the inactivation mechanism of microorganisms in water by plasma irradiation. However, the detailed mechanism of the decomposition is under investigation. Fig. 5(a) shows the survival curves of spores of B. thuringiensis with irradiation by the oxygen torch plasma. Dashed lines indicate survival curves of heat inactivation of 95 C and 100 C. In both
[1] Rutala WA, Weber DJ, the Healthcare Infection Control Practices Advisory Committee. Guideline for disinfection and sterilization in healthcare facilities. Centers for Disease Control and Prevention; 2008. [2] Molins RA. Food irradiation: principles and applications. New York: WileyIEEE; 2001. p. 23e36. [3] Brown AC. Understanding food: principles and preparation. 4th ed. Stanford: Cengage Learning; 2007. p. 537e548. [4] Teschke M, Kedzierski J, Finantu-Dinu EG, Korzec D, Engemann J. IEEE Trans Plasma Sci 2005;33:310e1. [5] Laroussi M, Lu X. Appl Phys Lett 2005;87:113902e4. [6] Stoffels E, Flikweert AJ, Stoffels WW, Kroesen GMW. Plasma Sources Sci Technol 2002;11:383e8. [7] Stoffels E, Sakiyama Y, Graves D. IEEE Trans Plasma Sci 2008;36:1441e57. [8] Ikawa S, Kitano K, Hamaguchi S. Plasma Process Polym 2010;7:33e42. [9] Korachi M, Aslan N. Plasma Sci Technol 2011;13:99e105. [10] Eliasson B. Electrical discharge in oxygen part 1: basic data. Brown Boveri; 1985. Research Report KLR 83/40 C.
Contents lists available at SciVerse ScienceDirect
Vacuum journal homepage: www.elsevier.com/locate/vacuum
Inactivation characteristics of Bacillus thuringiensis spore in liquid using atmospheric torch plasma using oxygen Nobuya Hayashi a, *, Yusuke Akiyoshi b, Yasuyo Kobayashi c, Kouzo Kanda c, Kazusato Ohshima c, Masaaki Goto d a
Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, 6-1 Kasuga-kouen, Kasuga-shi, Fukuoka 816-8580, Japan Faculty of Science and Engineering, Saga University, 1 Honjo-machi, Saga-shi, Saga 840-8502, Japan Faculty of Agriculture, Saga University, 1 Honjo-machi, Saga-shi, Saga 840-8502, Japan d Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga-shi, Saga 849-8501, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2011 Received in revised form 8 March 2012 Accepted 8 March 2012
The atmospheric plasma torch produced by a barrier discharge using air and oxygen as working gases is adopted to inactivate microorganisms in a liquid. The inactivation characteristics of bacillus spore are investigated and compared with the heat sterilization method, and sterilization factors are active species with short-lifetimes produced in water. The population of Bacillus thuringiensis spore in pure water decreases 103 times within 20 min, and the obtained result is equivalent to the heat sterilization of 95 C. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Atmospheric torch plasma Oxygen radicals Low temperature treatment Bacillus inactivation Spores in liquid
1. Introduction Disinfections of microorganisms in liquid is an important procedure in medicine [1] and the food Industry [2]. The unique method that has been adopted to inactivate the microorganisms in a liquid is the heat pulse method [3], which applies heat to the liquid for a short period of several minutes. The temperature of the pressurized water increases to 130 C at 200 kPa, and microorganisms including bacillus spores are inactivated. However, any liquid to be sterilized tends to suffer modification in taste and aroma, in spite of the short period of heat application. Another certain application of the low temperature sterilization of a liquid is for body fluids on wounds. The expected volume of the liquid to be sterilized in the medical treatment is the order of ml or ml. Recently, atmospheric barrier discharge plasma using He gas [4e7] has been adopted to inactivate microorganisms in water [8,9]. This method offers non-heat sterilization of a liquid. Since the cost of He gas used in the method is significantly high, the He torch plasma sterilization method is difficult to utilize practically. The torch plasma produced by the surface dielectric barrier discharge (SDBD) has been adopted for sterilization of water. There are three
* Corresponding author. Tel./fax: þ81 92 583 7649. E-mail address: [email protected] (N. Hayashi). 0042-207X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vacuum.2012.03.023
