Surface & Coatings Technology 204 (2010) 2954–2959

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Surface & Coatings Technology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s u r f c o a t

Bacterial sterilization by a dielectric barrier discharge (DBD) in air

K.G. Kostov a,⁎, V. Rocha a, C.Y. Koga-Ito b, B.M. Matos b, M.A. Algatti a, R.Y. Honda a, M.E Kayama a, R.P. Mota a
a
Faculty of Engineering in Guaratinguetá – FEG, São Paulo State University – UNESP Av. Dr. Ariberto Pereira da Cunha 333, SP 12516-410, Brazil
b
São José dos Campos Dental Faculty – FO, São Paulo State University – UNESP R. Eng. Francisco José Longo 777, SP 12245-000, Brazil

a r t i c l e i n f o a b s t r a c t

Available online 6 February 2010 Cold atmospheric plasma treatment of microorganisms and living tissues has become a popular topic in
modern plasma physics and in medical science. The plasma is capable of bacterial inactivation and non-
Keywords: inflammatory tissue modification, which makes it an attractive tool for treatment of skin diseases, open
Atmospheric plasma injuries and dental caries. Because of their enhanced plasma chemistry, Dielectric Barrier Discharges (DBDs)
Dielectric barrier discharge have been widely investigated for some emerging applications such as biological and chemical
Plasma sterilization
decontamination of media at ambient conditions. Despite the high breakdown voltage in air at atmospheric
E. coli
S. aureus
pressure, the average current of DBD discharges is low. Therefore, a DBD can be applied in direct contact with
biological objects without causing any damage. In this work a 60 Hz DBD reactor, which generates cold
atmospheric plasma inside Petri dishes with bacterial culture, is investigated. Samples of Staphylococcus
aureus, a Gram-positive bacterium and Escherichia coli a Gram-negative bacterium were selected for this
study. The bacterial suspensions were evenly spread on agar media planted in Petri dishes. The reactor
electrodes were placed outside the Petri dish, thus eliminating the risk of samples microbial contamination.
The covered Petri dish with agar medium in it serves as dielectric barrier during the treatment. The plasma
processing was conducted at same discharge power (∼ 1.0 W) with different exposure time. Sterilization of
E. coli and S. aureus was achieved for less than 20 min. Plasma induced structural damages of bacteria were
investigated by Scanning Electron Microscopy.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction The dielectric barrier discharge (DBD) is a non-equilibrium plasma

Nowadays, plasma processing is one of the leading technologies in
material engineering. Plasmas are generated in a wide range of gas
pressures and temperatures and also can be driven by different
sources such as microwave, RF, pulsed, ac and dc power supplies. In
recent years, non-thermal plasmas at or near atmospheric pressure
have attracted much attention because of their application in several
emerging technologies, such as the surface modification of polymers
and the biological and chemical decontamination of media [1].
Traditionally, the main methods of inactivation of microorganisms
are based on thermal treatment (dry or moist heat), chemical
treatment (H2O2 or ethylene oxide) or radiation (X-ray and γ-rays).
However, each of these conventional methods is either not suitable
for the treatment of temperature sensitive materials or produces
residue toxic gasses that raise significant safety concerns. On the other
hand, cold atmospheric plasmas are suitable for the treatment of heat-
sensitive and/or vulnerable objects that cannot withstand vacuum,
such as organic materials, foams, liquids, and living biological tissues
[2].
⁎ Corresponding author. Tel.: + 55 12 31232844; fax: + 55 12 31232840.
E-mail address: kostov@feg.unesp.br (K.G. Kostov).
source that can be operated with different gasses at elevated
pressures (up to atmospheric pressure). The plasma is created
between two conductive electrodes connected to an ac or pulsed
power supply. At least one of DBD electrodes is covered by dielectric
layer, which prevents the arc formation after breakdown. DBD
discharge usually consists of a large number of short-living micro-
channels (filaments) that are randomly distributed over entire area of
the dielectric barrier. Despite the high breakdown voltage in gasses at
atmospheric pressure (several kV), the average electric current is low.
Therefore, a DBD can be applied in direct contact with living tissues
and open injuries without causing any damage [3]. During the DBD
treatment the photons, electrons, ions and active chemical species
from the plasma reach the surface of a biologically contaminated
object and can eventually lead to its sterilization [4–9].
The contributions of the various agents emanating from plasma to
the bacterial killing process are still under discussion in the literature
[2,3]. From the plasma point of view, agents that cause bacterial death
or injury can be roughly classified as heat, UV radiation, free radicals
and charged particles [2]. Thermal damage is usually not an issue in
DBD discharges at atmospheric pressure since they are sources of non-
thermal plasmas where the electron temperature is between 1 and
10 eV, however the ion temperature is about the room temperature
[10,11]. UV radiation in the (220–280) nm wavelength range is a very
potent disinfectant, inhibiting the bacteria's ability to replicate.

