22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Influence of plasma gas temperature on inactivation effect to various bacteria
H. Kawano1, T. Oshita1, T. Takamatsu3, Y. Matsumura2, H. Miyahara1, A. Iwasawa2, T. Azuma3 and A. Okino1
1
2
Department of Energy Sciences, Tokyo Institute of Technology, Yokohama, Japan
Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Japan
3
Graduate School of Medicine, Kobe University, Kobe, Japan
Abstract: The influence of the plasma gas temperature on inactivation effects to various
bacteria and the amount of generated reactive oxygen species in plasma were investigated.
The 6 digits of surviving E.coli and S.aureus were inactivated by 20 sec plasma irradiation
at above 50 ºC. The concentration of reactive oxygen species increased as the plasma gas
temperature is raised.
Keywords: atmospheric plasma, plasma gas temperature, bacterial inactivation
1. Introduction
Atmospheric non-thermal plasma sources have attracted
attention in medical field as a new sterilization device
because it has a wide antibacterial spectrum at low
temperature and it does not have residual toxicity unlike
ethylene oxide gas [1]-[6]. In particular, studies of
sterilization on living bodies by direct plasma irradiation
has been active because plasma sources that do not give
discharge damages to the target were developed [7]-[10].
To irradiate living bodies with the plasma, accurate
temperature control is required to prevent the thermal
damage to the target. For example, to irradiate skin with
plasma, the plasma gas temperature should be controlled
below a denaturation temperature of proteins. In
conventional plasma sources, in order to reduce the
plasma gas temperature, the methods such as limiting
discharge power or increasing the gas flow rate are used.
However, in these methods, since the plasma density
decreases, therefore treatment effect also decreases. For
that reason it is difficult to generate high power plasmas
at low gas temperatures. To overcome these problems, we
proposed temperature-controllable plasma (PAT Japan:
4611409, U.S.: 8,866,389) and developed a plasma
source. This device can control the plasma gas
temperature from around -50 ºC to 160 ºC with a standard
deviation of 1 ºC independently from the discharge power
and the plasma gas flow rate. Since the discharge power is
constant by using this plasma source, a high bacterial
inactivation effect at low gas temperature is expected. In
this study, influence of the plasma gas temperature on
inactivation effect to various kind of bacteria was
investigated using the temperature-controllable plasma
source.
supplied from gas cylinder is cooled using a gas-cooling
2. Experimental section2.1. Temperature-controllable plasma source
As shown in Fig. 1(a), in general atmospheric plasma
sources, the plasma gas temperature is higher than the
room temperature because the plasma is generated from
the room temperature gas. As shown Fig. 1(b), in
temperature-controllable plasma source, the plasma gas
device that uses liquid nitrogen (-196 ºC). Then the gas is
heated using a heater to generate the plasma. Information
of the gas temperature is feed back to the heater, and the
plasma gas temperature can be controlled to the desired
value. The gas temperature was measured using
thermocouple. As shown in Fig. 2, a dielectric barrier
discharge (DBD) plasma jet was used as the plasma
P-III-10-13
(a) General plasma sources
(b) Temperature-controllable atmospheric plasma source
Figure 1. Comparison of general plasma sources and
temperature-controllable plasma source
Figure 2. Structure of DBD plasma jet
1
source. Two of 10 mm Ring-shaped copper electrodes
were placed keeping 10 mm space at the periphery of a
glass tube with 3 mm i.d. and 5 mm o.d., and 9 kV
electric power was applied at 16 kHz to generate the
plasma.
2.2. Experiment of bacterial inactivation
To evaluate bacterial inactivation effect of the plasma,
the number of the surviving bacteria after plasma
irradiation were counted. E.coli (ATCC25922) and
S.aureus (ATCC25923) were used as indicator bacteria to
investigate the inactivation effects on bacteria which have
different structure. E.coli is classified a gram-negative
bacterium, which has outer membrane. S.aureus is
classified a gram-positive bacterium which have
structureless of outer membrane. To isolate inactivation
effect from influence of pH, bacteria were suspended in
Dulbecco’s phosphate-buffer saline (DPBS) (-). Figure 3
shows the experimental setup for bacterial inactivation.
