Evidence of Temporal Postdischarge Decontamination of Bacteria by Gliding Electric Discharges: Application to Hafnia alvei

This study aimed to characterize the bacterium-destroying propertiesof a gliding arc plasma device during electric discharges andalso under temporal postdischarge conditions (i.e., when thedischarge was switched off). This phenomenon was reported forthe first time in the literature in the case of the plasma destructionof microorganisms. When cells of a model bacterium, Hafnia alvei,were exposed to electric discharges, followed or not followedby temporal postdischarges, the survival curves exhibited ashoulder and then log-linear decay. These destruction kineticswere modeled using GinaFiT, a freeware tool to assess microbialsurvival curves, and adjustment parameters were determined.The efficiency of postdischarge treatments was clearly affectedby the discharge time (t*); both the shoulder length and theinactivation rate k_max were linearly modified as a functionof t*. Nevertheless, all conditions tested (t* ranging from2 to 5 min) made it possible to achieve an abatement of at least7 decimal logarithm units. Postdischarge treatment was alsoefficient against bacteria not subjected to direct discharge,and the disinfecting properties of "plasma-activated water"were dependent on the treatment time for the solution. Watertreated with plasma for 2 min achieved a 3.7-decimal-logarithm-unitreduction in 20 min after application to cells, and abatementgreater than 7 decimal logarithm units resulted from the samecontact time with water activated with plasma for 10 min. Thesedisinfecting properties were maintained during storage of activatedwater for 30 min. After that, they declined as the storage timeincreased.

INTRODUCTION

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INTRODUCTION

The microbial contamination of liquids and surfaces is a majorsource of problems in numerous settings (food industry, hospitals).Various decontamination methods are available, and some of themare widely employed, including heat treatment, chemical disinfectionwith solutions or gases, filtration, and irradiation. Most conventionalmethods (autoclaving and dry heat) are associated with somedegree of damage to the material or medium. Another method,gas sterilization using ethylene oxide and compatible gaseouscompounds, allows low-temperature disinfection, but ethyleneoxide has recently been considered to be both mutagenic andcarcinogenic (26). Thus, the limitations of conventional techniqueshave motivated the search for alternative methods. Among theemerging techniques for decontamination, electric dischargesappear to be a promising alternative (11, 20), as they are usualsources of plasma gases.

The gliding electric discharge ("glidarc") device in humid airis an efficient and inexpensive source of nonthermal plasmawhich operates close to atmospheric pressure (it does not requirea vacuum) and at room temperature. This plasma exhibits characteristicsof both thermal and nonthermal plasma so that its interactionswith matter are those of quenched plasma (16). With humid airas the working gas, °OH and NO° were identified by emissionspectroscopy measurements as the principal radical species presentin the glidarc plasma plume (2), and they are probably precursorsof other active species. Such species endow the medium withvery high, enhanced chemical reactivity. As a result, the glidingdischarge device was first studied successfully for the treatmentof gases (5) and liquids (24) to abate chemical pollutants.More recently, it was also tested successfully for decontaminationof microorganisms (12, 22), which represents one of the mostinteresting applications of atmospheric pressure plasmas inrecent years.

This work dealt with inactivation of bacterial suspensions usinggliding arc plasma. In aqueous solution, a so-called temporalpostdischarge phenomenon was recently highlighted in a chemicalpollutant abatement setting. This phenomenon can simply be definedas the overall reactions that continue after the discharge isswitched off, and it probably involves the presence of long-livedreactive species. The postdischarge phenomenon may be of greatvalue in terms of environmental and industrial applicationsbecause the target continues to be treated without any contributionof energy. Costs are thus reduced. Although this phenomenonhas been observed in the oxidation of inorganic aqueous solutions(7) and in plasma chemical nucleophilic substitution reactions(6), no attempts to use plasma temporal postdischarge disinfectionhave been reported previously, as far as we know. In this study,as a first step toward full evaluation of the viability of glidarctechnology for industrial microbial decontamination, after characterizingthe destruction of bacterial cells by classical gliding electricdischarges, we focused on the study of temporal postdischargetreatment. Postdischarge efficiency was evaluated for cellsthat were present or not present in the fluid exposed to thedischarge. In the latter case, treatment consisted of disinfectionby plasma-activated water, the properties of which were studied.The entire study was conducted with Hafnia alvei, which wasselected as a bacterial model. This gram-negative bacteriumbelongs to the family Enterobacteriaceae. It has been foundin a number of foods, such as milk products and meats, and inhumans it may be associated with several intestinal disorders,including gastroenteritis (31).

