## 3 Results and Discussion
### Inactivation of E. coli  
There are numerous reports about cancer therapy, disinfection, and inactivation of fungi using PTW, and the high concentration of RONS in PTW produced by AALCA looks promising for many such applications. To confirm the bactericidal effect of direct treatment using AALCA, a suspension containing _E. coli_ in a concentration of 10\({}^{7}\) mL\({}^{-1}\) was treated with plasma. The number of surviving _E. coli_ was estimated using the colony counting method and the results are presented in Figure 8.  
It was found that the number of surviving _E. coli_ was about 2 \(\times\) 10\({}^{3}\) mL\({}^{-1}\) after direct plasma treatment for 10 s and decreased to the colony counting method detection limit of 10 mL 1 after 30 s, showing a 6-log reduction in the concentration of bacteria. As shown in Figure 8, the bactericidal effect of treatment with AALCA was significantly higher than the bactericidal effect of treatment with NEAPRS and APPJ, corona, or DBD discharges reported elsewhere [9; 43; 49; 50; 51; 52; 53; 54; 55; 56]. It was confirmed by thermographic observation that the suspension was heated by irradiation to temperatures up to 38 \({}^{\circ}\)C, which is below the temperature harmful for _E. coli_ reported elsewhere, suggesting that heating of the sample was not involved in the sterilization process [57]. On the other hand, the low pH value of about  
Figure 8: Number of surviving _E. coli_ after treatment by AALCA, NEAPRS, NEAPPJ [43], dielectric barrier discharges (DBD) [49] or corona [50], discharges for various time of irradiation. 3.4 after treatment of DI water by AALCA for 30 s could be effective for inactivation of bacteria [43]. To investigate the effect of temperature and pH on killing the bacteria, _E. coli_ was suspended for 30 s in DI water heated to 40 \({}^{\circ}\)C (to ensure that the temperature of the suspension was higher than the temperature of the sample treated by AALCA for 30 s), citrate-phosphate buffer solution with a pH of 3.3 (similar to the pH observed for DI water treated by AALCA for 30 s), and citrate-phosphate buffer solution with a pH of 3.3 heated to 40 \({}^{\circ}\)C. Then, the number of surviving bacteria was estimated using the colony counting method. It was found that the concentration of bacteria did not change after suspension for 30 s in DI water heated to 40 \({}^{\circ}\)C or citrate-phosphate buffer solution with a pH of 3.3; however, a 1-log reduction in the concentration of _E. coli_ was observed in the case of suspension in the citrate-phosphate buffer solution with a pH of 3.3 that was heated to 40 \({}^{\circ}\)C, indicating that the combination of heating the sample and reducing the pH value during AALCA treatment could contribute to observed bactericidal effect. On the other hand, the high concentration of RONS in the DI water treated by AALCA (Figure 7) could be the origin of the observed bactericidal effect. To investigate the effect of H\({}_{2}\)O\({}_{2}\), NO\({}_{2}\)\({}^{-}\), and NO\({}_{3}\)\({}^{-}\) on sterilization of the bacteria, a solution containing 7 mg/L of H\({}_{2}\)O\({}_{2}\), 8 mg/L of NO\({}_{2}\)\({}^{-}\), and 9 mg/L of NO\({}_{3}\)\({}^{-}\) (similar to the concentrations observed after treatment by AALCA for 30 s) was prepared using chemical reagents, and _E. coli_ was suspended for 30 s in the prepared solution. It was found by the colony counting method that the concentration of _E. coli_ did not change after suspension for 30 s in the solution containing H\({}_{2}\)O\({}_{2}\), NO\({}_{2}\)\({}^{-}\), and NO\({}_{3}\)\({}^{-}\), indicating that the high concentration of long-living RONS did not contribute to the strong bactericidal effect observed after 30 s of treatment.