advantages of the torch type SDBD for practical use: (i) The SDBD torch plasma is a kind of remote plasma where the treatment region separates from the discharge region. Therefore, low temperature treatment is realized and only energetic neutral particles and radicals with long lifetime are obtained at treatment region. (ii) The cost of torch electrodes and power supply is low because precise manufacturing of electrodes is not necessary. (iii) The discharge gap of SDBD is controlled spontaneously depending on circumstances such as material gas species, gas flow rate, humidity and temperature. Therefore, the SDBD is a significantly robust discharge and suitable for practical uses. In this study, the atmospheric plasma torch produced by the oxygen gas SDBD is studied to inactivate microorganisms in liquid. The inactivation characteristics of bacilli in spore and vegetative form in a liquid are investigated comparing the conventional heat sterilization method, and the sterilization factors are specified to be oxygen radicals with short lifetime. 2. Experimental procedure Fig. 1(a) shows the SDBD plasma torch used in this experiment. The copper thin sheet as a grounded electrode is wrapped around an alumina tube of the length of 100 mm and the inner diameter of 3 mm. The wound tungsten wire of diameter 0.23 mm is set in the inner surface of the alumina tube as a cathode. When a high voltage
174
N. Hayashi et al. / Vacuum 88 (2013) 173e176
with high frequency of 10 kHz is applied to the wire cathode electrode inside the alumina tube, the discharge occurs around the surface of the wire electrode. The pure oxygen, air, and Ar gases are used as the material gases of the plasmas. As the gas flows through the alumina tube, the ions and radicals are ejected from the opening edge of the alumina tube. The distance between the opening edge of the torch tube and the liquid surface is 15 mm, in order to prevent evaporation of the liquid by the gas flow. The applied voltage and the gas flow rate are kept constant at 6 kV and 1 slm, respectively. The bacilli utilized as sample microorganisms in the pure water are in spore and vegetative form of the Bacillus thuringiensis that is tolerant against heat and UV light. The spore with the population of order of 107 is dispersed in the water, and the water is pasteurized for the inactivation of unexpected microorganisms using a water bath for 20 min at 60 C. The torch plasma irradiates the surface of the water in the small petri dish. The amount and depth of the water are 20 ml and 3 mm, respectively. The treatment period (that is the plasma irradiation period) is 10, 20, or, 30 min. After the plasma irradiation, the bacilli in liquid are put on the agar in petri dishes and cultivate for 48 h. The number of surviving bacilli in petri dishes is determined using the colony count method.
3. Results and discussion 3.1. Production of active species The applied cathode voltage and the discharge current waveforms of the SDBD are shown in Fig. 1(b). The pulse-like waveforms indicate the typical barrier discharge, and the maximum discharge current is 23 mA. Typical power consumption of the torch obtained from the Lissajous figure is approximately 5 W in this experimental condition. The light emission of the air and oxygen torch plasmas is observed below the opening edge of the plasma torch with a length of approximately 5 mm, which originates mainly from the excited nitrogen. Fig. 2(a) and (b) illustrates the light emission spectra of the plasma torch using air and oxygen, respectively, measured horizontally using a multi-channel spectrometer. Fig. 2(a) indicates that the nitrogen second positive band is significant in the spectrum of the air plasma. The nitrogen metastable particles would affect the bacilli in the water. In the case of the oxygen plasma, the peak at 777 nm is significant in the light emission spectra as shown in Fig. 2(b), which is assigned as the triplet oxygen atom O(3P). In the atmospheric discharge generating high-energy electrons, the ground state O2 molecule is excited to B3S u , and then the reaction below occurs [10]:
B3
a
Gas
H.V. power supply
Tungsten wire
a Intensity (arb units)
b
u
40
6
30
0 0 -10 -2 -0.05
-20 0.00
0.05
0.10
0.15
Time (ms) Fig. 1. (a) Schematic diagram of atmospheric pressure plasma torch apparatus, and (b) typical cathode voltage and discharge current waveforms.
