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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 34, NO. 4, AUGUST 2006
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Bactericidal Action of the Reactive Species Produced
by Gas-Discharge Nonthermal Plasma at
Atmospheric Pressure: A Review
Lindsey F. Gaunt, Clive B. Beggs, and George E. Georghiou
Abstract—Biological decontamination using a nonthermal gas
discharge at atmospheric pressure in air is the subject of significant research effort at this time. The mechanism for bacterial deactivation undergoes a lot of speculation, particularly with regard
to the role of ions and reactive gas species. Two mechanisms have
been proposed: electrostatic disruption of cell membranes and
lethal oxidation of membrane or cytoplasmic components. Results
show that death is accompanied by cell lysis and fragmentation in
Gram-negative bacteria but not Gram-positive species, although
cytoplasmic leakage is generally observed. Gas discharges can be
a source of charged particles, ions, reactive gas species, radicals,
and radiation (ultraviolet, infrared, and visible), many of which
have documented biocidal properties. The individual roles played
by these in decontamination are not well understood or quantified.
However, the reactions of some species with biomolecules are documented otherwise in the literature. Oxidative stress is relatively
well studied, and it is likely that exposure to gas discharges in
air causes extreme oxidative challenge. In this paper, a review is
presented of the major reactive species generated by nonthermal
plasma at atmospheric pressure and the known reactions of these
with biological molecules. Understanding these mechanisms becomes increasingly important as plasma-based decontamination
and sterilization devices come closer to a wide-scale application
in medical, healthcare, food processing, and air purification applications. Approaches are proposed to elucidate the relative importance of reactive species.
Index Terms—Bacteria, nonthermal plasmas, reactive oxygen
species (ROS), sterilization, superoxide.
I. I NTRODUCTION
A
LMOST since the discovery of small air ions in the 1890’s
[1], their biological significance and impact has fascinated scientists. Whether air ions might influence physiological
processes has been discussed at length in the literature for over a
hundred years (for example see [2] and [3]). The study of ionic
action on microorganisms was pioneered in the 1930s when
bacteriostatic effects were observed with ion concentrations of
between 5 × 104 and 5 × 106 ions cm−3 [4].
Nonthermal plasma produced by a gas discharge produces a
cocktail of reactive molecules that continually react with other
Manuscript received November 21, 2005; revised April 10, 2006.
L. F. Gaunt is with the School of Electronics and Computer Science,
University of Southampton, SO17 1BJ Southampton, U.K. (e-mail: lfw1@
soton.ac.uk).
C. B. Beggs is with the School of Engineering, Design and Technology, University of Bradford, BD7 1DP Bradford, U.K. (e-mail: c.b.beggs@
bradford.ac.uk).
G. E. Georghiou is with the Department of Electrical and Computer Engineering, University of Cyprus, Nicosia 1678, Cyprus (e-mail: [email protected]).
Digital Object Identifier 10.1109/TPS.2006.878381
Fig. 1. Kinetics of the inactivation process. A multislope survivor curve for
Pseudomonas aeuroginosa on a nitrocellulose membrane filter exposed to glow
discharge at atmospheric pressure (from [16]).
molecules and particles present, often very rapidly. The use of
the gas discharge to generate ozone and thus disinfect water
has been practiced for over a 150 years [5]. However, there has
been a recent resurgence in the study of the gas discharge for
disinfection technologies, and more specifically using nonthermal plasma at atmospheric pressure [6]–[9]. The bactericidal
agents generated may include reactive oxygen species (ROS),
ultraviolet (UV) radiation, energetic ions, and charged particles.
Among the ROS reported in air are ozone, atomic oxygen,
superoxide, peroxide, and hydroxyl radicals.
In low-pressure plasma, UV radiation is thought to be the
most important factor in sterilization [10], [11], but in atmospheric pressure plasma most of the UV radiation produced
is reabsorbed in the plasma volume, and not delivered to the
treated surface. Studies have shown that the kinetics of cell
death during plasma exposure is not indicative of UV radiation
[12], [13].
Broad spectrums of microorganisms are susceptible to
plasma exposure, including Gram-negative and Gram-positive
bacteria, bacterial endospores, yeasts, viruses [14], and biofilms
[15]. Reductions in bacteria viability of over 6 log10 are reported from short exposures of less than 30 s [7], [14], [16], and
a total cell fragmentation is seen after longer exposures. In some
instances, the kinetics of cell death demonstrate single-slope
survivor curves [13], [16], while in others multislope curves are
seen, as shown in Fig. 1 [16], [17]. Multislope curves suggest
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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 34, NO. 4, AUGUST 2006
Fig. 2. Transmission electron micrograph images of Gram-negative
Escherichia coli [(a) and (b)] and Gram-positive Staphylococcus aureus [(c)
and (d)] bacteria before [(a) and (c)] and after [(b) and (d)] a 30-s exposure to
glow discharge at atmospheric pressure. Bar = 1 µm (from [7]).
different inactivation methods are in effect, possibly triggered
by different reactive gas species.
Typical of most sterilization techniques, the rates and magnitudes of cell killing in response to a particular treatment regime
differ for various species and strains of bacteria. Spores are
generally more resistant than vegetative or actively growing
bacteria, while there is no consensus on the relative susceptibility of Gram-negative or Gram-positive bacteria. The supporting
medium also influences killing times, with inactivation being
slower on agar than on polypropylene [6].
Bacterial inactivation is accompanied by leakage of proteins,
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from
the cellular cytoplasm [6]. These macromolecules are detectible
in the supernatant of a cell suspension of Gram-negative E. coli
treated with atmospheric pressure plasma after 10 s, and after
15 s of treatment for Gram-positive S. aureus. Observations of
the physical damage inflicted on Gram-negative and -positive
bacteria show marked differences. Transmission electron microscopy, such as shown in Fig. 2, reveal that the continuity
of the cellular envelope of E. coli is breached after 30 s of
treatment, while there is no apparent destruction of S. aureus
[6], [7]. Impairment of some metabolic function, not resulting
in cell death, has been reported from sublethal exposures,
indicative of changes in enzyme activity [18], [19]. There are
two main hypotheses for the mechanism of cell death caused by
gas discharge, both involving lethal damage to cell membrane
structures and ultimately leading to leakage of cyptoplasmic
contents or lysis. They are electrostatic disruption and oxidation of membrane components. The electrostatic disruption
mechanism [20] suggests that the total electric force caused by
accumulation of surface charge could exceed the total tensile
force on the membrane (Gram negative), the probability of
which is greater where some surface irregularities give regions
of higher local curvature. The tensile strength of the membrane
is conferred by a murein layer, which is thicker in Grampositive bacteria (∼ 15–18 nm) than Gram-negative bacteria
(∼ 2 nm), meaning a lower accumulated charge would be
required for lysis of Gram-negative bacteria than Gram positive.
