On the inactivation of Bacillus subtilis spores by surface streamer discharge in humid air caused by reactive species

The geometry of the CSDBD electrode allows for the production of thin plasma filaments distributed on the surface of a dielectric material [33, 34]. Figure 1 shows a simplified sketch of the CSDBD reactor used in this study. The reactor used to test the interaction between a multiple surface streamer micro-discharge and biologically contaminated samples consists of a concentric CSDBD electrode system placed in a compact 3D-printed flow-through chamber, designed in our lab and equipped with gas feed input/output ports, a high-voltage interface and a sample holder. The modular concept of the reactor allows for the quick insertion of treated samples and very straightforward replacement of the discharge electrode(s) to use alternative discharge and/or treatment geometries [35]. Multiple filaments were produced on the surface of the CSDBD electrode, a disc of diameter 25.4 mm thickness 5 mm, made from MACOR® machinable glass-ceramic material and sealed in a polyamide holder. Multiple-streamer micro-discharge was initiated on the ceramic surface. The driving electric field was induced by high-voltage (HV) waveforms imposed between two concentric ring-shaped silver electrodes, embedded approximately 0.4 mm below the MACOR® surface with a minimum distance of 1 mm between them. The reactor was powered by an amplitude-modulated AC HV power supply composed of a TG1010A Function Generator (TTi), a Powertron Model 250A RF amplifier and a HV step-up transformer, as in a previous study [36]. The micro-discharges were produced by a burst of four sine-waves (u_HV = 22 kV peak-to-peak, f_AC = 5 kHz) applied at a fixed repetition frequency of f_M = 500 Hz, with a resulting duty cycle of 0.4.

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A Tektronix oscilloscope (DPO5204, 2 GHz, 10 GS s⁻¹) was used to record the discharge characteristics, i.e. the HV, current and plasma-induced emission (PIE) waveforms. The discharge HV waveforms were sampled by a Tektronix P6015 HV probe. The current pulses produced by individual micro-discharges were monitored via the voltage drop across a non-inductive shunt resistor inserted between the grounded electrode and the grounding lead, and were measured by a Tektronix TPP1000 HV probe. The transferred charge was determined by inserting a non-inductive measuring capacitor (C = 0.47 μF) into the grounding lead and analysing the charge-voltage characteristic (Lissajous figures). The reactor was fed with humid synthetic air through a Bronkhorst HI-TEC model mass-flow controller (flow rate Q = 0.1 l min⁻¹). The vapour density was about 12.1 g m⁻³ (relative humidity of 70% measured at 20 °C). The distance between the surface of the CSDBD electrode and the exposed sample was adjustable, and was fixed at 3 mm in all of our experiments.

Two frontal windows allowed for direct visual control and optical emission diagnostics of the discharge area. A fast Hamamatsu R2949 photomultiplier (PMT) and Andor iStar ICCD DH740i-18U-03 camera were used to record the optical emission waveforms and the spectra collected from the whole surface of the CSDBD electrode. The ICCD was used to acquire the time-averaged emission spectra through the iHR-320 spectrometer.

The preparation of B. subtilis spore stock and deposition on the filters was carried out according to the protocol detailed by Dolezalova et al and Shaw et al [26, 27]. The advantage of this protocol lies in setting up of a universal biological reference protocol for the study and comparison of plasma treatments across laboratories. This protocol ensures (i) the production of microorganisms in a specific growth phase, and (ii) the standardised preparation of bacterial samples made of spores distributed in single monolayers. Both of these appear to be critical issues that can impede the direct comparison of inactivation results achieved in different labs. Spore resistance strongly depends on the conditions leading to sporulation, and the use of a 30 min thermal shock at 70 °C to initiate sporulation is therefore recommended.

