Bacterial inactivation using pulsed gliding arc discharges

Abstract. The classical inactivation methods for micro organisms use in present heat, toxic substances, UV light, and ionizing radiation that usually require long treatment times or use temperatures, which limits their application for temperature sensitive materials. Also, using toxic substances (as chlorine) have a major disadvantage due to the fact that they lead to the unwanted traces on the medium to be sterilized with negative implications when used on food or human contact objects. In the present study, low temperature at atmospheric pressure plasmas (produced by a pulsed gliding arc discharge) was utilized as a tool to deactivate a bacterial colony, E. Coli, growth on the surface of a nutrient substrate. The non-equilibrium plasmas produce high concentrations of radicals and other active species (especially hydrogen peroxide) that substantial decrease the bacterial concentration as was observed both in air and argon used as carrier gases. Together with the comparison of using directly on the surface contaminated with bacteria of a hydrogen peroxide solution indicates a major role of radicals and active species on the decontamination mechanisms.

1. Introduction

The cold plasma gliding arc discharge has been proved to be an effective method for gas and aqueous phase pollution control. However, recently the application of this technology has been expanded and is now being tested as an efficient anti-microbial agent. The aim of this paper is to both develop an understanding of the influence of the pulsed gliding arc reactor and also to determine the most significant chemical contributors to Escherichia Coli bacteria deactivation.

Many of the past bio-decontamination methods centered around chemical, physical, mechanical, and thermal (e.g. high pressure, high temperature, UV, and gamma irradiation) treatments.4 Techniques which based upon thermal processes, such as ovens or autoclaves, are particularly ineffective as many of the materials used today consist of polymers which are poorly resistant to heating, rendering these processes inapplicable [1]. Furthermore, non-thermal processes, such as exposure to ethylene oxide or chlorine among other chemicals, have inherent drawbacks as the treated object, as in the case of food, involves intimate contact with humans [1-3]. Techniques commonly designated as Advanced Oxidation Processes (AOP) have been emerging in the bio-decontamination field. Unlike other some other sterilization processes they are essentially “clean” and generate only small amounts of persistent chemical traces. Among others decontamination AOP techniques, one of the most effective is the pulsed gliding arc discharge [6]. Systems based on electrical discharge, especially those like the gliding arc that generate non-thermal plasma, appear to be very promising due to their low equipment and energy cost, their efficiency on multiple surfaces, and application to heat and chemically sensitive materials.

Inactivation of bacteria exposed to chemically reactive species has been observed by many research groups world wide [1-6]. Micro organisms can be exposed to radicals by exposing them directly to the plasma or by generating reactive species in a plasma and expose the micro organisms to the gas at the outlet of the discharge.The bacteria inactivation could be a process that leads to membrane lipid alteration caused by fatty acid peroxide formation and protein oxidation [14 -19].

The gliding arc reactor uses a discharge that glides between two divergent electrodes in a gas flow in order to maintain the non-thermal characteristic of plasma and leads to the formation of many different chemically reactive and oxidizing species including highly reactive radicals, positive ions, negative ions, electrons, photons [7]. Atomic oxygen, ozone, hydroxyl radicals, peroxyl, hydrogen peroxide and NOx radicals are some of the species that have been demonstrated to affect the integrity of the cells.

The original technique studied in this paper uses water spray injected directly in a gliding arc discharge for the treatment of contaminated surfaces, which eliminate the need of direct contact of the plasma while simultaneously sterilizing a larger area.

2. Experimental

The gliding arc reactor utilizes two stainless steel divergent electrodes attached to a ceramic support placed between two rectangular glass sheets. The reactor dimensions are shown in Fig. 1.