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strain, cultures, and the preparation of suspensions.
H. alvei CIP 5731 was purchased from Institut Pasteur (France)and stored frozen in cryotubes at –20°C in glycerol.The microbial strain was cultured on Trypticase soy broth (TSB)(bioMérieux, Marcy l'Etoile, France) at 30°C withoutshaking. After two subcultures (1 ml in 9 ml TSB) at intervalsof 8 and 24 h, 100 ml TSB was inoculated with 1 ml of a microbialsuspension and incubated for 17 h. The bacteria were then inearly stationary growth phase. After culture, microbial cellswere harvested by centrifugation (4°C, 7,000 x g, 10 min),washed twice, and resuspended in 1.5 x 10^–1 M NaCl toobtain concentrated suspensions (A₆₀₀, 1.1) and diluted suspensions(A₆₀₀, 0.08). Both types of suspensions were utilized for inactivationstudies.

Gliding discharge treatments.
The plasma device utilized was a nonthermal quenched plasmadevice of the gliding arc type operating at atmospheric pressurewith humid air as the plasma gas. The design of the device andthe procedure for gas discharge have previously been described(22). Under operating conditions, the feed gas was providedby an air compressor. The air supplied (550 liters h^–1)was bubbled through a water-filled Duran flask in order to saturateit with water vapor. The gap between the plasma source electrodeswas kept constant at 3.5 mm. The distance between the electrodetips and the top of the liquid target was 13 cm. The reactorwas fitted with a circulating water jacket to prevent any thermaleffect, so that the temperature of the treated suspensions remainedbelow 30°C. Valves and filters were adjusted to the reactorin order to allow sampling during plasma treatment, when necessary.For each series of experiments, 200-ml portions of stirred liquidsamples (1.5 x 10^–1 M NaCl or diluted suspensions) wereexposed to the plasma discharge. The treatment time under plasmadischarge was designated t*.

Temporal postdischarge treatments.
For temporal postdischarge treatments, diluted bacterial suspensionswere initially treated for various t* with the glidarc reactor,as described above. Once the discharge was stopped, the bacterialsuspension was left at the ambient temperature for various postdischargetimes (t_p) before sampling and determination of surviving cells.

During other experiments, 1.5 x 10^–1 M NaCl solutionswithout bacterial cells were treated with the glidarc reactorfor various t* and then referred to as "activated water." Concentratedbacterial suspensions (12 ml) were diluted in activated water(188 ml) immediately after plasma treatment or after a periodof storage. Thereafter, the suspensions were left at the ambienttemperature for a variety of postdischarge periods (referredto as "contact time" [t_c]) before samples were removed and survivorswere counted.

Evaluation of surviving cells.
Before and after treatment (discharge and/or postdischarge),the numbers of surviving cells were determined by duplicateplate counting on Trypticase soy agar (bioMérieux). Inorder to prevent any destruction after sampling, the lethaleffect of active species generated by glidarc plasma dischargewas stopped with a universal neutralizing solution (3 g liter^–1L- ${alpha}$ -phosphatidylcholine, 30 g liter^–1 Tween 80, 5 g liter^–1sodium thiosulfate, 1 g liter^–1 L-histidine, 30 g liter^–1saponine), as recommended in the NF EN 1040 European standard(1) with respect to disinfection. For this purpose, 1 ml ofthe suspension to be analyzed was diluted in 9 ml of neutralizingsolution at each time point. Thereafter, classic 1/10 dilutionin 1.5 x 10^–1 M NaCl was performed prior to plating. Asdescribed in the European standard, control experiments wereperformed to verify the inactivity of the neutralization procedurewith microbial cells and its efficiency against active speciesgenerated by glidarc plasma discharge. The number of cultivablebacteria (expressed as CFU) was counted after 2 days of incubationat 30°C. The limit of detection under our experimental conditionswas 5 CFU ml^–1. Each determination of the kinetics ofdestruction was performed with at least three independentlygrown cultures. The data presented below are the means of decimallogarithm CFU ml^–1 values ± standard deviations.