The outlet of the plasma was placed immediately above
the solution, and the distance between the outlet and the
surface of the solution was set to 5 mm. The solution was
irradiated with 3% oxygen-mixed helium plasma. The gas
flow rate was 10 slm. The plasma gas temperature was
controlled from 10 ºC to 80 ºC at an interval of 10 ºC.
The initial bacterial number was 108 cfu in 200 µL. After
the plasma irradiation, the solution was serially diluted
and plated on agar media. The agar media were incubated
for 18 hour at 37 ºC, after that the number of the glown
colony were counted.
Figure 3. Setup for plasma irradiation
2.3. Measurement of amount of The generated ROS
The factor of bacterial inactivation effect by plasma
irradiation is considered to be caused by oxidant stress of
reactive oxygen species (ROS), generated by plasma [11].
Hence, it is supposed that the amount of the generated
ROS affect the bacterial inactivation. Therefore, to
investigate the plasma gas temperature dependence on the
amount of generated ROS, the concentrations of ROS in
DPBS were measured. In this experiment, the 200-µl
DPBS (including the reagent in the detection of each
ROS) were irradiated with 3 % oxygen-mixed helium
plasma for 30 sec, and hydroxyl radicals (HO·), singlet
oxygen (1O 2 ), ozone (O 3 ) and hydrogen peroxide (H 2 O 2 )
in the solution were measured. The plasma gas
2
temperature was controlled under the same conditions as
the bacterial inactivation experiments.
Concentration of HO · and 1O 2 were measured by
Electron Spin Resonance (ESR). In this method, by
reacting the measuring ROS with spin-trapping agents,
spin-adducts that are chemically stable products are
formed and a magnetic resonance spectrum of formed
spin-adducts are obtained by ESR spectroscopy (JESFA100, JEOL Ltd., Tokyo, Japan). The ROS
concentrations are calculated from the magnetic
resonance spectrum.
As the spin-trapping agents of HO·, 5,5-dimethyl-1pyrroline-N-oxide (DMPO) was used [12], and for 1O 2 ,
2,2,5,5,-tetramethyl-3-pyrroline-3-carboxamide
(TPC)
was used [13]. These reagents were dissolved in a DPBS
(-) solution, and the concentrations of DMPO, TPC were
fixed at 200 mM and 75 mM, respectively. The ESR was
set at a 9.424818-GHz microwave frequency, 100 kHz
modulation frequency, 2 min sweep time, a 335.5 ± 5 mT
magnetic field, a 0.7 mT modulation width and a 0.1 s
time constant.
Concentration of O 3 and H 2 O 2 were measured by
absorption spectrophotometry using a double beam
spectrophotometer [U-2900, Hitachi High-Technologies
Co.]. In this method, the concentration of each ROS was
calculated from the absorbance of the reagents that react
with the ROS to give a color. Concentration of O 3 was
measured from the absorbance of a 350 nm wavelength
that decreases by reaction of O 3 with indigo reagent
(Ozone AccuVac® Ampules, MR, pk/25, Hach Company
(USA)). Concentration of H 2 O 2 was measured from the
absorbance of a 200 nm wavelength that increases by the
reaction of H 2 O 2 with the solution including xylenol
orange 200 µM, ammonium iron (II) sulfate 150 mM,
sulfuric acid 150 mM and sorbitol 200 mM [14]. The
absorbance of a 440 nm wavelength was measured.
3. Result and discussion
3.1. Influence of plasma gas temperature on bacterial
inactivation
To investigate the influence of the plasma gas
temperature on the bacterial inactivation effect, the
bacteria in solution were irradiated with the plasma at
various gas temperature. Figure 4 and 5 show the number
of surviving E.coli and S.aureus after plasma irradiation.
With 120 sec. plasma irradiation at 10 ºC, the number of
surviving E.coli was decreased by 3 digits, and the
number of surviving S.aureus was decreased by 1 digit.
