Efficient Sterilization with Reactive Oxygen Produced By Radical Vapor Reactor (RVR)
Authors: Ishikawa Shoko , Miyori Nobuhiro , Haruyama Tetsuya 
Published: February 2020

Reactive Oxygen Species (ROS, e.g., ･OH, 1O2, O･2
-) are that the oxygen species with higher reactivity than oxygen molecules (O2) in the atmosphere. ROS provide high reactivity without residual elements except entire O2 and H2O. The advantages of ROS are practically applicable for various purposes, such as solid surface reformation and chemical oxidation. Among them effectiveness of ROS on microorganism sterilizing has been investigated. ROS produced by the macrophages in the body shows a bactericidal effect on bacteria invading from the outside. Thereby we can prevent infection and food poising. As described above, ROS are well known to have a bactericidal action. However, there are not practical ways to produce and expose ROS to objectives. The reason for this may be that the Half-life of ROS is an ephemeral time. Therefore ROS has to be produced high concentration in order to effective exposure.
Our laboratory have uniquely conducted research for ROS produce reaction locus, and innovated Radical vapor reactor (RVR) which produces ROS, especially hydroxyl radical (.OH) and singlet oxygen (1O2) with high concentration and instantly. Previous and existing ROS reactors for the production and treatment of these species have been used only in dry atmospheres or in the solution phase. However, in order to produce the hydroxyl radical and related reactive oxygen, water is required as a proton donor. It is important to use the phase interface between the gas (e.g., the plasma phase) and the solution phase (e.g., water) as a reaction locus for the reactive gas and ion(s). In the RVR, ozone was produced by electrical discharge, and the atmosphere in RVR chamber which were containing dispersed water mist and oxygen species (e.g., O2, O3) was irradiated by UV light and maintained at a specific temperature and normal pressure. This RVR reaction is an interfacial reaction between ozone phase gas and water phase with ultraviolet (UV) irradiation. In the interface, produced atomic oxygen abstract hydrogen from water and produce hydroxyl radical and flowing singlet oxygen is produced.[1] RVR can produce ROS instantly and quantitatively without any chemical regents using Radical vapor reaction. Also ROS exposure can be performed with RVR. We have developed some different application of ROS exposing reaction with RVR. It is clean and sustainable non-catalytic reaction that is widely applicable.

In this study, we have tried to employ RVR for sterilization of microorganisms. RVR sterilization was evaluated in comparison with other existing sterilizations, e.g., ozone exposure, and UV irradiation. In order to clarify an impact of ROS for sterilization, ROS concentration was assayed quantitatively by spin trap electron spin resonance (st-ESR). 2,2,5,5-tetrametyl-3-pyrroline-3-carboxamide (TPC: highly selective for 1O2) and 5,5-dimetyl-1-pyrroline N-oxide (DMPO : analysis for ･OH and other ROS) was employed as spin trap solutions. These solutions were placed in the RVR reaction chamber. After the RVR reaction by each condition, the spin trap regent solution was analyzed by ESR spectroscopy.

Escherichia coli and Bacillus subtilis were employed in order to examine the bactericidal effect of each condition. Bactericidal effect was researched by colony counting after sterilizing treatment. Survival rate was calculated by colony counting. Revealing survival rate and concentration of ROS leads to know the required time for sterilization by ROS. In the results, the RVR sterilization was successfully performed when singlet oxygen and hydroxyl radical were produced and exposed by RVR. Hydroxyl radical was produced much when UV irradiated in RVR, and it was also effective in sterilization.
Taking into account of above results, RVR sterilization, i.e. ROS sterilization, is effective to sterilize bacteria.
[1] Keishi Matsuo, Yoshiyuki Takatsuji, Masahiro Kohno, Toshiaki Kamachi, Hideco Nakata, and T. Haruyama, Dispersed-phase interfaces between mist water particles and oxygen plasma efficiently produce singlet oxygen (1O2) and hydroxyl radical (･OH), Electrochemistry, 83 (9) , 721-724 (2015) doi: http://dx.xoi.org/10.5796/electrochemistry.83.721