The protocol also includes a novel technique for the deposition of spore samples on the filter membrane. Until now, pipetting a bacterial solution onto a substrate has been one of the most commonly used techniques in the study of the bactericidal properties of various plasmas. Unfortunately, the microbial distribution produced by pipetting a bacterial suspension as a droplet is neither homogeneous nor reproducible. Furthermore, heterogeneously distributed bacteria can migrate, creating clusters and consequently forming cell refuges that are not exposed to plasma. Pipetting is therefore unsuitable for plasma-assisted sporicidal testing.

In this procedure, vegetative cells of B. subtilis (ATCC 6633, Czech Collection of Microorganisms, Brno, Czech Republic) were pipetted onto Petri dishes containing sporulation agar (Himedia, Mumbai, India). Petri dishes were incubated at 30 °C for three weeks. Sterile physiological solution (5 ml) was added to the Petri dishes in order to harvest the spores. The surface was carefully scraped with a sterile inoculation loop to separate the spores from the surface of the agar, and the suspension was centrifuged in order remove agar debris. The supernatant was poured away and 10 ml of fresh sterile physiological solution was added, followed by vigorous shaking in order to re-suspend the spore pellets. This process was then repeated twice, in order to remove the majority of the remaining debris. The resulting spore suspension, contained in a sterile glass bottle, was transferred to a water bath at 70 °C for 30 min. After the sporulation process, the suspension was stored at 4 °C until handling, without loss of viability.

Before any individual plasma-assisted treatment of spores was carried out, 1.2 ml of the initial bacterial suspension (0.5 × 10⁸ CFU ml⁻¹) was filtered through a sterile polycarbonate filter membrane (with diameter and porosity of 13 mm and 0.4 μm, respectively; Whatman, GE Healthcare Life Sciences, Tonglu, China) in order to deposit a single monolayer of spores. Filtration was carried out through a rubber bung on a top of a Büchner flask using a vacuum pump. Once the spores were deposited on the filter, the filter was removed from the filter holder using tweezers, placed on the autoclaved glass slide and inserted in the CSDBD reactor on the sample holder at a fixed distance from the surface of the CSDBD electrode.

After treatment in the reactor, both exposed (discharge on) and control (discharge off) spore samples were recovered by transferring the filter into a sterile bottle containing physiological solution and six ballotini beads; the tube was then placed into an Eppendorf shaker for 45 s. The obtained spore suspension was diluted to the desired concentration and 100 μl was spread onto a tryptone soya agar plate with a disposable spreader. The Petri dishes containing B. subtilis were incubated at 30 °C for 24 h and the colony forming units (CFU ml⁻¹) were counted.

As the principal gaseous discharge products, ozone and N_xO_y species were sampled at the output port of the CSDBD reactor. A non-dispersive UV absorption ozone monitor API 450 and chemiluminiscence NOx analyser model 200EM (both Teledyne Instruments) were used, with a minimum airflow rate of 0.5 l min⁻¹, to quantify the discharge products in both dry and humid air.

Chemical measurements of H₂O₂, NO₂⁻ were performed using the sample holder, which was designed to contain 2.5 ml volume of liquid. The water surface was placed bellow the discharge at a distance of 3 mm from the electrode, maintaining similar conditions as in the case of the treatment of the spores.

The concentration of hydrogen peroxide was measured using a titanyl sulfate reagent solution, which reacts selectively with H₂O₂ [37]. Sodium azide was added prior to the mixing of the sample with the titanyl sulfate reagent [38]. The absorbance at 407 nm of the yellow complex formed with H₂O₂ was recorded using a microplate reader (Varioskan® Flash).

The concentration of nitrites was measured using Griess reagent. The Griess reaction is based on the diazotisation reaction of sulphanilamide and N-1-napthylelthylenediamine dihydrochloride under acidic conditions [39, 40]. The absorbance at 540 nm of the pink complex formed with NO₂^– was recorded using a microplate reader (Varioskan® Flash).