In the second mechanism, oxidation and damage of membrane or cellular components are suggested to be caused by
the energetic ions, radicals, and reactive species generated by
gas discharge. Active radicals are generated directly in plasma
and diffuse to the cell surface, while reaction chemistry in
a moisture layer on the cell surface can produce secondary
radicals. It is well documented that ROS have profoundly
damaging effects on cells through reactions with various biomacromolecules [21]–[24]. The involvement of superoxide in
the bactericidal effects of a corona discharge is alluded to by
the protective effects demonstrated by superoxide dismutase
(SOD) enzymes [25]. Ozone has well-recognized properties as
a disinfectant while hydrogen peroxide acts as a bacteriostatic
agent at concentrations of 25–50 µm [23].
ROS are generated by the normal respiratory process of aerobic organisms. Hence, virtually all aerobic organisms, including
bacteria, demonstrate oxidative defense and repair mechanisms
on exposure to oxidizing agents such as those generated by
air plasma [21], [22]. Indeed, both plant and animal hosts
have adopted defense strategies which utilize ROS (superoxide, hydrogen peroxide, hydroxyl radical) against invading
microorganisms [24]. Oxidative stress occurs when the levels
of exposure exceed the capacity of the cell defense systems.
The mechanism of microorganism inactivation by the gas
discharge is undoubtedly complex and heterogeneous in nature.
In microbiology, even the process of determining cell death is
not straightforward. Inactivation or loss of viability occurs at
the point in time when vital cellular components suffer certain
levels of irreversible damage. Often this can initiate a chain
reaction of supplementary damage unrelated to that directly
caused by the exposure. For this reason, the initial reactions
in the sequence leading to the death of the study organisms
should be identified in order to truly elucidate the disinfection
mechanism. Post hoc observations of bacteria eradicated by
specific treatments are not necessarily indicative. Chemical
reactions between the reactive species generated will continue
after death, while compounds released by dead cells may cause
further physical damage. Thus, while cell lysis may be correctly
observed, loss of membrane integrity may have played no active
role in cell death. This point is exemplified in a study of Escherichia coli inactivation by ozone in which scanning electron
microscopy (SEM) images show that noticeable changes to cell
interiors occur only after most of the cells in the sample have
become nonviable [26]. Decomposition of dissolved ozone
measurements suggested that only 25% of the ozone demand
was responsible for inactivation of practically all microorganisms present and that it was subsequent ozone exposure that led
to structural changes, membrane deterioration, and cell lysis.
This review of biological decontamination by gas-discharge
nonthermal plasma at atmospheric pressure will focus on
the killing of microorganisms by oxidation and damage to
membrane or cellular components by the reactive gas species
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GAUNT et al.: BACTERICIDAL ACTION OF THE REACTIVE SPECIES PRODUCED BY NONTHERMAL PLASMA
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generated. A discussion of the electrostatic disruption mechanism is given otherwise [20], [27]. An overview of the proposed
inactivation agents generated by the gas discharge is presented.
Their reactions with biomolecules and the implications of these
for bacterial viability are discussed, drawing on the literature at
large. Also discussed are the defense mechanisms demonstrated
by bacteria on exposure to some reactive species.
II. N ONTHERMAL -P LASMA P RODUCTION AT
A TMOSPHERIC P RESSURE T HROUGH G AS D ISCHARGES
Nonthermal plasma is increasingly playing a central part in
many areas of modern technology. To date, nonthermal, “cold,”
or nonequilibrium plasma (operating at low neutral gas temperature, while the electron temperature can be much higher)
has been successfully utilized in the area of material processing
as well as pollution control, sterilization, disinfection, ozone
production, and surface processing [28].
The production and maintenance of plasma constitutes one of
the main challenges in the area of plasma technology. Plasma
is generated when enough energy is supplied to a neutral gas to
cause charge production. This is achieved by ionization or photoionization when electrons or photons with sufficient energy
collide with the neutral atoms or other molecules. The most
widely used method for plasma generation utilizes the electrical
breakdown of a neutral gas (gas discharge) in the presence of an
external electric field. In this case, electrons and other charged
particles accelerate under the externally applied electric field
and transfer their energy through inelastic collisions to other
particles and atoms [29]. Discharges are classified as dc, ac,
or pulsed discharges on the basis of the temporal behavior of
the sustaining electric field. The spatial and temporal characteristics of plasma depend to a large degree on the particular
application for which the plasma is utilized.
The majority of gas-discharge nonthermal-plasma processes
has, so far, operated at low pressure. Nevertheless, the latest
developments in plasma science have opened up a way to create
nonthermal plasma at atmospheric pressure. The study of this
plasma has received much interest in recent years because of
the need to replace expensive vacuum systems by simpler ones,
thus leading to higher throughput and cost reduction [30]. The
current major challenge is to produce, stabilize, and control
such plasma at atmospheric pressure.
Typical examples of the utilization of gas-discharge nonthermal plasma at atmospheric pressure include the production of
ozone for air cleaning [31], sterilization and gas treatment [32],
pollution control, CO2 lasers [28], [33]–[35], surface treatment,
high-efficiency excimer UV light sources, and plasma display
panels [36]–[38].
Central to the production of nonequilibrium plasma at atmospheric pressure lies the dielectric barrier discharge (DBD)
(Fig. 3). DBDs, also known as silent discharges, provide a
simple technology to establish nonthermal-plasma conditions
in atmospheric pressure gases [39]. As the emergence of the
DBD is responsible for the renewed interest in atmospheric
pressure discharges a brief overview of the DBDs and their
operation is given next. More details and a review of their
potential applications are outlined in [40].
Fig. 3. Some typical configurations of the DBD.
