Bactericidal Action of the Reactive Species Produced by Gas-Discharge Nonthermal Plasma at Atmospheric Pressure: a Review Lindsey F

IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 34, NO. 4, AUGUST 2006 1257 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 de- activation 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 Fig. 1. Kinetics of the inactivation process. A multislope survivor curve for with biological molecules. Understanding these mechanisms be- Pseudomonas aeuroginosa on a nitrocellulose membrane filter exposed to glow comes increasingly important as plasma-based decontamination discharge at atmospheric pressure (from [16]). 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 impor- molecules and particles present, often very rapidly. The use of tance of reactive species. the gas discharge to generate ozone and thus disinfect water has been practiced for over a 150 years [5]. However, there has Index Terms—Bacteria, nonthermal plasmas, reactive oxygen species (ROS), sterilization, superoxide. 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 I. INTRODUCTION agents generated may include reactive oxygen species (ROS), LMOST since the discovery of small air ions in the 1890’s ultraviolet (UV) radiation, energetic ions, and charged particles. A [1], their biological significance and impact has fasci- Among the ROS reported in air are ozone, atomic oxygen, nated scientists. Whether air ions might influence physiological superoxide, peroxide, and hydroxyl radicals. processes has been discussed at length in the literature for over a In low-pressure plasma, UV radiation is thought to be the hundred years (for example see [2] and [3]). The study of ionic most important factor in sterilization [10], [11], but in at- action on microorganisms was pioneered in the 1930s when mospheric pressure plasma most of the UV radiation produced bacteriostatic effects were observed with ion concentrations of is reabsorbed in the plasma volume, and not delivered to the between 5 × 104 and 5 × 106 ions cm−3 [4]. treated surface. Studies have shown that the kinetics of cell Nonthermal plasma produced by a gas discharge produces a death during plasma exposure is not indicative of UV radiation cocktail of reactive molecules that continually react with other [12], [13]. Broad spectrums of microorganisms are susceptible to Manuscript received November 21, 2005; revised April 10, 2006. plasma exposure, including Gram-negative and Gram-positive L. F. Gaunt is with the School of Electronics and Computer Science, bacteria, bacterial endospores, yeasts, viruses [14], and biofilms University of Southampton, SO17 1BJ Southampton, U.K. (e-mail: lfw1@ [15]. Reductions in bacteria viability of over 6 log10 are re- soton.ac.uk). C. B. Beggs is with the School of Engineering, Design and Technol- ported from short exposures of less than 30 s [7], [14], [16], and ogy, University of Bradford, BD7 1DP Bradford, U.K. (e-mail: c.b.beggs@ a total cell fragmentation is seen after longer exposures. In some bradford.ac.uk). instances, the kinetics of cell death demonstrate single-slope G. E. Georghiou is with the Department of Electrical and Computer Engi- neering, University of Cyprus, Nicosia 1678, Cyprus (e-mail: [email protected]). survivor curves [13], [16], while in others multislope curves are Digital Object Identifier 10.1109/TPS.2006.878381 seen, as shown in Fig. 1 [16], [17]. Multislope curves suggest

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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 Gram- positive 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 bio- macromolecules [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 Fig. 2. Transmission electron micrograph images of Gram-negative Escherichia coli [(a) and (b)] and Gram-positive Staphylococcus aureus [(c) bacteria, demonstrate oxidative defense and repair mechanisms and (d)] bacteria before [(a) and (c)] and after [(b) and (d)] a 30-s exposure to on exposure to oxidizing agents such as those generated by glow discharge at atmospheric pressure. Bar =1µm (from [7]). air plasma [21], [22]. Indeed, both plant and animal hosts have adopted defense strategies which utilize ROS (super- different inactivation methods are in effect, possibly triggered oxide, hydrogen peroxide, hydroxyl radical) against invading by different reactive gas species. microorganisms [24]. Oxidative stress occurs when the levels Typical of most sterilization techniques, the rates and magni- of exposure exceed the capacity of the cell defense systems. tudes of cell killing in response to a particular treatment regime The mechanism of microorganism inactivation by the gas differ for various species and strains of bacteria. Spores are discharge is undoubtedly complex and heterogeneous in nature. generally more resistant than vegetative or actively growing In microbiology, even the process of determining cell death is bacteria, while there is no consensus on the relative susceptibil- not straightforward. Inactivation or loss of viability occurs at ity of Gram-negative or Gram-positive bacteria. The supporting the point in time when vital cellular components suffer certain medium also influences killing times, with inactivation being levels of irreversible damage. Often this can initiate a chain slower on agar than on polypropylene [6]. reaction of supplementary damage unrelated to that directly Bacterial inactivation is accompanied by leakage of proteins, caused by the exposure. For this reason, the initial reactions deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from in the sequence leading to the death of the study organisms the cellular cytoplasm [6]. These macromolecules are detectible should be identified in order to truly elucidate the disinfection in the supernatant of a cell suspension of Gram-negative E. coli mechanism. Post hoc observations of bacteria eradicated by treated with atmospheric pressure plasma after 10 s, and after specific treatments are not necessarily indicative. Chemical 15 s of treatment for Gram-positive S. aureus. Observations of reactions between the reactive species generated will continue the physical damage inflicted on Gram-negative and -positive after death, while compounds released by dead cells may cause bacteria show marked differences. Transmission electron mi- further physical damage. Thus, while cell lysis may be correctly croscopy, such as shown in Fig. 2, reveal that the continuity observed, loss of membrane integrity may have played no active of the cellular envelope of E. coli is breached