A DPA assay was carried out following Warth [41]. In brief, the spores from the filter were suspended in a 5 mM Ca²⁺, Tris buffer at pH 7.6 and centrifuged. The DPA concentration was recorded based on the difference in the two peaks at 276 nm (maximum) and 280 nm (minimum) using a microplate reader (Varioskan® Flash). The concentration of DPA was calculated based on the prepared standard curve in the range from 0 to 10 μg ml⁻¹ of purified DPA (Sigma, St. Luis, USA)

The leaking of intracellular components (DNA, RNA, and proteins) can be revealed by UV absorption. We selected a wavelength of 260 nm, corresponding to the peak absorbance of DNA. For these tests, plasma treated spores were suspended in 250 μl of physiological solution and centrifuged (3 min at 12 000 rpm) to remove any cells or cellular debris. The absorbance of supernatant was then recorded by a microplate reader (Varioskan® Flash) at 260 nm (i.e. the wavelength of maximum absorption for DNA) [42–44]. Assuming the presence of the DNA in the liquid, its concentration was estimated assuming that an absorbance of one is equivalent to 50 μg ml⁻¹ of pure DNA.

The CSDBD driven in the amplitude-modulated regime at a fixed AC HV amplitude of 22 kV (peak-to-peak) and a duty cycle of 0.4 produces between seven and nine strong micro-discharge filaments during both the positive and negative half period of any AC cycle, as evidenced by the snapshot in figure 1(c). Figure 2 shows the typical voltage-current-photomultiplier waveforms that can be observed during a single burst. All PMT waveforms were acquired with no spectral filter, and the PMT signal is therefore given by the total UV–vis-NIR discharge emission modulated by the efficiency of the PMT's photocathode. In addition, figure 2(b) clearly shows the synchronous occurrence of the current and PMT pulses.

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The production of micro-discharges occurs with a certain distribution [33] along the rising slope of a positive half-period with a typical pulse-to-pulse separation of few microseconds. A similar trend can be observed during any negative half-cycle. Figures 3(a) and (b) show the characteristic distributions of micro-discharges during both the positive (a) and negative (b) half-cycles captured by the PMT waveforms for a given HV amplitude of 22 kV (peak-to-peak), acquired as an average of 1024 samples (DPO5204 triggered on the HV signal). The highest probability of micro-discharge onset occurs for positive AC voltages between 6.6 and 7 kV, while for negative AC voltages, the best conditions occur between −8.5 and −9.5 kV.

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Further insight can be obtained by analysing the voltage, current and PMT waveforms for micro-discharges characterised by onset at the defined HV amplitude. Examples of such waveforms are shown in figure 3(c) and (d) (obtained by employing the A → B sequence trigger mode of the DPO5204). Figure 3(c) captures micro-discharges occurring at a HV level of 6.33 kV and causing sudden distortion of the regular AC waveform, characterised by a drop in the voltage of Δu⁺ ≅ 45 V. Similar behaviour can be observed during the negative half-cycle, as shown in (d) for an HV level of −9.18 kV and Δu^- ≅ 15 V. In both cases, the duration of the current pulses is very short (≈5 ns) and the PMT signal is slightly delayed (a shift of ≈25 ns caused by the time response of R2949).

Since the CSDBD electrode with HV coaxial cable represents a ≈50 pF capacitor in the AC HV circuit, the voltage drop Δu⁺/ Δu⁻ then reflects the variation in the energy stored in the capacitor. Assuming that the energy lost from the capacitor correlates with the energy dissipated in the discharge filament, we can roughly estimate the maximum mean energy released in a single micro-discharge filament. In the case of the events captured in figure 3(c) and (d), the maximum of estimated energy is about 15 ± 5 and 6.5 ± 2.2 μJ per filament for the events occurring during the positive and negative half-cycles, respectively.