Fig.1

The sterilized water is sprayed directly into the plasma formed between the electrodes through an injection nozzle equipped with one port for the gas injection and one port for the liquid. The nozzle consists of two SS tubes attached to a Swagelok Tee (1/16 inch). The two ports of the Tee are used for gas and water injection. The water is carried with the gas flow (Qg=2L/min) through the outlet port and it is atomized due the high kinetic energy of the carrier gas, which assures the disintegration of the liquid. The very small diameter of the inner tube (Ø=0.15 mm) led to an increase in relative exit velocity of the gas. The gas was injected to the straight port of the Swagelok Tee and the water was injected by a pump (Harvard Apparatuses, PHD 2000 Infusion) through the perpendicularly port of the tee [7], [10].

The aerosolized water droplets formed in the flowing gas provide a large contact surface area with the plasma, enhancing the chemical reactions between the active species formed in the gas and water droplets. The input energy is not sufficient to vaporize the water droplets and can lead to only a few degrees rise in water temperature. The electrical discharge forms at the minimum distance between the electrodes and “glides” along their edges in a mixture of gas (e.g., air or argon) containing the very fine droplets of water. In the present study the gas flow rate, Qg, is approximately 2L/min and the water flow rate, Qw, varies for different experimental situations between 0.5 and 2 mL/min.

In testing the efficiency of the new reactor design several chemical tests including conductivity, pH, hydrogen peroxide concentration, and nitrate concentrations were measured. For measuring pH, conductivity, and nitrate formation three individual Oakton Acorn 6 Series meters were used. In the case of the nitrate/ion meter a Cole Parmer nitrate combined electrode was used in tandem with the Oakton meter. All other probes were included with the Oakton meters themselves. For the measurement of hydrogen

peroxide (H2O2) concentration, a spectrophotmetric method involving the reaction of H2O2 with titanyl ions was used. In this reaction, titanyl ions react with the hydrogen peroxide in the following reaction:

Ti4+ + H2O2 + 2H2O → TiO2•H2O2 + 4H+

As the two components react they form pertitanic acid which gives off a distinct yellow color which has a peak absorbance at 410nm. The discharge formed between the divergent electrodes leads to the formation of many different chemically reactive and oxidizing species including highly reactive radicals, positive ions, negative ions, electrons, photons. For example, it has been well documented that hydrogen peroxide and its precursors are formed from the following chemical reaction [5], [10],[11],[13]:

H2O + e H2O+ +2eH2O+ OH +H3O+

2OH H2O2

H2O + e H2 + O+ +2eH2O + e H+ + O + H +2eHO2 + HO2 H2O2 + O2

H2O + Heat H + OH

As one of the goals of the sterilization process is to maximize the concentration of oxidizing species, specifically hydroxyl radicals and hydrogen peroxide, an appropriate carrier gas must be selected. Although air is the most abundant and consequently the cheapest gas available, there are some inherent drawbacks associated with its use. When air is used as the carrier gas nitrogen oxides are formed by the following gas-phase chemical reactions:

N2 + e 2N + eO2 + e 2O + eN + O NONO + O NO2

2NO2 N2O4 + H2O HNO3 + HNO2

HNO2 H+ NO2-These side reactions are somewhat undesirable as the nitrogen oxides will react with hydrogen

peroxide in the following reaction:NO2

- + H2O2 NO3- + H2O

The air leads to formation of nitrites which eventually react with and degrade hydrogen peroxide, therefore a higher concentration of hydrogen peroxide with argon versus air have been observed, where nitrogen and its derivatives were not present. Thus, for bacteria inactivation argon and air have been studied in order to determine the economic viability of each gas. In the case of either gas, the abundance of the various radicals and oxidative species leads to the formation of larger molecules resulting in a largely acidic environment.