after 30 s of role in cell death. This point is exemplified in a study of Es- treatment, while there is no apparent destruction of S. aureus cherichia coli inactivation by ozone in which scanning electron [6], [7]. Impairment of some metabolic function, not resulting microscopy (SEM) images show that noticeable changes to cell in cell death, has been reported from sublethal exposures, interiors occur only after most of the cells in the sample have indicative of changes in enzyme activity [18], [19]. There are become nonviable [26]. Decomposition of dissolved ozone two main hypotheses for the mechanism of cell death caused by measurements suggested that only 25% of the ozone demand gas discharge, both involving lethal damage to cell membrane was responsible for inactivation of practically all microorgan- structures and ultimately leading to leakage of cyptoplasmic isms present and that it was subsequent ozone exposure that led contents or lysis. They are electrostatic disruption and oxi- to structural changes, membrane deterioration, and cell lysis. dation of membrane components. The electrostatic disruption This review of biological decontamination by gas-discharge mechanism [20] suggests that the total electric force caused by nonthermal plasma at atmospheric pressure will focus on accumulation of surface charge could exceed the total tensile the killing of microorganisms by oxidation and damage to force on the membrane (Gram negative), the probability of membrane or cellular components by the reactive gas species

Authorized licensed use limited to: Univ of Calif Los Angeles. Downloaded on April 13,2010 at 16:42:56 UTC from IEEE Xplore. Restrictions apply. GAUNT et al.: BACTERICIDAL ACTION OF THE REACTIVE SPECIES PRODUCED BY NONTHERMAL PLASMA 1259 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. NONTHERMAL-PLASMA PRODUCTION AT ATMOSPHERIC PRESSURE THROUGH GAS DISCHARGES 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 Fig. 3. Some typical configurations of the DBD. 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 pho- DBDs have been known for more than a century and were toionization when electrons or photons with sufficient energy originally developed for large-scale industrial ozone production collide with the neutral atoms or other molecules. The most [41]. In its simplest configuration, a DBD is the gas discharge widely used method for plasma generation utilizes the electrical between two electrodes, separated by one or more dielectric breakdown of a neutral gas (gas discharge) in the presence of an layers and a gas-filled gap. When high ac voltage is applied external electric field. In this case, electrons and other charged to the electrodes, the resulting electric field is adequate to particles accelerate under the externally applied electric field produce ionization of the gas in the gap. The radicals, ions, and transfer their energy through inelastic collisions to other and electrons produced are attracted toward the electrodes of particles and atoms [29]. Discharges are classified as dc, ac, opposite polarity and form a charge layer on the dielectric or pulsed discharges on the basis of the temporal behavior of surface. These charges cancel the charge on the electrodes so the sustaining electric field. The spatial and temporal charac- that the electric field in the gap falls to zero, the discharge stops, teristics of plasma depend to a large degree on the particular and the current is limited. A weak current and hence low-power application for which the plasma is utilized. discharge is achieved. However, the activation of the working The majority of gas-discharge nonthermal-plasma processes gas in the discharge space enables the generated radicals and has, so far, operated at low pressure. Nevertheless, the latest atoms to be applied to treat the surface layer. The early phases developments in plasma science have opened up a way to create of breakdown observed in barrier discharges are similar to those nonthermal plasma at atmospheric pressure. The study of this without a dielectric, namely avalanche, streamer formation, plasma has received much interest in recent years because of and propagation between the electrodes [42]–[46] followed by the need to replace expensive vacuum systems by simpler ones, streamer decay. Fig. 4 shows the early phases of breakdown thus leading to higher throughput and cost reduction [30]. The between two parallel electrodes for different instances. The current major challenge is to produce, stabilize, and control presence of the dielectric barriers ensures that the current is such plasma at atmospheric pressure. limited so that the discharge does not transit to a spark or arc Typical examples of the utilization of gas-discharge nonther- and the plasma therefore remains cold [40], [46]. mal plasma at atmospheric pressure include the production of Depending on the gas mixture, dielectric surface proper- ozone for air cleaning [31], sterilization and gas treatment [32], ties, and operating conditions, vastly different modes, ranging pollution control, CO2 lasers [28], [33]–[35], surface treatment, from filamentary to completely diffuse barrier discharges, have high-efficiency excimer UV light sources, and plasma display been observed [28]. In most DBD configurations operating in panels [36]–[38]. atmospheric pressure gases, miniature filamentary discharges Central to the production of nonequilibrium plasma at at- called microdischarges are formed [48] (Fig. 5). Most of the mospheric pressure lies the dielectric barrier discharge (DBD) industrial applications of DBDs operate in this filamentary (Fig. 3). DBDs, also known as silent discharges, provide a mode [28]. simple technology to establish nonthermal-plasma conditions Under controlled operating conditions, stable, uniform, non- in atmospheric pressure gases [39]. As the emergence of the filamentary, or glow discharges can be produced which are DBD is responsible for the renewed interest in atmospheric often more effective for surface modification compared to pressure discharges a brief overview of the DBDs and their filamentary discharges [49]. In recent years, a uniform mode of operation is given next. More details and a review of their the barrier discharge [50], the so-called atmospheric pressure potential applications are outlined in [40]. glow (APG) discharge, has been reported for several gases and

