Decontamination of biological suspensions by pulsed corona discharge:
Contribution of UV light to overall bacterial inactivation
E. Spetlikova1, H. Shejbalova1, V. Janda1, M. Clupek2 and P. Lukes2
1
Institute of Chemical Technology Prague, Technicka 5, Prague 6, 166 28, Czech Republic
Institute of Plasma Physics AS CR, v.v.i., Za Slovankou 3, Prague 8, 182 00, Czech Republic
2
Abstract: The contribution of UV light emitted by the pulsed corona
discharge generated in water in the plasma induced bacterial inactivation was
investigated. The effect was studied in dependence on the solution
conductivity (200 and 500 μS.cm-1) and the type of microorganism. Role of
UV photolysis was evaluated using an UV light transparent spectrometric
cell, which was irradiated by underwater discharge generated using a needle
to plate geometry of electrodes fully immersed in the water. We investigated
the role of UV photolysis in inactivation of several microorganisms of
different cell wall structure as Gram-negative bacteria Escherichia coli,
Gram-positive bacteria Enterococcus faecalis, vegetative cells and
endospores of Bacillus subtilis. Depending on the type of microorganism up
to 50% contribution of UV radiation to the overall plasma inactivation
induced by the electrical discharge in water was estimated.
Keywords: underwater plasma, UV radiation, solution conductivity, bacteria
1. Introduction
Previous research has demonstrated that a
high voltage pulse electrical discharges generated
directly in a liquid phase initiate a variety of
chemical and physical processes. These processes
include a high electric field, intense ultraviolet
radiation, overpressure shock waves and formation
of various highly reactive chemical species such as
radicals and ions. It was shown that these
processes are capable to efficiently destroy or
inactivate a number of organic compounds and
microorganisms [1, 2]. However, in comparison
with destruction of organic compounds, where the
degradation mechanism is attributed mainly to the
oxidation by OH radicals, detailed mechanism of
plasma-induced microbial inactivation in water is
still widely not known. In general, it is expected
that the inactivation effect of electrical discharges
on microorganisms proceeds due to the cell wall
damage and by changes in DNA [3, 4]. In addition
to the chemical effects induced by reactive oxygen
species (e.g. OH radical, atomic oxygen, ozone
and hydrogen peroxide) physical effects such as
heat, high electric field, UV radiation and shock
waves, all commonly present in the electrical
discharge plasma generated in water, may
contribute in the plasma inactivation process.
Concerning the bactericidal effects of UV
radiation, UV light (200–300 nm) with dose of
several mW.cm−2 is known to cause lethal damage
to cells. UV radiation affects the cells of bacteria
by inducing the formation of thymine dimmers in
DNA. It suppresses replication of DNA that causes
lethal effect to bacteria. UV photolysis is generally
not considered to participate significantly in the
inactivation process in the atmospheric-pressure
plasmas due to low intensity of UV radiation
emitted by these plasmas (below 50 μW.cm2 [5]).
On the other hand, electrical discharges
generated in liquid phase can emit significant
intensity of UV light [6]. Research obtained using
the emission spectroscopy has showed a radiation
from the pulsed corona discharge in liquid phase in
a wide range of wavelengths (200–1000 nm),
which is dominated by the spectral lines of
hydrogen (peaks at 434, 486, 656 nm) and oxygen
atom (777 nm) and by emission from OH. radical
(309 nm) [7, 8]. Consequently, Lukes et al. [6]
have determined that pulse radiant power of the
corona discharge in water (190–280 nm) could
reach in dependence on the solution conductivity
levels of the order of tens to hundreds of watts
during the pulse, which corresponds to UV
radiation intensity of the order 0.1–10 mW.cm−2.
In this work the role of UV light emitted by
the pulsed corona discharge generated in water in
the plasma induced microbial inactivation was
investigated on several model microorganisms of
different cell wall structure as Gram-negative
bacteria Escherichia coli, Gram-positive bacteria
Enterococcus faecalis, vegetative cells and
endospores of Bacillus subtilis.
2. Experimental
The reactor for generating pulsed corona
discharges in liquid phase was described in detail
previously [8]. A needle to plate geometry of
electrodes both immersed in a cylindrical glass
vessel was used. Needle to plate distance was
52 mm. A pulsed high voltage applied to the
needle was provided by a pulse power supply. All
experiments were conducted with fixed applied
voltage of 27 kV, pulse repetition frequency of
35 Hz and charging capacitance of 7 nF. The mean
electrical power P applied to the reactor was 90 W.