In order to study the effect of the plasma treatment on bacteria is to create a medium for them to thrive and reproduce. The chosen media was a dilute water solution consisting of 10% Brain-Heart Infusion (BHI) additives. This medium was produced by dissolving 10% by weight of BHI into a solution of distilled water. After the solution was prepared, it was sent through autoclave in order to ensure complete sterilization. After the sterilization was complete, the solution was inoculated with a strain of E. Coli. Once this solution had been inoculated, the strain was allowed to grow for 24 hours in an incubator held a constant temperature of

37 Celsius. After this initial growing period, the BHI solution had reached “terminal concentration” based on the amount of nutrients available resulting in roughly 109 cells per mL. In order quantify the amount of bacterial inactivation achieved from treatment by the gliding arc, the colony counting method was utilized. Consequently, the creation of an agar plating medium was essential in order to plate the treated bacterial. This is a necessary step because if the bacteria are plated on a surface on which there are no nutrients for them to reproduce, the remaining cells/colonies will generally not be visible to the naked eye and thus render the colony counting method ineffective.

After being plated, the bacterium was given between 60 to 120 minutes to settle and adhere to the agar surface, after which it was subjected to the treated spray from the reactor. The treated plates were then placed in the incubator and given 24 hours to grow and after which they would be counted. In many of the tests, the concentration of surviving bacteria was near zero and thus counting could be done with the naked eye. In the event that the remaining bacterium density was estimated to be on the order of 1000 a different technique would be required. In this case, a portion of the plate that was indicative of the whole sample would be chosen. The bacteria in this region have been counted and then multiplied by the fractional area and thus the total concentration would be estimated.

3. Results and discussions

Prior to conducting trials involving bacteria, it was of interest to complete a series of chemical tests on the reactor concerning hydrogen peroxide generation. Preliminary tests have been performed in order to emphasize what persistent chemical species were produced as well as how certain parameters (power, frequency, flow rate) affect these production rates. Specifically, it was of interest to measure hydrogen peroxide concentration, pH, and nitrate formation. These results are shown in Fig. 7 and Fig.8.

Fig. 7 - Hydrogen Peroxide Concentration (Air)

Fig. 8 - Hydrogen Peroxide Formation (Argon)

Table 1Species Formation

Air Argon

pH 3.8-3.9 3.6-3.7

Nitrates (ppm) 230-100 320-120

Conductivity (µS) 50-70 50-70

As it was hypothesized that the hydrogen peroxide concentration was an essential factor in bacterial inactivation, the reactor should be run at a very low liquid flow rate. Secondly, it appears from the H 2O2 and other chemical results that the choice of gas plays a very limited role in chemical species production. This is mostly likely due to the reactor being virtually open to the environment.

To determine the effectiveness of the gliding arc reactor on bacteria and to see the effect of hydrogen peroxide, a series of controls were put into place. In each trial, 5 different types of plates have been produced. For each trial there was a full control plate, a water spray plate, a hydrogen peroxide control plate (pipetted), a hydrogen peroxide control plate (sprayed), and a treated plate. The full control plate would be prepared by simply plating the desired concentration of E. Coli on the agar Petri dish and then placed in the incubator. This plate is invaluable to the results as it would confirm that the plating technique was adequate as well as confirming the bio-viability of the E. coli sample.

The next plate produced in each trial was the “water spray plate”. Like the full control plate, this plate was made by plating the desired concentration of bacteria on the plate. After completing that step, the plate would then be treated with water sprayed out of the gliding arc apparatus (with the power off). The spray was pure deionized water and not activated in any way by electrical discharge. The advantage of having this plate is it allows the experimenter to determine if the physical effect of high gas flow rates

combined with water spray contributes in any way to the inactivation of the E. Coli cells.The following “pipetted” peroxide control plate was prepared just as the previous two plates.

However, unlike the water spray plate; this plate was treated with a hydrogen peroxide control solution. This control was initially delivered via pipette versus spray because it was thought to be best to minimize the amount of hydrogen peroxide in direct contact with the reactor. Because the experiments involving the treated spray are conducted using two types of carrier gases (air and argon), two different concentrations of control H2O2 solution were prepared. The H2O2 control solution was at exactly the same concentration of the treated solution, chemical testing would be completed after each trial. The second of the peroxide control plates was the H2O2 control produced via spray. As noted above, in the preliminary tests this plate was initially excluded. Aside from possibly damaging the reactor, it was believed that this control would be unnecessary as the water control plate would reveal the effect, if any, of the high velocity gas /water mixture.

