States of Biological Components in Bacteria and Bacteriophages during Inactivation by Atmospheric Dielectric Barrier Discharges Hachiro Yasuda, Mai Hashimoto, Md. Masudur Rahman, Kazunori Takashima, Akira Mizuno * Introduction Non-thermal atmospheric pressure plasmas have recently been applied in the biomedical field. Several types of devices for low temperature atmospheric plasma have been developed [1–7] and applied to medical research such as wound healing, [8–11] transformation, tissue engineer- ing, [12,13] and sterilization, [3,14–19] which deal with living organisms, cultured animal cells, tissue, and bacteria. For carrying forward these applications, it is very important to know the effects caused by plasma on the cells and cellular components, especially membranes, proteins, and DNA at the molecular level. [20] But the complexity of both the donor plasma source and the recipient cell components have so far made it difficult to analyze inside the cells. The dielectric barrier discharge (DBD) is a promising device for generating stable plasma at atmospheric pres- sure. The dielectric barrier inserted in between two parallel electrodes prevents arcing, and an AC voltage application generates non-thermal plasma. [3,38] We have previously reported on the sterilization of Bacillus subtilis spores using DBD at atmospheric pressure. [21] The results showed that the plasma sterilization method is accomplished at a relatively high speed for Bacillus subtilis spore compared with conventional methods. Plasma treatment with H 2 O addition showed a higher performance compared with the dry method. Taking the advantages of cell wall destruction by DBD application, [3,30] a universal DNA extraction method from the microbes, which does not use cell wall digesting enzymes, has also been proposed. [22] The expanding field of plasma application to living cells such as wound healing, transformation, and tissue engineer- ing mostly deal with wet animal cells and does not dose Full Paper H. Yasuda, M. Hashimoto, M. M. Rahman, K. Takashima, A. Mizuno Department of Ecological Engineering, Toyohashi University of Technology, Hibarigaoka, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan Fax: þ81 532 44 6904; E-mail: mizuno@eco.tut.ac.jp Atmospheric DBD has been applied to the wet state of Escherichia coli and bacteriophage-l. Upon DBD treatment, both E.coli and lphage were immediately inactivated. The states of different biological components were monitored during the course of inactivation. Only minor and slow degradation of proteins, DNA, and mem- branes was observed, a remarkable degradation was seen only after the completion of steriliza- tion. Analysis of GFP recombinantly introduced into E.coli cells proved that the DBD has a promi- nent protein denaturation activity without affect- ing peptide bonds. The irreversible denaturation of proteins, seen in the early stage of DBD appli- cation, may play a central role in inactivation of both the bacteria and bacteriophages. Plasma Process. Polym. 2008, 5, 615–621 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ppap.200800036 615