There is a growing body of literature that recognizes the importance of plasma treated water (PTW) for inactivation of microorganism. However, very little attention has been paid to the role of reactive nitrogen species (RNS) in deactivation of bacteria. The aim of this study is to explore the role of RNS in bacterial killing, and to develop a plasma system with increased sterilization efficiency. To increase the concentration of reactive oxygen and nitrogen species (RONS) in solution, we have used vapor systems (DI water/HNO3 at different wt%) combined with plasma using N2 as working gas. The results show that the addition of the vapor system yields higher RONS contents. Furthermore, PTW produced by N2 + 0.5 wt% HNO3 vapor comprises a large amount of both RNS and ROS, while PTW created by N2 + H2O vapor consists of a large amount of ROS, but much less RNS. Interestingly, we observed more deactivation of E. Coli with PTW created by N2 + 0.5 wt% HNO3 vapor plasma as compared to PTW generated by the other plasma systems. This work provides new insight into the role of RNS along with ROS for deactivation of bacteria.

Heavy water (D 2 O) is introduced into a non-thermal plasma jet (NTPJ) device to generate deuterium monoxide (OD) radicals instead of hydroxyl (OH) radicals. An NTPJ generated from a vapor mixture of N 2 /H 2 O and N 2 /D 2 O is applied to a cell membrane component and its effects are analyzed by means of 1 H NMR, GC-FID and TOF-SIMS spectroscopies. The results show that OH and OD radical species induce similar levels of oxidative breakage of lipid molecules. In addition, the 2 H NMR spectra show that deuteriums are incorporated into the lipid oxidative products. In order to trace these effects in vivo, E. coli bacteria are treated with an NTPJ and analyzed using NanoSIMS. Deuterium is observed in both the cytoplasm and membrane, which are colocalized well with nitrogen and phosphorus atoms. The high colocalization of D atoms inside E. coli provides the first direct and visual evidence of the role of OD radicals, which may be utilized to visualize OH radical interactions inside cells.