0257-8972/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.surfcoat.2010.01.052
 K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959
However its role in air plasma sterilization at atmospheric pressure is
considered to be modest [11], since no significant UV emission occurs
below 285 nm. In high-pressure non-equilibrium plasma discharges,
reactive species are generated through various collisional ways, such
as electron impact excitation and dissociation [11]. The active free
radicals abundant in DBD plasma, particularly the reactive oxygen
species (O, OH, O− 2 , O3) are considered to be the prime atmospheric
plasma disinfectants [11–13]. Finally, the exact role of charged
particles in bacterial inactivation is not yet fully resolved, but there
are some indications of their importance [2,9]. At atmospheric
pressure DBD discharges the average energy of the ions is quite low
(on the order of 10−2 eV). Therefore the cell physical damage induced
by bombardment with low energy charged particles is expected to
play no direct role in the destruction of microorganisms. In analogy to
the charging of dust in complex plasmas one can consider a negative
charging of the bacterial cells during the plasma exposure. The
authors [2] suggest that the mechanical stress induced by electrostatic
repulsion forces of the charged surface may under certain conditions
cause cell rupture in Gram-negative bacteria because they possess fine
membrane and irregular shape. Some researchers [14] reported cells
morphological changes after direct plasma exposure, which were
attributed to the charge accumulation on bacteria. Furthermore,
bacteria are very sensitive to changes in their surface charges.
Therefore disturbing the surface charge equilibrium of bacteria by
charged particle bombardment may actually kill the cell without its
lysis, what probably happen with more robust Gram-positive bacteria
[2,14].
The nature of bacteria inactivation by cold atmospheric plasma is
rather complex and the exact mechanisms of cellular death are still
under discussion [3]. However, the kinetics of the bacterial inactiva-
tion can provide some important information for the process. A simple
and reliable approach is based on the dynamical studies of bacterial
growth after plasma treatment, that involve planting and counting the
number of colony forming units (CFU). Typically, dose–response (or
survivors) curves are determined, i.e., the numbers of CFUs are
measured in function of the plasma exposure time. The most common
forms of the survivor's curves for air plasma treatment are
exponentially decaying functions with a single or multiple time
constants [2,3]. These curves are usually plotted in semi-logarithmic
scales and the time for a one-log10 reduction is referred to as D-value
(Decimal value) — the time required to reduce an original concen-
tration of microorganisms by 90%. The D-values of air plasma
deactivation can range from couple of seconds up to several minutes
depending on the type of microorganisms, the kind of medium
supporting the microorganisms, the method of plasma exposure
(remote or direct) and the treatment parameters [2,11,14].
In this work we describe a 60 Hz DBD reactor, which generates
cold atmospheric plasma inside a Petri dish with bacterial culture.
Samples of Staphylococcus aureus, a Gram-positive bacterium and
Fig. 1. Experimental set-up.
2955
Escherichia coli a Gram-negative bacterium were selected for this
study. Closed Petri dishes, planted with agar media containing E. coli
or S. aureus, were placed between two metal electrodes connected to a
high-voltage transformer. All plasma treatments were conducted at
same discharge power (∼1.0 W) with different exposure time. All
bacterial cells were killed for less than 20 min of DBD treatment.
Plasma induced structural damages of bacteria were investigated by
Scanning Electron Microscopy (SEM).
2. Experimental procedure
The experimental arrangement used for DBD bacterial sterilization
is shown in Fig. 1. Low-temperature plasmas at atmospheric pressure
were generated inside covered Petri dishes (90 × 15 mm, Inlab, São
Paulo, Brazil) that were partially filled with 20 ml culture medium
(Triptic soy agar, Oxoid, Hampshire, England) leaving air spacing of
about 5 mm.
Reference strains E. coli ATCC 23922 and S. aureus ATCC 6538 were
used in this study. These two species are commonly employed as the
reference microorganisms in the development of new sterilization
techniques, representing Gram-negative and Gram-positive bacteria,
respectively. Suspensions of S. aureus or E. coli were standardized to
concentration of 1 × 104 cells/ml by optical spectrophotomety (the
values of respective wavelengths and optical densities were: 490 nm
and 0.366 for S. aureus and 590 nm and 0.319 for E. coli). Portions of
0.1 ml of the initial suspensions were evenly spread on the surface of
the agar medium, then left to dry for 15 min under controlled
conditions inside a laminar flow chamber and, finally treated by
plasma. The discharge was driven by a low-frequency high voltage
power supply operating at 60 Hz. Two 75-mm-diam, aluminum