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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. BREAKDOWN MECHANISMS 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 detach- ment 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]. Fig. 5. Photograph depicting microdischarge formations (from [28]). 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 gas mixtures. The APG is preferred to the filamentary mode for life of the avalanche, secondary electrons are released, then applications that require uniform plasma production such as the new avalanches will be created and the total current will be deposition of uniform thin films or surface treatment [51]. Their amplified. Secondary electrons are released either by positive spatial appearance is characteristically diffuse and uniform, and ions or UV photons hitting the cathode, or by photoionization their temporal features are reproducible and stable [47]. of the gas behind an avalanche. In this way, the current grows Both discharge types, filamentary and diffuse, have common exponentially by what is known as the “Townsend breakdown features: the generation of cold nonequilibrium plasmas at mechanism” [25]. The Townsend mechanism is the first model atmospheric pressure and the strong influence of local field proposed to explain the initial phase of electrical breakdown in distortions caused by space charge accumulation. gases at high pressures. Diffuse discharges, recently observed in

DBDs, can be obtained at breakdown when there is a sufficient TABLE I overlap of simultaneously propagating electron avalanches to TYPICAL DENSITIES OF OXYGEN IONS,OXYGEN ATOMS,OZONE, AND CHARGED SPECIES IN PLASMA DISCHARGES [56] 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]. Essen- istry is dominated by reactive neutral species such as oxygen tially a streamer is an ionization wave. In front of the wave (the atoms, singlet oxygen, and ozone rather than ions [57], [58]. In streamer head) the separation of positive and negative charged addition to gas-phase processes, surface reactions should also particles shield the interior, and cause a sharp enhancement of be taken into account when considering the species to which the electric field over a limited region just outside the streamer biological samples are exposed. As this paper is concerned head. For fields near the breakdown threshold, the ionization with oxidative damage caused by gas discharges at atmospheric coefficient is a strong function of the electric field, so that even pressure in air or the presence of oxygen, the most significant − modest field enhancements can result in substantial increases reactive species are ozone (O3), atomic oxygen (O or O• ), •− −2 in the ionization rate. If a mechanism such as transport, photo- superoxide (O2 ), peroxide (O2 or H2O2), and hydroxyl emission, or photoionization exists that places a few free seed radicals (OH•). A full description of air plasma chemistry, electrons just in front of the streamer head, avalanching in the including tables of relevant collision processes, is provided locally enhanced field can cause the streamer to propagate with otherwise [59]. velocities much higher than the peak electron drift velocity. Table I summarizes the typical densities of some ion and Additionally, the ionization density in the streamer body can charge species in various plasma devices. In corona and DBD build up to values considerably larger than that necessary to ozone is the main reaction product, while in other plasma initiate streamer formation. This explains why at atmospheric sources, oxygen atoms represent a larger proportion of the pressures the breakdown mechanism in air and other gases is reactive species. A classical application of corona and DBD dis- found to be very fast (of the order of an avalanche transit time) charges are in the commercial production of ozone for air and and to consist of a filamentary discharge channel, rather than a water purification. In a numerical investigation, the distribution diffuse distribution of avalanches. of oxygen species generated by a coaxial corona potential was At the time the streamer bridges the gap, a cathode-fall modeled [60]. Ozone was found to be the dominant species at a region of high electric field and high positive-ion densities is concentration of 5 × 1018 cm−3 with singlet oxygen and atomic established within a fraction of a nanosecond. Glow discharges oxygen being about five to six orders of magnitude lower are characterized by a localized high-field cathode-fall region. in concentration. Ionized species were present at an average At atmospheric pressure, the thickness of this high-field region density of only 1010 cm−3. In another example, a model of is about 10 µm. The current peak in a microdischarge is reached ozone generation in a DBD calculated that most of the charged at the time of cathode-layer formation. Immediately afterward, oxygen species formed were short lived, with O3 being the charge accumulation at the dielectric surface leads to a local enduring reactive species [61]. collapse of the electric field in the area defined by the surface In another study, time-resolved UV absorbance spectroscopy, charge. This self-arresting effect of the dielectric barrier limits optical emission spectroscopy, and numerical modeling meth- the duration of a microdischarge to a few nanoseconds, and ods were used to investigate the reaction chemistry of at- hence the dissipated energy and temperature rise remain very mospheric pressure discharges [62]. The concentrations of low [46]. ground state molecular oxygen, ground state oxygen atoms, metastable molecular oxygen, and ozone were quantified, considering the process conditions of gas pressure and the IV. CHEMISTRY OF ELECTRIC DISCHARGES distance from the plasma. The plasma generated 0.2−1.0 × The properties of nonthermal plasmas are determined by 1016 cm−3 ground state oxygen atoms, between 2.0 × 1014 and collisions between electrons and other plasma constituents. In 1 × 1016 cm−3 metastable molecular oxygen, and 0.1−4.0 × air, chemical reactions are mainly initiated by the impact of 1015 cm−3 ozone. Ozone concentration increased after the electrons with oxygen and nitrogen. Thus, among the primary plasma was extinguished due to reactions between oxygen products of electron collision are atomic and metastable oxygen atoms and oxygen molecules. This research is in agreement and nitrogen, with subsequent reactive collisions producing a with studies of DBD discharges showing that the charged cocktail of neutral and ionic species. Atmospheric pressure oxygen species are rapidly extinguished within micro- or mil- discharges differ from low-pressure plasma in that the chem- liseconds.