A precise determination of average energy per burst released by the discharge was made by inserting a non-inductive capacitor (C = 0.47 μF) into the ground lead to determine the transferred charge [45]. Figure 4(a) shows characteristic charge-voltage parallelograms (Lissajous figures) acquired under the discharge conditions used for the treatment. Figure 4(b) illustrates the dependence of the average energy per burst on the amplitude (peak-to-peak) of the applied HV waveforms. All treatment experiments were performed under discharge conditions characterised by an average energy of ∼3.5 mJ per burst, which corresponds to an average power of ∼1.7 W. Based on the CSDBD geometry and gas flow, the estimated average surface power density of ∼1.5 W cm⁻² and energy density of ∼0.3 Wh l⁻¹ fall into a range of conditions that are suitable for the efficient generation of ozone with minor concentrations of nitrogen oxides by surface streamers in air [45, 46].

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The time-averaged emission spectra produced by the CSDBD filaments and illustrated in figures 5 and 6 show all the major characteristics of streamer-based discharges [47, 48]. The spectra of filamentary CSDBD revealed strong bands of the second positive system (SPS) (${C^3}{{{\Pi }}_u} \to {B^3}{{{\Pi }}_g}$) of N₂ and weak first negative system (FNS) (${B^2}{{\Sigma }}_u^ + \to {X^2}{{\Sigma }}_g^ +$) of N₂⁺ in the UV spectral range, while in the vis-NIR range, we observed characteristic sequences of bands of the first positive system (FPS) (${{\rm B}^3}{{{\Pi }}_g} \to {A^3}{{\Sigma }}_u^ +$) of N₂ and atomic lines of neutral oxygen O^I.

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The partially-resolved rotational structures of the SPS, FNS and FPS bands displayed in figure 5 were analysed by means of the synthetic models detailed in [47, 49, 50]. The FNS(0,0) band can be fitted using a given instrumental function by fixing the rotational temperature at 315 ± 20 K. The rotational temperature of the N₂(B³Π_g, v = 2) vibronic state was determined using a peak method described in [47, 50]. This method utilises the temperature-sensitive ratio of three selected sub-band heads; in the case of the FPS(2,0) band, these are formed from the P₁₁ + Q₁₂, P₂₂ + Q₂₃ and Q₃₃ branches (indicated in figure 6(b)), and the T_rot determined from the time-averaged spectra reaches 325 ± 15 K.

Membranes inoculated with the B. subtilis spores were exposed to reactive species, and the dependence of the number of surviving bacteria on the treatment time was studied under fixed treatment conditions (discharge power, CSDBD plasma-to-sample distance, fixed airflow through the discharge chamber, sample and air humidity). In this work, we tailored the discharge duty cycle and maintained constant airflow through the discharge in order to keep the gas temperature low enough to exclude partial thermal inactivation. The rotational temperature obtained from two different excited states of molecular nitrogen indicated a low gas temperature in the plasma filaments (about 325 K), and hence also a low temperature at the position of the sample with the spores. The distribution of spores on the filter was homogeneous, as verified by the SEM images [51] (a heterogeneous distribution of the spores on the sample holder can cause the formation of cells refuges, and can shield a certain fraction of spores from the reactive species).

We verified that the airflow through the discharge led to progressive drying (thereby changing the treatment conditions) of the bacterial samples. In order to maintain the humidity of exposed samples during the whole exposure, it was necessary to add 10 μl of sterile water to the sample holder prior to switching on the discharge. Losses due to the evaporation of water from filter samples under the given exposure geometry were verified gravimetrically. The rate of evaporation losses in the case of dry airflow through the chamber with the discharge switched on was about 3.9 μl min⁻¹ (compared with 2.9 μl min⁻¹ with the discharge off). In the case of humid airflow with the discharge switched on, the evaporation rate was about 2.1 μl min⁻¹ (compared with 0.22 μl min⁻¹ with the discharge off). This means that in the case of the discharge with humid airflow and 10 μl of sterile water, the filter with bacterial samples becomes completely dry within few minutes, completely changing the initial treatment conditions (wet → dry).

