# Biological Decontamination by Microplasma
## 4 Discussions
aureus_ has a larger cellular volume and the peptidoglycan structure is also thicker; thus, when the cell envelope is compromised, there are minimal changes in particle size and, consequently, the likelihood of cytoplasmic leakage is reduced. Microplasma treatment causes damage to the intracellular macromolecules due to the excessive accumulation of intracellular ROS that furthermore causes cellular death. For _P. aeruginosa_, a Gram-negative bacteria, the inactivation mechanism is similar to _E. coli_. According to [PERSON] et al. [13], a thinner peptidoglycan layer of _P. aeruginosa_, compared with Gram-positive bacteria and the presence of an outer membrane that contains proteins and lipopolysaccharidesaccharide, which are sensitive to ROS and RNS in _P. aeruginosa_, will cause cell breakage after microplasma treatment. In various research papers, the use of argon in mixtures with air or oxygen is investigated as a discharge gas for the plasma treatment of bacteria [16,26,31]. The discharge voltage is lowered when argon is used; thus, the power supply and the electrical insulation is reduced, but argon is expensive compared with air. Moreover, the use of room-temperature air is a more economical and practical solution, since it is freely available; thus, a portable micoplasma device only needs a pump to generate a gas flow. The value of the air-flow rate was set considering our previous research [16]. In this research, the value of the gas-flow rate was set at 0.6 L/min due to the effect observed at a flow rate of 2 L/min [16]. The gas flowing to the holes, as shown in Figure 2b, has to have an optimum velocity, thus transporting the active species to the target but not flowing directly to the target through the hole without taking the active species from the microplasma generated area near the grid of the electrode. Although the velocity in the gas was higher compared with this research, the area on the agar directly under the electrode had spots of bacteria growing after the treatment. These spots were facing the electrode holes; thus, we concluded that the flow was too high and the gas was flowing directly to the agar without taking active species. Thus, after trials, we reached the value of 0.6 L/min for the gas flow, which allowed the gas to gather the active species generated by microplasma and transport them to the agar surface. Besides the gas flow from the pump, the air is also directed to the target by the vortexes induced by microplasma electrode due to the electrohydrodynamic (EHD) phenomenon [47]. At a gas flow of 0.6 L/min, the calculated time for the gas to pass through the microplasma electrode's holes and cross the distance of 2 mm to reach the agar that contains bacteria was calculated to be at least 5 ms. The lifetime of the highly reactive neutral species O, OH, and NO is about 0.1 ms and about 1 ms for N, O\({}_{2}\)(a\({}^{1}\)A\({}_{\text{g}}\)), and HO\({}_{2}\) in the afterglow region [48]. According to [PERSON] et al., these reactive species could be delivered to a surface to be treated, with a gap of under 1 mm and an introduced convective flow of at least 10 m/s. Considering this, we can conclude that the inactivation of bacteria was mostly due to the long-lived neutrals such as H\({}_{2}\)O\({}_{2}\), N\({}_{2}\)O, NO\({}_{2}\), HNO\({}_{2}\), O\({}_{3}\), NO\({}_{3}\), HNO\({}_{3}\), and N\({}_{2}\)O\({}_{5}\), although the simulation results shown in [48] indicate that the interpretation of results can be affected due to a significant evolution in the reactive species density over time. To also benefit from the effect of the reactive neutral species with a shorter lifetime, the distance between microplasma and bacteria should be decreased, as [PERSON] et al. [12] reported results with a bacteria-inoculated glass coverslip in direct contact with plasma. This is possible with our microplasma electrode system, since microplasma is generated on the grounded electrode side. These types of experiments will be carried out in our future research.