electrodes were attached outside the Petri dish, thus preventing
microbial contaminations of the samples. The high voltage electrode
was placed on the Petri dish's cover and the electrode under the Petri
dish was grounded.
Applied voltage was measured by a high-voltage probe Tektronix
TDS 2024B. The current waveforms were recorded by measuring the
voltage across a 1200 Ω resistor connected in series with the reactor.
The discharge current and the applied voltage are shown in Fig. 2. To
obtain the charge flowing through the gap the resistor was
substituted by a 0.91 μF capacitor. From the Q–V Lissajous figure
(see Fig. 3) the discharge energy per cycle is calculated using the
standard method based on the figure's area. The mean power is
obtained by multiplying the energy by the source frequency.
During all experiments the magnitude of the applied voltage was
fixed at 20 kV, which corresponded to mean discharge energy per
cycle of about 17 mJ (electric power of 1.0 W). To examine the DBD
sterilization efficiency the plasma exposure time was varied from
1 min to 20 min.
2956 K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959
Fig. 2. Waveforms of the discharge current – CH1 and the applied voltage – CH2.
After the treatment with plasma, the Petri dishes were incubated
at 37 °C for 24 hours. Then, the number of colonies was counted by
naked eyes. The results were expressed in values of colonies forming
units (CFU) and the mean values were used in the bacterial survivor
curves.
To verify whether the nutrition properties of agar media were
somehow compromised by the plasma exposure, two standard Petri
dishes with same quantity of agar (20 ml) were prepared. One of
them was DBD treated for 30 min and the other served as control
sample. After that both Petri dishes were inoculated with bacterial
suspensions with the same concentration and incubated at 37 °C.
After 24 h the CFU in both dishes were counted and no visual
difference in the bacterial growth due to the agar plasma exposure
was detected.
3. Results and discussion
Same quantities of the initial bacterial suspension (concentration
104 cell/ml) were spread on agar inside six Petri dishes, of which 5
were DBD treated and one was kept for control (without plasma
exposure). The gap spacing of 5 mm left inside the Petri dishes was
filled with air at atmospheric pressure and temperature of 20 °C. DBD
generated inside the Petri dishes consisted of large number of short-
living micro-discharges, randomly distributed over the agar surface.
Five treatment time intervals of 2, 5, 10, 15 and 20 min were chosen at
a fixed applied voltage of 20 kV. After the plasma treatment all sample
Fig. 3. Q–V Lissajous figure for calculation of the discharge energy.
were incubated for 24 h at 37 °C and the visible bacterial colonies
were counted. All experiments were performed in duplicate.
Photographs of E. coli colonies inside Petri dishes are shown in
Fig. 4(A) while Fig. 4(B) presents the bacterial survival rate as a
function of the plasma exposure time. As can be seen from this figure
bacterial sterilization was achieved in less than 20 min.
The kinetics of E. coli cell death shown in Fig. 4(B) is characterized
by a double-slope exponential decay that was also observed in some
cases of direct plasma exposure of microorganisms [3]. The time
interval necessary to reduce the bacterial population by 90%, i.e. the
D1 value for the first phase of E. coli elimination was 2.5 min. It was
followed by a second phase of bacterial killing with a considerably
higher D2 value of about 15 min.
In case of S. aureus, both, the plasma treatment conditions and the
procedures adopted for the preparation of microbial cultures, were the
same. The results of cells plasma treatment are presented in Fig. 5(A–B)
and a similar bacterial inactivation behavior can be observed. Again, the
time required for sterilization was on the order of 20 min. Similarly to
the previous experiment, the S. aureus surviving curve (Fig. 5(B)) can be
fitted by a two-slope exponential decay. The D-value of the first slope
(D1 ≈ 2.1 min) was much shorter than the second D-value — D2, which
is about 18 min. Such disproportionably long treatment times required
for inactivation of the remained low bio-burdens were also observed by
other authors [15]. It can be noted in Figs. 4 and 5 that during the first
few minutes of the treatment there is nearly no difference in
inactivation kinetics between both test strains. One recent study of
bacterial inactivation [16] revealed that the S. aureus and E. coli planted
on agar media are rather resistant to the plasma treatment and also both
species showed similar surviving rates.
The sterilization times obtained by us seem to be quite long when
compared to the typical sterilization times reported in the literature
(∼10 s) [6]. However one should take into account that in this work
we used a 60 Hz power supply instead of an intermediate frequency
source operated in the kHz range, as employed by most authors.
Considering that about the same energy density is probably required
for microbial inactivation at different treatment frequencies then the
sterilization time should be inversely proportional to the source