Counting bacterial populations reveals a roughly biphasic shape for the inactivation kinetics [52], before a plateau is reached (after approximately 2 min exposure) with as much as 97% (1.5 log) of spores inactivated during plasma-assisted treatment of humidified samples (figure 7). The D-value (inactivation 1.0 log₁₀) determined in this work was 1.17 min, which is very similar to the D-value of 1.21 min reported by Hertwig et al [14], or the value found by Wang et al [15], which was even below 1 min. It is clear that the rate of inactivation slows down significantly with progressive drying of the samples. This is indirect evidence for the important role of the dissolved reactive species. It is also important to emphasise here that due to the technique of the amplitude modulation technique used for the driving AC HV (duty cycle of 0.4), a two-minute exposure means 48 s of plasma ON and 72 s of plasma OFF. Furthermore, assuming monolayer exposure of the bacteria and considering the mean discharge power, we can estimate an upper limit of 2.5 × 10⁻⁶ mJ CFU⁻¹ (or 1.5 × 10¹⁰ eV CFU⁻¹) for the mean energy necessary to inactivate one bacterium.

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Hertwig et al [14] treated inoculated glass beads by diffuse coplanar surface barrier discharge, which was continuously excited (20 kV peak-to-peak at 15 kHz). The discharge chamber was continuously shaken to obtain homogeneous treatment. Consequently, at any instant, part of the inoculated surface of each bead was in direct contact with the discharge filaments, while the other part of the surface was treated indirectly (as in the case of this work). The authors claim that the air-plasma temperature did not exceed 63 °C, even though the treatment was performed in the static gas at a power input of 350 W (compared with the average power of ∼1.7 W used in this work). However, they measured only the average gas temperature with a fiber-optic temperature sensor (based on a GaAs crystal with a response time of 2 s), which cannot reflect fast temperature variations inside plasma filaments on a μs-ms timescale. It is therefore very likely that the inoculated beads were locally in contact with plasma filaments characterised by significantly higher temperatures, and partial thermal inactivation cannot be excluded, especially at longer exposures (3.5 log₁₀ inactivation for 7 min exposures).

Wang et al [15] used volume DBD with air gap of 1.5 mm (continuous AC discharge, 14 kV peak-to-peak at 60 Hz) to treat directly inoculated microscope glass cover slips. These authors used an infrared thermometer to measure the temperature of inoculated cover slips after the DBD treatment, which, again, cannot be used as proof of the gas temperature of DBD plasma filaments during the treatment. Due to the much longer treatment times (up to 30 min) under static gas conditions, partial thermal inactivation cannot be excluded, as in [14]. In fact, Wang et al [15] report 5.5 log₁₀ inactivation for 3 min exposures, which clearly indicates that the temperature in a volume DBD configuration was even higher than that reported by Hertwig et al [14].

When treating homogeneously distributed spores, one might expect linear kinetics of inactivation; however, multiphasic kinetics [52] is observed instead (see figure 7). Bayliss et al suggested that a biphasic kinetics is caused by the response of the bacterial population, which tend to resist to the plasma treatment. This response includes rearranging cells into aggregates, preventing plasma-generated species from reaching the individual cells [32]. On the other hand, Moisan et al proposed that the plasma inactivation of B. subtilis spores displayed two or three different phases in the survival curve, mainly caused by a shift in the plasma-generated processes predominating at each given moment. According to the author, the complex shape of inactivation curve was probably caused by the shift from the UV caused to reactive species killing [53]. The change in the dominant plasma-assisted processes (wet treatment → dry treatment) seems to be the cause of the multiphasic kinetics observed in this work. The effects of plasma-generated UV and reactive species are discussed later in the text.