Modeling of bacterial destruction.
GinaFiT, a freeware add-in for Microsoft Excel (9), was utilizedto model inactivation kinetics. This tool enables testing ofnine different types of microbial survival models, and the choiceof the best fit depends on five statistical measures (i.e.,sum of squared errors, mean sum of squared errors and its root,R², and adjusted R²). During this study, the different modelswere applied to the mean values obtained from experimental data.The "shoulder + log-linear" inactivation model exhibited thebest fit with our experimental data and was thus considered.It took account of a latency time before a decrease in the populationsize. The kinetics of destruction were expressed as follows(9):

Formula
where N is the cellconcentration (CFU ml^–1) after a treatment time t (min),N₀ is the initial cell concentration (CFU ml^–1), k_maxis the maximum inactivation rate (min^–1), and Sl is theshoulder length (min) (i.e., the length of the lag phase).

RESULTS

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RESULTS

Destruction of bacteria under direct discharge.
The effect of the glidarc decontamination technique on the cultivabilityof planktonic cells of H. alvei was first studied under directdischarge. In this setting, suspensions of bacterial cells weresubjected to the electric discharge and reactions were neutralizedimmediately after sampling, as described in Materials and Methods.Under these conditions, the treatment time corresponded to thet*. As illustrated in Fig. 1, the kinetics of destruction ofthe bacterial concentration versus the treatment time presentedtwo phases: an initial phase, during which the number of cultivablebacteria remained almost steady, followed by a rapid decrease.The reduction in the initial population was completed withinless than 4 min, and after gliding discharge treatment for 10min, no cultivable cells could be detected. The "shoulder +log-linear" GInaFiT model was applied to our experimental dataand revealed satisfactory adjustment (R² = 0.9902), showingthat decay followed a first-order kinetic law with an inactivationrate (k_max) of 4.51 ± 0.30 min^–1. The Sl was estimatedto be 5.57 ± 0.17 min by this model. A control experimentwas also carried out with nonionized gas, and the results showedthat the gas had no biocidal effect unless it was activatedby the electric discharge (Fig. 1).

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FIG. 1. Survival curve for H. alvei treated with gliding arc plasma discharge (x). The values are the means ± standard deviations for at least three experiments conducted with independently grown cultures. +, blank (treatment with un-ionized humid air); solid line, GInaFiT inactivation model; dashed and dotted line, detection threshold.

Destruction of bacteria during direct discharge followed by temporal postdischarge.
The effect of the glidarc decontamination technique on the cultivabilityof planktonic cells of H. alvei was also studied under temporalpostdischarge conditions. In this case, bacterial cells suspendedin the aqueous media were subjected to the electric dischargefor t*. Then, after extinction of the discharge, the cells wereleft in the aqueous media without neutralization in order toreveal destruction by temporal postdischarge. Under these conditions,the treatment time was equal to the plasma discharge time (t*)plus the postdischarge time (t_p). Neutralization was performedafter the postdischarge time.