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The presence of significant concentrations of atomic oxy- treated with an electroeffluvial ionizer was also measured using gen in a uniform glow discharge was demonstrated through electron paramagnetic resonance and found to be in the region the oxidation of oil of wintergreen by discharge effluvia [6]. of 0.5−1.0 µm/min [67]. Oxidation of this agent would not be expected through reaction Hydrogen peroxide is a product of negative air ionization in with ozone. the presence of water or water vapor, and is thus generated by Hydroxyl radicals OH• are highly reactive species that can most ionizers [66], [68]. It can also be formed on the surface cause significant damage to most biological molecules, and are of cells in the presence of both positive and negative air ions reported amongst the reactive species in many types of plasma [69]. The concentration of hydrogen peroxide generated by [6], [7], [16]. The presence of water vapor in a discharge feed the corona discharge in air under conditions of increasing RH gas results in the formation of OH• by electron impact dissoci- has been measured using Fourier-transform infrared (FTIR) ation of H2O and by reactions of electronically excited oxygen spectroscopy, showing that H2O2 concentration increases with atoms and nitrogen molecules [9]. It is probable that hydroxyl RH [70]. In ordinary air at 40% RH the concentration was 0.46 radicals also form during surface reactions, for example through rising to 936 µg · l−1 at 96% RH. Only at 10% RH was the the reaction (1) between hydrogen peroxide and superoxide H2O2 concentration below the detectable limit. − − O2 • +H2O2 → O2 + OH • +HO . (1) V. O XIDATIVE DAMAGE IN BACTERIAL CELLS The presence of hydroxyl radicals in the effluent of an RF- A great deal of damage can be done to bio macromolecules driven plasma has been observed using laser-induced fluo- by oxygen radicals, but the damage that leads to cell death is not rescent spectroscopy [63]. However, as this discharge was in always clear. The consequences of oxidative damage include saline, the outcome is likely to be different from a discharge the following. in air. The presence of OH• in the microdischarges of a silent 1) Adaptation of the cell by upregulation of defense systems. discharge plasma reactor has also been imaged using OH• 2) Cell injury involving damage to molecular targets such as fluorescence [64]. It was demonstrated that OH• only exists lipids, DNA, protein, and carbohydrate. in the channels formed by the transient discharges, and did 3) Cell death usually arising due to excessive unrepaired not diffuse out. The presence of water vapor in the feed gas damage triggering cell death. for the device promotes OH• production while simultaneously reducing ozone concentration [65]. As the water vapor reduces It is important to consider that cell injury is not necessarily the surface resistance and increases the dielectric capacity, total caused directly by oxidative damage, but stress-related changes charge transfer is reduced. Dry air is used in the majority in ion levels or activation of proteases for example may them- of plasma devices as water vapor reduces the number of mi- selves be damaging. Damage to DNA, RNA, proteins, and crodischarges, and thus the plasma volume. For example, a lipids has been demonstrated in vivo and in vitro. glow discharge is operated below 14% relative humidity (RH) ROS are used by the immune systems of multicelled or- to obtain the desired uniform discharge across the electrode ganisms to counteract microbial growth [71]. Consequently, gap [6], [7]. microorganisms have evolved specific multigene antioxidant − defense systems to neutralize their effects [72]. In many ways, The presence of superoxide (O2• ) in the effluvia of a discharge is difficult to detect because it is short-lived and the bactericidal action of ROS in cold plasma, mimics that does not accumulate. Its presence in ionized air is more often experienced by bacteria undergoing phagocytosis in the human assumed where hydrogen peroxide is detected, as superoxide is body. Phagocytic cells utilize respiratory bursts to produce •− a common precursor for this species. The radical can be gen- O2 and H2O2, which kill opsonized (made more suscep- erated through the combination of an electron with an oxygen tible to phagocytes) bacteria [73], [74]. Hydrogen peroxide molecule, and its chemistry can involve reaction with water to is a bacteriostatic rather that bactericidal agent, dramatically form hydrated cluster ions. Spontaneous decomposition occurs increasing the mean generation time of bacteria at micromolar through a dismutation reaction concentrations [23]. The toxicity of superoxide radicals, however, derives from their great instability, which causes them to − + 2O2 • +2H → H2O2 + O2. (2) scavenge electrons from neighboring molecules, which in turn become radicals. The highly destructive OH• is also known Evidence for the generation of superoxide during negative to be a product of stimulated phagocytes [73], and is thought air ionization is given by several authors [25], [66], [67]. to be generated by a metal-catalyzed reaction between super- Its generation in the corona discharge was suggested by the oxide and hydrogen peroxide, known as the Fenton reaction protection conferred by the enzyme SOD to bacteria exposed (3) [22], [75] to negative corona [25]. Exposing the bacteria Staphylococcus albus in solution to the products of a negative corona, with •− −−−−−−Fe/Cu→ • − O2 +H2O2 2OH +O2. (3) an estimated generation rate of 0.3 mmol O2• per hour, substantial loss of viability was observed after 2 h and total loss Since the dismutation of superoxide produces hydrogen seen after 5 h. Addition of SOD provided complete protection peroxide, it is likely that as the concentration of superoxide from the corona treatment such that total viability was retained, increases, so concentrations of hydrogen peroxide and hydroxyl suggesting the superoxide radical to be largely responsible radicals will also increase [22]. The hydroxyl radical is a − for bacterial death. The accumulation of O2• in a solution highly reactive species, having extremely high rate-constants

