Plasma Current Voltage and Emission Spectroscopy
The SDBD was operated in humid ambient air (25°C, 40% RH) at atmospheric pressure. The applied AC voltage (20.6 kVpp (peak-to-peak), see Fig. 1) consisted of repetive burst (repetition rate 500Hz), each consisting of nine AC cycles (fAC = 19.6 kHz) with a repetition rate of 500 Hz (duty cycle of 0.25). In this way we obtained a homogeneous distribution of microdischarges. The actual discharge ON-time is shorter than the duration of the AC cycles. We can estimate an effective duty cicle of the order of 0.1 of the nominal one53. This ensures that biological samples are not exposed to excessively damaging temperatures. Typical voltage, current, and charge characteristics are shown in Fig. 1A-C, along with the typical Lissajous figure showing the charge voltage characteristic (Fig. 1D) used to determine the discharge energy. The estimated energy per burst is about 4.5 mJ, i.e. an average power of 2.25 W, with an energy density of 3.8 10− 2 Wh L− 1. The air-SDBD operated in ozone mode, and we can expect a concentration on the order of 200–300 ppm, well above the concentration of nitrogen oxide products13,53.
Figure 2 shows low-resolution emission spectra of filamentary SDBD operating in humid air, and reveals strong bands of the second positive system (SPS) (C3Πu → B3Πg) of N2 and the first negative system (FNS) (B2Σu+ → X2Σg+) of N2+ in the UV spectral region. In the Vis-NIR region, we observed characteristic sequences of bands of the first positive system (FPS) (B3Πg → A3Σu+) of N2. The emission line for atomic oxygen was observed at 777 nm, indicating the production of atomic oxygen during the plasma phase. The partially resolved structures of SPS (0,0) shown in Fig. 3 were analysed using the Massive OESspectroscopy tool65 − 67. The SPS (0,0) band can be fitted for a specific instrumental function by setting the rotational temperature to 350 ± 25 K. This temperature represents the gas temperature during the discharge on phase near the dielectric surface, where plasma is confined in SDBDs. Thus, it does not represent the temperature of the gas in direct contact with the treated substrate. Also, considering that the gas flows at 7 slm, which gives a residence time in the discharge volume of 64 ms, we can rule out heat accumulation within the gap.
Figure 3 shows the experimental SPS(0,0) band profile along with three synthetic profiles (simulated for rotational temperatures of 300, 350 and 400 K) on a logarithmic scale to demonstrate the sensitivity of the band tail of the band (formed by the overlap of the R1, R2 and R3 branches) to the rotational temperature of the C3Πu state.
Figure 4, shows the emission formed by the Δv = -3 and − 2 sequences of the SPS and by the Δv = 0 sequences of the FNS. From Fig. 4, the ratio between FNS(0,0) and SPS(0,3) is equal to 0.6 ± 0.02. From the FNS/SPS calibration curves66 we can estimate that the averaged reduced electric field E/N is more than 900 Td.
Figure 5 shows the characteristics of the vibrational distributions of N2(C3Π u) obtained from the time- and space-averaged spectra shown in Fig. 4, together with three Boltzman vibrational distribution functions (VDFs) corresponding to the vibrational temperature of 2500K, 2750K and 3000K. We can conclude that the experimental VDF is characterized by a clear non-Boltzman behaviour.
PAW Characterization
Chemical properties of PAW
The content of nitrate and nitrite (mg/L) of PAW was measured and their stability in water after plasma activation was evaluated. The mean value of nitrate (25.7 mg/L) in water sample exposed to 15 min of SDBD plasma activation was kept almost constant (22.8 mg/L), while the nitrite content was drastically reduced (started from 26.9 mg/L to 16.4 mg/L) after 24 h storage at 4°C from the activation. The pH value increased from an initial mean value of 3.4 to 4.3. The level of hydrogen peroxide measured through a colourimetric test decreased from 5–10 mg/L to 2–5 mg/L.
Electrical Impedance (EIS) and Broadband Dielectric Spectroscopy (BDS) results.
The results of EIS and BDS allowed rapid monitoring of PAW properties. The effect of plasma treatment was evaluated by ion density n and estimation of ionic conductivity. In this way, it was possible to predict the effectiveness of the PAW treatment on the XfDD.
In Fig. 6, we show the comparison between NPs (Fig. 6A) as calculated from the impedance of untreated deionized water (DIW) and PAW. The latter refers to two representative plasma treatment times of 5 min and 15 min, which are of interest for this work. The inset shows the magnified view of the region in the high-frequency region.