Figure 2 shows the survival curves for H. alvei planktonic cellssubjected to temporal postdischarge for different periods ofglidarc plasma treatment. All these curves presented the sameprofile, which is similar to that obtained with direct discharge,with an initial lag phase followed by decay of the cultivablepopulation. For all the different curves, the "shoulder + log-linear"GInaFiT model fitted the data relatively satisfactorily (R²> 0.9733). Inactivation constants, as well as the lengthsof the shoulder phase, are shown in Table 1. All the conditionstested made it possible to achieve more than 7 decimal logarithmunits of destruction, but the efficiency of postdischarge treatmentswas clearly affected by the discharge duration. Postdischargetreatment was more efficient after a longer t*, and no detectablecells (from the initial population containing 4 x 10⁷ bacteriaper ml) remained after 13 min for a t* of 5 min, after 16 minfor a t* of 4 min, after 21 min for a t* of 3 min, and after28 min for a t* of 2 min. As illustrated in Fig. 2 and Table1, both the length of the lag phase and the inactivation ratedepended on t*. More precisely, it could even be consideredthat there was a positive linear correlation between k_max andt* and a negative linear correlation between Sl and t* (Fig.3). Within the domain studied, the k_max and the Sl could thenbe expressed as k_max = 0.37t* + 0.56 (R² = 0.9776) and Sl =–3.36t* + 21.68 (R² = 0.9654), respectively.

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FIG. 2. Survival curves for H. alvei subjected to temporal postdischarge time (tp) after different t*, including ( ${blacklozenge}$ ) 5 min, ( ${blacksquare}$ ) 4 min, ( ${blacktriangleup}$ ) 3 min, and (•) 2 min. For each curve, the open symbol indicates the beginning of postdischarge treatment. The values are means ± standard deviations for at least three experiments conducted with independently grown cultures. Solid lines, GInaFiT; dashed and dotted line, detection threshold.

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TABLE 1. "Shoulder + log-linear" model parameters for survival curves for H. alvei subjected to temporal postdischarge after different t*^a

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FIG. 3. Correlation between model temporal postdischarge kinetic parameters, including k_max ( ${blacksquare}$ ) and Sl ( ${blacklozenge}$ ), and t*.

Destruction of bacteria by activated water.
The efficiency of bacterial destruction by temporal postdischargetreatments was clearly demonstrated for bacteria exposed toelectric discharge. The postdischarge behavior of bacteria withoutprecontact with the discharge was then considered. For thispurpose, as described in Materials and Methods, 1.5 x 10^–1M NaCl was exposed to the discharge for 5 min, and the bacteriawere then immediately placed in contact with the plasma-treatedsolution (referred to as activated water) for a period calledthe contact time (t_c) before neutralization. The survival curveobtained under these conditions for H. alvei cells is shownin Fig. 4 and compared to that obtained previously for exposureof bacteria to direct discharge for a t* of 5 min. Even if comparable,the destruction was slightly less efficient when cells werenot subjected to the discharge, due to the existence of a tailin destruction kinetics or to log-linear destruction with alower inactivation rate. Given the number of points in the log-lineardestruction phase and the variability of bacterial concentrationsobtained after 10 and 15 min of exposure to activated water,more precise analysis of the activated water inactivation curvewas not performed. However, it can be pointed out that reductionsof more than 5 decimal logarithm units were obtained in lessthan 15 min with activated water. This shows that exposure ofbacteria to direct discharge was not a prerequisite for theirdestruction by plasma technology.

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FIG. 4. Kinetics of destruction of H. alvei cells exposed for a t* of 5 min to the glidarc plasma discharge and then to postdischarge (during t_p; ${blacklozenge}$ ) or exposed to activated water for a contact time (t_c; ${blacksquare}$ ). In the latter case, activation was achieved by exposure for 5 min of 1.5 x 10^–1 M NaCl without cells to gliding arc plasma discharge. The values are means ± standard deviations for at least three experiments conducted with independently grown cultures. Dotted and dashed line, detection threshold. tc, contact time; tp, postdischarge time.

Characterization of the disinfecting properties of activated water.
Activated water was thus intrinsically efficient, and next characterizationof its disinfecting properties was attempted. First, the effectof the t* used to treat the solutions on subsequent bacterialcell destruction by activated water was evaluated. Various t*were used with 1.5 x 10^–1 M NaCl solutions just priorto mixing with bacteria, and surviving cells were counted aftercontact times of 10 and 20 min. All conditions tested resultedin significant reductions in the initial cultivable population(Table 2). However, no detectable cells were obtained only fora t* of 10 min and a contact time of 20 min. Table 2 shows thatthe disinfecting efficiency of activated water increased withthe plasma electric discharge time, and it was again shown thatthe temporal postdischarge effect was controlled by the durationof the discharge.

