## Results and Discussion  
### Characteristics of the Plasma-Activated Water Produced by the Soft Plasma Jet  
In this research, a nonthermal atmospheric pressure air plasma jet (soft plasma jet) was used to prepare PAW for treating _A. brasiliensis_ spores. The soft plasma jet exhibited typical output characteristics of a nonthermal atmospheric pressure plasma jet, as reported previously [22]. Because oxygen molecules could receive sufficient amounts of electrons by the plasma, they could be transformed to reactive oxygen intermediates (\(OH\), \(O_{2}^{-}\) and \(H_{2}O_{2}\)) through the \(O_{2}\)-reduction pathway: \(O_{2}\to O_{2}^{-}\to H_{2}O_{2}\to OH\to H_{2}O\)[28, 29]. When the soft plasma jet entered the aqueous solution, additional RONS could be induced from the interaction between plasma radicals and water molecules, presented in the following reactions [30, 31, 32, 33, 34, 35, 36, 37, 38]:  
\[N_{2}+e\to N+N+e\,, \tag{1}\]  
\[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]  
\[2H_{2}O+M^{+}\to H^{+}(H_{2}O)+OH+M\,, \tag{3}\]  
\[N+O_{2}\to NO+O\,, \tag{4}\]  
\[NO+O_{2}^{-}\to ONOO^{-}\,, \tag{5}\]\[NO+HO_{2}\to ONOOH, \tag{6}\]  
\[4 NO+O_{2}+2H_{2}O\to 4 NO_{2}^{-}+4H^{+}, \tag{7}\]  
\[3 NO_{2}^{-}+3H^{+}\to 2 NO+NO_{3}^{-}+H^{+}(H_{2}O). \tag{8}\]  
where \(e\) and \(e_{aq}\) are dry (non-hydrated) and wet (hydrated) electrons, respectively, \(M^{+}\) is any plasma ion with ionization energy above the \(H_{2}O\) ionization threshold, and \(H^{+}(H_{2}O)\) represents the hydrated hydrogen ion. \(O_{2}^{-}\) and \(HO_{2}\) are converted to stable end products (\(H_{2}O_{2}\)) [39; 40]. \[HO_{2}+HO_{2}\to H_{2}O_{2}+O_{2}, \tag{9}\]  
\[HO_{2}+O_{2}^{-}+H_{2}O\to H_{2}O_{2}+O_{2}+OH^{-}. \tag{10}\]  
\(O_{2}^{-}\), \(NO_{2}^{-}\), and \(NO_{3}^{-}\) anions exist in equilibrium with their conjugate acids (\(HO_{2}\), \(HNO_{2}\), and \(HNO_{3}\)), respectively [39]. \[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]0  
\[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]1  
\[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]2  
The concentrations of \(NO_{2}^{-}\), \(HNO_{2}\), \(NO_{3}^{-}\), \(HNO_{3}\), \(O_{2}^{-}\), and \(HO_{2}\) in the PAW depend on the acidity of the PAW [39]. When DI water was activated for 1, 3, 6, and 10 min by the soft plasma jet, the pH values of PAW (1 min), PAW (3 min), PAW (6 min), and PAW (10 min) were 3.53, 3.24, 3.10, and 3.01, respectively. This indicates that the acidity of PAW becomes stronger with the increase of plasma activation time. Meanwhile, \(NO\) can react with \(O_{2}^{-}\) or \(HO_{2}\) or oxygenated aqueous solution, generating peroxynitrite (\(ONOO^{-}\)) or peroxynitrous acid (\(ONOOH\)) or nitrous acid (\(HNO_{2}\)). As with oxidative stress by reactive oxygen species [41; 42], these reactive nitrogen species could also react with the biological components of cells [43]. Among the reactive species derived from the interaction of plasma radicals with an aqueous solution, hydrogen peroxide (\(H_{2}O_{2}\)) and nitric acid (\(HNO_{3}\)) are more stable when compared to free radicals such as \(OH\), \(O_{2}^{-}\), \(NO\), etc. Experimentally, the concentrations of \(H_{2}O_{2}\) in the PAW were measured using a QuantiChrom\({}^{\text{TM}}\) Peroxide Assay Kit (DIOX-250). When DI water was activated for 3 min by the soft plasma jet, the concentration of \(H_{2}O_{2}\) in the PAW was measured to be 50 uM. This confirms that reactive oxygen species are present in the PAW. The concentration of plasma-induced \(H_{2}O_{2}\) increased with longer plasma activation time, as shown in Figure 1a. Next, the concentrations of \(NO_{x}\) (\(NO_{2}^{-}\) and \(NO_{3}^{-}\)) in the PAW were measured using a QuantiChrom\({}^{\text{TM}}\) Nitric Oxide Assay Kit (DIOX-100). The concentration of \(NO_{x}\) in the PAW with a plasma activation time of 3 min was measured to be 1.8 mM. The plasma-induced \(NO_{x}\) concentration also increased with longer plasma activation time, as shown in Figure 1b. [PERSON] et al.