It is worth noting that there are significant differences between the NPs associated with the DIW (black curves) and the NPs from PAW.
We obtained the best fit with the circuit (see Fig. 6B and Table 1) consisting of the series of two components, namely the parallel R1//ZCPE related to the response in the low-frequency region and specifically in the f < 100 Hz region, and a modified Randle circuit related to the f > 100 Hz frequency region.
Table 1
EIS parameters extracted by the best fit of the experimental Nyquist plots in Fig. 7A with that one of the equivalent circuits represented in Fig. 7B.
ITEMτ | DIW | PAW 5min | PAW 15min | PAW 15 min ag |
R1(Ω) | 10956 | 1.95x105 | 1.34x105 | 3.84x105 |
ZCPE (Ω1−nFn) n | 6.7x10− 10 0.95 | 9.10x10− 6 0.88 | 8.10x10− 6 0.96 | 5.18x106 0.84 |
CDL | 2.9x10− 11 | 2.70x10− 11 | 2.72x10− 11 | 2.88x10− 11 |
ZW(Ω s− 0.5) | 1.4x105 | 25811 | 41011 | 19989 |
Rct(Ω) | 4.1x105 | 15151 | 5414 | 6772 |
The R1//ZCPE component is assigned to the interaction of the solution at the interface with the electrodes (electrode polarization, EP effect), while the Randle circuit corresponds to the electrochemical processes within the solution69. Following the theory of Bard and Faulkner, the processes involve redox reactions whose effectiveness can be evaluated by the value of the charge transfer resistance Rct, which is also an index of the magnitude of the charge transfer current Ict (Ict∝Rct −1), and then for the ions and free electrons involved in the redox process70.
It is noteworthy that the impedance ZR= Rct+ Zw as a function of the treatment time τ is related to the concentration of ionic species determined by the optical absorption methods ρabs (Fig. 6B) and represents a fingerprint of the production and total evolution of NO3− and NO2− radicals and hydrogen peroxide concentration with treatment time. On the one hand, the decrease of ion diffusivity underlined by the increase of ZW (ZW ∝D− 1) is due to the increase of ion concentration; on the other hand, the Rct shows the increase of charge transfer current (Ict∝Rct−1) and then the presence of free redox electrons and underlines the onset of a dominant oxidant environment.
We gain further information on ionic diffusivity D and species concentration n determined via the analysis of dielectric permittivity returned from BDS data. In Fig. 7 we summarize the AC conductivity (left axis) (σ(ω ) vs ω) and the loss factors tan δ frequency dispersion (tan δ vs ω) (right axis) together with the values of the ionic diffusivity, D, and concentration, n, calculated with expr. 5,6,7. Interestingly, the value of the continuous part of the AC conductivity σDC evidenced by the plateau starting at around f > 100Hz (Fig. 7A) is increasing with the treatment time. Moreover, the σDC PAW response is not much affected by an ageing time of 24 h (15 min ag) thus confirming the stability in a time of the oxidant environment.
We also compare the tanδ in PAW with that one of the well-known chemical reactions in a solution H2O2 + HNO3 (1:1) progressively diluted in DIW (data not shown). This is because in both cases the reaction products are the same (i.e., NO3−, NO2− and H2O2). Moreover, the dilution in DIW allowed the use of the known ionic charges’ concentration of the chemical reaction to calibrate those observed in PAW vs treatment time (data not shown). The cross-checks of the tanδ of the chemical reaction and PAW spectra allowed us to conclude that BDS results on PAW are consistent with those of the chemical reaction either because of the similar spectral features or the concentration of the ionic charge whose value was found furthermore in agreement with those derived from optical methods.
We can observe (Fig. 7A) that the position tan δ peak of PAW shifts to a higher frequency than that of DIW, which is associated with a decrease in peak intensity compared to treatment time. In addition, the 15min ag PAW item shows a slight increase in tan δ peak intensity with no change in frequency and similar values of σDC. Consequently, we observed a decrease in the concentration of ionic charges and a corresponding increase in the diffusivity D. This behaviour could be related to the decomposition of hydrogen peroxide and nitrogen peroxyacids52. However, the results of EIS and BDS show that the pro-oxidant environment can still be effective after 24 hours.
Although we can only quantify the total concentration of ionic species at this time, we found a linear correlation between the ion diffusivity D or the total concentration of PAW ionic species n and the total concentration ρabs (A) measured by chemical methods.
XfDD growth conditions and plasma treatments
Treatment on agar surface-grown bacteria
Preliminary investigations showed that the plasma pre-treatment of the agar plates did not alter the growth of XfDD cells (data not showed).