N2 2nd positive
6000 5000 4000
N2 1st negative
3000 2000 1000 300
400
b Intensity (arb units)
Applied voltage (kV)
10
Discharge current (mA)
20
7000
0 200
4
2
/Oð3 PÞ þ Oð1 DÞ
The O(3P) is excited and emits light of wavelength 777 nm. On the other hand, the light emission of O(1D) is significantly small due to its long lifetime. Therefore, the light emission of O(3P) is the index of the excited singlet oxygen atom O(1D). Fig. 2(b) suggests that the O(1D) is the major active species in the oxygen torch plasma. The O(1D) is a candidate for the inactivation factor of bacilli, because the lifetime of O(1D) is the order of milliseconds and significantly larger than that of other active oxygen species such as OH with a lifetime of ms. The chemical indicator which is specialized for oxygen radical detection shows the generation of active oxygen
Alumina tube
Copper sheet
X
500
600
700
v800
Wavelength [nm] 160 140 120 100 80 60 40 20 0 200
3
O(P)
1
O( D)
300
400
500
600
700
800
Wavelength [nm] Fig. 2. Typical light emission spectra of (a) air plasma, and (b) oxygen plasma generated inside the plasma torch.
N. Hayashi et al. / Vacuum 88 (2013) 173e176
175
Fig. 3. Oxygen radical detection in gas phase and H2O2, NO 2 , O3 measurement in liquid.
species at the downstream of the torch plasma for air and oxygen plasmas, as shown in Fig. 3. When the SDBD torch plasma using air or Ar irradiates the water surface, hydrogen peroxide (H2O2) is generated in the water due to the dissociation of H2O by high-speed neutral particles. The concentration of H2O2 is measured using an H2O2 test strip (Merquant), as illustrated in Fig. 3. The H2O2 concentration increases monotonically with the discharge period, and reaches 5.0 mg/l after 20 min. Also nitric oxide is generated in the water with concentration of 25 mg/l by a discharge of 20 min. In the case of oxygen plasma, both particle density and energy of radicals ejected from the torch would be small and H2O2 would not be detected throughout the 20 min plasma irradiation using H2O2 test strips. Also, the dissolved nitric oxide is detected with concentration of 1 mg/l for both air and Ar discharges using a pack test, and is below the measurement limit (0.02 mg/l) in the water after the oxygen discharge for 20 min, as shown in Fig. 3. The high density Ar plasma induces the discharge of surrounding air and the produced nitric oxide is dissolved in the water. On the other hand, the density of oxygen ions and radicals generated by the oxygen discharge is relatively small when comparing air and Ar discharges. Therefore, generation and solution of nitric oxide in water would be significantly small. In the case of air and Ar discharges, the ozone generation in the gas phase is below approximately 50 ppm, whereas the oxygen discharge generates ozone with concentration over 600 ppm and ozone dissolved into water at a concentration of 2.0 mg/l. The ultra violet (UV) light emission from the SDBD torch plasma to the water is measured utilizing the UV detection label, which is located at the bottom of the water vessel. When the UV level is irradiated by the plasma torch, the UV light emission intensity from the plasma torch is negligibly smaller than that of the UV light tube for microorganism inactivation. The gas temperature in the plasma generation region is approximately 50 C, and the water surface is more than 10 cm apart from the plasma production region. The temperature on the surface of the water is measured using both the IR thermometer and the thermolabel. After the plasma irradiation for 30 min, the temperature on the water surface is below 40 C, which is approximately 15 C higher than the room temperature. This temperature is acceptable even for the vegetative state of B. thuringiensis. Therefore, this sterilization method is categorized as a low temperature sterilization. The above chemical species of H2O2, N2O, O3 in water have typical half-lives of at least several tens of minutes and they remain in the water. However, their concentration of below several ten mg/ l is too low to inactivate bacilli in water. Therefore, active species with short lifetime generated by the torch plasma, such as O and OH radical are candidates for causing the sterilization.
3.2. Inactivation of bacillus in liquid Fig. 4(a) illustrates the residual number of vegetative form of bacilli as a function of the oxygen plasma irradiation period, that is, the survival curves of vegetative state of B. thuringiensis with irradiation of oxygen torch plasma. Dashed lines indicate survival curves of heat inactivation. The effect of the plasma irradiation is equivalent to the heat inactivation with a temperature of 50 C in
Fig. 4. Survival curves of the vegetative state of Bacillus thuringiensis applying plasma and heat treatment, and (b) microscope images of Bacillus thuringiensis before (left) and after (right) the plasma irradiation.