By increasing the plasma gas temperature above 50 ºC,
the numbers of surviving bacteria in both the cases after
the plasma irradiation for 20 sec were decreased by 6
digits. These results show that the inactivation effects on
both bacteria increased with the plasma gas temperature.
Even low gas temperature plasmas could also decrease the
number of the surviving bacterium in both cases by
extending the irradiation time.
Figure 6 shows the number of surviving E. coli and S.
aureus after plasma irradiation for 40 sec. With the
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plasma at 10 ºC the number of surviving E.coli was
almost the same as S.aureus. However, with the plasma
irradiation at above 20 ºC, the number of surviving E.coli
was less than that of S.aureus. Therefore, it is indicated
that inactivation effect on E.coli with irradiation of high
temperature plasmas is higher than that on S.aureus. This
result shows that the influence of the plasma gas
temperature on bacterial inactivation effect differs
according to the bacterial species.
As the cause of high inactivation effects on various
bacteria with high gas temperature plasmas, the heat or
the amount of the generated ROS could be considered.
However, it is rejected that the heat directly affects
inactivation effects because the number of the surviving
bacteria of both bacteria sustained with only gas at 80 ºC
in 2 min as shown in figure 3 and 4. The influence of
generated ROS is described in the next section.
3.2. Influence of plasma gas temperature on amount of
the generated ROS
To investigate the influence of the plasma gas
temperature on the amount of generated ROS, each ROS
concentration in DPBS was measured after the plasma
irradiation. Figure 7 to 10 show the concentration of
HO·, 1O 2 , O 3 and H 2 O 2 in DPBS, respectively. By
increasing the plasma gas temperature, the concentration
of HO·, 1O 2 and H 2 O 2 increased. The concentration of
O 3 was below the detection limit at above 50 ºC. These
results indicate that the plasma gas temperature affects the
amount of generated ROS. Particularly the increase rate
of 1O 2 concentration was relatively high in measured
ROS and it became 17 times from 10 ºC to 80 ºC. This
result suggests that the amount of the generated 1O 2
strongly affect the bacterial inactivation effect.
Figure 6. Comparison between number of surviving
E.coli and S. aureus for 40 sec of plasma irradiation
Figure 4. Number of surviving E.coli irradiated with
various plasma gas temperature
Concentration [µM]
30
25
20
15
10
5
0
0 10 20 30 40 50 60 70 80 90
Plasma gas temperature [ºC]
Figure 7. Influence of plasma gas temperature
on HO· concentration
Figure 5. Number of surviving S. aureus irradiated with
various plasma gas temperature
Concentration [µM]
160
120
80
40
0
0 10 20 30 40 50 60 70 80 90
Plasma gas temperature [ºC]
Figure 8. Influence of plasma gas temperature
1
on O2 concentration
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3
Concentration [µM]
20
15
10
5
0
0 10 20 30 40 50 60 70 80 90
Plasma gas temperature [ºC]
Figure 9. Influence of plasma gas temperature
on H2O2 concentration
Concentration [µM]
3
2
5. Acknowledgement
This work was partially supported by Plasma Concept
Tokyo, Inc. The authors would like to thank them for their
support and collaboration.
1
0
0 10 20 30 40 50 60 70 80 90
Plasma gas temperature [ºC]
Figure 10. Influence of plasma gas temperature
on O3 concentration
4. Summary
Using the temperature-controllable plasma jet, influence
of the plasma gas temperature on inactivation effects on E.
coli and S. aureus were investigated. The bacteria in
DPBS (-) were irradiated with various gas temperature
plasmas and the number of surviving bacteria was
measured. As a result, with 120 sec plasma irradiation at
10 ºC, the number of surviving E.coli was decreased by 3
digits, and the number of surviving S.aureus was
decreased by 1 digit. By increasing the plasma gas
temperature above 50 ºC, the numbers of surviving
bacterium in both cases were decreased by 6 digits. These
results show that the bacterial inactivation effect
increased with the plasma gas temperature and even low
gas temperature plasmas could decrease the number of
surviving bacterium in both cases by extending the
irradiation time.