frequency. Therefore for low frequency power sources, bacterial
inactivation time in the order of several min should be expected. For
instance, the authors in [17] utilized a 50 Hz power supply for DBD
formation and sterilization of E. coli and S. aureus was achieved after
couple of min plasma treatment, which is very close to the
characteristic D1 time obtained in our experiments. However instead
of cells planted on agar, in this work bacterial strains in liquid
suspension were spread on cover glasses and treated with plasma. As
suggested by some authors the kind of species being treated
determines the D1 value, while the D2 is dependent on the type of
surface or medium supporting the microorganisms. The comparative
study in [18] examined the microbial inactivation on different
supporting surfaces and revealed that the longest D-values were
achieved for the bacteria inoculated on agar medium. This finding is
coherent with the elevated D2 value obtained in our experiments.
Differently from some previous works [3,16] the obtained D2-values
in the experiment (N15 min) are higher than the respective D1-values
(in the order of 2 min). Some authors [15] argued that this kind of
double-phase survivor curves with D1 b D2 might be a general
characteristic of non-thermal plasma treatments when the process
efficiency is dependent on the number of targets.
It is now widely accepted that in case of atmospheric pressure air
plasma in DBD discharges the contribution of the heat and the UV
radiation for the bacterial killing is insignificant [3], and plasma
induced physical and chemical processes perform the sterilization.
The physical process proceeds by positive and negative ions in the
discharge streamer and the chemical process is done by the action of
active neutral species. Since the voltage applied in our experiment is
quite high (20 kV), the kinetic energy of the ions may become high
 K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959
Fig. 4. (A) Images of Petri dishes showing E. coli colonies after air plasma treatment for 0; 2; 5; 10; 15 and 20 min and (B) the E. coli survivor curve.
enough to produce some structural damage on the cells membrane
upon hitting a bacterium. Besides the bombardment with charged
particles, the accumulation of electric charge on the cell membrane
induces electrostatic stress, which can eventually rupture cells [2].
The air plasmas are excellent sources of reactive oxygen and
nitrogen species such as atomic oxygen, ozone, hydroxyl group, NO,
NO2 etc. These highly reactive species have direct impact on the
microorganisms, and especially on their outmost membranes by
chemical etching [3]. Although, the exact sterilization mechanisms
using plasma is still unclear most researchers have concluded that the
simultaneous action of both processes — the chemical etching
performed by reactive neutral species as well as the physical damages
of the cells membrane due to the ion bombardment and the charge
accumulation, are important for the cells inactivation [2,6,13].
To investigate the effects of plasma exposure on the bacterial
structure Scanning Electron Microscopy (SEM) was used to inspect
E. coli and S. aureus morphology before and after the DBD treatment.
Suspensions of E. coli and S. aureus were inoculated on sterile glass
slides (5 × 5 mm), which were put inside a covered Petri dish to form a
DBD reactor similar to that used in the previous experiment. The
samples were treated for 15 min at applied voltage of 20 kV. Prior the
SEM analysis plasma treated samples and untreated controls were
subjected to the following procedures to develop adhesion between
2957
the cells and the plates. The fixation process included immersion in
2.5% glutaraldehyde for 1 h. Afterwards the slides were dehydrated by
submission to ethylic alcohol solutions with sequential concentra-
tions of (25%, 20 min; 50%, 20 min; 75%, 20 min; 90%, 20 min; and
100%, 60 min).
As processed glass plates containing S. aureus and E. coli cells were
coated with an ultra-thin layer of gold and micrographs were
subsequently taken for morphological inspection. SEM images of the
treated and control samples are shown in Figs. 6 and 7.
As can be seen in Fig. 6, the Gram-positive bacteria S. aureus
suffered structural damage when exposed to the DBD plasma. Many
cells have their outer membrane disrupted leading to release of their
cytoplasm. The Gram-positive bacteria have thicker membranes
providing them with higher tensile stress and rigidity in comparison
to the G-negative bacteria. Probably, that is why some cells in Fig. 6(B)
exhibit no visible morphological damages in their structure although
they may have lost their vitality.
Gram-negative bacteria have thinner outer membrane and
therefore they are more susceptible to the attack of charged particles
and active radicals abundant in plasmas. Apparently the cells of
plasma treated Gram-negative bacteria E. coli in Fig. 7(B) do not
represent apparent cytoplasm leakage. On the other hand, some cell
fragments can be seen and the membranes of all bacteria in Fig. 7(B)
2958 K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959