176
N. Hayashi et al. / Vacuum 88 (2013) 173e176
cases of the plasma irradiation with the oxygen gas flow rates of 1 l/ min and 3 l/min, the inactivation effect of the plasma irradiation is equivalent to the heat inactivation at a temperature of 95 C. Fig. 5(b) illustrates the microscope images of the spores of B. thuringiensis after the plasma irradiation. The significant variation of shape of the spore is not observed. Therefore, the decomposition of substances in a spore occurs by the oxygen plasma. All tendencies of the survival curves with the plasma treatments described above are similar to those with the heat treatment. This fact implies that the inactivation mechanism by the plasma irradiation would be same as the heat treatment that is modification and decomposition of proteins containing inside the cell wall. The expected volume of water to be sterilized by one SDBS plasma torch is typically several hundred ml/day. In order to apply the system to a beverage factory, the electrode array system should have hundreds of thousands of torches, which is difficult to realize under the present situation. If the torch electrode is improved and the number of torches can be reduced to 1/10, the SDBS plasma torch system could be adapted for use in a beverage factory. On the other hand, sterilization of body fluids on wounds is possible. The treatment of liquids of several ml is achieved by one plasma torch. 4. Conclusions
Fig. 5. (a) Survival curves of the spore of Bacillus thuringiensis using plasma and heat treatment, and (b) microscope image of the spore after the plasma irradiation.
The effects of the plasma irradiation are equivalent to heat inactivation at a temperature of 50 C and 95 C in the case of vegetative state and for spores of bacilli, respectively. All tendencies of the survival curves with the plasma treatments are similar to those with the heat treatment. This fact implies that the inactivation mechanism by the plasma irradiation would be same as the heat treatment, that is modification and decomposition of proteins contained inside the cell wall. References
the case of vegetative state and spore of bacilli. Fig. 4(b) shows the microscope images of the vegetative state of B. thuringiensis before (left photograph) and after (right photograph) the oxygen plasma irradiation, respectively. The length of the bacilli becomes shorter by plasma irradiation of the liquid surface. Therefore, oxygen radicals are dissolved in the liquid and decompose the chain structure of the bacilli. This phenomenon suggests that radicals in water damage the cell wall protein of the bacilli, and this is one of candidates for explaining the inactivation mechanism of microorganisms in water by plasma irradiation. However, the detailed mechanism of the decomposition is under investigation. Fig. 5(a) shows the survival curves of spores of B. thuringiensis with irradiation by the oxygen torch plasma. Dashed lines indicate survival curves of heat inactivation of 95 C and 100 C. In both
[1] Rutala WA, Weber DJ, the Healthcare Infection Control Practices Advisory Committee. Guideline for disinfection and sterilization in healthcare facilities. Centers for Disease Control and Prevention; 2008. [2] Molins RA. Food irradiation: principles and applications. New York: WileyIEEE; 2001. p. 23e36. [3] Brown AC. Understanding food: principles and preparation. 4th ed. Stanford: Cengage Learning; 2007. p. 537e548. [4] Teschke M, Kedzierski J, Finantu-Dinu EG, Korzec D, Engemann J. IEEE Trans Plasma Sci 2005;33:310e1. [5] Laroussi M, Lu X. Appl Phys Lett 2005;87:113902e4. [6] Stoffels E, Flikweert AJ, Stoffels WW, Kroesen GMW. Plasma Sources Sci Technol 2002;11:383e8. [7] Stoffels E, Sakiyama Y, Graves D. IEEE Trans Plasma Sci 2008;36:1441e57. [8] Ikawa S, Kitano K, Hamaguchi S. Plasma Process Polym 2010;7:33e42. [9] Korachi M, Aslan N. Plasma Sci Technol 2011;13:99e105. [10] Eliasson B. Electrical discharge in oxygen part 1: basic data. Brown Boveri; 1985. Research Report KLR 83/40 C.