DBDs have been known for more than a century and were
originally developed for large-scale industrial ozone production
[41]. In its simplest configuration, a DBD is the gas discharge
between two electrodes, separated by one or more dielectric
layers and a gas-filled gap. When high ac voltage is applied
to the electrodes, the resulting electric field is adequate to
produce ionization of the gas in the gap. The radicals, ions,
and electrons produced are attracted toward the electrodes of
opposite polarity and form a charge layer on the dielectric
surface. These charges cancel the charge on the electrodes so
that the electric field in the gap falls to zero, the discharge stops,
and the current is limited. A weak current and hence low-power
discharge is achieved. However, the activation of the working
gas in the discharge space enables the generated radicals and
atoms to be applied to treat the surface layer. The early phases
of breakdown observed in barrier discharges are similar to those
without a dielectric, namely avalanche, streamer formation,
and propagation between the electrodes [42]–[46] followed by
streamer decay. Fig. 4 shows the early phases of breakdown
between two parallel electrodes for different instances. The
presence of the dielectric barriers ensures that the current is
limited so that the discharge does not transit to a spark or arc
and the plasma therefore remains cold [40], [46].
Depending on the gas mixture, dielectric surface properties, and operating conditions, vastly different modes, ranging
from filamentary to completely diffuse barrier discharges, have
been observed [28]. In most DBD configurations operating in
atmospheric pressure gases, miniature filamentary discharges
called microdischarges are formed [48] (Fig. 5). Most of the
industrial applications of DBDs operate in this filamentary
mode [28].
Under controlled operating conditions, stable, uniform, nonfilamentary, or glow discharges can be produced which are
often more effective for surface modification compared to
filamentary discharges [49]. In recent years, a uniform mode of
the barrier discharge [50], the so-called atmospheric pressure
glow (APG) discharge, has been reported for several gases and
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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 34, NO. 4, AUGUST 2006
Fig. 4. Contours of electron density in a 0.1-cm gap at 5.6-kV applied voltage. Critical avalanche and cathode streamer formation and propagation. The four
instances correspond to the following times: (a) t = 3.64 ns, (b) t = 4.13 ns, (c) t = 6.32 ns, and (d) t = 6.49 ns (from [30]).
III. B REAKDOWN M ECHANISMS
Fig. 5. Photograph depicting microdischarge formations (from [28]).
gas mixtures. The APG is preferred to the filamentary mode for
applications that require uniform plasma production such as the
deposition of uniform thin films or surface treatment [51]. Their
spatial appearance is characteristically diffuse and uniform, and
their temporal features are reproducible and stable [47].
Both discharge types, filamentary and diffuse, have common
features: the generation of cold nonequilibrium plasmas at
atmospheric pressure and the strong influence of local field
distortions caused by space charge accumulation.
At atmospheric pressure, breakdown occurs in the form of
microdischarges for the majority of cases in DBD configurations. Under certain circumstances, however, diffuse as well as
glow discharges can be obtained [41]. The form of the discharge
plays an important role in the application and utilization of
plasma.
In any volume of gas, there exist free electrons that are
caused by cosmic rays, natural radioactivity, and the detachment of negative ions. Thus, if the electric field is high enough,
these will accelerate and collide with molecules of the gas,
thereby releasing more electrons, which in turn will do the
same, creating what is known as an electron avalanche. In this
way, electron numbers multiply [52], [53].
As long as the net charge is not sufficient to distort the field
appreciably, the center of the avalanche moves with the electron
drift velocity appropriate to the applied field. If, during the
life of the avalanche, secondary electrons are released, then
new avalanches will be created and the total current will be
amplified. Secondary electrons are released either by positive
ions or UV photons hitting the cathode, or by photoionization
of the gas behind an avalanche. In this way, the current grows
exponentially by what is known as the “Townsend breakdown
mechanism” [25]. The Townsend mechanism is the first model
proposed to explain the initial phase of electrical breakdown in
gases at high pressures. Diffuse discharges, recently observed in
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GAUNT et al.: BACTERICIDAL ACTION OF THE REACTIVE SPECIES PRODUCED BY NONTHERMAL PLASMA
DBDs, can be obtained at breakdown when there is a sufficient
overlap of simultaneously propagating electron avalanches to
cause smoothing of transverse field gradients. It has repeatedly
been demonstrated that diffuse discharges can also be obtained
in barrier discharge configurations at about atmospheric pressure and gap widths up to several centimeters [54]. Low-current
diffuse barrier discharges are more like Townsend discharges,
in which the charge density is so low that it has practically no
influence on the applied field.
At the high pressures usually encountered in a DBD, however, a different kind of breakdown takes place (streamer),
due to the considerable space charge generated during the first
avalanche’s transit through the gap. The streamer mechanism
was initially proposed to explain the electrical breakdown of
overvolted gaps at near-atmospheric pressure [55], [56]. Essentially a streamer is an ionization wave. In front of the wave (the
streamer head) the separation of positive and negative charged
particles shield the interior, and cause a sharp enhancement of
the electric field over a limited region just outside the streamer
head. For fields near the breakdown threshold, the ionization
coefficient is a strong function of the electric field, so that even
modest field enhancements can result in substantial increases
in the ionization rate. If a mechanism such as transport, photoemission, or photoionization exists that places a few free seed
electrons just in front of the streamer head, avalanching in the
locally enhanced field can cause the streamer to propagate with
velocities much higher than the peak electron drift velocity.
Additionally, the ionization density in the streamer body can
build up to values considerably larger than that necessary to
initiate streamer formation. This explains why at atmospheric
pressures the breakdown mechanism in air and other gases is
found to be very fast (of the order of an avalanche transit time)
and to consist of a filamentary discharge channel, rather than a
diffuse distribution of avalanches.
At the time the streamer bridges the gap, a cathode-fall
region of high electric field and high positive-ion densities is
established within a fraction of a nanosecond. Glow discharges
are characterized by a localized high-field cathode-fall region.
At atmospheric pressure, the thickness of this high-field region
is about 10 µm. The current peak in a microdischarge is reached
at the time of cathode-layer formation. Immediately afterward,
charge accumulation at the dielectric surface leads to a local
collapse of the electric field in the area defined by the surface
charge. This self-arresting effect of the dielectric barrier limits
the duration of a microdischarge to a few nanoseconds, and
hence the dissipated energy and temperature rise remain very
low [46].