Fig. 5. (A) Images of Petri dishes showing the viable S. aureus colonies after plasma exposure for 0; 2; 5; 10; 15 and 20 min and (B) the S. aureus survivor curve.

look somehow eroded when compared to the cells membrane on the
control sample (Fig. 7(A)). Based on the SEM micrographs we can
infer that the DBD treatment induces some structural changes in the
bacterial surface leading to lysis of the S. aureus cells and to the
membrane erosion of E. coli.
The electric power applied in this experiment was quite low
(∼ 1.0 W) therefore longer treatment times were required to
inactivate the bacterial strains. One may speculate that such a plasma
treatment in which the mean energy (∼ 17 mJ) is delivered during
long time (tens of min.), does not necessarily compromise the entire

Fig. 6. SEM images of (A) untreated S. aureus and (B) S. aureus cells treated for 15 min.
 K.G. Kostov et al. / Surface & Coatings Technology 204 (2010) 2954–2959 2959

Fig. 7. SEM images of (A) untreated E. coli and (B) E. coli treated for 15 min.

structure of bacteria. That is probably why the SEM micrographs species and the physical damage of the bacteria outer membranes
demonstrate that some S. aureus cells look intact and the membranes induced by charged particles are involved in the sterilization process.
of E. coli seem eroded but not fully disrupted. Despite of this, both
bacterial strains were inactivated for about the same time of 20 min. References
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[2]
4. Conclusion [3]
[4]
In this paper, bacterial inactivation using an atmospheric pressure,
[5]
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