Comparison of The Real-Time And Long Last Disinfection Abilities Of A
Direct-Current Cold Atmospheric-Pressure Micro-Jet
Qian Zhang, Peng Sun, Haiyan Wu, Ruixue Wang, Ruonan Ma, Jue Zhang, Jing Fang
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
WeiDong Zhu
Saint Peter’s College, Jersey City, New Jersey, USA
Abstract: A direct-current, atmospheric pressure, cold plasma microjet (PMJ) has been sustained in distilled water
in a quasi-steady gas cavity [1]. PMJ with various argon based gas mixtures (Ar, Ar/O2 (2%), Ar/O2 (2%)/N2
(10%), Ar/O2 (2%)/N2 (50%)) was used to inactive S. aureus suspended in liquid. LIVE/DEAD Baclight bacterial
viability test (fluorescence microscopy) was used to confirm the inactivation of S.aureus. Reactive oxygen species
and reactive nitrogen species (ROS/RNS) were measured by Electron Spin Resonance spectroscopy. The role of
ROS in inactivation was evaluated by the scavengers of co-responding radicals. Plasma activated water (PAW)
was applied to S.aureus to evaluate the effect of long-lived species in water. The prosperities of the PAW such as
pH, temperature, and the concentrations of NO2-, NO3-, Cu+, Cu2+, H2O2, and O3 were detected and analyzed
throughout. Possible disinfection pathways of reactive oxygen species and reactive nitrogen species (ROS/RNS)
will be discussed at the conference.
Keywords: S. aureus, Cold plasma microjet (PMJ), Reactive oxygen/nitrogen species, Plasma Activated Water
(PAW)
1. Introduction
Water pollution and waterborne diseases are still big
problems in both developed and developing
countries [1][2].Over 100 different types of bacteria,
protozoa, and viruses exist in contaminated water.
These microorganisms are responsible for various
serious illnesses such as kidney failure and
degenerative heart disease. According to statistics,
water related causes occupied approximately 80% of
all communicable diseases [3].
Traditional water treatments are still limited and
cannot meet the demands in many cases. For
example, adding chemicals (such as chlorine) into
water, which is the most widely used water
treatment method, have problems of chemicals
residual as well as potential health issues.[4] Ozone
and Ultra Violet light started to gain popularity in
recent years. However, Ozone is too expensive and
bacterial with Ultra Violet light treatment can
survive through photo-reactivation [5].
More recently, attempts have been made to
inactivate bacteria in water with non-thermal
plasmas (e.g., pulsed streamer discharge plasma,
gliding arc discharge plasma, etc.) because of the
simplicity and efficiency of plasma-based methods.
A direct-current, atmospheric pressure, cold plasma
microjet (PMJ) can be sustained in distilled water in
a quasi-steady gas cavity and can effectively
inactivation of bacteria in an aqueous environment
[6][7][8][9][10][11]. However, few had reported the
comparision of the real-time and long last
disinfection of inactivation of bacteria by
non-thermal atmospheric pressure plasmas in
aqueous environment.
In this paper, PMJ with various argon based gas
mixtures (Ar, Ar/O2 (2%), Ar/O2 (2%)/N2 (10%),
Ar/O2 (2%)/N2(50%)) was used to inactive S.aureus
suspended in liquid. Unlike our previous studies
which only discussed the PMJ real-time disinfection
ability [6] [11] [13], we compared the real-time and
long last disinfection abilities of PMJ. Possible
disinfection pathways of reactive oxygen species and
reactive nitrogen species (ROS/RNS) will be
discussed.
2. Experimental Part
The plasma device used in this study comprised two
copper tubes as electrodes separated by a ceramic
tube. The device was driven by a direct current
negative-polarity high-voltage power supply
(Matsuada AU5R120) through a 5 kΩ ballast resistor.
Detailed schematic diagram of the device as well as
the electrical circuit can be found in references [6]
[11] [13].
A quick increase of
inactivation rate from 0% to 85.5% was
observed when Ar/O2 (2%) was used as the
working gas within the first 6 minutes. The
inactivation rate of Ar/O2 (2%)/N2 (10%)PMJ was
Various argon based gas mixtures (Ar, Ar/O2 (2%),
Ar/O2 (2%)/N2 (10%), Ar/O2(2%)/N2(50%)) was
used as the working gas. S.aureus was used as a
model in the inactivation experiments. The bacterial
is purchased from China General Microbiological
Culture Collection Center.
around 35% within the first 10 minutes while its’
inactivation rate can increase uo to 93.3 % in 20min
(data not show here). The real time inactivation
capabilities of the PMJ with various gas mixtures
were
in
the
following
order:
Ar/O2
(2%)>Ar/O2(2%)/N2(10%)>Ar.
disinfection
ability.
Colony-forming unit (CFU) counts and inactivation
calculated by the formula below:
For bacteria viability test, the PMJ treated solution
were collected, diluted to the same optical density
value and prepared for the viability detection
according to the protocol of LIVE/DEAD®
BacLight™ Bacterial Viability kit (Invitrogen,
L7012).