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TABLE 2. Effects of the length of plasma activation of 1.5 x 10^–1 M NaCl solutions on the temporal postdischarge destruction of H. alvei cells

The influence of the length of storage of activated water wasalso examined. NaCl solutions (1.5 x 10^–1 M) were treatedwith plasma discharge for a t* of 5 min and then stored at theambient temperature for increasing periods of time from 5 minto 24 h before they were mixed with bacteria. H. alvei survivingcells were detected as described above after 10 and 20 min ofcontact time with stored activated water. The results obtainedunder these conditions are shown in Table 3 and can be comparedwith those obtained when activated water was not stored beforeuse (Table 2). The disinfecting properties of activated waterwere not affected by storage for 30 min, but thereafter theefficiency decreased with the storage duration. Nevertheless,a residual disinfecting efficiency was sustained, even after24 h of storage. Contact for 20 min with 24-h-old activatedwater resulted in a decimal logarithm reduction of approximately2.5 U.

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TABLE 3. Effects of the resting time of activated water on the temporal postdischarge destruction of H. alvei cells^a

DISCUSSION

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DISCUSSION

The main goal of this work was to demonstrate the effectivenessof glidarc technology in microbial decontamination, especiallyby means of temporal postdischarge (i.e., after the electricdischarge is switched off). It was shown previously that theglidarc technique, like other plasma techniques (14, 20), wasable to destroy planktonic, adherent, and biofilm bacteria underdirect discharge (12, 22). However, this study was the firstinvestigation of bacterial destruction by temporal postdischarge,whatever the plasma technology considered. This temporal postdischargehas to be distinguished from the postdischarges or afterglowsusually used for plasma treatment that are actually spatialpostdischarges, inducing the destruction of microorganisms disposedof in the afterglow plume of an operating discharge (23, 32).The occurrence of such a phenomenon is of considerable economicinterest in terms of its industrial and environmental applications,as the inactivation initiated by plasma discharge requires noextra energy supply to continue and has displayed efficiencywhich is comparable to that achieved during plasma discharges(Fig. 1 and 2). Current development of plasma devices is focusedon decontamination of medical supplies and military equipment(30). Decontamination of food product surfaces (meats, vegetables)is also under study (10, 33). In all cases, careful attentionhas to be paid to product deterioration. As temporal postdischargewas shown to be efficient even when microorganisms were notexposed to the plasma plume (Fig. 3 and Tables 2 and 3), theproducts to be decontaminated do not need to be subjected tothe discharge, which should limit their alteration by the treatment.This is interesting as the lethal effect of temporal postdischargeis sustained (at a lower degree of efficacy) for several hoursafter the plasma treatment ends. Plasma-activated water couldthus be introduced as a sanitizer and potentially used for industrialcleaning in place. Nevertheless, as discussed below, this requiresfurther characterization of active species.

The kinetics of destruction were studied under various conditions,including direct discharge followed or not followed by temporalpostdischarge. In both cases, the kinetics of destruction fittedwell with the "shoulder + log-linear" GInaFiT model (Fig. 1and 2). In previous studies, the kinetics of plasma treatmentwere often polyphasic (14, 20). Biphasic curves with a lag phasewere previously observed during the destruction of planktoniccells by direct discharges using the same type of plasma equipment(22), as well as with other types of discharges (17), and thereported values for shoulder lengths and inactivation rateswere comparable to those obtained during the present study.For example, Maeda et al. (17) obtained a lag phase of 14 minand an inactivation rate of 1 min^–1. Our study showedthat these two parameters were contrastingly linearly modifiedwith discharge time t*. This situation can be compared withthe results of thermal destruction, where biphasic curves havealso been observed, and both shoulder lengths and inactivationrates have been clearly related to the destruction temperatureby some authors (4, 8). Thus, the quantity of energy injected(as heat or electricity) is a key parameter in bacterial inactivation,as it is for chemical compound abatement using glidarc techniques(13). The energy provided to the discharge was used to createactive (and/or lethal) species. In this context, we can assumethat the lag phase was a manifestation of accumulated damagewhich became irreparable (and thus lethal) above a criticallevel. However, from results shown in Fig. 4, it can be deducedthat this level depended not only on the amount of active speciesbut also on the contact time between cells. This correspondsto the CT parameter (concentration x contact time) frequentlyemployed in the case of disinfection with classical chemicalcompounds (25).