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 con- taining (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 Fig. 6. Basic reaction sequence for lipid (L) peroxidation by free radicals. 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 for reactions with almost every type of molecule found in degraded [85], [86]. living cells [21]. For example, the rate constant for its reac- Another target for ROS is DNA. ROS can cause numerous tion with lecithin (an important membrane phospholipid) is types of DNA lesions, through reactions with both bases and 5.0 × 108 m1−s−1 [21], [76]. Consequently, OH• has an av- sugar moieties [22]. The hydroxyl radical is the most likely erage diffusion distance of only a few nanometers, so is likely candidate to produce DNA damage due to its extreme reactivity to react with cell membrane components in gas exposure [22], [87], attacking the sugar moiety and leading to DNA strand [77]. Secondary radicals produced through the reactions of breaks [22]. As OH• cannot diffuse through cells to reach the OH• are inevitably less reactive. DNA due to its reactivity, it is likely that hydrogen peroxide A further reaction of superoxide in aqueous solution is to may serve as a diffusible “latent” form, reacting with metal ions act as a base and accept a proton to form hydroperoxyl rad- close to DNA molecules to liberate the highly reactive OH• ical (HO2•) (4). Since HO2• will also dissociate to release [75], [87]. In addition to the DNA damage caused directly by the proton an equilibrium is set up, the position of which is ROS, intermediate radicals formed during lipid peroxidation pH dependent. Within the physiological range of 6.4–7.5 the also react with DNA [22]. For example, decomposition of superoxide radical remains almost entirely in this form rather purine may occur as a result of H+ abstraction by fatty acid free than becoming protonated [21] radicals or the fatty acid free radicals may add to the purines to form bulky adducts [22]. Some of the stable end products − + O2 • +H ↔ HO2 • . (4) of lipid peroxidation, such as aldehydes have been shown to be reactive with DNA, either by alkylating bases [88] or by ROS can cause a great amount of damage to macromolecules. forming intra- and interstrand cross-links [89]. Biological targets for ROS include DNA, proteins, and lipids Criteria have been proposed [90] for the implication of [78]. Although oxidative damage may be extensive, it is not reactive species in tissue injury in human disease. The criteria always clear the precise nature of the damage that leads to are as follows. cell death [22]. Lipids in particular, are major targets during 1) The reactive species should be demonstrable at the site of oxidative stress [78]. Radicals attack the polyunsaturated fatty injury. acids in the cell membrane, initiating lipid peroxidation, setting 2) The time course for the formation of reactive species off a destructive chain reaction, as shown in Fig. 6. Of the should be consistent with the time course of cell injury, ROS formed during oxidative stress, hydroperoxyl radicals precede or accompany it. (HOO•), superoxide radicals, singlet oxygen, and ozone can 3) Direct application of the reactive species over the relevant all initiate lipid peroxidation [22]. The lipid peroxidation chain time course, at concentrations within the relevant range reaction is initiated when a hydrogen atom is abstracted from should reproduce most or all of the injury observed. an unsaturated fatty acid to form a lipid radical, which in turn 4) Removing or inhibiting formation of the reactive species reacts with molecular oxygen to form a lipid peroxyl radical should diminish the cell injury to an extent related to the (L-OO•). These radicals attack other unsaturated fatty acids degree of inhibition of the oxidative damage caused. and abstract hydrogen atoms to form fatty acid hydroperoxides These criteria could equally be used to implicate ROS in (L-OOH), thus perpetuating the chain reaction. Lipid perox- the current subject. For example, reporting of the densities idation generates products, which are shorter than the initial of reactive species at the exposed sample would be helpful unsaturated fatty acids [22], with the result that their ability in determining the species influential in killing [criteria 1)]. to rotate within the cell membrane is altered [79]–[81] and Similarly, the impact of specific reactive species could be the structural integrity of the membrane is compromised. Loss implied where cell death and damage is inhibited by the use

