# Biological Decontamination by Microplasma
## 4 Discussions
The values of ozone concentration, as shown in Figure 4, are 12.8 ppm and 19.2 ppm at \(-\)1.5 kV and \(-\)1.7 kV, respectively. These values are lower than what we reported on with microplasma electrodes energized at an AC voltage. Among long-lived neutrals, ozone plays a major role in the sterilization process. The lower concentration obtained with pulse microplasma compared with AC-driven microplasma recommend microplasma energized by pulse power as a safer alternative for the biological decontamination due to ozone hazards for human health at higher concentrations. For experimental design 1, the inhibition zone as shown in Figure 6, corresponding to the microplasma treatment at \(-\)1.7 kV, shows an area of bacterial inhibition larger than the microplasma electrode area of 3 cm in diameter. Due to the gas flow, the active species are disseminated; thus, for all bacterial strains, an inhibition area with a diameter of at least 4.5 cm was measured. The colony-forming-unit counts, as shown in Table 1, represent all the bacteria grown on the Petri dish. Because the Petri dish diameter is larger than the microplasma electrode diameter, for a more accurate assessment of the inhibition efficiency of microplasma, the area below the electrode should be considered. In this case, as shown in Figure 5, at \(-\)1.7 kV discharge voltage, the microplasma inhibition efficiency could be considered as 100% for all bacterial strains. Experimental design 2, in which the bacteria was inoculated on agar plates and incubated for 24 h, treated with microplasma at \(-\)1.7 kV for 5 min, and re-incubatedafterwards for an additional 24 h, showed no visible effect on bacteria after microplasma treatment. The bacteria was inhibited 100% on the surface of agar below the electrode, as shown in Figure 5. Thus, the CFU count was carried out for the bacteria that appeared mostly on the sides of the agar plates not reached by the gas active species generated by microplasma. The discharge power of the microplasma electrode is proportional with the surface area of the electrode. It can be considered that in the case of swabbing 1 mL of bacterial liquid, the surface of the bacterial spread on agar will increase 20 times compared with 50 \(\upmu\)L of bacterial liquid and, consequently, to inactivate this area, an electrode 20 times larger will be necessary. Thus, discharge power will increase 20 times. According to this, the D values calculated for 1 mL of bacterial liquid are the D values calculated for 50 \(\upmu\)L multiplied with 20. The D values are shown in Figure 6:0 For _E. coli_, the initial concentration of \(6.96\times 10^{3}\) CFU/mL was reduced to \(5.58\times 10^{3}\) CFU/mL at \(-1.5\) kV and \(4.45\times 10^{3}\) CFU/mL at \(-1.7\) kV after 5 min of treatment. The experiments were carried out using 50 \(\upmu\)L of bacterial liquid; thus, the D values were \(157.5\) kJ/mL at \(-1.5\) kV and \(89.9\) kJ/mL at \(-1.7\) kV. Considering only the inactivation of 50 \(\upmu\)L of bacterial liquid, the D values were \(7.88\) kJ and \(4.49\) kJ at \(-1.5\). kV and \(-1.7\) kV, respectively. The experiments carried out with _S. aureus_ had an initial concentration of \(4.1\times 10^{3}\) CFU/mL. After 5 min of treatment time, the concentration was reduced to \(0.71\times 10^{3}\) CFU/mL at \(-1.5\) kV. This corresponds to a D value of \(19.9\) kJ/mL and a D value considering 50 \(\upmu\)L of bacterial liquid of \(0.99\) kJ. The calculated D values in Joule, based on the D values reported by [PERSON] et al. [9], were 18 kJ/mL and 40 kJ/mL for _E. coli_ and _S. aureus_, respectively, after 50 s of treatment time considering 1000 W input power and a 4 cm distance between the plasma jet and the bacterial sample. [PERSON] et al. [17] reported a 4.95 log CFU/mL reduction in _S. aureus_ after 45 s of treatment time at 60 W input power. This corresponds to a D value of \(0.55\) kJ/mL. In another study from [PERSON] et al. [30], a 0.85 log CFU/mL reduction was reported for _S. aureus_, as well as a 1.72 log CFU/mL reduction for _E.