When bacteria (107 CFU ml-1) were plated out on Buffered Charcoal Yeast Extract (BCYE) agar and exposed to SDBD for 200 s, complete inhibition of cell growth was observed, whereas after 100 s and 10 s of exposure (Fig. 8) there was less pronounced but time-dependent inhibition.
Since it is difficult to quantify the effect of plasma treatment by counting colonies at a concentration of 107 CFU ml− 1 (Fig. 9), decimal dilutions from 105 to 103 CFU ml− 1 of XfDD inoculum were applied to the BCYE agar plates in subsequent experiments (trials). Final SDBD plasma treatment conditions were therefore performed on cells grown 1–5 days after seeding, with a maximum exposure time of 200 s.
Table 2 shows the results for different three dilutions (103, 104, 105) for untreated and 200 s treated plates pre-cultured for one or five days before LTP exposure. The treatment was effective and complete removed of all bacteria by plasma, at both 1 and 5 days.
Table 2
Results of the experiment performed on agar surface grown XfDD. Treatment time was fixed at 200 s following the finding of the previous experiment. We performed experiments in triplicate for a total of 9 dishes for each experiment. The experiments were conducted on plates just at T0 = 0 and T5 = 5 days after XfDD seeding.
Concentration | Time | N of replicas | 11 days CFU ml− 1 | 13 days CFU ml− 1 |
105 | untreated | 3 | 3.8x108 | 9.8x108 |
104 | 3 | 4x105 | 2.1x107 |
103 | 3 | 0 | 2x105 |
105 | T0 | 3 | 0 | 0 |
104 | 3 | 0 | 0 |
103 | 3 | 0 | 0 |
105 | T5 | 3 | 0 | 0 |
104 | 3 | 0 | 0 |
103 | 3 | 0 | 0 |
When plasma interacts with a surface, its energy (in the form of ions, RONS, and UV radiation) is deposited in the exposed matrix. Multiple exposures result in a cumulative effect of absorbed doses leading to progressive destruction of the microorganism. To evaluate the effect of the LTP dose-effect, we performed a repeated plasma treatment of 200 s with two different frequencies: 1 treatment per day for a period of 10 days (Trial a) and once per week for a period of three weeks (Trial b).
The results of these experiments are given in Table 3 and show that in Trial a) a strong effect of cumulative dose was observed, since at the lower concentration (103 CFU ml− 1) the bacteria were completely killed by the multiple exposures, resulting in a reduction of at least log 5, while at higher CFU the reduction reached at least a log 2 value. In Trial b), cells were counted 20 days after plating. The concentration-dependent effect was less pronounced. Treatment resulted in a reduction of at least log 1 for the lower concentrations (103 CFU ml− 1, 104 CFU ml− 1), whereas at 105 CFU ml− 1 counting was not possible for the untreated sample, making it difficult to estimate the reduction.
Table 3
Results of the dose dependence experiment performed on BYCE agar surface grown XfDD
Treatment frequency | Initial Concentration (CFU mL− 1) | CFU ml− 1 at day |
11 | 13 | 15 | 20 |
untreated | 105 | 1,1x109 | 1,9x109 | | not count |
| 104 | 1,2x106 | 6,3x107 | | 7,5x107 |
| 103 | 0 | 6x105 | | 2,8x106 |
Trial a) 1 per day | 105 | 3.3x106 | 4.1x106 | | |
104 | 8.3x104 | 3.3x105 | | |
103 | 0 | | | |
Trial b) 1 per week | 105 | | | 3.5X107 | 1.6x108 |
104 | | | 3.3x105 | 1.7x106 |
103 | | | 0 | 1x105 |
Treatment of cells with Plasma Activated Water (PAW)
To test the efficacy of plasma treatment and its potential for use in vivo, bacteria were suspended in PAW previously treated in the SDBD discharge chamber for 15 minutes. To decipher the exact mode of action of the plasma treatment, its effect on XfDD was monitored using viability assays with fluorescence live/dead staining.
Fluorescent probes were used to assess cell membrane integrity and XfDD viability after incubation in PAW. The untreated control cells were almost all stained green with SYTO 9, indicating that they were viable. A small number of cells were stained red with propidium iodide (PI), indicating that they were probably dead (Fig. 10A). The number of cells stained red with PI increased dramatically after treatment with PAW, while cells stained with SYTO 9 decreased as expected and only a few fluorescent cells were visible after treatment (Fig. 10 (B)). This indicates that the integrity of the cell membrane of the bacteria was damaged by the treatment, affecting their viability.