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H2O2, 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 H2O2 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). − −9 The steady-state concentration of O2• is about 10 to 10−10 M in wild-type aerobically growing E. coli. In mutant strains, which lack SOD activity, the concentration is much Fig. 7. Cell survival for three strains of Bacillus subtilis exposed to increasing higher at around 5 × 10−6 M [91]. The presence of SOD in a doses of H2O2. Open symbols show naive cells while closed symbols show cells demonstrating the peroxide stress response induced by preexposure to cell is therefore thought to reduce the steady-state concentration 50-µmH2O2. The three strains are YB886 (wild-type) circles, YB1015 (repair- by up to three orders of magnitude. deficient strain) triangles and YB2001 (catalase deficient) squares (from [72]). Based on the metal ligands there are three types of SOD: CuZnSOD, FeSOD, and MnSOD. FeSOD is primarily found of spin traps to intercept radicals or of enzymes to neutralize in prokaryotic cells, MnSOD is found in both prokaryotes specific species [criteria 4)]. The latter approach was used to and eukaryotes, while CuZnSOD is generally not found in suggest a critical role for superoxide in the bactericidal effect bacteria [21], [22]. The rate constant for MnSODs is about of corona, where SOD conferred complete protection [25]. 1.8 × 109 m−1s−1 at pH 7.8 and decreases as pH becomes The presence of atomic oxygen at the reaction site of glow more alkaline. In this respect, it is unlike the CuZnSODs of discharge plasma was implied in a similar way, through a study eukaryotes, which catalyze the same reaction, because the rate of the oxidation of the chemical warfare agent simulant: oil of constant increases with alkalinity [21]. wintergreen [7]. One of the products of superoxide dismutation, hydrogen The reactions between ROS and the various classes of peroxide, is itself a reactive species. Although thought to be biomolecules are relatively well understood, principally as a less toxic than superoxide, hydrogen peroxide is nonetheless result of studies into oxidative stress in aerobic organisms noxious, and therefore bacteria have developed enzymes to and of bacteria challenged by ROS by the immune systems neutralize it. The most familiar of these is catalase, which of multicelled organisms. These reaction schemes demonstrate converts hydrogen peroxide into water and oxygen (5). In the fatal nature of ROS exposure at micro- and nanomolar bacteria, catalase destroys hydrogen peroxide with remarkable concentrations, suggesting a definite role for ROS in biological rapidity [22]. The reaction is a disproportionation (i.e., an decontamination using nonthermal gas discharges. Principal oxidation–reduction) reaction, in which hydrogen peroxide is differences are likely to arise in the concentrations of ROS to the electron source. Consequently, the reaction does not require which bacteria are exposed and from the simultaneous exposure an exogenous reducing source. The reaction is also exothermic, to a cocktail of reactive species from air plasma. meaning that catalase can protect against the action of hydrogen peroxide even in energy-depleted cells

VI. OXIDATIVE STRESS AND BACTERIAL DEFENCE 2H2O2 → 2H2O + O2. (5) Aerobic bacteria have developed a complex array of preven- tative and reparative mechanisms to protect themselves from the Bacteria also utilize peroxidases to neutralize hydrogen deleterious effects of oxidative damage. These cellular defenses peroxide. Unlike catalase, however, peroxidases require nicoti- include both enzymatic and nonenzymatic components. Two namide adenine dinucleotide (NADH) or nicotinamide adeno- main oxidative stress responses of bacteria are the peroxide sine dinucleotide phosphate (NADPH) as an electron source to stress response and the superoxide stress response. Although reduce H2O2 to water. totally independent of each other, both are accompanied by On the reparative side, bacteria possess catalysts involved in increased DNA repair capacity [22]. The peroxide stress re- the repair of some protein and DNA damage. Bacteria possess sponse to challenge by H2O2 is characterized by elevated catalysts that are able to repair some covalent modiﬁcations concentrations of about 30 proteins above the basal level. A to the primary structure of proteins. For example, oxidation level of resistance is acquired by cells through preexposure to of the amino acid methionine to methionine sulfoxide can be

Authorized licensed use limited to: Univ of Calif Los Angeles. Downloaded on April 13,2010 at 16:42:56 UTC from IEEE Xplore. Restrictions apply. GAUNT et al.: BACTERICIDAL ACTION OF THE REACTIVE SPECIES PRODUCED BY NONTHERMAL PLASMA 1265 repaired by methionine sulfoxide reductase [78], [92]. A num- photoreactivation, nucleotide excision repair, recombination ber of DNA repair enzymes are also induced by oxidative stress repair, and SOS repair (i.e., error-prone repair). Prokaryotes in bacteria (reviewed in [93]). They are primarily involved employ are range of enzymes to facilitate these various repair in two types of repair: base excision repair and nucleotide mechanisms. Although not all bacteria species exhibit photore- excision repair [75], [94]. Base excision repair is achieved pair mechanisms [97], photoreactivation is one of the principal through the action of DNA glycosylase, which recognizes dam- mechanisms used by many species to repair DNA damage. aged bases and cleaves their respective glycosylic bonds [75]. Bacterial species which exhibit photoreactivation posses an en- Nucleotide