## 4 Discussion  
Different discharge modes above liquids can realise reactive chemical species specificity. The impact of PAW on the reduction of bacterial and mammalian growth depends on its chemical composition. The bactericidal effects of PAW generated by SD and GD revealed that both of these PAW were more effective against _E. coli_ than _S. aureus_. Gram-positive bacteria have been reported to be more resistant than Gram-negative bacteria when exposed directly to plasma discharges [26; 27]. Comparing SD and GD PAW, for the same plasma activation times, GD PAW caused higher microbial inactivation, with spark discharge resulting in generation of hydrogen peroxide and nitrate in PAW, whereas glow discharge contained nitrite, nitrate, and other oxidative species. In the present work, the bactericidal effects are attributed to nitrites, nitrates, ROS, and acidic pH for PAW made by glow discharge. Regarding SD PAW, high concentrations of hydrogen peroxide and nitrates, and acidic pH are the main chemical species to inactivate bacteria. The existence of other chemical species, both short and long lived, in PAW and their role in bactericidal effects should not be excluded. Synergistic effects of ROS and RNS are important for bactericidal activity [16] and the chemical composition of PAW influences its antimicrobial efficacy in different ways. Studies on the bactericidal effects of PAW on Gram-negative cells showed that nitrates, nitrites, and H\({}_{2}\)O\({}_{2}\) in acidic solution can lead to lethal activity [3]. PAW caused damage to the outer and cytoplasmic membrane of _Pseudomonas_, which was accompanied by leakage of intracellular components such as nucleic acids and proteins. Plasma-induced chemical species can cause bacterial death through inhibition of the antioxidant machinery, which disrupts the membrane protein repair chaperone mechanism, and also DNA repair cascade [28]. Interaction between nitrite, nitrate, and hydrogen peroxide generated in PAL, showed strong antibacterial effects against _E. coli_[29]. In our study, _E. coli_ was reduced by up to 6 log which was attributed to effects of both ROS and RNS and/or potential synergies between reactive species, by using either of the setups. Therefore, the plasma-induced effects on living organisms are likely to be caused by several agents with multiple targets which possibly lead to synergistic inactivation effects. Further studies are required to better understand the bactericidal inactivation mechanisms of PAW. Peroxynitrite concentration in PAW was reported to have a crucial role in the microbial inactivation of _E. coli_, where despite the short half-life, this was sufficient to diffuse into cells and lead to bactericidal effects [17; 30]. In our study this conclusion could corelate with PAW made by glow discharge, which was more efficient than SD PAW and lacked hydrogen peroxide, whereas measurements with potassium iodide indicated high concentrations of ROS which could be peroxpiritrite. According to [PERSON] et al. peroxpiritrite or peroxynitrous acid as transient products could be formed at some stage of the chemical reactions in the liquid phase [31]. With regards to Gram-positive bacteria, simulation studies have shown that plasma generated chemical species such as hydrogen peroxide, atomic oxygen, and hydroxyl radicals can interact with the bacterial peptidoglycan of _S. aureus_ and lead to breakage of bonds of peptidoglycan of the cell wall, and result in its destruction [32]. Bactericidal solutions to be used as antiseptics should possess a potent activity and a long-lasting effect. According to [PERSON] et al., 20 min PAW, generated by a surface micro-discharge plasma system in air, retained its antibacterial activity for 7 days [33]. Our study is in accordance with these results as we reported that SD PAW and GD PAW plasma treated for 15 min, retained their antimicrobial activity after 1 week of storage at 4 \({}^{\circ}\)C, thus making PAW a candidate for biomedical use. Plasma treatment of a liquid can cause acidification of solution [31; 33].