23P023
Inactivation Characteristics of Bacillus Thuringiensis Spore in Liquid
Using Atmospheric Torch Plasma Generated by Low-frequency Discharge
大気圧トーチプラズマによる液中BT菌芽胞の不活化特性
Nobuya Hayashi1, Yusuke Akiyoshi1, Yasuyo Kobayashi2, Kouzo Kanda2,Kazusato Ohshima2 and Masaaki Goto3
林 信哉1, 秋吉雄介1，小林康子2，神田康三2，大島一里2，後藤昌昭3
1

Faculty of Science and Engineering, Saga University, 1 Honjo-machi, Saga-shi, Saga 840-8502, Japan
佐賀大学大学院工学系研究科 〒840-8502 佐賀市本庄町12
Faculty of Agriculture, Saga University, 1 Honjo-machi, Saga-shi, Saga 840-8502, Japan
佐賀大学農学部 〒840-8502 佐賀市本庄町13
Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga-shi, Saga 849-8501, Japan佐賀大学医学部 〒849-8501 佐賀市鍋島5-1-1

The atmospheric plasma torch produced by barrier discharge using air as working gas is adopted to sterilize
microorganisms in liquid. The inactivation characteristics of bacillus spore are investigated comparing heatsterilization method, and sterilization factors are active species with short-lifetime produced in water. The
population of Bacillus Thuringiensis spore in PBS solution decreases 10-3 times within 20 min, and theobtained result is equivalent to the heat sterilization of 95oC.

1. Introduction
Disinfection of microorganism in liquid is
important procedure in medicine and foodIndustry. The unique method that has been
adopted to inactivate the microorganism in liquid
is the heat pulse method, which applies heat toliquid for short period of several minutes. The
temperature of water increase to 130 oC, andmicroorganism including bacillus spores areinactivated. However, liquid to be sterilized tend
to suffer modification in taste and aroma, in spiteof short period of heat application.
Recently, atmospheric torch plasma using He
gas has been adopted to inactivate microorganismin water[1,2]. This method serves the non-heatsterilization of liquid. Since the cost of He gas
used in the method is significantly high, the He
torch plasma sterilization method is difficult to beutilized practically. In this study, the atmosphericplasma torch using air is adopted to sterilize
liquid. The inactivation characteristics of bacillus
spore are clarified, and sterilization factors areattempted to be specified.

applied to the wire cathode electrode inside thealumina tube, the discharge occurs around thesurface of the wire electrode. The air or pureoxygen gas is used as material gas of plasma. Asthe gas flows through the alumina tube, the ions andradicals are ejected from the opening edge of thealumina tube. The distance between the openingedge of the torch tube and the liquid surface is15mm, in order to prevent evaporation of the liquidby the gas flow. The applied voltage and the gas
flow rate are kept constant, 6 kV and 1 slm,respectively.
The bacillus spore utilized as sample
microorganism in the PBS is the Bacillus
Thuringiensis that is tolerant against heat and UVlight. The vegetative state of the Bacillus
Thuringiensis is also served, and its inactivation
characteristics are compared with those of the spore.
The population of the spore is order of 106, which isdispersed in pure water. The torch plasma irradiates
to the surface of the water in the small petri dish.
The amount and depth of the water are 20ml and3mm, respectively.

2. Experimental procedure
The plasma torch adopted in this experiment iscategorized into the surface discharge electrodes.
The tungsten wire as a grounded electrode is woundaround an alumina tube of the length of 100 mm
and the inner diameter of 3 mm, as illustrated in Fig.
1. Also, another tungsten wire is set inner surface of
the alumina tube as a cathode. When the highvoltage with the high frequency of 10 kHz is

3. Results and discussion
3.1 Production of active species
Figure 2 shows the typical open-shutter
photograph of the air torch plasma. The lightemission of the plasma torch is observed with thelength of approximately 5 mm, which is mainlyoriginated from the excited nitrogen. Figure 3illustrates the light emission spectra measured using
a multi-channel spectrometer. The nitrogen second

GasAlumina
tubeTungsten
wire

H.V. power～ supplyFig.1. Schematic of atmospheric pressure torch
plasma apparatus.

Nornalized population of bachilli

(a)

010

plasma treatmentheat
oheat treatment at 45 C
oheat treatment at 50 C-1

10-2

10-3

10-4

10-5

1005(b) 10 0
Normalized number of bacilli

In general, when the atmospheric air or Ar
plasma irradiates to the water surface, the hydrogen
peroxide (H2O2) is generated in the water due to thedissociation of H2O by high-speed neutral particles.
In the case of oxygen plasma, velocity of particles
ejected from the torch would be small, and H2O2
has not been detected throughout the plasma
irradiation for 20 min using H2O2 test strips(Merquant). Also, the dissolved nitric oxide is not
obtained in the water after the discharge for 20 min.
The Ultra violet (UV) light emission from theplasma torch is measured utilizing the UV detection
label, which is located at the bottom of the liquidvessel. The UV label is irradiated by the plasma
torch, and indicates that the UV light emission
intensity from the plasma torch is significantly
smaller than that of the UV light tube formicroorganism inactivation. The temperature ofboth of the water and the gas on the surface of thewater is below 40 oC, which is 15 oC higher than theroom temperature. This temperature is acceptable
for the vegetative state of Bacillus Thuringiensis.
Above results imply that the sterilization factor is
not active species of H2O2, N2O- and O3 and UVlight, but unidentified active species with short
lifetime, such as OH radical.

101520Treatment period (min)2530

plasma treatment
oheat treatment at 95 C

10-1

10-2

10-3

10-410-50510
1520
Treatment period (min)2530Figs.2. Survival curves of the vegetative (a) andspore (b) of Bacillus Thuringiensis using plasma andheat treatment.

positive band is significant in the spectra. Therefore,
the nitrogen meta-stable particles would affect thebacillus in the solution. Also, a chemical indicatorfor oxygen radical detection indicates thegeneration of active oxygen species at the down-stream of the torch plasma.

3.2 Inactivation of bacillus in liquid
Figures 2(a) and (b) illustrate survival curves ofvegetate state and spore of bacilli with irradiation of
torch plasma, respectively. Dashed lines indicate
survival curves of heat inactivation. The effects ofthe plasma irradiation is equivalent to the heat
inactivation with the temperature of 50oC and 95oC
in the case of vegetate state and spore of bacilli,respectively. When the oxygen flow rates are 1l/min and 3 l/min, the inactivation effects of theplasma irradiation are equivalent to the heat
inactivation with the temperature of 95oC and 85oC,respectively. Above all tendencies of the survival
curves with the plasma treatments are similar to
those with the heat treatment. This fact implies thatthe inactivation mechanism by the plasma
irradiation would be same as the heat treatment thatis modification and decomposition of proteins
containing inside the cell wall.
References[1] M. Korachi and N. Aslan, Plasma Sci. Technol. 13,
99 (2011).
[2] S. Ikawa, K. Kitano, and S. Hamaguchi, Plasma
Process. Polym. 7, 33 (2010).