With 40 sec plasma irradiation at 10 ºC, the number of
surviving E.coli was almost same as the number of
surviving S.aureus. However, with the plasma at above 20
ºC, the number of surviving E.coli was less than S.aureus.
Therefore, it is indicated that inactivation effect on E.coli
with irradiation of high plasma gas temperatures is higher
than that on S.aureus. This result shows that the influence
of the plasma gas temperature on bacterial inactivation
effect differs according to the bacterial species.
To investigate the influence of a change of the
generated ROS, the concentration of HO·, 1O 2 , O 3 and
H 2 O 2 in DPBS was measured after the plasma irradiation.
As a result, increasing plasma gas temperature, the
concentration of HO·, 1O 2 and H 2 O 2 were increased and
the O 3 was below minimum detection limit at above 50
4
ºC. These results indicate that the plasma gas temperature
affects the amount of the generated ROS. Particularly the
increase rate of 1O 2 concentration was relatively high in
measured ROS and it became 17 times from 10 ºC to 80
ºC. This result suggests that the amount of the
generated 1O 2 strongly affect the bacterial inactivation
effect.
These results show a possible that the amount of the
generated ROS could be controllable with controlling
plasma gas temperature and the effective ROS differs
according to the bacterial species. It is considered that
specific bacterium is inactivated by controlling plasma
gas temperature. Further study, changes of the amount of
generated various active species such as reactive nitrogen
species will be investigated.
6. References
[1] D. Dobrynin, G. Fridman, Y. V. Mukhin, M. A.
Wynosky-Dolfi, J. Rieger, R. F. Rest, A. F. Gutsol, and
A. Fridman, IEEE Trans. on Plasma Science, 38, 1878
(2010).
[2] T. Iwai, K. Okumura, K. Kakegawa, H. Miyahara and
A. Okino, Journal of Analytical Atomic Spectrometry, 29,
2108 (2014).
[3] T. Takamatsu, A. Kawate, K. Uehara, T. Oshita, H.
Miyahara, D. Dobrynin, G. Fridman, A. Fridman and A.
Okino, Plasma Medicine, 2, 4, 237 (2012).
[4] T. Tamura, Y. Kaburaki, T. Sasaki, H. Miyahara and
A. Okino, IEEE Trans. on Plasma science, 39, 1684
(2011).
[5] Y. Kaburaki, A. Nomura, Y. Ishihara, T. Iwai, H.
Miyaahara and A. Okino, Anal. Sci., 29, 1147 (2013).
[6] K. Shigeta, Y. Nagata, T. Iwai, H. Miyahara and A.
Okino, IEEF Trans. on Fundamentals and Marerials, 130,
955 (2010).
[7] S. kanazawa, M. Mriwaki, and S. Okazaki, J. Phys,
Appl. Phys, 21, 838 (1988).
[8] T. Takamatsu, H. Hirai, R. Sasaki, H. Miyahara and
A. Okino, IEEE Trans. On Plasma Science, 41, 119
(2013).
[9] T. Takamatsu, H. Miyahara, T. Azuma and A. Okino,
J. Toxicolo. Sci., 39, 281 (2014).
[10] T. Iwai, Y. Takahashi, H. Miyahara and A. Okino,
Analytical Sciences, 29, 1141 (2013).
[11] T. Takamatsu, K. Uehara, Y. Sasaki, H. Miyahara, Y.
Matsumura, A. Iwasawa, N. Ito, T. Azuma, M. Kohno
and A. Okino, RSC Advances, 4, 39901 (2014)
[12] M. Kohno, M. Yamada, K. Mitsuta, Y. Mizuta, and
T. Yoshikawa, Bull. Chem. Soc. Jpn, 64, 1447 (1991).
[13] Y. Matsumura, A. Iwasawa, T. Kobayashi, T.
Kamachi, T. Ozawa and M. Kohno, Chem. Lett., 42, 10,
1291 (2013).
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[14] H. Ikai, K. Nakamura, T. Kanno, M. Shirato, L.
Meirelles, K. Sasaki and Y. Niwano, Biocontrol Sci. 18, 3,
137, (2013).
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