Plasma-induced inactivation may be caused mainly by UV light, heat, chemically reactive species and energetic charged particles [54]. Concerning the possible effects of UV light on spore inactivation (via DNA damage enabled by UV light), CSDBD driven in wet air strongly emits UV-A photons (315–380 nm), less intense UV-B photons (280–315 nm) and negligible UV-C photons (bellow 280 nm), as evidenced in figure 5. Nevertheless, both UV-A and UV-B photons are able to induce various forms of damage in bacterial DNA, such as spore photoproducts or cyclobutane pyrimidine dimers [55, 56]. However, we do not expect any significant contribution from UV irradiation of the spores, since a strong source of UV-C is missing under humid air discharge conditions; in addition, a significant part of the UV-B radiation (produced by the N₂-SPS emission) is absorbed by O₂, O₃ and H₂O species between the discharge filaments and the remote (3 mm) spores. In a prior study, we demonstrated that the contribution of UV light generated by the air CSDBD has a negligible antibacterial effect [36]. In addition, the observation of a significant slow-down in the inactivation kinetics after 2 min provides independent evidence of the negligible influence of UV photons in the present work. An earlier study by Deng et al [57] tested the influence of UV emission produced by an atmospheric-helium plasma plume. These authors performed experiments with the spore samples that were protected from all plasma products except for UV photons (given in this case by the NO-γ bands in UV-C, N₂-SPS and OH emission at 310 nm in UV-B, and N₂-SPS and N₂⁺-FNS in UV-A [47]), and they concluded that the ROS generation in a comparable atmospheric helium–oxygen plasma plume was the primary process of killing bacteria, whereas the contributions from UV photons (inspected without an oxygen admixture), charged particles and heat were of minor importance. Since the emission in a helium plume is a significantly stronger source of UV-C and UV-B compared with the SPS produced by the CSDBD in humid air, we must expect negligible UV-assisted inactivation under the present conditions.

It has been proposed that the contribution of reactive species to the plasma-caused bacterial inactivation is significant [58, 59]. We tried to identify species that might participate in microbial inactivation under the present conditions. Previous studies have concluded that the effects of post-discharge chemistry, including ozone, nitrates, nitrites and hydrogen peroxides, might significantly contribute to plasma-assisted inactivation [36, 44, 60]. The main focus was on the detection of the chemically reactive products generated by the discharge and transferred to the liquid.

As a result of the experimental procedure used here, some residual liquid water remains on the filter, and we must expect the formation of H₂O₂ and NO₂⁻ in the wet environment surrounding the cells. The residual water was introduced by moistening the filter with 10 μl of sterile water in addition to the humidity present in the air flux. Unfortunately, such a small volume was insufficient for the H₂O₂ and NO₂⁻ analysis, and we therefore performed a complementary measurement using a larger H₂O volume. We determined the formation of H₂O₂ and NO₂⁻ in 2.5 ml of deionised water, placed under the discharge at the same distance as the filter with the spores. The concentration of hydrogen peroxide and nitrite ions formed in exposed water increased with the duration of treatment (figure 8). Moreover, the pH of the exposed water decreased from 6 to 3.7 after 6 min of treatment. However, it was found that the concentration of RONS dissolved in water depends on the volume and the area of the water surface in contact with the plasma [61, 62]. Hence, we must expect much higher concentrations of RONS dissolved in 10 μl of water during the treatment of bacterial samples. This also means that dissolved RONS are expected to contribute significantly to the inactivation of humid samples.

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Gaseous ozone and N_xO_y species were monitored at the CSDBD reactor output port in both dry and humid air without bacterial samples. The results corresponding to an airflow rate of 0.5 l min⁻¹ and at various energies are shown in figure 9. At the discharge conditions used for the treatment, the concentration of produced NO was always below the detection threshold, for the mass flow Q in the range 0.5–1 l min⁻¹, while some PPM of NO₂ were always detected in both dry and humid air. Concentrations of ozone of several hundreds of PPM were obtained, with notably higher densities obtained in dry air. Taking into consideration the very high ratio of O₃/NO_x ≫ 1, we can expect that the CSBDB produces mainly O₃ with relatively high energy efficiency for a flow rate of Q = 0.1 l min⁻¹ in humid air. The findings suggest that the CSDBD reactor works in ozone 'mode' which should favour bactericidal effects [63]. The observation of NO₂^– formation confirms the reaction via which nitrogen oxides generated in the gas phase dissolve in the aqueous phase [38]. Nitrites can also be formed by chain reactions, directly in the humid-air plasma, and subsequently transformed into liquid [64].