Bacterial suspensions of Escherichia coli
CCM 3954, Enterococcus faecalis CCM 4224 and
vegetative cells and endospores of Bacillus subtilis
CCM 4062 were prepared by preculturing
lyophilized bacteria in gelatin discs in growth
medium. Endospores were prepared in sporulating
solution, which was heated in boiling water bath
for 30 minutes to kill vegetative cells. Electrolytic
conductivity of bacterial suspensions was adjusted
by NaCl to 200 or 500 µS.cm-1 before each
experiment. The number of bacteria in the solution
was assayed by counting colony forming units
(CFUs). The initial amount of bacteria was about
105 CFU in 1 ml except solution with endospores
(103 CFU in 1 ml). The viability of the bacteria
was determined as the ratio of the concentration of
surviving bacteria to the total concentration.
The contribution of UV light was determined
using method developed in previous work [6].
Briefly, spectrometric cell (Starna Scientific Ltd.,
cell type 35, path length 10 mm, diameter 50 mm)
was filled with bacterial suspension, placed into
the discharge gap between the needle electrode and
grounded electrode and irradiated by the light
emitted from the discharge. Comparison of
irradiation experiments was performed in two
types of cells of different transmittance. One of
them was made from Pyrex glass (35/PX/10) with
cutoff at λ = 280 nm and the other one was from
Spectrosil Quartz (35/Q/10) with transmission
below λ = 200 nm.
UV contribution was calculated as
UV contribution 
1  UV VDZ

,
1 T
VT
(1)
where VDZ is the volume of active zone (408 ml),
VT is the volume of the treated zone in the reactor
(1250 ml), τUV time constant of bacterial
inactivation determined in the spectrometric cell,
τT time constant of bacterial inactivation
determined in total volume of bacterial suspension
treated in the discharge reactor.
3. Results and discussion
Figures 1 and 2 show comparison of the effect
of electric discharge on bacteria E. faecalis and
E. coli in the water of solution conductivities
200 μS.cm-1 and 500 μS.cm-1. Gradual decrease in
survival bacteria was observed with increasing
time of discharge treatment in all experiments.
Figure 1. Kinetics of inactivation of Enterococcus faecalis in water
(U = 27 kV, C = 7 nF, V = 1250 ml).
Figure 2. Kinetics of inactivation of Escherichia coli in water
(U = 27 kV, C = 7 nF, V = 1250 ml).
Number of bacteria decreased faster in
solutions with higher conductivity. 3-log reduction
in number of E. faecalis and 2-log reduction in
number of E. coli was obtained in 1250 ml volume
solution within 6 min. From presented data follows
that E. coli was somewhat more resistive species to
the effects of pulse corona discharge than E.
faecalis under both solution conductivities. Such
difference is more likely related to the different
composition of cell wall of both bacteria. Cell wall
of E. coli as a gram-negative bacterium is
composed
from
murein
layer
and
lipopolisacharides. E. faecalis is a gram-positive
bacterium, which lacks outer membrane, but
possess thicker murein layer, which might
contribute to the lower resistance of E. faecalis to
the bactericidal effects (both chemical and
physical) induced by plasma agents generated by
electrical discharge in water. From these effects
contribution of UV light was further investigated.
these conditions. From time constants of bacterial
inactivation determined in the spectrometric cell
τUV and in the solution τT a contribution of UV photolysis was estimated using Eq. 1. The results are
summarized in Table 1, which shows that up to 4050% from total bacterial inactivation might be
attributed to the UV radiation from the discharge.
Consequently, E. faecalis exhibited slightly higher
sensitivity to the UV radiation than E. coli.
Table 1. Contribution of UV radiation in bacterial inactivation by the
pulsed corona discharge.
Figure 3. Kinetics of inactivation of Enterococcus faecalis in
spectrometric cell (U = 27 kV, C = 7 nF, V = 17,1 ml).
Figure 4. Kinetics of inactivation of Escherichia coli in spectrometric
cell (U = 27 kV, C = 7 nF, V = 17,1 ml).