Reactive oxygen species and reactive nitrogen
species (ROS/RNS) were detected by electron spin
resonance spectroscopy. The role of ROS in
inactivation was evaluated by co-responding radical
scavengers. The overall pH and temperature of water
were recorded during the span of the experiments;
while the concentrations of NO2-, NO3-, Cu+, and
Cu2+ in water were evaluated with HPLC. At the
end of each experiment, the concentration of H2O2
was evaluated with Hydrogen Peroxide Test Kit
Model HYP-1 Cat.No.22917-00. Detail information
can be found in our previous works [6][11][12][13]
3. Results and Discussions
The real-time disinfection abilities of PMJ
S. aureus in water was treated by PMJ with
different gases input (Ar, Ar/O2 (2%), Ar/O2
(2%)/N2 (10%)) from 0 to 10 min and the
inactivation rates were plotted in figure 1. When
Ar was used as the input gas, the PMJ almost had no
Ar
Inactivation Rate(%)
rate are used to demonstrate the inactivation of
bacterial. The inactivation rate of bacteria is
Ar/O2(2%)
100
Ar/O2(2%)/N2(10%)
80
60
40
20
0
0
2
4
6
8
10
Treatment Time(mins)
Figure 1. Inactivation rate of S.aureus treated with Ar, Ar/O2
(2%) and Ar/O2 (2%)/N2 (10%) plasma in water separately.
To verify the inactivation of S.aureus in water by
PMJ treatment, we evaluated the viability of the
cells by monitoring the membrane integrity of the
cells through fluorescence microscopy. Live cells
are stained green and dead cells are stained red. The
ratio of green to red fluorescence intensities provides
a quantitative index of bacterial viability.
Majority of the cells stained red after 8min Ar/O2
(2%) PMJ treatment (Figure 2(A)) or 16min Ar/O2
(2%)/N2 (10%) PMJ treatment (Figure 2(B)). An
even clearer situation was observed in the samples
treated for 16min Ar/O2 (2%) PMJ treatment or
32min Ar/O2 (2%)/N2 (10%) PMJ treatment,
indicating the death of the bacterial cells in the
system.
The short lived species generated in PMJ-liquid
system
Short life reactive oxygen species and reactive
nitrogen species (such as •OH, 1O2 and NO) were
Figure 2. Fluorescence microscope images of S.aureus. S.aureus suspended liquid was treated with PMJ in deferent times. (A) Ar/O2 (2%)
PMJ; (B) Ar/O2 (2%)/N2 (10%) PMJ
measured by Electron Spin Resonance spectroscopy.
DMPO was used as the spin-trap reagent of •OH[14],
while TEMP was used as the spin-trap reagent
of1O2[15].
Both DMPO-OH and TEMPO signal intensity were
detected in Ar/O2 (2%) and Ar/O2 (2%)/N2 (10%)
plasma-liquid environments. However, at the same
experimental conditions, the relative intensity of
DMPO-OH signal in Ar/O2 (2%)/N2 (10%)
PMJ-liquid was much higher than that in Ar/O2 (2%)
PMJ-liquid while the TEMPO signal was on the
contrary.
(DETC)2-Fe2+ -NO signal was only detected in
Ar/O2 (2%)/N2 (10%) PMJ, which was attributed to
the involvement of N. Both •OH and 1O2 are very
reactive and toxic ROS [16]. They can oxidize
unsaturated fatty acid on cell membrane, causing
membrane damage and cell rupture [17][18]. To
further verify the role of 1O2 and •OH in real time
inactivation, we added L-His (a 1O2 quencher) and
mannitol(a OH quencher) into the system prior to the
PMJ treatment. The inactivation rate decreased
significantly in Ar/O2 (2%) PMJ when L-His added
in the system. The inactivation ability of Ar/O2
(2%)/N2 (10%) plasma in liquid also decreased
whenever L-His or mannitol involved.
The long-last disinfection abilities of PMJ
To demonstrate the long-last disinfection abilities of
PMJ, distilled water was treated by PMJ (refer as
Plasma Activated Water (PAW)) for 20 minutes, and
immediately used to treat S.aureus. The treatment
time was from 4 to 20 minutes. Based on the present
data, the inactivation capability of PAW treated with
various gas mixtures is probably in a different order.
Detail information will be discussed in the
conference.
The long lived species generated in PMJ
The half-life of 1O2 is 2μs and the half-time of .OH
is approximately 10−9 seconds.[19] H2O2 half-life in
freshwater ranged from 8 hours to 20 days, in air
from 10-20 hrs and in soils from minutes to hours
depending upon microbiological activity and metal
contaminants. O3 can live in air for 3 days at room
temperature but has a half-life of about 1000 seconds
in distilled water at pH ~ 7and room temperature.
[20]
The concentration of H2O2, and O3 were evaluated at
the end of each experiment. H2O2 and O3
concentrations increased with the rising of N2 when
detected in the air. The H2O2 concentration could
reach 25.33mg/L in 20 min Ar/O2 (2%)/N2 (10%)
PMJ treated water, while 3.4mg/L in that of Ar/O2
(2%)/N2 (10%) PMJ treated water. According to our
previous study, these H2O2, concentrations do not
reach the lethal value to inactive the bacterial in
liquid [6].
Detailed
information
about
disinfection
mechanisms will be discussed on the conference.
Funding
This research is supported by National Natural
Science Foundation of China (30970131) and
Beijing Natural Science Foundation (7102149) to Dr.
Wei Liu, and is also sponsored by Bioelectrics Inc.
(U.S.A.), MST Program of International Science and
Technology
Cooperation
(under
Grant
#
2009DFB30370: ‘‘Cold Plasma induced biological
effect and its clinical application studies’’) and
National
Basic
Research
Program
(No.
2007CB935602)
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