By comparing the results of the H2O2 control plates with that of the treated spray plates, one could better determine the role of hydrogen peroxide in bacterial lethality. The final plate created in each trial was the treated plate. As the name suggests this plate was treated with the electrically activated spray produced in the gliding arc. In effort to collect the most accurate data, a repeat of each treated plate was produced. Each plate was treated under the following conditions, presented in Table 2: Table 2

Trial Conditions

Variable Value

Water Flow Rate (mL/min) 0.5

Carrier Gas Flow Rate (L/min) 2.5

Distance between plate and electrode (cm) 3

Spray duration (min) 2

Duration between plating and treating (min) 90

The water flow rate was chosen to be as small as possible (roughly 0.5 mL/min) in order to yield the highest concentration of hydrogen peroxide (see figs 7,8). The initial chemical testing for the glide arc revealed that for this flow rate, our H2O2 concentration would be approximately 2.8 mM and 3.2 mM respectively for air and argon as the carrier gas.

Next, the gap distance between the electrodes and the Petri dish was set at 3 cm to avoid that the high gas velocity to displace the bacteria medium and push it to the sides of the Petri dish. Consequently, 3 cm was the shortest gap distance that would minimize this displacement.

Ultimately, after treatment is was desired to only spray just enough water to cover the entire surface area of the plate. Consequently the treatment time would be a direct function of the water flow rate (0.5 mL/min) and it was adjusted accordingly to roughly 2 minutes. The disadvantage of increasing the treatment time too much was that if the plate becomes over saturated with water, the bacteria will have a tendency to detach from the solid substrate and to migrate to the liquid phase.

Finally, the time between plating and treatment was set at 60-120 minutes. This was mainly due to the constraints of the equipment and experimental procedures.

Three sets of experiments have been performed for 104 colonies, 105 colonies and 106 colonies on the plate. In Fig.9-16 are presented the results for 104 colonies on the Petri dish.

Trial 1: Inactivation of Escherichia coli (approximate concentration: 104 colonies)

Fig. 9 – Full Control 104 Fig. 10 – Water Spray Control 104

Fig. 11 – Treated Spray (Air) 104 Fig. 12 – Treated Spray (Ar) 104

Fig. 13 – H2O2 Pipette Control (2.3 mM, Air proxy)

Fig. 14 – H2O2 Pipette Control (2.8 mM, Ar proxy)

Fig. 15 – H2O2 Sprayed Control (2.3 mM, Air proxy)

Fig. 16 – H2O2 Sprayed Control (2.8 mM, Ar proxy)

For the first trial, the results clearly show that for a concentration of approximately 10,000 (104) Escherichia Coli cells per mL, the treated sprayed water was completely effective. For both the air and argon plates there were essentially 4 logarithmic units of removal. It is also important to note from the previous photos that the water control plate did not yield any significant differences when compared to the full control plate. This implies that the physical effect of high gas flows coupled with sterile water had no noticeable effect on the bacteria. Perhaps the most interesting of all of the above results is the seemingly highly significant effect of the spraying mechanism when used in conjunction with hydrogen peroxide. In figures 13-14 the hydrogen peroxide control solution was not delivered to the plates with spray but instead via pipette. As a result, the concentration of surviving bacteria was effectively 100%. However, in figures 15-16 when the same concentration of hydrogen peroxide (2.3 / 2.8 mM) was delivered to the plate via the spray, the surviving number of colonies was 134 and 112 respectively, yielding 2 log units of removal. From the results of the water control, we expect that the physical effect of the gas/water mixture alone should play no role in the bacteria inactivation. Thus, the fact that the hydrogen peroxide is mixed with the gas and subsequently delivered at a relatively high velocity plays an important role in the inactivation. These results lead to several conclusions. First, it is apparent that while the physical effect of the high velocity spray is negligible (see figure 10), the spray plays a large role in enhancing hydrogen peroxides lethality to bacteria. Secondly, from the data above, it would appear that hydrogen peroxide as expected, is a significant contributor to bacterial deactivation. These results presented lead to the idea that the mechanism between hydrogen peroxide (hydroxyl radicals) and DNA (among other proteins) is suspected to be the main contributor to bacterial inactivation. The Table 3 summarizes the results from the Trial 1.Table 3

