## 3 Results and Discussion
### Treatment of Flowing and Circulating Liquid
To investigate the bactericidal effect of treatment of flowing liquid using AALCA, an _E. coli_ suspension with a concentration of bacteria of 10\({}^{7}\) mL\({}^{-1}\) was treated with AALCA at an input power of 28 W and a flow rate of 10 mL/min. It was found by the colony counting method that the concentration of bacteria decreased to the detection limit of 10 CFU/mL after treatment by AALCA at a flow rate of 10 mL/min, showing a 6-log reduction in the concentration of bacteria. This result demonstrates that treatment of a continuous flow of liquid by AALCA is applicable to disinfection of larger volumes of liquids. To investigate the bactericidal effect of PTW produced by treatment of flowing liquid using AALCA, DI water was treated with AALCA at an input power of 28 W and a flow rate of 10 mL/min  
Figure 10: Schematic of experimental setup for treatment of continuous flow of liquid, thermographic image of flowing DI water treated with AALCA (right, bottom), and comparison of water temperatures in tubes before and after treatment (right, top). and collected in a clean vial. Then, 0.3 mL of the _E. coli_ suspension was mixed with 2.7 mL of collected PTW and incubated for various times. After incubation, the number of surviving bacteria was estimated using the colony counting method. The estimated values of _E. coli_ after various durations of incubation are presented in Figure 9. It can be clearly observed that the concentration of bacteria gradually decreased as the incubation time increased after suspension of _E. coli_ to produce PTW, and it reached the detection limit of 10 CFU/mL at 180 min, showing a 6-log reduction in the concentration of bacteria. Considering that the concentration of _E. coli_ did not change in the control sample of _E. coli_ suspended in pristine DI water, it can be concluded that PTW produced by treatment of a continuous flow of liquid with AALCA has a strong bactericidal effect originating from the high concentration of RONS. The difference in bactericidal effect observed for the 5 mL sample and the flowing DI water can be explained by the difference in concentrations of RONS and the higher pH. In the case of flowing DI water, the concentration of NO\({}_{2}\)\({}^{-}\) was about 10 times lower and the pH was higher than the 5 mL of DI water treated by AALCA. Moreover, in the case of a flowing liquid, the concentrations of short-living reactive species could be different, resulting in a reduced bactericidal effect. We found in this work that AALCA is effective at generating RONS and features low production and operation costs, which helps to overcome a number of problems in existing systems for plasma treatment of liquids. However, a major drawback of AALCA is the heating and evaporation of the liquid. We demonstrated that these problems can be solved by treatment of a continuous flow of liquid, which avoids changes in the discharge gap by evaporation and achieves a temperature safe for the human body that is below 37 \({}^{\circ}\)C after treatment [60]. The low temperature and high concentration of RONS in PTW produced by treatment of flowing liquid looks promising for biomedical and agricultural applications. Furthermore, production of PTW using a continuous flow of liquid could be easily scaled to large volumes, which looks promising for water disinfection and purification, as well as other applications requiring low-cost treatment of large volumes of liquid. Treatment of a liquid flow using AALCA allows for the low-cost production of PTW and solves several problems associated with using a stationary solution, however, further tuning of the discharge conditions and development of a custom pumping system is necessary for precise control of RONS concentrations in the produced PTW. Additionally, the stable generation of AALCA was confirmed using liquids with various conductivities, such as DI water, tap water, ethanol, propylene glycol, phosphate buffer, phosphate saline buffer, and phosphate buffer containing amino acids.