IV. C HEMISTRY OF E LECTRIC D ISCHARGES
The properties of nonthermal plasmas are determined by
collisions between electrons and other plasma constituents. In
air, chemical reactions are mainly initiated by the impact of
electrons with oxygen and nitrogen. Thus, among the primary
products of electron collision are atomic and metastable oxygen
and nitrogen, with subsequent reactive collisions producing a
cocktail of neutral and ionic species. Atmospheric pressure
discharges differ from low-pressure plasma in that the chem-
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TABLE I
TYPICAL DENSITIES OF OXYGEN IONS, OXYGEN ATOMS, OZONE,
AND C HARGED S PECIES IN P LASMA D ISCHARGES [56]
istry is dominated by reactive neutral species such as oxygen
atoms, singlet oxygen, and ozone rather than ions [57], [58]. In
addition to gas-phase processes, surface reactions should also
be taken into account when considering the species to which
biological samples are exposed. As this paper is concerned
with oxidative damage caused by gas discharges at atmospheric
pressure in air or the presence of oxygen, the most significant
reactive species are ozone (O3 ), atomic oxygen (O or O•− ),
superoxide (O2 •− ), peroxide (O−2
2 or H2 O2 ), and hydroxyl
radicals (OH•). A full description of air plasma chemistry,
including tables of relevant collision processes, is provided
otherwise [59].
Table I summarizes the typical densities of some ion and
charge species in various plasma devices. In corona and DBD
ozone is the main reaction product, while in other plasma
sources, oxygen atoms represent a larger proportion of the
reactive species. A classical application of corona and DBD discharges are in the commercial production of ozone for air and
water purification. In a numerical investigation, the distribution
of oxygen species generated by a coaxial corona potential was
modeled [60]. Ozone was found to be the dominant species at a
concentration of 5 × 1018 cm−3 with singlet oxygen and atomic
oxygen being about five to six orders of magnitude lower
in concentration. Ionized species were present at an average
density of only 1010 cm−3 . In another example, a model of
ozone generation in a DBD calculated that most of the charged
oxygen species formed were short lived, with O3 being the
enduring reactive species [61].
In another study, time-resolved UV absorbance spectroscopy,
optical emission spectroscopy, and numerical modeling methods were used to investigate the reaction chemistry of atmospheric pressure discharges [62]. The concentrations of
ground state molecular oxygen, ground state oxygen atoms,
metastable molecular oxygen, and ozone were quantified,
considering the process conditions of gas pressure and the
distance from the plasma. The plasma generated 0.2−1.0 ×
1016 cm−3 ground state oxygen atoms, between 2.0 × 1014 and
1 × 1016 cm−3 metastable molecular oxygen, and 0.1−4.0 ×
1015 cm−3 ozone. Ozone concentration increased after the
plasma was extinguished due to reactions between oxygen
atoms and oxygen molecules. This research is in agreement
with studies of DBD discharges showing that the charged
oxygen species are rapidly extinguished within micro- or milliseconds.
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The presence of significant concentrations of atomic oxygen in a uniform glow discharge was demonstrated through
the oxidation of oil of wintergreen by discharge effluvia [6].
Oxidation of this agent would not be expected through reaction
with ozone.
Hydroxyl radicals OH• are highly reactive species that can
cause significant damage to most biological molecules, and are
reported amongst the reactive species in many types of plasma
[6], [7], [16]. The presence of water vapor in a discharge feed
gas results in the formation of OH• by electron impact dissociation of H2 O and by reactions of electronically excited oxygen
atoms and nitrogen molecules [9]. It is probable that hydroxyl
radicals also form during surface reactions, for example through
the reaction (1) between hydrogen peroxide and superoxide
O2 •− +H2 O2 → O2 + OH • +HO− .
(1)
The presence of hydroxyl radicals in the effluent of an RFdriven plasma has been observed using laser-induced fluorescent spectroscopy [63]. However, as this discharge was in
saline, the outcome is likely to be different from a discharge
in air. The presence of OH• in the microdischarges of a silent
discharge plasma reactor has also been imaged using OH•
fluorescence [64]. It was demonstrated that OH• only exists
in the channels formed by the transient discharges, and did
not diffuse out. The presence of water vapor in the feed gas
for the device promotes OH• production while simultaneously
reducing ozone concentration [65]. As the water vapor reduces
the surface resistance and increases the dielectric capacity, total
charge transfer is reduced. Dry air is used in the majority
of plasma devices as water vapor reduces the number of microdischarges, and thus the plasma volume. For example, a
glow discharge is operated below 14% relative humidity (RH)
to obtain the desired uniform discharge across the electrode
gap [6], [7].
The presence of superoxide (O2 •− ) in the effluvia of a
discharge is difficult to detect because it is short-lived and
does not accumulate. Its presence in ionized air is more often
assumed where hydrogen peroxide is detected, as superoxide is
a common precursor for this species. The radical can be generated through the combination of an electron with an oxygen
molecule, and its chemistry can involve reaction with water to
form hydrated cluster ions. Spontaneous decomposition occurs
through a dismutation reaction
2O2 •− +2H+ → H2 O2 + O2 .
(2)
Evidence for the generation of superoxide during negative
air ionization is given by several authors [25], [66], [67].
Its generation in the corona discharge was suggested by the
protection conferred by the enzyme SOD to bacteria exposed
to negative corona [25]. Exposing the bacteria Staphylococcus
albus in solution to the products of a negative corona, with
an estimated generation rate of 0.3 mmol O2 •− per hour,
substantial loss of viability was observed after 2 h and total loss
seen after 5 h. Addition of SOD provided complete protection
from the corona treatment such that total viability was retained,
suggesting the superoxide radical to be largely responsible
for bacterial death. The accumulation of O2 •− in a solution
treated with an electroeffluvial ionizer was also measured using
electron paramagnetic resonance and found to be in the region
of 0.5−1.0 µm/min [67].
Hydrogen peroxide is a product of negative air ionization in
the presence of water or water vapor, and is thus generated by
most ionizers [66], [68]. It can also be formed on the surface
of cells in the presence of both positive and negative air ions
[69]. The concentration of hydrogen peroxide generated by
the corona discharge in air under conditions of increasing RH
has been measured using Fourier-transform infrared (FTIR)
spectroscopy, showing that H2 O2 concentration increases with
RH [70]. In ordinary air at 40% RH the concentration was 0.46
rising to 936 µg · l−1 at 96% RH. Only at 10% RH was the
H2 O2 concentration below the detectable limit.