The course of the kinetics of destruction of microorganismsby any lethal agent can be related to the rate of the reactionbetween the agent and the microorganism under study and alsoto the variable resistance of individual cells in a given population(25, 27). For example, the fact that a small fraction of anypopulation can survive treatments that kill the majority ofthat population has frequently been reported (which leads toa survival curve with a tail, as shown in Fig. 4), and intrapopulationdiversity has been postulated to be a mechanism to ensure survivalfollowing exposure to stress (3). Moreover, in the specificcase of plasma treatment, polyphasic curves could result fromthe successive actions of several lethal agents that dependon the plasma device (15, 19). In the present study, the pHof H. alvei suspensions fell from 6.1 to 3.6 with only 1 minof treatment, and HNO₃ was detected in the suspended fluids.But acidification alone cannot explain the cell mortality observedduring discharge and postdischarge treatments, as immersionfor 30 min of an H. alvei suspension in acidified NaCl (HNO₃,pH 3.6) induced a reduction in the number of cultivable cellswhich was less than 2 decimal logarithm units (data not shown).Glidarc destruction is probably also based on the chemical processesof oxidation. As parallel experiments conducted in NaCl solutionsand deionized water produced the same level of destruction (datanot shown), plasma treatment could not be suspected to inducethe formation of chlorine active species, which are potent oxidizersused as sanitizers (18). On the other hand, various free radicals(e.g., °OH and NO°) are formed in the plasma plume (2).These radicals are highly active in the inactivation of microorganisms(20, 29) and possibly involved in direct discharge treatments.Through recombination and collisions, these labile free radicalsmay give rise to new longer-life-span species that would beresponsible for the lethal effects of temporal postdischarge.In agreement, H₂O₂ (a molecule derived from °OH) was proposedto be the principal agent for oxidation in the temporal postdischargeof chemical compounds (7), and NO° was oxidized to HNO₂and subsequently to HNO₃, both of which were detected in plasma-treatedfluids (data not shown). The evolution over time of discharge-generatedactive species led to evolution of activated water efficiency(Table 2). Together with lesions induced directly by plasmadischarges on bacterial cells (due to, for example, an electricfield, as in the case of destruction by a pulsed electric field[28]), the evolution of active species was also probably responsiblefor the difference in efficiency observed when the cells werein the fluid or not in the fluid during the discharge (Fig.4).

To summarize, for discharge alone or under in postdischargeconditions, the efficiency probably results from a combinationof different effects, principally of a chemical nature. We planto pursue our studies to determine the implications for differentchemical active species, particularly in the setting of thelethal postdischarge effect of glidarc. Even though preliminarystudies have shown that the glidarc treatment of water doesnot generate genotoxic compounds (21), the production of potentialtoxic by-products also needs to be carefully examined beforeany practical applications can be proposed, especially in thefood and water industries.

ACKNOWLEDGMENTS

We thank Laurent Guillier for introducing us to the GInaFiTadd-in for Microsoft Excel and for stimulating discussions onthe analysis of destruction kinetics.

Georges Kamgang-Youbi's thesis received financial support fromthe "Service de la Coopération et d'Action Culturelle(SCAC)" at the French Embassy in Cameroon via an EGIDE scholarship.

FOOTNOTES

* Corresponding author. Mailing address: UMR763 BHM, AgroparisTech, INRA, 25 Avenue de la République, F-91300 Massy, France. Phone: 33 1 69 53 64 73. Fax: 33 1 69 93 51 44. E-mail: murielle.naitali{at}agroparistech.fr

Published ahead of print on 8 June 2007.

REFERENCES

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