excision repair differs from base excision repair zyme, DNA photolyase, which absorbs photons of visible light insomuch that several consecutive nucleotide bases (including to facilitate DNA repair [97]. Consequently, these species repair any oxidized bases) are removed in a single action. Nucleotide DNA damage much more efficiently under light conditions than excision repair is facilitated by the enzymes endonuclease and they do in the dark, where excision repair is thought to be the exonuclease [75]. principle mechanism employed. In bacteria, the various repair Repair of membrane lipids is less well documented. It is mechanisms are generally very efficient and rapid, meaning that possible that some repair may be effected by reduction of fatty cell death only occurs when the UV photon hits are so numerous acid hydroperoxides [22]. that the bacterial repair mechanisms are overwhelmed. The discussion above has so far focused on the phenotypical In low-pressure plasma, UV radiation is thought to be the response of cells to oxidative stress. At a genomic level, bacte- most important factor in sterilization, but in the atmospheric ria possess sets of genes that regulate response to environmental pressure plasma under consideration here, UV radiation gener- stresses. At least 13 different multigene systems are known to ated is reabsorbed such that lethal doses are not delivered. For be induced by various stress stimuli [22]. Although phenotyp- example, the UV component of an atmospheric glow discharge ically the peroxide and superoxide stress response systems are was not considered a significant agent of lethality because the interrelated, at a genomic level the two systems appear to be decontamination efficiency of the discharge was significantly separate and distinct. The expression of H2O2-induced defense greater than that arising from the same duration of UV expo- proteins is mediated by the transcriptional activator OxyR. sure [12]. In another report, the role of UV was discounted Superoxide stress induces the production of defense proteins, on the basis of killing kinetics. Similar killing kinetics were which are for the most part different from those of the peroxide recorded from an atmospheric glow discharge when Staphylo- stress response. Positive regulation of these is mediated by the coccus aureus was exposed wrapped in semipermeable bags two-stage sox RS system. 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]. VII. UV DAMAGE AND REPAIR IN BACTERIA UV radiation has well-known bactericidal properties. When photons of UV light strike biological cells, the energy in the VIII. OZONE DAMAGE OF BACTERIA photons is absorbed by chromophores at discrete wavelengths. The biological impact of UV radiation is primarily due to the Ozone is a powerful oxidizing agent, which readily forms absorption of photons by nucleic acids. DNA has an absorption reactive OH and HOO radicals in aqueous solution. By all spectrum with a maximum in the region 260–265 nm and which accounts, ozone disinfection is a complex heterogeneous phe- rapidly declines toward longer wavelengths [94]. Specific UV nomenon, the mechanism of which remains little understood disinfection devices emit upward of 86% of their light at despite decades of research effort. Ozone itself reacts with 254 nm, to coincide with this germicidal peak as closely as many biomolecules. Protein damage is caused by reactions with possible [96]. Many researchers have demonstrated that when dienes, amines, and thiols [22], and by cross-linking tyrosine UV light at 253.7 nm is absorbed by nucleic acids, pyrimidine residues by oxidizing the –OH groups [21]. Cell wall targets lesions are readily formed [97]. Although purine bases are also such as fatty acids and peptides appear among the most likely strong absorbers of the UV photons, the quantum yield required targets for gaseous ozone [99], [100]. Lipid peroxidation can to create dimmers is an order of magnitude higher for purines be stimulated by oxidation by ozone, resulting in a reduction of than pyrimidine bases [98]. While other photoproducts may be membrane fluidity. Single-strand DNA breaks have also been created through UV irradiation, pyrimidine dimers are gener- observed, which unrepaired can lead to extensive DNA damage ally considered to be the most important class. They are formed and death [101], [102]. in double stranded DNA when two adjacent pyrimidine bases Disinfection of E. coli in wastewater with ozone has been fuse together to form a dimer. Three types of pyrimidine dimers studied in various semibatch and continuous-flow configura- can be formed, thymine–thymine (T <> T), thymine–cytosine tions. Bactericidal concentrations are in the 0.1–0.2 ppm range (T <> C), and cytosine–cytosine (C <> C) dimers. During [103]. Although cell lysis is often observed following pro- UV irradiation the frequency with which each type is formed, longed ozoneation, is not necessarily a characteristic of the depends on the DNA base composition of the irradiated process [26]. Bacterial inactivation by ozone is a function of bacteria [95]. ozone concentration per viable bacteria, meaning that below a Bacteria possess a range of DNA repair systems, permitting threshold concentration ozone has no effect on survival. This rapid recovery from sublethal UV damage [70]. In bacteria, UV- can be complicated by the presence of other compounds with induced DNA lesions can be repaired by four main processes: which ozone can react [26].