Figures 3 and 4 show the effect of UV light
emitted from the discharge in water on suspensions
of E. faecalis and E. coli, which were irradiated in
the Quartz spectrometric cell placed in the
discharge gap. Preliminary experiments were
performed with Pyrex spectrometric cell, (i.e.,
which do not transmit UV light), which revealed
no inactivation of bacteria. Thus, any other
processes produced by electrical discharge than
UV radiation did not influenced inactivation of
bacterial suspension in the spectrometric cell [6].
Results presented in Figures 3 and 4 indicate
that pulsed corona discharge in water emits UV
light with germicidal effect. Higher efficiency of
E. faecalis and E. coli inactivation was obtained in
solution with conductivity of 500 μS.cm-1 than in
200 μS.cm-1. This corresponds to a higher intensity
of UV radiation emitted from the discharge with
higher solution conductivity and, thus, to the
higher contribution of UV in the inactivation
process of bacteria induced by the discharge under
E. coli
E. faecalis
B. subtilis
Contribution of UV,
200 μS/cm [%]
42
40
5
Contribution of UV,
500 μS/cm [%]
39
51
5
Figure 5 shows the effect of corona discharge
in water on bacteria Bacillus subtilis (in
vegetative state). Concentration of Bacillus subtilis
in water decreased exponentially with increasing
discharge treatment time. Significant difference in
inactivation was observed in dependence on the
solution conductivity. 4-log reduction in number of
B. subtilis was obtained in the solution with
conductivity of 500 μS.cm-1 after 6 min of
discharge treatment compared to 1.5-log reduction
in 200 μS.cm-1 performed under the same
experimental conditions. Compared to E. faecalis
and E. coli, B. subtilis was more sensitive to the
effects of corona discharge induced in dependence
on the solution conductivity giving significantly
higher inactivation in conductivity of 500 μS.cm-1
than 200 μS.cm-1 (see Fig. 1, 2, 5).
However, results from UV irradiation
experiments performed with B. subtilis in the
spectrometric cells showed only very small effect
of UV radiation on B. subtilis (Fig. 6) compared to
E. faecalis and E. coli (Figs. 3, 4). This
indicates that UV photolysis did not contributed
significantly in the inactivation of B. subtilis
(Table 1) and, thus, some other effects participated
in inactivation process of this bacterium.
Since B. subtilis exhibit ability to form
a tough, a highly protective endospore, comparison
experiments were made with B. subtillis in
vegetative and endospore state in order to discern
difference in the effects of corona discharge in
water induced on both forms of B. subtilis.
Figure 7 shows that discharge did not significantly
influenced the concentration of endospores of
bacteria B. subtilis in water. Less than 1-log
reduction in number of endospores was obtained in
the solution treated by the discharge for 6 min and
only slight difference in inactivation was observed
in solution with conductivity of 500 μS.cm-1
compared to 200 μS.cm-1. Also negligible effect of
endospores
to
unfavorable
environmental
conditions. Both forms of B. subtilis evidenced
only a small response to UV radiation. Vegetative
cells of B. subtilis were, however, more sensitive
to the effects induced by the electric discharge in
water than endospores.
4. Conclusion
Figure 5. Kinetics of inactivation of Bacillus subtilis in water
(U = 27 kV, C = 7 nF, V = 1250 ml).
The inactivation effect of pulsed corona
discharge in water in dependence on the solution
conductivity was investigated. Inactivation rate
depended on type of bacteria. Important role of the
UV radiation in bactericidal effects was
determined. Higher intensity of UV radiation from
the discharge with higher solution conductivity
was observed. UV radiation emitted from the
discharge in water contributed up to 40-50% to the
overall inactivation in dependence on type of
bacteria.
Acknowledgments. The present work was supported by the
Grant Agency of the Academy of Sciences of the Czech
Republic (No. IAAX00430802) and the Czech Science
Foundation (No. 104/09/H080).
References
Figure 6. Kinetics of inactivation of Bacillus subtilis in spectrometric
cell (U = 27 kV, C = 7 nF, V = 17,1 ml).
Figure 7. Kinetics of inactivation of endospores of Bacillus subtilis in
water (U = 27 kV, C = 7 nF, V = 1250 ml).
UV radiation was observed in the inactivation of
endospores of bacteria B. subtilis. This is in
agreement with the known high resistivity of
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