V. O XIDATIVE D AMAGE IN B ACTERIAL C ELLS
A great deal of damage can be done to bio macromolecules
by oxygen radicals, but the damage that leads to cell death is not
always clear. The consequences of oxidative damage include
the following.
1) Adaptation of the cell by upregulation of defense systems.
2) Cell injury involving damage to molecular targets such as
lipids, DNA, protein, and carbohydrate.
3) Cell death usually arising due to excessive unrepaired
damage triggering cell death.
It is important to consider that cell injury is not necessarily
caused directly by oxidative damage, but stress-related changes
in ion levels or activation of proteases for example may themselves be damaging. Damage to DNA, RNA, proteins, and
lipids has been demonstrated in vivo and in vitro.
ROS are used by the immune systems of multicelled organisms to counteract microbial growth [71]. Consequently,
microorganisms have evolved specific multigene antioxidant
defense systems to neutralize their effects [72]. In many ways,
the bactericidal action of ROS in cold plasma, mimics that
experienced by bacteria undergoing phagocytosis in the human
body. Phagocytic cells utilize respiratory bursts to produce
O2 •− and H2 O2 , which kill opsonized (made more susceptible to phagocytes) bacteria [73], [74]. Hydrogen peroxide
is a bacteriostatic rather that bactericidal agent, dramatically
increasing the mean generation time of bacteria at micromolar
concentrations [23]. The toxicity of superoxide radicals, however, derives from their great instability, which causes them to
scavenge electrons from neighboring molecules, which in turn
become radicals. The highly destructive OH• is also known
to be a product of stimulated phagocytes [73], and is thought
to be generated by a metal-catalyzed reaction between superoxide and hydrogen peroxide, known as the Fenton reaction
(3) [22], [75]
Fe/Cu
O2 •− +H2 O2 −−−−−−→ 2OH • +O2 .
(3)
Since the dismutation of superoxide produces hydrogen
peroxide, it is likely that as the concentration of superoxide
increases, so concentrations of hydrogen peroxide and hydroxyl
radicals will also increase [22]. The hydroxyl radical is a
highly reactive species, having extremely high rate-constants
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GAUNT et al.: BACTERICIDAL ACTION OF THE REACTIVE SPECIES PRODUCED BY NONTHERMAL PLASMA
Fig. 6.
Basic reaction sequence for lipid (L) peroxidation by free radicals.
for reactions with almost every type of molecule found in
living cells [21]. For example, the rate constant for its reaction with lecithin (an important membrane phospholipid) is
5.0 × 108 m1− s−1 [21], [76]. Consequently, OH• has an average diffusion distance of only a few nanometers, so is likely
to react with cell membrane components in gas exposure [22],
[77]. Secondary radicals produced through the reactions of
OH• are inevitably less reactive.
A further reaction of superoxide in aqueous solution is to
act as a base and accept a proton to form hydroperoxyl radical (HO2 •) (4). Since HO2 • will also dissociate to release
the proton an equilibrium is set up, the position of which is
pH dependent. Within the physiological range of 6.4–7.5 the
superoxide radical remains almost entirely in this form rather
than becoming protonated [21]
O2 •− +H+ ↔ HO2 • .
(4)
ROS can cause a great amount of damage to macromolecules.
Biological targets for ROS include DNA, proteins, and lipids
[78]. Although oxidative damage may be extensive, it is not
always clear the precise nature of the damage that leads to
cell death [22]. Lipids in particular, are major targets during
oxidative stress [78]. Radicals attack the polyunsaturated fatty
acids in the cell membrane, initiating lipid peroxidation, setting
off a destructive chain reaction, as shown in Fig. 6. Of the
ROS formed during oxidative stress, hydroperoxyl radicals
(HOO•), superoxide radicals, singlet oxygen, and ozone can
all initiate lipid peroxidation [22]. The lipid peroxidation chain
reaction is initiated when a hydrogen atom is abstracted from
an unsaturated fatty acid to form a lipid radical, which in turn
reacts with molecular oxygen to form a lipid peroxyl radical
(L-OO•). These radicals attack other unsaturated fatty acids
and abstract hydrogen atoms to form fatty acid hydroperoxides
(L-OOH), thus perpetuating the chain reaction. Lipid peroxidation generates products, which are shorter than the initial
unsaturated fatty acids [22], with the result that their ability
to rotate within the cell membrane is altered [79]–[81] and
the structural integrity of the membrane is compromised. Loss
1263
of membrane integrity leads to an osmotic imbalance, which
may ultimately result in cell lysis. During lipid peroxidation
aldehydes can also be formed [78]. These in themselves are
very reactive and can damage proteins [77]. However, unlike
ROS, aldehydes are longer lived and thus can attack targets,
which are more distant from the initial free-radical event.
When proteins are oxidized, modifications occur to the
amino acid side chains, with the result that the protein structure is altered [78], [82], [83]. These modifications may lead
to functional changes in the proteins, which can disrupt cell
metabolism, with potentially catastrophic effects. Proteins containing (Fe-S)4 clusters appear to be particularly sensitive to
attack by superoxide radicals [84]. In general, metal-binding
sites in proteins appear to be especially sensitive to attack by
ROS [22]. When proteins are damaged it is necessary that they
be removed in order to prevent accumulation, which might
otherwise compromise cell metabolism [78]. There is evidence
to suggest that heavily oxidized proteins can inhibit the action
of protease, with the result that the oxidized proteins cannot be
degraded [85], [86].
Another target for ROS is DNA. ROS can cause numerous
types of DNA lesions, through reactions with both bases and
sugar moieties [22]. The hydroxyl radical is the most likely
candidate to produce DNA damage due to its extreme reactivity
[87], attacking the sugar moiety and leading to DNA strand
breaks [22]. As OH• cannot diffuse through cells to reach the
DNA due to its reactivity, it is likely that hydrogen peroxide
may serve as a diffusible “latent” form, reacting with metal ions
close to DNA molecules to liberate the highly reactive OH•
[75], [87]. In addition to the DNA damage caused directly by
ROS, intermediate radicals formed during lipid peroxidation
also react with DNA [22]. For example, decomposition of
purine may occur as a result of H+ abstraction by fatty acid free
radicals or the fatty acid free radicals may add to the purines
to form bulky adducts [22]. Some of the stable end products
of lipid peroxidation, such as aldehydes have been shown to
be reactive with DNA, either by alkylating bases [88] or by
forming intra- and interstrand cross-links [89].