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IX. CONCLUSION with the time course of cell injury, by demonstrating that direct application of relevant concentrations of the reactive species In this paper, the role of oxidative stress and damage by ROS over a comparable time period reproduces most or all of the in bacterial neutralization by air plasma has been considered. damage observed or by demonstrating that the extent of injury There is strong evidence from research into physiological expo- is diminished by removal of or inhibition of the formation of sures that the ROS formed in nonthermal plasma at atmospheric the reactive. pressure would cause various forms of cellular damage. If Use could also be made of experimental bacterial strains that unrepaired, such damage could be lethal. The concentration of may exhibit different phenotypical responses to the gas dis- ROS and the cocktail of species implicated during gas discharge charge. For example, SOD-deficient mutants of E. coli would exposure differ considerably from that of even the most extreme be more susceptible to gas discharge exposure than wild-type physiological conditions. Only further research will elucidate strains if superoxide were a significant reactive species. Like- the extent to which the reactions discussed here are involved in wise, up regulation of superoxide or hydrogen peroxide stress the bactericidal action. responses (defense and repair) by preexposure to nonlethal Biological and engineering challenges are still to be over- doses should confer a degree of protection. Indeed, studies come in the development of effective atmospheric pressure of the effects of sublethal exposures are likely to shed more discharges for decontamination and disinfection applications. light on killing mechanisms because the complete pathway of On the biological side, it remains critical to understand the events may be identified and not masked by damage caused pathway of damage leading to death for bacteria, viruses, and after the cells are actually dead. For this reason, it is important fungi. Without this knowledge, optimization of the decontami- to quantify the plasma dose required to achieve lethality in nation process is reduced to an empirical affair. In elucidating various bacteria species. Some species and strains may be the processes leading to cell death, the actual cause must be more resistant than others to damage caused by ROS. During separated from any physical changes that occur postmortem. a disinfection process it is essential that pathogenic microbial One approach would be to study the sublethal effects of plasma species receive a lethal dose; otherwise the oxidative damage on cells, while the effects on individual cellular components may be repaired in time, invalidating the disinfection process. such as cell membranes, nucleic acids, proteins, and enzymes It is clear that a truly multidisciplinary approach is needed could also be considered. Furthermore, the relative effects of in order to fully understand the biophysical and biochemical plasma exposure on microbial and human tissues should be fur- processes. Evidence strongly suggests that the vulnerability of ther compared where applications in patient care are considered cells and microorganisms to oxidation lies at the root of the such as for dentistry and wound treatment. mechanism for cell death during exposure to nonthermal gas On the chemical side, a deeper appreciation regarding the discharges at atmospheric pressure. Further studies will show relative importance of the various reactive species involved whether it is possible for oxidative stress to be exploited as a in bacterial neutralization is urgently needed. More detailed weapon for biological decontamination. 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 REFERENCES abundant ROS would be influential, this may not necessarily [1] J. Elster and H. Geitel, Wein Ber., vol. 94, 1890. [2] A. P. Krueger and E. J. 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Clive B. Beggs received the B.Sc. degree in building George E. Georghiou received the B.A., M.Eng., engineering from Leeds Polytechnic, Leeds, U.K., in M.A., and Ph.D. degrees from the University of 1981, the M.Phil. degree in refrigeration engineering Cambridge, Cambridge, U.K., in 1995, 1996, 1997, from the University of Sheffield, Sheffield, U.K., and 1999, respectively. in 1991, and the Ph.D. degree in energy engineer- He is currently an Assistant Professor at the ing from DeMontfort University, Leicester, U.K., Department of Electrical and Computer Engineer- in 1995. ing, University of Cyprus, Cyprus. Prior to this, he He is currently Professor of medical technology at was the undergraduate course leader in electrical the University of Bradford, Bradford, U.K., and was engineering at the Department of Electronics and formally, in 1996–2005, a Senior Lecturer in aerobi- Computer Science, University of Southampton, and ological engineering at the University of Leeds. He a Research Advisor for the Electricity Utilization, has spent many years researching the use of engineering methods to control the University of Cambridge. He continued his work at the University of Cam- spread of infection and has investigated the effects of ultraviolet light and air bridge in the capacity of a Fellow at Emmanuel College for three more ions on airborne bacteria. He has a particular interest in the epidemiology of years (1999–2002). He has a special interest in the development of numerical infection and the strategic use of interventions to break transmission pathways. 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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