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Although spores are extremely resistant to various chemicals, they are susceptible to nitrous acid, ozone and hydrogen peroxide, which we detected in the plasma-treated water. The plasma-assisted generation of hydrogen peroxide may make a contribution to the killing of spores; however, the mechanism via which hydrogen peroxide may affect the spore cell remains uncertain. It has been shown that small acid-soluble proteins (SASPs) play a significant role in the resistance of spores to hydrogen peroxide, and it is therefore likely that hydrogen peroxide kills the spore likely at other sites than DNA and that the mechanism is different from the killing of vegetative cells [17, 21, 24]. Melly et al [65] proposed that spores killed by hydrogen peroxide maintain their membrane permeability, blocking the core contents from leaking out. This persistent membrane stability may be caused by the fact that hydrogen peroxide attacks polyunsaturated fatty acids as the usual target in the membrane, while the spore has an extremely low level of polyunsaturated fatty acids. Additionally, it assumes that the proteineous coat acts as a barrier or target to lower H₂O₂ concentration which can react with the core structure [23, 66]. The effect of hydrogen peroxide is likely to be caused by defects in a healthy metabolism [65]. Based on the investigations mentioned above, we are of the opinion that plasma-generated hydrogen peroxide contributes to spore killing, although the specific mechanism is unknown.

Furthermore, nitrous acid, which is generated in liquid treated by plasma above the surface, is a well-known mutagen. The study of Tennen et al [67] evidenced high levels of DNA damage after the exposure of spores to nitrous acid. We therefore expect that nitrous acid formed by plasma may cause the same defects in spores, although further specific tests of the extent and type of this DNA damage need to be carried out. Moreover, a systematic study by Hertwig et al [14] investigated the effects of plasma on isogenic mutants of the B. subtilis strain without (i) genes encoding SASPs, (ii) DPA-deficient spores, and (iii) spore outer protein. It appeared that Bacilllus spores without SASPs were inactivated more effectively.

Spore decontamination was reported after treatment by ozone and peroxynitrous acid, which are both commonly generated by discharge reactions [68, 69]. The formation of peroxynitrous acid in the post-discharge chemistry involves hydrogen peroxide and nitrites in the plasma treated water [38]:

$$\begin{equation}{\rm{NO}}_2^ - {\rm{ }} + {\rm{ }}{{\rm{H}}_2}{{\rm{O}}_2}{\rm{ }} + {\rm{ }}{{\rm{H}}^ + }{\rm{ }} \to {\rm{ ONOOH }} + {\rm{ }}{{\rm{H}}_2}{\rm{O}}.\end{equation} \tag{ 1 }$$

The formation of high concentrations of ozone was detected in the output from the reactor. Ozone and peroxynitrous acid affect spores by damaging inner membrane of the cell, and this can be supported by observing DPA release. Although the treatment of spores with peroxynitrous acid and ozone does not cause straightforward leaking out of DPA [68, 69], the subsequent heat treatment initiates the loss of DPA. We observed an increasing concentration of free DPA with an increase in the time of treatment, as illustrated in figure 10. We therefore suggest that DPA release can contribute significantly to plasma-assisted inactivation, as also observed in other studies [15, 16, 70].

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Finally, we detected DNA leakage into the surrounding suspension (an increase of 1.96 μg ml⁻¹ after 5 min), although the detected concentration and increase was very small, probably due to the extremely good protection of the spores in the area around the DNA. Nevertheless, an increase in the free intracellular components, including the DNA, can be considered as an important sign of a leaking membrane, cortex, and/or coat structure. Thus, free DNA occurring in the spore suspension after plasma treatment may be caused by the destabilisation of the crucial biochemical structure of the spore.