Criteria have been proposed [90] for the implication of
reactive species in tissue injury in human disease. The criteria
are as follows.
1) The reactive species should be demonstrable at the site of
injury.
2) The time course for the formation of reactive species
should be consistent with the time course of cell injury,
precede or accompany it.
3) Direct application of the reactive species over the relevant
time course, at concentrations within the relevant range
should reproduce most or all of the injury observed.
4) Removing or inhibiting formation of the reactive species
should diminish the cell injury to an extent related to the
degree of inhibition of the oxidative damage caused.
These criteria could equally be used to implicate ROS in
the current subject. For example, reporting of the densities
of reactive species at the exposed sample would be helpful
in determining the species influential in killing [criteria 1)].
Similarly, the impact of specific reactive species could be
implied where cell death and damage is inhibited by the use
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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 34, NO. 4, AUGUST 2006
Fig. 7. Cell survival for three strains of Bacillus subtilis exposed to increasing
doses of H2 O2 . Open symbols show naive cells while closed symbols show
cells demonstrating the peroxide stress response induced by preexposure to
50-µm H2 O2 . The three strains are YB886 (wild-type) circles, YB1015 (repairdeficient strain) triangles and YB2001 (catalase deficient) squares (from [72]).
of spin traps to intercept radicals or of enzymes to neutralize
specific species [criteria 4)]. The latter approach was used to
suggest a critical role for superoxide in the bactericidal effect
of corona, where SOD conferred complete protection [25].
The presence of atomic oxygen at the reaction site of glow
discharge plasma was implied in a similar way, through a study
of the oxidation of the chemical warfare agent simulant: oil of
wintergreen [7].
The reactions between ROS and the various classes of
biomolecules are relatively well understood, principally as a
result of studies into oxidative stress in aerobic organisms
and of bacteria challenged by ROS by the immune systems
of multicelled organisms. These reaction schemes demonstrate
the fatal nature of ROS exposure at micro- and nanomolar
concentrations, suggesting a definite role for ROS in biological
decontamination using nonthermal gas discharges. Principal
differences are likely to arise in the concentrations of ROS to
which bacteria are exposed and from the simultaneous exposure
to a cocktail of reactive species from air plasma.
VI. O XIDATIVE S TRESS AND B ACTERIAL D EFENCE
Aerobic bacteria have developed a complex array of preventative and reparative mechanisms to protect themselves from the
deleterious effects of oxidative damage. These cellular defenses
include both enzymatic and nonenzymatic components. Two
main oxidative stress responses of bacteria are the peroxide
stress response and the superoxide stress response. Although
totally independent of each other, both are accompanied by
increased DNA repair capacity [22]. The peroxide stress response to challenge by H2 O2 is characterized by elevated
concentrations of about 30 proteins above the basal level. A
level of resistance is acquired by cells through preexposure to
H2 O2 , leading to enhanced survival ratios, as shown in Fig. 7.
The superoxide stress response to O2 •− is also mediated by
elevated levels of about 30 proteins, which are mostly different
to those in the peroxide stress response. Again, preexposure
leads to enhanced survival rates by means of acquired resistance
and repair capacity. The two mechanisms being independent
confer no cross-resistance, so that preexposure to H2 O2 does
not enhance survival ratios on exposure to O2 •− and visa versa.
Probably the most important group of enzymes employed by
bacteria to defend against oxidation are the SODs. Superoxide
radicals are formed in small amounts during normal bacterial
respiration and by virtually all aerobic cells. O2 •− is so toxic
that all organisms attempting to grow in atmospheric oxygen
produce SOD to neutralize it. Aerobic bacteria and facultative
anaerobes (growing aerobically) produce SOD, with which
they convert superoxide into molecular oxygen and hydrogen
peroxide according to (2).
The steady-state concentration of O2 •− is about 10−9 to
−10
M in wild-type aerobically growing E. coli. In mutant
10
strains, which lack SOD activity, the concentration is much
higher at around 5 × 10−6 M [91]. The presence of SOD in a
cell is therefore thought to reduce the steady-state concentration
by up to three orders of magnitude.
Based on the metal ligands there are three types of SOD:
CuZnSOD, FeSOD, and MnSOD. FeSOD is primarily found
in prokaryotic cells, MnSOD is found in both prokaryotes
and eukaryotes, while CuZnSOD is generally not found in
bacteria [21], [22]. The rate constant for MnSODs is about
1.8 × 109 m−1 s−1 at pH 7.8 and decreases as pH becomes
more alkaline. In this respect, it is unlike the CuZnSODs of
eukaryotes, which catalyze the same reaction, because the rate
constant increases with alkalinity [21].
One of the products of superoxide dismutation, hydrogen
peroxide, is itself a reactive species. Although thought to be
less toxic than superoxide, hydrogen peroxide is nonetheless
noxious, and therefore bacteria have developed enzymes to
neutralize it. The most familiar of these is catalase, which
converts hydrogen peroxide into water and oxygen (5). In
bacteria, catalase destroys hydrogen peroxide with remarkable
rapidity [22]. The reaction is a disproportionation (i.e., an
oxidation–reduction) reaction, in which hydrogen peroxide is
the electron source. Consequently, the reaction does not require
an exogenous reducing source. The reaction is also exothermic,
meaning that catalase can protect against the action of hydrogen
peroxide even in energy-depleted cells
2H2 O2 → 2H2 O + O2 .
(5)
Bacteria also utilize peroxidases to neutralize hydrogen
peroxide. Unlike catalase, however, peroxidases require nicotinamide adenine dinucleotide (NADH) or nicotinamide adenosine dinucleotide phosphate (NADPH) as an electron source to
reduce H2 O2 to water.