Summary of results – Trial 1 (104)Initial colonies Surviving Colonies Log Reduction

Treated (Air) 104 1 4Treated (Ar) 104 4 4H2O2 (spray 2.3 mM) 104 176 2H2O2 (spray 2.8 mM) 104 118 2

Trial 2: Inactivation of Escherichia coli (approximate concentration: 105 colonies)The results for an E. Coli concentration of approximately 105 (100,000)

colonies/mL in trial two was nearly identical to the results produced in trial one the gliding arc was successful at removal up to 4 logarithmic units of E. Coli. In both cases, with air and argon, there were 11 and 20 colonies respectively left on the plates, suggesting the carrier gas in this scenario may be insignificant. It is also important to notice that again the water control yielded no noticeable difference when compared to the full control plate. The Table 4 summarizes the results from the Trial 2.

Table 4

Summary of results – Trial 2 (105)Initial colonies Surviving Colonies Log Reduction

Treated (Air) 104 12 4Treated (Ar) 104 15 4H2O2 (spray 2.3 mM) 104 950 2H2O2 (spray 2.8 mM) 104 145 3

As the concentration is increased, the differences between the treated plates and the H2O2 spray plates are becoming more visible. The H2O2 control spray revealed that after treatment approximately 1200 and 180 cells were remaining for the 2.3 and 2.8 mM concentrations respectively where as the treated sprays yielded 10-20 survivors. Finally, as expected, the hydrogen peroxide controls which were simply plated via a pipette had seemingly no effect on bacterial removal, which again is to be expected as it had no effect on the lower bacteria concentration in Trial 1.

Trial 3: Inactivation of Escherichia coli (approximate concentration: 106 colonies)In trial three, the concentration of plated E. Coli was again increased by a factor

of 10 from 105 to 106 colonies/mL. As the concentration continues to increase, the untreated bacteria no longer forms individual colonies but instead multiples into a mass known as “bio-film”. Unlike the previous two trials conducted at lower concentrations, the treated plates are now showing a higher concentration of surviving bacteria. In the first two trials the glide arc successfully reduced the surviving number of colonies by 4 log units. However, as the concentration has been increased the reduction was only 3 log units. This provides valuable data for the upper limit operating range of the glide arc apparatus under these conditions. The Table 5 summarizes the results from the Trial 3.

Table 5

Summary of results – Trial 3 (106)Initial colonies Surviving Colonies Log Reduction

Treated (Air) 104 1000 3Treated (Ar) 104 800 3H2O2 (spray 2.3 mM) 104 10 000 2H2O2 (spray 2.8 mM) 104 20 000 2

4. Conclusions

The physical effect of a high gas flow rate plays a significant role in the sterilization potential of hydrogen peroxide on adherent phase E. Coli. At concentrations of 1 – 10 mM, hydrogen peroxide alone is not sufficient to successfully treat bacteria in the adherent and exponential growth phase without the presence of a high flow carrier gas. However, these concentrations were effective on planktonic bacteria.

The pulsed gliding arc apparatus can successfully reduce the surviving number of Escherichia Coli colonies by 4 logarithmic units after 2 minutes of treatment with air. The pulsed gliding arc reactor generates highly oxidative chemical species other than hydrogen peroxide which enhance bacterial degradation.

In the presence of a high flowing carrier gas (air), low concentrations (2 mM) of hydrogen peroxide are capable of up to 3 logarithmic units of removal in E. Coli.

The anti-microbial resistance of Escherichia coli bacteria increases as the time between plating and treatment grows. The effect of carrier gas selection on Escherichia coli lethality appears to be minimal provided sufficient hydrogen peroxide is delivered.

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