On the reparative side, bacteria possess catalysts involved in
the repair of some protein and DNA damage. Bacteria possess
catalysts that are able to repair some covalent modifications
to the primary structure of proteins. For example, oxidation
of the amino acid methionine to methionine sulfoxide can be
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GAUNT et al.: BACTERICIDAL ACTION OF THE REACTIVE SPECIES PRODUCED BY NONTHERMAL PLASMA
repaired by methionine sulfoxide reductase [78], [92]. A number of DNA repair enzymes are also induced by oxidative stress
in bacteria (reviewed in [93]). They are primarily involved
in two types of repair: base excision repair and nucleotide
excision repair [75], [94]. Base excision repair is achieved
through the action of DNA glycosylase, which recognizes damaged bases and cleaves their respective glycosylic bonds [75].
Nucleotide excision repair differs from base excision repair
insomuch that several consecutive nucleotide bases (including
any oxidized bases) are removed in a single action. Nucleotide
excision repair is facilitated by the enzymes endonuclease and
exonuclease [75].
Repair of membrane lipids is less well documented. It is
possible that some repair may be effected by reduction of fatty
acid hydroperoxides [22].
The discussion above has so far focused on the phenotypical
response of cells to oxidative stress. At a genomic level, bacteria possess sets of genes that regulate response to environmental
stresses. At least 13 different multigene systems are known to
be induced by various stress stimuli [22]. Although phenotypically the peroxide and superoxide stress response systems are
interrelated, at a genomic level the two systems appear to be
separate and distinct. The expression of H2 O2 -induced defense
proteins is mediated by the transcriptional activator OxyR.
Superoxide stress induces the production of defense proteins,
which are for the most part different from those of the peroxide
stress response. Positive regulation of these is mediated by the
two-stage sox RS system.
VII. UV D AMAGE AND R EPAIR IN B ACTERIA
UV radiation has well-known bactericidal properties. When
photons of UV light strike biological cells, the energy in the
photons is absorbed by chromophores at discrete wavelengths.
The biological impact of UV radiation is primarily due to the
absorption of photons by nucleic acids. DNA has an absorption
spectrum with a maximum in the region 260–265 nm and which
rapidly declines toward longer wavelengths [94]. Specific UV
disinfection devices emit upward of 86% of their light at
254 nm, to coincide with this germicidal peak as closely as
possible [96]. Many researchers have demonstrated that when
UV light at 253.7 nm is absorbed by nucleic acids, pyrimidine
lesions are readily formed [97]. Although purine bases are also
strong absorbers of the UV photons, the quantum yield required
to create dimmers is an order of magnitude higher for purines
than pyrimidine bases [98]. While other photoproducts may be
created through UV irradiation, pyrimidine dimers are generally considered to be the most important class. They are formed
in double stranded DNA when two adjacent pyrimidine bases
fuse together to form a dimer. Three types of pyrimidine dimers
can be formed, thymine–thymine (T <> T), thymine–cytosine
(T <> C), and cytosine–cytosine (C <> C) dimers. During
UV irradiation the frequency with which each type is formed,
depends on the DNA base composition of the irradiated
bacteria [95].
Bacteria possess a range of DNA repair systems, permitting
rapid recovery from sublethal UV damage [70]. In bacteria, UVinduced DNA lesions can be repaired by four main processes:
1265
photoreactivation, nucleotide excision repair, recombination
repair, and SOS repair (i.e., error-prone repair). Prokaryotes
employ are range of enzymes to facilitate these various repair
mechanisms. Although not all bacteria species exhibit photorepair mechanisms [97], photoreactivation is one of the principal
mechanisms used by many species to repair DNA damage.
Bacterial species which exhibit photoreactivation posses an enzyme, DNA photolyase, which absorbs photons of visible light
to facilitate DNA repair [97]. Consequently, these species repair
DNA damage much more efficiently under light conditions than
they do in the dark, where excision repair is thought to be the
principle mechanism employed. In bacteria, the various repair
mechanisms are generally very efficient and rapid, meaning that
cell death only occurs when the UV photon hits are so numerous
that the bacterial repair mechanisms are overwhelmed.
In low-pressure plasma, UV radiation is thought to be the
most important factor in sterilization, but in the atmospheric
pressure plasma under consideration here, UV radiation generated is reabsorbed such that lethal doses are not delivered. For
example, the UV component of an atmospheric glow discharge
was not considered a significant agent of lethality because the
decontamination efficiency of the discharge was significantly
greater than that arising from the same duration of UV exposure [12]. In another report, the role of UV was discounted
on the basis of killing kinetics. Similar killing kinetics were
recorded from an atmospheric glow discharge when Staphylococcus aureus was exposed wrapped in semipermeable bags
and unwrapped. As the porous bag blocked UV radiation yet
allowed the passage of small reactive species, UV exposure was
assumed not to contribute to cell death [17].
VIII. O ZONE D AMAGE OF B ACTERIA
Ozone is a powerful oxidizing agent, which readily forms
reactive OH and HOO radicals in aqueous solution. By all
accounts, ozone disinfection is a complex heterogeneous phenomenon, the mechanism of which remains little understood
despite decades of research effort. Ozone itself reacts with
many biomolecules. Protein damage is caused by reactions with
dienes, amines, and thiols [22], and by cross-linking tyrosine
residues by oxidizing the –OH groups [21]. Cell wall targets
such as fatty acids and peptides appear among the most likely
targets for gaseous ozone [99], [100]. Lipid peroxidation can
be stimulated by oxidation by ozone, resulting in a reduction of
membrane fluidity. Single-strand DNA breaks have also been
observed, which unrepaired can lead to extensive DNA damage
and death [101], [102].
Disinfection of E. coli in wastewater with ozone has been
studied in various semibatch and continuous-flow configurations. Bactericidal concentrations are in the 0.1–0.2 ppm range
[103]. Although cell lysis is often observed following prolonged ozoneation, is not necessarily a characteristic of the
process [26]. Bacterial inactivation by ozone is a function of
ozone concentration per viable bacteria, meaning that below a
threshold concentration ozone has no effect on survival. This
can be complicated by the presence of other compounds with
which ozone can react [26].
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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 34, NO. 4, AUGUST 2006
IX. C ONCLUSION
In this paper, the role of oxidative stress and damage by ROS
in bacterial neutralization by air plasma has been considered.
There is strong evidence from research into physiological exposures that the ROS formed in nonthermal plasma at atmospheric
pressure would cause various forms of cellular damage. If
unrepaired, such damage could be lethal. The concentration of
ROS and the cocktail of species implicated during gas discharge
exposure differ considerably from that of even the most extreme
physiological conditions. Only further research will elucidate
the extent to which the reactions discussed here are involved in
the bactericidal action.
Biological and engineering challenges are still to be overcome in the development of effective atmospheric pressure
discharges for decontamination and disinfection applications.
On the biological side, it remains critical to understand the
pathway of damage leading to death for bacteria, viruses, and
fungi. Without this knowledge, optimization of the decontamination process is reduced to an empirical affair. In elucidating
the processes leading to cell death, the actual cause must be
separated from any physical changes that occur postmortem.
One approach would be to study the sublethal effects of plasma
on cells, while the effects on individual cellular components
such as cell membranes, nucleic acids, proteins, and enzymes
could also be considered. Furthermore, the relative effects of
plasma exposure on microbial and human tissues should be further compared where applications in patient care are considered
such as for dentistry and wound treatment.
On the chemical side, a deeper appreciation regarding the
relative importance of the various reactive species involved
in bacterial neutralization is urgently needed. More detailed
reporting on the nature and quantity of reactive species to
which test organisms are exposed would greatly assist in this.
Although it is intuitive that the most reactive species or the most
abundant ROS would be influential, this may not necessarily
be so. The importance of understanding the role of specific
reactive species in cell killing is highlighted by the case of
hydroxyl radicals. These are significantly implemented in the
killing of microorganisms, yet many plasma are operated in
dry air, where OH production is minimized. It can therefore be
hypothesized that the use of humidified air in plasma reactors,
where this is possible, may promote bactericidal efficiencies.
Implication of a reactive species in cell damage and death is
importantly not restricted to those species directly present in
gas discharge effluent. The absence of hydroxyl radicals in
gas discharge effluent does not preclude its involvement in the
killing process. Superoxide radicals in the effluent will react
with moisture in and around a bacterium to form hydrogen peroxide. This can form hydroxyl radicals on the surface of cells
or diffuse through the membrane and cytoplasm to form hydroxyl radicals in proximity to nucleic acids, ultimately causing
DNA damage.
A role for specific ROS in bactericidal activity could be implicated by demonstrating the presence of the reactive species
at the site of injury. This can be done either by establishing that
the time course of formation of the reactive species is consistent
with the time course of cell injury, by demonstrating that direct
application of relevant concentrations of the reactive species
over a comparable time period reproduces most or all of the
damage observed or by demonstrating that the extent of injury
is diminished by removal of or inhibition of the formation of
the reactive.
Use could also be made of experimental bacterial strains that
may exhibit different phenotypical responses to the gas discharge. For example, SOD-deficient mutants of E. coli would
be more susceptible to gas discharge exposure than wild-type
strains if superoxide were a significant reactive species. Likewise, up regulation of superoxide or hydrogen peroxide stress
responses (defense and repair) by preexposure to nonlethal
doses should confer a degree of protection. Indeed, studies
of the effects of sublethal exposures are likely to shed more
light on killing mechanisms because the complete pathway of
events may be identified and not masked by damage caused
after the cells are actually dead. For this reason, it is important
to quantify the plasma dose required to achieve lethality in
various bacteria species. Some species and strains may be
more resistant than others to damage caused by ROS. During
a disinfection process it is essential that pathogenic microbial
species receive a lethal dose; otherwise the oxidative damage
may be repaired in time, invalidating the disinfection process.
It is clear that a truly multidisciplinary approach is needed
in order to fully understand the biophysical and biochemical
processes. Evidence strongly suggests that the vulnerability of
cells and microorganisms to oxidation lies at the root of the
mechanism for cell death during exposure to nonthermal gas
discharges at atmospheric pressure. Further studies will show
whether it is possible for oxidative stress to be exploited as a
weapon for biological decontamination.
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Lindsey F. Gaunt was born in East Sussex, U.K., in
1974. She received the B.Sc. degree in biology and
the Ph.D. degree in applied electrostatics, both from
the University of Southampton, Southampton, U.K.,
in 1995 and 2000, respectively.
She joined the School of Electronics and Computer Science, University of Southampton in 1995
and now holds a post as School Research Fellow.
Her current research interests include bioelectrostatics, the biological effects of electric fields and
discharges, and electrical disinfection methods.
Dr. Gaunt is a member of the Institute of Physics (IOP) and Honorary
Secretary of the Electrostatics Group of the IOP.
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GAUNT et al.: BACTERICIDAL ACTION OF THE REACTIVE SPECIES PRODUCED BY NONTHERMAL PLASMA
Clive B. Beggs received the B.Sc. degree in building
engineering from Leeds Polytechnic, Leeds, U.K., in
1981, the M.Phil. degree in refrigeration engineering
from the University of Sheffield, Sheffield, U.K.,
in 1991, and the Ph.D. degree in energy engineering from DeMontfort University, Leicester, U.K.,
in 1995.
He is currently Professor of medical technology at
the University of Bradford, Bradford, U.K., and was
formally, in 1996–2005, a Senior Lecturer in aerobiological engineering at the University of Leeds. He
has spent many years researching the use of engineering methods to control the
spread of infection and has investigated the effects of ultraviolet light and air
ions on airborne bacteria. He has a particular interest in the epidemiology of
infection and the strategic use of interventions to break transmission pathways.
1269
George E. Georghiou received the B.A., M.Eng.,
M.A., and Ph.D. degrees from the University of
Cambridge, Cambridge, U.K., in 1995, 1996, 1997,
and 1999, respectively.
He is currently an Assistant Professor at the
Department of Electrical and Computer Engineering, University of Cyprus, Cyprus. Prior to this, he
was the undergraduate course leader in electrical
engineering at the Department of Electronics and
Computer Science, University of Southampton, and
a Research Advisor for the Electricity Utilization,
University of Cambridge. He continued his work at the University of Cambridge in the capacity of a Fellow at Emmanuel College for three more
years (1999–2002). He has a special interest in the development of numerical
algorithms (both serial and parallel codes) applied to the characterization of
multiphysics problems, such as deoxyribonucleic acid sequencing, electronic
manipulation of nanoparticles, lightning, and gas discharges. His research interests lie predominantly in the utilization of electromagnetic fields and plasma
processes for environmental, food processing and biomedical applications,
biomicroelectromechanical systems, nanotechnology, and power systems.
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